WO2005049072A2 - Therapeutic cancer vaccine with fragments of trag-3 - Google Patents

Therapeutic cancer vaccine with fragments of trag-3 Download PDF

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Publication number
WO2005049072A2
WO2005049072A2 PCT/DK2004/000798 DK2004000798W WO2005049072A2 WO 2005049072 A2 WO2005049072 A2 WO 2005049072A2 DK 2004000798 W DK2004000798 W DK 2004000798W WO 2005049072 A2 WO2005049072 A2 WO 2005049072A2
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seq
trag
cancer
amino acid
cells
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PCT/DK2004/000798
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French (fr)
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WO2005049072A3 (en
Inventor
Mads Hald Andersen
Per Thor Straten
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Cancer Research Technology Ltd
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Publication of WO2005049072A3 publication Critical patent/WO2005049072A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination

Definitions

  • the present invention relates to polypeptide fragments of Taxol Resistance Associ- ated Gene-3 (TRAG-3) (SEQ ID NO:1) and its splice variant TRAG-3L (SEQ ID NO:1) and its splice variant TRAG-3L (SEQ ID NO:1)
  • the present invention also relates to a therapeutic vaccine comprising one or more polypeptide fragments of TRAG-3 and/or TRAG-3L.
  • the vaccine can be used for prophylactic, ameliorating and/or curative treatment of e.g. cancers and autoimmune diseases.
  • TAA tumor-associated antigens
  • CTLs cytotoxic T lymphocytes
  • tumor antigens the expression of which is associated with resistance to chemotherapy in cancer represents a strategy for successive treatment of cancers, particularly combined with chemotherapy.
  • CT cancer/testis
  • CT antigens is a group of proteins expressed in male germline cells and certain tumor types, but not, or at least a 1000 fold less, in other healthy tissues. Because of the blood-testis barrier and the immune-privileged status of germline cells, these antigens can be considered functionally tumor-specific, and hence, represent attractive targets for immunotherapy 14 .
  • the main additional characteristics of this group are mapping to the X chromosome, the presence of multigene families, and immunogenicity in cancer patients 15 . Subsequent studies have identified additional characteristics, like heterogenous antigen expression in cancer, and association with tumor progression and the metastatic potential of cancer cells, but also exceptions to all of these characteristics 16"18 .
  • Taxol Resistance Associated Gene-3 represents one example of a CT antigen the expression of which is essential for the survival of tumor cells.
  • Taxol is a natural product derived form the bark of Taxus brevafolio, and taxanes represent one of the most important anti-tumor agents introduced in cancer chemotherapy over the past decade 19 . Taxanes inhibits microtubule depolymerization and demonstrate a broad spectrum of anti-tumor activity in various cancers including cancers of the breast, ovary, and lung 20"22 . However, despite effective initial responses for these malignancies, taxol treatment is rarely a curative intervention for the treatment of metastatic disease due to the development of taxol- resistance 23 .
  • drug resistance represents a general characteristic observed in virtually every type of tumor (with few exceptions), with every known type of anticancer chemotherapeutic drug 24 .
  • Several lines of research suggests that the development of drug resistance is associated with the expression of proteins not expressed in sensitive cells, underlining the potential of immunological targeting of proteins associated with drug resistance.
  • TRAG-3 was identified by comparing mRNA expression profiles of the taxol- sensitive ovarian cancer line SKOV3, to a taxol-resistant daughter line 25"26 . Thus, TRAG-3 was identified as a transcript associated with taxol resistance in cancer.
  • TRAG-3 encodes a 110 amino acid protein (SEQ ID NO:1) whereas the cDNA of an identified splice variant TRAG-3L encodes a protein of 127 amino acids (SEQ ID NO:2) with an insert between position 88 and 89 in the TRAG-3 amino acid sequence.
  • TRAG-3 refers to all TRAG-3 gene products, regardless of splice variants and hence, includes TRAG-3 sequences such as set out in SEQ ID NO:1 and SEQ ID NO:2 as well as TRAG-3 sequences such as described in US 6,362,321 and in WO 2003004615
  • TRAG-3 are expressed in cancers of the colon, CNS, ovary, kidney, prostate, and breast, as well as non-small-cell lung cancers, melanomas and leukemias, but not in normal non-testis tissue 25"27 .
  • TRAG-3-derived peptides have previously been characterized by means of establishing specific CTL by repeated in vitro peptide stimulation 28 .
  • the present invention demonstrates that T cells infiltrating the tumor environment or circulating in the peripheral blood of cancer patients specifically recognize TRAG-3- derived peptides.
  • TRAG-3 is on the one hand important for the resistance of the cancer cell against taxol and on the other hand a target for immunological effector cells.
  • the present invention is in one aspect directed to fragments of TRAG-3 and of
  • TRAG-3L capable of eliciting a specific T-cell response.
  • the present invention is directed to functional equivalents and variants of TRAG-3 and TRAG-3L.
  • the present invention is directed to a therapeutic vaccine comprising TRAG-3 and/or TRAG-3L, and/or one or more fragments of TRAG-3 and/or TRAG-3L capable of eliciting a specific T-cell response, including a response involving the activation of CTL and/or T helper (Th) cells.
  • the vaccine composition preferably further comprises an adjuvant and/or a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising TRAG-3 and/or TRAG-3L, and/or one or more fragments of of TRAG-3 and/or TRAG-3L capable of eliciting a specific T-cell response, including a response involving the activation of CTL and/or Th cells, and a bioactive compound selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent, and a second cancer vaccine composition.
  • the pharmaceutical composition can further comprise an adjuvant and/or a pharmaceutically acceptable carrier.
  • kits of parts comprising TRAG-3 and/or TRAG-3L, and/or one or more fragments of TRAG-3 and/or TRAG- 3L capable of eliciting a specific T-cell response, including a response involving the activation of CTL and/or Th cells, and a bioactive compound selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent, and a second cancer vaccine composition, wherein the one or more fragments of TRAG-3 and/or TRAG-3L and the bioactive compound can be administered simultaneously, or sequentially in any order.
  • the kit of parts can optionally comprise a manual comprising information on the dosage regime or the administration of the TRAG-3 fragment and the bioactive compound.
  • a method for treatment of a human or animal comprising the step of administering to an individual in need of said treatment the pharmaceutical composition or the components of the kit of parts according to the invention.
  • Said treatment can be a curative, ameliorating or prophylactic treatment.
  • a fragment of TRAG-3 in combination with a bioactive agent in the manufacture of a pharmaceutical composition for treatment of a cancer and/or an auto-immune disease in an individual in need of said treatment.
  • the treatment can be a curative, ameliorating or prophylactic treatment.
  • antibodies being capable of specific recognition of TRAG-3 fragments.
  • a method for activating and expanding T-cells specific for TRAG-3 or fragments thereof comprising the steps of co-cultivating T cells and TRAG-3, and/or at least one fragment thereof, thereby activating the T cells, and isolating activated TRAG-3-specific T cells and/or TRAG-3 fragment-specific T cells.
  • a method for treating an individual diagnosed with a cancer, or a patient at risk of developing a cancer comprising the steps of administering to said individual at least one isolated and activated TRAG-3-specific T cell, and/or at least one isolated and activated TRAG-3 fragment-specific T cell.
  • Stabilization of HLA-A*0201 by TRAG-3 derived peptides Examples of quantification of binding affinity for the TRAG-3 derived peptides.
  • X-axis represents the pep- tide concentration assayed, Y-axis the arbitrary value of number of decays for the given peptide's band.
  • T cell that has released IFN- ⁇ Due to limited patient material, it was not possible to analyze for reactivity against the full panel of peptides in all patients. The number of antigen specific spots formed per 10 5 lymphocytes is shown, with error bars marking the range. In the empty slots, no response was detected.
  • TIL Tumor infiltration lymphocytes
  • ME TIL from metastasis
  • PBL Peripheral blood lymphocytes from blood samples.
  • the C-terminal amino acid of a polypeptide of the invention exists as the free carboxylic acid, this may also be specified as "-OH".
  • the N-terminal amino acid of a polypeptide comprises a free amino-group, this may also be specified as "H-”.
  • the group comprises: Alanine (a), Cysteine (c), Aspartic acid (d), Glu- tamic acid (e), Phenylalanine (f), Glycine (g), Histidine (h), Isoleucine (i), Lysine (k), Leucine (I), Methionine (m), Asparagine (n), Proline (p), Glutamine (q), Arginine (r), Serine (s), Threonine (t), Valine (v), Tryptophan (w), and Tyrosine (y).
  • nucleic acid is meant to encompass DNA and RNA as well as derivatives thereof such as peptide nucleic acids (PNA) or locked nucleic acids (LNA) throughout the description.
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • Fragments of TRAG-3 and therapeutical vaccine compositions comprising such fragments or full length TRAG-3
  • the present invention is directed to fragments of TRAG-3 and therapeutical vaccine compositions comprising one or more fragments of TRAG-3, and a pharmaceutically acceptable carrier.
  • the vaccine composition can further comprise a carrier and/or an adjuvant compound.
  • a vaccine composition comprising full length TRAG-3 is also provided.
  • Preferred fragments of consecutive TRAG-3 amino acid residues according to the present invention can be selected from the sequence of amino acid residues of TRAG-3 as listed herein below as SEQ ID NO:1 :
  • fragments of consecutive TRAG-3 amino acid residues can also be selected from the sequences of amino acid residues of full lengthTRAG-3 as listed in SEQ ID NO:2 and SEQ ID NO:3
  • TRAG-3 refers to all TRAG-3 gene products mentioned above, regardless of splice variants.
  • fragments of TRAG-3 are tested for binding affinity to HLA molecules, elicited CTL response and/or any other antigenic properties to identify those fragments of TRAG-3 which constitute good antigens. Fragments comprising or consisting of a seguence of 9 consecutive amino acid residues of TRAG-3
  • the present invention in one preferred embodiment is directed to fragments of TRAG-3 comprising or consisting of at least 9 consecutive amino acid residues and vaccine compositions comprising fragments of TRAG-3 comprising or consisting of at least 9 consecutive amino acid residues, including the fragments of TRAG-3 as represented by SEQ ID NO:1 listed below:
  • mwmgliqlv (SEQ ID NO:3); wmgliqlve (SEQ ID NO:4); mgliqlveg (SEQ ID NO:5); gliqlvegv (SEQ ID NO:6); liqlvegvk (SEQ ID NO:7); iqlvegvkr (SEQ ID NO:8); qlvegvkrk (SEQ ID NO:9); Ivegvkrkd (SEQ ID NO:10); vegvkrkdq (SEQ ID NO:11); egvkrkdqg (SEQ ID NO:12); gvkrkdqgf (SEQ ID NO:13); vkrkdqgfl (SEQ ID NO:14); krkdqgfle (SEQ ID NO: 15); rkdqgflek (SEQ ID NO: 16); kdqgfleke (SEQ ID NO: 17); dqgf
  • tnikmhcef SEQ ID NO:30
  • nikmhcefh SEQ ID NO:31
  • ikmhcefha SEQ ID NO: 32
  • kmhcefhac SEQ ID NO:33
  • mhcefhacw SEQ ID NO:34
  • hcefhacwp SEQ ID NO:
  • SEQ ID NO:50 awrdqvdws (SEQ ID NO:51); wrdqvdwsr (SEQ ID NO:52); rdqvdwsrl (SEQ ID NO:53); dqvdwsill (SEQ ID NO:54); qvdwsillr (SEQ ID NO:55); vdwsillrd (SEQ ID NO:56); dwsillrda (SEQ ID NO:57); wsillrdag (SEQ ID NO:58); sillrdagl (SEQ ID NO:59); illrdaglv (SEQ ID NO:60); llrdaglvk (SEQ ID NO:61); Irdaglvkm (SEQ ID NO:62); rdaglvkms (SEQ ID NO:63); daglvkmsr (SEQ ID NO:64); aglvkmsrk (SEQ ID NO:65);
  • the fragment is preferably not SEQ ID NO:6, SEQ ID NO:39, SEQ ID NO:59, or SEQ ID NO:60.
  • the present invention is also directed to fragments of TRAG-3L comprising or consisting of at least 9 consecutive amino acid residues and vaccine compositions comprising of fragments of TRAG-3L comprising or consisting of at least 9 consecutive amino acid residues, including the fragments of TRAG-3L as represented by SEQ ID NO:2, which are not identical to the above, listed below:
  • nhpptpkrr (SEQ ID NO:105); hpptpkrrg (SEQ ID NO:106); pptpkrrgs (SEQ ID NO:
  • Fragments comprising or consisting of a seguence of 10 consecutive amino acid residues of TRAG-3
  • the present invention also relates to fragments of TRAG-3 comprising or consisting of at least 10 consecutive amino acid residues and vaccine compositions comprising fragments of TRAG-3 comprising or consisting of at least 10 consecutive amino acid residues.
  • the present invention is directed to fragments comprising or consisting of 10 consecutive amino acid residues of TRAG-3 as represented by SEQ ID NO:1 , such as e.g. the 10 most N-terminal amino acid residues of SEQ ID NO:1, i.e. mwmgliqlve (SEQ ID NO:130), as well as vaccine compositions comprising such fragments. Additional fragments comprising or consisting of 10 consecutive amino acid residues of TRAG-3 represented by SEQ ID NO:1 can thus, by way of example, be obtained in the following way:
  • fragments comprising or consisting of 10 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2 can be obtained.
  • the present invention is directed to such fragments comprising or consisting of 10 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2, as well as vaccine compositions comprising such fragments, including the fragments of TRAG-3L as represented by SEQ ID NO:2, which are not identical to the ones obtained above, listed below:
  • nnhpptpkrr (SEQ ID NO:131); nhpptpkrrg (SEQ ID NO:132); hpptpkrrgs (SEQ ID NO:133); pptpkrrgsg (SEQ ID NO:134); ptpkrrgsgr (SEQ ID NO:135); tpkrrgsgrh (SEQ ID NO:136); pkrrgsgrhp (SEQ ID NO:137); krrgsgrhpl (SEQ ID NO:138); rrgsgrhpln (SEQ ID NO:139); rgsgrhplnp (SEQ ID NO:140); gsgrhplnpg (SEQ ID NO:141); sgrhplnpgp (SEQ ID NO:142); grhplnpgpe (SEQ ID NO:143); rhplnpgpea (SEQ
  • Fragments comprising or consisting of a seguence of 11 consecutive amino acid residues of TRAG-3
  • the present invention also relates to fragments of TRAG-3 comprising or consisting of at least 11 consecutive amino acid residues and vaccine compositions comprising fragments of TRAG-3 comprising or consisting of at least 11 consecutive amino acid residues.
  • the present invention is directed to fragments comprising or consisting of 11 consecutive amino acid residues of TRAG-3 as represented by SEQ ID NO:1 , such as e.g. the 11 most N-terminal amino acid residues of SEQ ID NO:1 , i.e. mwmgliqlveg (SEQ ID NO: 157), as well as vaccine compositions comprising such fragments. Additional fragments comprising or consisting of 11 consecutive amino acid residues of TRAG-3 represented by SEQ ID NO:1 can thus be obtained in the following way:
  • fragments comprising or consisting of 11 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2 can be obtained.
  • the present invention is directed to such fragments comprising or consisting of 11 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2, as well as vaccine compositions comprising such fragments, including the fragments of TRAG-3L as represented by SEQ ID NO:2, which are not identical to the ones obtained above, listed below: snnhpptpkrr (SEQ ID NO: 158); nnhpptpkrrg (SEQ ID NO: 159); nhpptpkrrgs (SEQ ID NO:160); hpptpkrrgsg (SEQ ID NO:161); pptpkrrgsgr (SEQ ID NO:162); ptpkrrgsgrh (SEQ ID NO:163); tpkrrgsgrhp (SEQ ID NO:164); pkrrgsgrhpl (SEQ ID NO:165); krrgsgrhpln (S
  • Fragments comprising or consisting of a seguence of 12 consecutive amino acid residues of TRAG-3
  • the present invention also relates to fragments of TRAG-3 comprising or consisting of at least 12 consecutive amino acid residues and vaccine compositions comprising fragments of TRAG-3 comprising or consisting of at least 12 consecutive amino acid residues.
  • the present invention is directed to fragments comprising or consisting of 12 consecutive amino acid residues of TRAG-3 as represented by SEQ ID NO:1 , such as e.g. the 12 most N-terminal amino acid residues of SEQ ID NO:1 , i.e. mwmgliqlvegv (SEQ ID NO:185), as well as vaccine compositions comprising such fragments. Additional fragments comprising or consisting of 12 consecutive amino acid residues of TRAG-3 represented by SEQ ID NO:1 can thus be obtained in the following way: selecting any one of the above fragments defined by SEQ ID NO:4 to SEQ ID NO:101 (i.e.
  • fragments comprising or consisting of 12 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2 can be obtained.
  • a preferred embodiment of the present invention is directed to such fragments comprising or consisting of 12 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2, as well as vaccine compositions comprising such fragments, including the fragments of TRAG-3L as represented by SEQ ID NO:2, which are not identical to the ones obtained above, listed below:
  • Fragments comprising or consisting of a seguence of 13 consecutive amino acid residues of TRAG-3
  • the present invention also relates to fragments of TRAG-3 comprising or consisting of at least 13 consecutive amino acid residues and vaccine compositions comprising fragments of TRAG-3 comprising or consisting of at least 13 consecutive amino acid residues.
  • the present invention is directed to fragments comprising or consisting of 13 consecutive amino acid residues of TRAG-3 as represented by SEQ ID NO:1 , such as e.g. the 13 most N-terminal amino acid residues of SEQ ID N0:1 , i.e. mwmgliqlvegvk (SEQ ID NO:214), as well as vaccine compositions comprising such fragments. Additional fragments comprising or consisting of 13 consecutive amino acid residues of TRAG-3 represented by SEQ ID NO:1 can thus be obtained in the following way:
  • fragments comprising or consisting of 13 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2 can be obtained.
  • the present invention is also directed to such fragments comprising or consisting of 13 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2, as well as vaccine compositions comprising such fragments, including the fragments of TRAG-3L as represented by SEQ ID NO:2, which are not identical to the ones obtained above, listed below:
  • plsnnhpptpkrr (SEQ ID NO:215); Isnnhpptpkrrg (SEQ ID NO:216); snnhpptpkrrgs (SEQ ID NO:217); nnhpptpkrrgsg (SEQ ID NO:218); nhpptpkrrgsgr (SEQ ID NO:219); hpptpkrrgsgrh (SEQ ID NO:220); pptpkrrgsgrhp (SEQ ID NO:221); ptpkrrgsgrhpl (SEQ ID NO:222); tpkrrgsgrhpln (SEQ ID NO:223); pkrrgsgrhplnp (SEQ ID NO:224); krrgsgrhplnpg (SEQ ID NO:225); rrgsgrhplnpgp (
  • the present invention also relates to fragments of TRAG-3 comprising or consisting of at least 14 consecutive amino acid residues and vaccine compositions comprising fragments of TRAG-3 comprising or consisting of at least 14 consecutive amino acid residues.
  • the present invention is directed to fragments comprising or consisting of 14 consecutive amino acid residues of TRAG-3 as represented by SEQ ID NO:1 , such as e.g. the 14 most N-terminal amino acid residues of SEQ ID NO:1 , i.e. mwmgliqlvegvkr (SEQ ID NO:244), as well as vaccine compositions comprising such fragments. Additional fragments comprising or consisting of 14 consecutive amino acid residues of TRAG-3 represented by SEQ ID NO:1 can thus be obtained in the following way:
  • fragments comprising or consisting of 14 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2 can be obtained.
  • the present invention is directed to such fragments comprising or consisting of 14 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2, as well as vaccine compositions comprising such fragments, including the fragments of TRAG-3L as represented by SEQ ID NO:2, which are not identical to the ones obtained above, listed below: splsnnhpptpkrr (SEQ ID NO:245); plsnnhpptpkrrg (SEQ ID NO:246); Isnnhpptpkrrgs (SEQ ID NO:247); snnhpptpkrrgsg (SEQ ID NO:248); nnhpptpkrrgsgr (SEQ ID NO:249); nhpptpkrrgsgrh (SEQ ID NO:250); hpptpkrrgsgrhp (SEQ ID NO:251); pptpkrrgsgrhpl (SEQ
  • rrgsgrhplnpgpe SEQ ID NO:257); rgsgrhplnpgpea (SEQ ID NO:258); gsgrhplnpgpeal (SEQ ID NO:259); sgrhplnpgpeals (SEQ ID NO:260); grhplnpgpealsk (SEQ ID NO:261); rhplnpgpealskf (SEQ ID NO:262); hplnpgpealskfp (SEQ ID NO:263); plnpgpealskfpr (SEQ ID NO:264); Inpgpealskfprq (SEQ ID NO:265); npgpealskfprql (SEQ ID NO:266); pgpealskfprqlg (SEQ ID NO:267); gpealskfprqlgr (SEQ ID NO:268); peals
  • Fragments comprising or consisting of a seguence of 15 consecutive amino acid residues of TRAG-3
  • the present invention also relates to fragments of TRAG-3 comprising or consisting of at least 15 consecutive amino acid residues and vaccine compositions comprising fragments of TRAG-3 comprising or consisting of at least 15 consecutive amino acid residues.
  • the present invention is directed to fragments comprising or consisting of 15 consecutive amino acid residues of TRAG-3 as represented by SEQ ID NO:1 , such as e.g. the 15 most N-terminal amino acid residues of SEQ ID NO:1 , i.e. mwmgliqlvegvkrk (SEQ ID NO:275), as well as vaccine compositions comprising such fragments. Additional fragments comprising or consisting of 15 consecutive amino acid residues of TRAG-3 represented by SEQ ID NO:1 can thus be obtained in the following way:
  • fragments comprising or consisting of 15 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2 can be obtained.
  • the present invention is directed to such fragments comprising or consisting of 15 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2, as well as vaccine compositions comprising such fragments, including the fragments of TRAG-3L as represented by SEQ ID NO:2, which are not identical to the ones obtained above, listed below:
  • ssplsnnhpptpkrr (SEQ ID NO:276); splsnnhpptpkrrg (SEQ ID NO:277); plsnnhpptpkrrgs (SEQ ID NO:278); Isnnhpptpkrrgsg (SEQ ID NO:279); snnhpptpkrrgsgr (SEQ ID NO:280); nnhpptpkrrgsgrh (SEQ ID NO:281 ); nhpptpkrrgsgrhp (SEQ ID NO:282); hpptpkrrgsgrhpl (SEQ ID NO:283); pptpkrrgsgrhpln (SEQ ID NO:284); ptpkrrgsgrhplnp (SEQ ID NO:285); tpkrrgsgrhplnpg
  • N is an integer of from 4 to preferably less than 104, such as 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 (fragments comprising or consisting
  • the present invention is directed to fragments of TRAG-3 as represented by SEQ ID NO:1 comprising or consisting of more than 9 consecutive amino acid residues.
  • Such fragments can comprise for example 10 amino acid residues, such as 11 amino acid residues, for example 12 amino acid residues, such as 13 amino acid residues, for example 14 amino acid residues, such as 15 amino acid residues, for example 16 amino acid residues, such as 17 amino acid residues, for example 18 amino acid residues, such as 19 amino acid residues, for example 20 amino acid residues, such as 21 amino acid residues, for example 22 amino acid residues, such as 23 amino acid residues, for example 24 amino acid residues, such as 25 amino acid residues, for example 26 amino acid residues, such as 27 amino acid residues, for example 28 amino acid residues, such as 29 amino acid residues, for example 30 amino acid residues, such as 31 amino acid residues, for example 32 amino acid residues, such as 33 amino acid residues, for example 34 amino acid residues, such as 35 amino acid residues.
  • N-terminal amino acid residue of each of the above fragments is for example residue 1 of SEQ ID NO:1 , such as residue 2 of SEQ ID NO:1 , for ex- ample residue 3 of SEQ ID NO:1 , such as residue 4 of SEQ ID NO:1 , for example residue 5 of SEQ ID NO:1 , such as residue 6 of SEQ ID NO:1 , for example residue 7 of SEQ ID NO:1, such as residue 8 of SEQ ID NO: 1, for example residue 9 of SEQ ID NO:1 , such as residue 10 of SEQ ID NO:1 , for example residue 11 of SEQ ID NO:1 , such as residue 12 of SEQ ID NO:1 , for example residue 13 of SEQ ID NO:1 , such as residue 14 of SEQ ID NO:1 , for example residue 15 of SEQ ID NO:1 , such as residue 16 of SEQ ID NO: 1 , for example residue 17 of SEQ ID NO:1 , such as residue 18 of SEQ ID NO:1 , for example residue 19
  • SEQ ID NO:1 such as residue 48 of SEQ ID NO:1 , for example residue 49 of SEQ ID NO:1 , such as residue 50 of SEQ ID NO: 1 , for example residue 51 of SEQ ID NO:1 , such as residue 52 of SEQ ID NO:1 , for example residue 53 of SEQ ID NO:1 , such as residue 54 of SEQ ID NO:1 , for example residue 55 of SEQ ID NO:1 , such as residue 56 of SEQ ID NO:1 , for example residue 57 of SEQ ID NO:1 , such as residue 58 of SEQ ID NO:1 , for example residue 59 of SEQ ID NO:1 , such as residue 60 of SEQ ID NO:1 , for example residue 61 of SEQ ID NO:1 , such as residue 62 of SEQ ID NO:1 , for example residue 63 of SEQ ID NO:1 , such as residue 64 of SEQ ID NO:1 , for example residue 65 of SEQ ID NO:1 , such as residue 66 of SEQ
  • SEQ ID NO:1 such as residue 80 of SEQ ID NO:1 , for example residue 81 of SEQ ID NO:1 , such as residue 82 of SEQ ID NO:1 , for example residue 83 of SEQ ID NO:1 , such as residue 84 of SEQ ID NO: 1 , for example residue 85 of SEQ ID NO:1 , such as residue 86 of SEQ ID NO:1 , for example residue 87 of SEQ ID NO:1 , such as residue 88 of SEQ ID NO:1 , for example residue 89 of SEQ ID NO:1 , such as residue 90 of SEQ ID NO:1 , for example residue 91 of SEQ ID NO:1 , such as residue 92 of SEQ ID NO:1 , for example residue 93 of SEQ ID NO:1 , such as residue 94 of SEQ ID NO:1 , for example residue 95 of SEQ ID NO:1 , such as residue 96 of SEQ ID NO:1 , for example residue 97 of SEQ ID NO
  • the present invention is directed to fragments of TRAG-3L as represented by SEQ ID NO:2 comprising or consisting of more than 9 consecutive amino acid residues.
  • Such fragments can comprise for example 10 amino acid residues, such as 11 amino acid residues, for example 12 amino acid residues, such as 13 amino acid residues, for example 14 amino acid residues, such as 15 amino acid residues, for example 16 amino acid residues, such as 17 amino acid residues, for example 18 amino acid residues, such as 19 amino acid residues, for example 20 amino acid residues, such as 21 amino acid residues, for example 22 amino acid residues, such as 23 amino acid residues, for example 24 amino acid residues, such as 25 amino acid residues, for example 26 amino acid residues, such as 27 amino acid residues, for example 28 amino acid residues, such as 29 amino acid residues, for example 30 amino acid residues, such as 31 amino acid residues, for example 32 amino acid residues, such as 33 amino acid residues, for example 34 amino acid residues, such as 35 amino acid residues.
  • amino acid residues such as 83 amino acid residues, for example 84 amino acid residues, such as 85 amino acid residues, for example 86 amino acid residues, such as 87 amino acid residues, for example 88 amino acid residues, such as 89 amino acid residues, for example 90 amino acid residues, such as 91 amino acid residues, for example 92 amino acid residues, such as 93 amino acid residues, for example
  • amino acid residues such as 95 amino acid residues, for example 96 amino acid residues, such as 97 amino acid residues, for example 98 amino acid residues, such as 99 amino acid residues, for example 100 amino acid residues, such as 101 amino acid residues, for example 102 amino acid residues, such as 103 amino acid residues, for example 104 amino acid residues, such as 105 amino acid residues, for example 106 amino acid residues, such as 107 amino acid residues, for example 108 amino acid residues, such as 109 amino acid residues, for example 110 amino acid residues, such as 111 amino acid residues, for example 112 amino acid residues, such as 113 amino acid residues, for example 114 amino acid residues, such as 115 amino acid residues, for example 116 amino acid residues, such as 117 amino acid residues, for example 118 amino acid residues, such as 119 amino acid residues, for example 120 amino acid residues, such as 121 amino acid residues, for example 122 amino acid residues, such as
  • the most N-terminal amino acid residue of each of the above fragments is for example residue 1 of SEQ ID NO:2, such as residue 2 of SEQ ID NO:2, for example residue 3 of SEQ ID NO:2, such as residue 4 of SEQ ID NO:2, for example residue 5 of SEQ ID NO:2, such as residue 6 of SEQ ID NO:2, for example residue 7 of SEQ ID NO:2, such as residue 8 of SEQ ID NO:2, for example residue 9 of SEQ ID NO:2, such as residue 10 of SEQ ID NO:2, for example residue 1 1 of SEQ ID NO:2, such as residue 12 of SEQ ID NO:2, for example residue 13 of SEQ ID NO:2, such as residue 14 of SEQ ID NO:2, for example residue 15 of SEQ ID NO:2, such as residue 16 of SEQ ID NO:2, for example residue 17 of SEQ ID NO:2, such as residue 18 of SEQ ID NO:2, for example residue 19 of SEQ ID NO:2, such as residue 20 of SEQ ID NO:2, for example residue 21 of SEQ ID NO:2, such as residue 22
  • residue 50 of SEQ ID NO:2 for example residue 51 of SEQ ID NO:2, such as residue 52 of SEQ ID NO:2, for example residue 53 of SEQ ID NO:2, such as residue 54 of SEQ ID NO:2, for example residue 55 of SEQ ID NO:2, such as residue 56 of SEQ ID NO:2, for example residue 57 of SEQ ID NO:2, such as resi- due 58 of SEQ ID NO:2, for example residue 59 of SEQ ID NO:2, such as residue
  • polypeptides comprising a sequence of 9 or more consecutive amino acid residues of TRAG-3, such as the polypeptide fragments mentioned above, wherein the polypeptide comprises more than 9 amino acid residues.
  • polypeptides can comprise for example no more than 10 amino acid residues, such as no more than 11 amino acid resi- dues, for example no more than 12 amino acid residues, such as no more than 13 amino acid residues, for example no more than 14 amino acid residues, such as no more than 15 amino acid residues, for example no more than 16 amino acid residues, such as no more than 17 amino acid residues, for example no more than 18 amino acid residues, such as no more than 19 amino acid residues, for example no more than 20 amino acid residues, such as no more than 21 amino acid residues, for example no more than 22 amino acid residues, such as no more than 23 amino acid residues, for example no more than 24 amino acid residues, such as no more than 25 amino acid residues, for example no more than 30
  • TRAG-3 Much preferred fragments of TRAG-3 are listed herein below in Table 1 and characterised by their C 50 value ( ⁇ M) as the concentration of the peptide required for half maximal binding to HLA-A2. The value listed after the designation T_ (TRAG-3) or
  • T_L (TRAG-3L) indicates the position of the first amino acid in the sequence. The value listed after the . indicates the number of consecutive amino acid residues in the peptide fragments.
  • C2 is a high affinity, positive control peptide from Epstein Barr Virus (EBV).
  • the present invention thus relates to a polypeptide fragment capable of raising a specific T-cell response, said fragment comprising a peptide consisting of at least 9 consecutive amino acid residues of TRAG-3, preferably of SEQ ID NO:1 or SEQ ID NO:2, wherein said peptide is selected from the group consisting
  • NIKMHCEFHA SEQ ID NO:230
  • VLGEAWRDQV SEQ ID NO:232
  • SILLRDAGLV SEQ ID NO:233
  • ALSKFPRQL ALSKFPRQL
  • LLRDAGLVKM preferably from the group consisting of NIKMHCEFHA (SEQ ID NO:230), VLGEAWRDQV (SEQ ID NO:232), SILLRDAGLV (SEQ ID NO:233), ALSKFPRQL (SEQ ID NO:126); and wherein said polypeptide fragment consists of at the most 100 amino acids.
  • the polypeptide fragment consists of at the most 90, more preferably at the most 80, even more preferably at the most 70, yet more preferably at the most 60 even more preferably at the most 70, yet more preferably at the most 60, even more preferably at the most 50, yet more preferably at the most 40, even more preferably at the most 30, yet more preferably at the most 20, even more preferably at the most 18, yet more preferably at the most 16, even more preferably at the most 15, yet more preferably at the most 14, even more preferably at the most 13, yet more preferably at the most 12, such as at the most 11 , for example at the most 10, such as at the most 9 amino acids.
  • the polypeptide fragment comprises at least 9, such as at least 10, for example at least 11 , such as at least 12, for example at least 13, such as at least 14, for example at least 15 consecutive amino acids of TRAG-3, preferably of SEQ ID NO:1 or SEQ ID NO:2.
  • the polypeptide fragment may consist of 9, such as 10, for example 11 , such as 12, for example 13, such as 14, for example 15 consecutive amino acids of SEQ ID:1 , comprising (SEQ ID NO:230), (SEQ ID NO:232), (SEQ ID NO:233), (SEQ ID NO:126) or (SEQ ID NO:234), preferably,
  • Very preferred fragments of TRAG-3 are fragments of TRAG-3 capable of eliciting a specific T-cell response, which may be either a cytotoxic T-lymphocyte response or a helper T-cell response or both, but which preferably is a cytotoxic T-lymphocyte response.
  • a specific T-cell response which may be either a cytotoxic T-lymphocyte response or a helper T-cell response or both, but which preferably is a cytotoxic T-lymphocyte response.
  • Several different state-of- the-art methods may be used to identify peptide fragments capable of eliciting a specific T-cell response. However it is difficult to predict whether a given peptide will be capable of eliciting a specific T-cell response. Even though computer models may predict functional epitopes, experimental evidence is usually required in order to verify whether a predicted epitope indeed is a functional epitope. Even fragments that associate with MHC molecules with high affinity will not necessarily give rise to a T-cell
  • the TRAG-3 fragments are capable of association with an MHC molecule, even more preferred that the fragment is capable of association with a class I MHC molecule.
  • Preferred MHC class I molecules are frequently occuring MHC class I molecules, such as for example the MHC class I molecules described herein below.
  • Association between TRAG-3 and an MHC molecule may for example be determined using the assembly assay described herein below.
  • Preferred TRAG-3 fragments according to the invention may be characterised by having a C 50 value, measured as the concentration ( ⁇ M) of the polypeptide fragment required for half maximal binding to an MHC molecule, preferably to an MHC class I molecule, in the range of from 500 to 1000, such as in the range of 200 to 500, for example in the range of 100 to 200, such as in the range of 50 to 100, for example in the range of 25 to 50, such as in the range of 10 to 25, for example in the range of 5 to 10 such as in the range of 1 to 5, for example in range of 0,1 to 1 such as in the range of 0,05 to 0,1 , for example less than 0,05.
  • ⁇ M concentration
  • Said MHC class I molecule may be any MHC class I molecule, for example an HLA-A molecule, such as HLA-A2, for example the subtype HLA-A*0201.
  • Preferred MHC class I molecules are frequently occuring MHC class I molecules, for example the MHC class I molecules described herein below.
  • More preferred fragments of TRAG-3 includes fragments of TRAG-3 wherein the C 50 value is less than 1000 ⁇ M, even more preferably less than 200 ⁇ M, yet more preferably less than 100 ⁇ M, even more preferably less than 75 ⁇ M, yet more preferably less than 50 ⁇ M, even more preferably less than 40 ⁇ M, yet more preferably less than 31 ⁇ M, even more preferably less than 25 ⁇ M, yet more preferably less than 10 ⁇ M, even more preferably less than 5 ⁇ M, yet more preferably less than 1 ⁇ M, even more preferably less than 0,5 ⁇ M, yet more preferably less than 0,2 ⁇ M, even more preferably less than 0,1 ⁇ M, yet more preferably less than 0,05 ⁇ M, wherein the C 50 value is the concentration of the peptide required for half maximal binding to an MHC molecule, preferably to an MHC class I molecule.
  • Said MHC class I molecule may be any MHC class I molecule, such as an HLA-A molecule, for example HLA-A2, such as the subtype HLA-A*0201.
  • Preferred MHC class I molecules are frequently occuring MHC class I molecules, for example the MHC class I molecules described herein below.
  • the MHC class I molecules are selected from a group comprising the highly prevalent MHC class I alleles HLA-A2, HLA-A3, HLA-A1 , HLA-A11 , HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61 , and HLA-B62.
  • These MHC class I alleles have been reported to cover more than 95% of the Caucasian population 58 . It is preferred that the C 50 value is determined according to the assembly assay described herein below.
  • preferred fragments of TRAG-3 may be selected from the group consisting of T_57.10 (SEQ ID NO:310), TJ.102 (SEQ ID NO: 126), T_59 (SEQ ID NO:311) , and T_45 (SEQ ID NO:309). More preferred fragments of TRAG-3 may be selected from the group consisting of
  • T_59 (SEQ ID NO:311) and T_45 (SEQ ID NO:309).
  • Transporter Associated with antigen Processing (TAP) deficient cells T2 were metabolically labeled with [ 35 S]-methionine (Amersham, Freiburg, Germany). The cells were lyzed in 0.5 ml lysis buffer (150 mM NaCI, 50 mM TrisHCI, 0.5% NP-
  • M0736 was added at a final concentration of 10 ⁇ g/ml, incubated for 90 min, followed by addition of protein A-Sepharose (75 ⁇ l, 10% v/v) and incubation for 1 h. The beads were washed 4 times and stored at -20°C until analysis by electrophoresis.
  • a modified assembly can be used as described previously by us 30 .
  • B*2705 and K are unusually stable in the absence of added peptide.
  • a mild heating step was introduced in order to preferentially destabilize MHC molecules which remain empty after peptide incubation. Briefly, the cell lysates are incubated with peptide for 2 hours at 4°C, allowing the binding of peptide to empty class I molecules. Next, the cell lysates were heated (60°C for 5 min. for T2-B * 2705 or 55°C for 2 min. for T2-2K k ). Pansorbin was then added to the samples as in the conventional as- sembly assay.
  • Samples from assembly assays with allele-specific antibodies can be eluted by boiling (5 min.) in SDS reducing buffer (50 mM Tris-HCI pH 6.8, 2% SDS, 5% 2- mercaptoethanol, 10% blycerol, 2.5% bromophenol blue) and electrophorezed on 12% SDS-PAGE gels (1 hour at 200V). All gels are fixed in 10% acetic acid with 5% methanol and dried onto 3 MM paper (Whatman, Maidstone, UK). MHC heavy chain bands were quantified using the Im- agequant Phosphorimager program (Molecular Dynamics, Sunnyvale, CA, USA). The intensity of the band is directly related to the amount of peptide-bound class I MHC complex recovered during the assay.
  • C 50 value ( ⁇ M) as the concentration of the peptide required for half maximal binding to MHC may thus be determined.
  • the binding affinities of the analysed peptides are determined according to their efficiency of stabilising the HLA class I molecules.
  • the binding affinity is represented as the peptide concentration required to reach half- maximal stabilistation of appropriate HLA-molecule. Previous analyses have shown that the C 50 values measured in this assay fits well with the dissociation constant (Kd) of the complex 32 .
  • preferred TRAG-3 fragments are TRAG-3 fragments predicted to be epitopes, in particular HLA-restricted epitopes.
  • Epitope prediction may be performed using any suitable algorithm, preferably an algorithmus based on the book "MHC Ligands and Peptide Motifs" by H.G. Ram- mensee, J.Bachmanna and S.Stevanovic.
  • MHC Ligands and Peptide Motifs by H.G. Ram- mensee, J.Bachmanna and S.Stevanovic.
  • epitope prediction is not reliable, and thus it is preferred within the present invention that TRAG-3 fragments are identified using ELISPOT assays as described herein elsewhere.
  • One preferred method of predicting epitopes is using the SYFPEITHI epitope prediction algorithm. Further explanations on the algorithm can be found in HG Rammensee, J Bach- mann, NPN Emmerich, OA Bachor and S Stevanovic (1999) SYFPEITHI: database for MHC ligands and peptide motifs.
  • the algorithm calculates the predicted ligation strength to a defined HLA type for a sequence of amino acids.
  • Preferred TRAG-3 fragments has a predicted ligation strength to a given HLA molecule as calculated using said algorithm of at least 10, preferably at least 12, more preferably at least 15, even more preferably at least 16, such as at least 18, for example at least 20.
  • fragments according to the present invention are TRAG-3 fragments capable of raising a specific T-cell response as determined by an ELISPOT assay, for example the ELISPOT assay described herein below.
  • Some fragments of TRAG-3 although not binding MHC with high affinity still may give rise to a T-cell response as determined by ELISPOT.
  • Other fragments of TRAG-3 may be capable of binding MHC with high affinity and gives rise to a T-cell response as determined by ELISPOT. Both kinds of fragments are preferred fragments according to the invention.
  • preferred fragments according to the present invention are TRAG-3 fragments capable of raising a specific T-cell response as measured by an ELISPOT assay, wherein more than 50 fragment specific spots per 10 8 cells, more preferably per 10 7 cells, even more preferably per 10 6 cells, yet more preferably per 10 5 cells, for example per 10 4 cells are measured
  • preferred TRAG-3 fragments may be selected from the group consisting of T_29 (SEQ ID NO:307), T_45 (SEQ ID NO:309), T_57.10 (SEQ ID NO:310), and TJ.102 (SEQ ID NO:126), or more preferably from the group consisting of T_45 (SEQ ID NO:309), T_57.10 (SEQ ID NO:310), and TJ.102 (SEQ ID NO:126).
  • PBL peripheral blood lymphocytes
  • Peripheral blood was obtained from patients suffering from breast cancer, melanoma, and hematopoetic malignancies before vaccination and subsequent to a series of vaccinations.
  • peripheral blood lymphocytes PBL
  • ELISPOT designated direct ELISPOT
  • PBL peripheral blood lymphocytes
  • the ELISPOT assay used to quantify peptide epitope specific interferon- ⁇ releasing effector cells was adapted from Lalvani et al. 35 , and Scheibenbogen et al. 36 . Briefly, nitrocellulose bottomed 96-well plates (Multiscreen MAIP N45, Millipore) were coated overnight at room temperature with 7.5 ⁇ g/ml of anti-IFN- ⁇ antibody (1-D1 K, Mabtech, Sweden) in 75 ⁇ l sterile PBS. Subsequently, the wells were washed six times with PBS, and non-specific binding were blocked by X-vivo medium for 2 hours at 37°C.
  • the plates were washed twice and enzyme substrate (DAKO, Carpinteria; CA) was added to each well, and incubated for 5-10 min. at room temperature. The reaction was terminated by washing with tap-water upon the emergency of dark purple spots. Spots were quantitated using a computerized ELISPOT counter (Immunospot, CTL inc., CA, USA) and the peptide specific CTL frequency could be calculated as the number of spot-forming cells.
  • enzyme substrate DAKO, Carpinteria; CA
  • the ELISPOT assay is performed using PBL derived from an indi- vidual that has not previously been immunised with TRAG-3 or a fragment thereof. More preferably, said individual has not been subjected to any kind of immune therapy against a neoplastic disease. Hence it is for example preferred that the individual has not been immunised with tumor cells previously.
  • PBL from individuals that have been subjected to immune therapy, in particular immune therapy comprising TRAG-3 may give a positive result against a given peptide in an ELISPOT assay, even though PBL from a naive person would not have given a positive result.
  • preferred TRAG-3 fragments are fragments capable of activating T-cell growth in vitro.
  • preferred TRAG-3 fragments induce expansion of antigen-specific CTL using DC loaded with said fragments.
  • a method of expanding antigen-specific CTLs is described herein below.
  • very preferred TRAG-3 fragments include fragments, wherein more than 10 5 antigen specific CTLs, more preferably 10 6 , even more preferably 10 7 antigen specific CTLs may be harvested after 4 stimulation cycles starting with 10 4 PBMC as described herein below.
  • PBMC dendritic cells
  • PBMC dendritic cells
  • 85 mm dishes either bacteriological, Primaria or Tissue culture dishes, Falcon, Cat. No. 1005, 3038 or 3003; Becton Dickinson, Hershey, USA
  • RPMI 1640 Prod. Nr.
  • MCM mono- cyte conditioned medium
  • MCM was prepared in the following way: Ig coated bacteriological plates (85 mm,
  • CD8 + T lymphocytes were enriched from PBMC by depletion of CD4 + , CD11 b + , CD16 + , CD19 + , CD36 + and CD56 + cells with magnetic cell sorting using a midiMACS device (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The resulting population consisted of >90% CD8 + T cells.
  • the CD8-enriched T cells were transferred to 25-cm 2 flasks coated with anti-CD3/anti-CD28 mAbs, as described previously 38 .
  • 25-cm 2 flasks (Falcon, Heidelberg, Germany) were coated with anti-human CD3 mAb (OKT3, Ortho Pharmaceutical Corp., Raritan, NJ) and anti-human CD28 mAb (L293, Becton Dickinson) at a concentration of 1 ⁇ g/ml in PBS/100 mM HEPES buffer (pH 9). After incubation overnight at 4°C, coated flasks were washed twice with PBS.
  • CD8 + T cells were placed on the precoated and washed flasks at 5 * 10 5 cells/ml in 10 ml MCM medium supplemented with 10% human AB serum (PAA Laboratories GmbH, Coelbe, Germany) and 100 IU IL-2/ml (EuroCetus, Amsterdam, Netherlands). Cells were restimulated with anti-CD3/anti-
  • CD28 mAbs once a week, culture medium and IL-2 (100 lU/ml) was changed twice a week. Part of CD8-purified CTL were expanded by weekly restimulation with antigen-loaded DC or PBL.
  • preferred TRAG-3 fragments are fragments capable of being presented by a specific MHC molecule.
  • very preferred TRAG-3 fragments may be fragments capable of raising a specific T-cell response in an individual with a specific tissue type.
  • preferred fragments of TRAG-3 are fragments which may give rise to a specific T-cell response without leading to antibody production. Epitopes only leading to a T-cell response, but not an IgG response have been described in the prior art 39"40 . Methods of identifying fragments
  • the present invention also relates to methods of selecting a peptide comprising a fragment of TRAG-3 for use in a vaccine composition comprising the steps of
  • the T-cell response may be tested according to any suitable method, it is however preferred that testing said T-cell response comprises an ELISPOT assay.
  • ELISPOT assay is preferably the assay described herein above.
  • fragments giving rise to more than 50 peptide specific spots per 10 6 cells in an ELISPOT assay are selected.
  • the present invention in one embodiment is directed to an immunogenic composition such as a vaccine composition capable of raising e.g. a specific T-cell response.
  • the vaccine composition comprises full length TRAG-3 and/or one or more fragments thereof.
  • the vaccine compositions comprise isolated TRAG-3 and/or one or more isolated fragments thereof.
  • the terms "TRAG-3 peptide” and "TRAG-3 fragments" as used herein below refers to full length TRAG-3 and fragments thereof and to full length TRAG-3L and fragments thereof as described herein elsewhere. Any of the TRAG-3 fragments described herein above may be comprised within said vaccine, in particular the preferred TRAG-3 fragments described above may be comprised within a vaccine.
  • the vaccine compositions according to the invention may comprise more than one different TRAG-3 fragment, such as 2, for example 3, such as 4, for example 5, such as 6, for example 7, such as 8, for example 9, such as 10, such as a number of fragments in the range of from 5 to 10, for example in the range of from 10 to 15, such as in the range of from 15 to 20, for example in the range of from 20 to 30, such as in the range of from 30 to 40, for example in the range of from 40 to 60, such as in the range of from 60 to 100, for example in the range of from 100 to 200.
  • TRAG-3 fragment such as 2, for example 3, such as 4, for example 5, such as 6, for example 7, such as 8, for example 9, such as 10, such as a number of fragments in the range of from 5 to 10, for example in the range of from 10 to 15, such as in the range of from 15 to 20, for example in the range of from 20 to 30, such as in the range of from 30 to 40, for example in the range of from 40 to 60, such as in the range of from 60 to 100, for example in the
  • the different TRAG-3 fragments are selected so that one vaccine composition comprises fragments capable of associating with different MHC molecules, such as different MHC class I molecule, i.e. the TRAG-3 fragments are restricted to specific HLA's.
  • one vaccine composition comprises fragments capable of associating with the most frequently occurring MHC class I molecules.
  • preferred vaccine compositions comprises different fragments capable of associating with at least 2 preferred, more preferably at least 3 preferred, even more preferably at least 4 preferred MHC class I molecules.
  • the vaccine composition comprises one or more fragments capable of associating to an MHC class I molecule and one or more fragments capable of associating with an MHC class II molecule.
  • Peptide vaccine preparations capable of being used in accordance with the present invention may thus comprise a class l-restricted TRAG-3 peptide and/or a class II-TRAG-3 peptide and/or fusion peptides comprising both peptides.
  • a vaccine composi- tion is preferably capable of raising a specific cytotoxic T-lymphocyte response and/or a specific helper T-cell response.
  • the vaccine composition can further comprise an adjuvant and/or a carrier.
  • an adjuvant and/or a carrier examples of useful adjuvants and carriers are given herein below.
  • TRAG-3, or the fragment thereof, present in the composition can be associated with a carrier such as e.g. a protein or an antigen-presenting cell such as e.g. a dendritic cell (DC) capable of presenting TRAG-3 or a fragment thereof to a T cell.
  • a carrier such as e.g. a protein or an antigen-presenting cell such as e.g. a dendritic cell (DC) capable of presenting TRAG-3 or a fragment thereof to a T cell.
  • DC dendritic cell
  • Adjuvants are any substance whose admixture into the vaccine composition in- creases or otherwise modifies the immune response to TRAG-3 or a TRAG-3 frag- ment.
  • Carriers are scaffold structures, for example a polypeptide or a polysaccha- ride, to which TRAG-3, or the fragment thereof is capable of being associated.
  • Adjuvants could for example be selected from the group consisting of: AIK(S0 4 ) 2 , AINa(S0 4 ) 2 , AINH 4 (S0 ), silica, alum, AI(OH) 3 , Ca 3 (P0 4 ) 2 , kaolin, carbon, alumi- num hydroxide, muramyl dipeptides, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'- dipalmitoyl-sn -glycero-3-hydroxphosphoryloxy)-ethylamine (CGP 19835A
  • lipid A lipid A
  • FCA Freund's Complete Adjuvant
  • FCA Freund 's Incomplete Adjuvants
  • Merck Adjuvant 65 polynucleotides (for example, poly IC and poly AU acids), wax D from Mycobacte- rium, tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, liposomes or other lipid emulsions, Titermax, ISCOMS, Quil A, ALUN (see US 58767 and 5,554,372), Lipid A derivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrixes or GMDP, Interleukin 1 , Interleukin 2, Montanide ISA-51 and QS-21.
  • Montanide ISA-51 (Seppic, Inc.) is a mineral oil-based adjuvant analogous to incomplete Freund's adjuvant, which must be administered as an emulsion.
  • QS-21 Anti- genics; Aquila Biopharmaceuticals, Framingham, MA
  • QS-21 and Montanide ISA-51 adjuvants can be provided in sterile, single-use vials.
  • the adjuvant is a Montanide adjuvant (all available from Seppic Inc., Belgium), which may be selected from the group consist- ing of Montanide ISA-51 , Montanide ISA-50, Montanide ISA-70, Montanide ISA-206,
  • Additional preferred adjuvants capable of being used in vaccine compositions comprising TRAG-3, and/or one or more fragments thereof, are e.g. any substance which promote an immune response.
  • the adjuvant of choice is Freund's complete or incomplete adjuvant, or killed B. pertussis organisms, used e.g. in com- bination with alum precipitated antigen.
  • adjuvants A general discussion of adjuvants is provided in Goding, Monoclonal Antibodies: Principles & Practice (2nd edition, 1986) at pages 61-63. Goding notes, however, that when the antigen of interest is of low molecular weight, or is poorly immunogenic, coupling to an immunogenic carrier is recommended. Examples of such carrier molecules include keyhole limpet haemocya- nin, bovine serum albumin, ovalbumin and fowl immunoglobulin.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • cytokine a well known cytokine
  • Immunomodulafion Generally small molecules or proteins which modify the Upregulatio ⁇ of immune response. Selection of Th1 or cytokine network Th2 2. Presentation Generally amphipathic molecules or complexes which Increased neutralizing antibody response. Greater interact with immunogen in its native conformation duration of response 3. CTL induction , Particles which can bind or enclose immunogen and Cytosotic processing of protein yielding correct c'ass which can fuse with or disrupt cell membranes 1 restricted peplides , /o emulsions for direct attachment of peptide to cell Simple process if promiscuous peptidefs) known surface HC-1 4. Targeting .
  • Particulat ⁇ adjuvants which bind Immunogen.
  • Adjuvants Efficient uso of adjuvant and immunogen which saturate Kupffer cells .
  • Carbohydrate adjuvants which target leclin receptors on As above. May also determine type of response if macrophages and DCs targeting selective 5.
  • Depot generation . w/o emulsion for short term Efficiency .
  • Microspheres or nanospheres lor long term Potential for single-dose vaccine
  • a vaccine composition according to the present invention may comprise more than one different adjuvant.
  • the invention encompasses a therapeutic composition further comprising any adjuvant substance including any of the above or combinations thereof. It is also contemplated that TRAG-3, or one or more fragments thereof, and the adjuvant can be administered separately in any appropriate sequence.
  • a carrier may be present independently of an adjuvant. The function of a carrier can for example be to increase the molecular weight of in particular TRAG-3 fragments in order to increase their activity or immunogenicity, to confer stability, to increase the biological activity, or to increase serum half-life. Furthermore, a carrier may aid presenting TRAG-3, or the fragments thereof to T cells.
  • the carrier may be any suitable carrier known to the person skilled in the art, for example a protein or an antigen presenting cell.
  • a carrier protein could be but is not limited to keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid.
  • the carrier must be a physiologically acceptable carrier acceptable to humans and safe.
  • tetanus toxoid and/or diptheria toxoid are suitable carriers in one embodiment of the invention.
  • the carrier may be dextrans for example sepharose.
  • the vaccine composition may comprise dendritic cells.
  • the dendritic cells may be prepared and used in therapeutic procedure according to any suitable protocol, for example as described herein below.
  • the protocol may be adopted to use with patients with different HLA type and different diseases.
  • the peptide in the procedure described in example 3 can be any fragment of TRAG-3 such as e.g. fragments of TRAG-3 described herein, in particular preferred fragments of TRAG-3 described herein.
  • Cancer patients such as e.g. stage IV metastatic melanoma patients with progressive disease were entered into a DC-based vaccination trial e.g. after having failed to respond to chemotherapy. All patients provided informed consent to participate in the experimental vaccination and to donate blood for immunological monitoring.
  • CD25+, CD14- More than 80% of the cells expressed the CD83 antigen as marker for mature DC.
  • the peptide used in the vaccination trial were synthesized at a GMP quality by Clinalfa (purity >98%).
  • PBMC peripheral blood mononuclear cells
  • the non-adherent fractions were centrifuged and plated once more in new 85 mm tissue-culture-dishes for readherence.
  • the non-adherent fraction from these 'replate' dishes was discarded after 1hour adherence.
  • All adherent fractions were cultured until day 1 , then culture medium was taken off carefully so that loosely adherent cells were not removed, and new culture medium containing GM-CSF (800 U/ml final concentration) and IL-4 (1000 U/ml final concentration) was added. Cytokines were added again on day 3 in 3 ml fresh medium (containing 8000 U GM-CSF and 10,000 U IL-4) per dish.
  • MCM monocyte conditioned medium
  • Coating was performed with 10 ml of diluted (with PBS without calcium or magnesium, Bio Whittaker) immu- noglobulin (10 ⁇ g/ml) for 10 min at room temperature. After the coating procedure plates were rinsed twice with PBS without calcium or magnesium (Bio Whittaker). 50 ⁇ 10 6 PBMC were plated on these dishes in complete medium without cytokines and incubated at 37°C, 5% C0 2 for 20 h. Then the monocyte conditioned medium was harvested, centrifuged at 1360 g for 10 min (22°C), sterile filtered (0.22 ⁇ m fil- ters, Millipore, Molsheim, France) and frozen down in aliquots at -20°C.
  • Vaccine compositions may be prepared and administered using any conventional protocol known by a person skilled in the art. Below a non-limiting example of preparation of a vaccine composition according to the invention is given as well as a non- limiting example of adminstration of such as a vaccine. It will be appreciated by the person skilled in the art that the protocol may be easily adapted to any of the vac- cine compositions described herein.
  • TRAG-3 peptides can e.g. be synthesized e.g. at the UVA Biomolecular Core Facility with a free amide NH 2 terminus and free acid COOH terminus. Each was provided as a lyophilized peptide, which was then reconstituted in sterile water and diluted with Lactated Ringer's solution (LR, Baxter Healthcare, Deerfield, IL) as a buffer for a final concentration of 67-80% Lactated Ringer's in water. These solutions were then sterile-filtered, placed in borosilicate glass vials, and submitted to a series of quality assurance studies including confirmation of identity, sterility, general safety, and purity, in accordance with FDA guidelines, as defined in IND 6453. Tests of pep- tide stability demonstrated no decrease in purity or in the peptide concentration, when these peptide solutions were stored at -20°C for 3 years.
  • LR Lactated Ringer's solution
  • patients will receive a vaccine comprising about 100 ⁇ g of a class I HLA-restricted TRAG-3 peptide with or without a class II HLA-restricted TRAG-3 helper peptide.
  • the patients are vaccinated with e.g. about 100 ⁇ g of the class I HLA peptide in adjuvant alone, or were vaccinated with e.g. about 100 ⁇ g of the HLA class l-restricted peptide plus 190 ⁇ g of the class ll-restricted helper peptide.
  • the higher dose of the helper peptide was calculated to provide equimolar quantities of the helper and cytotoxic epitopes.
  • patients can be vacci- nated with a longer peptide comprising the amino acid sequences of both peptides.
  • the above peptides, in 1-ml aqueous solution can be administered either as a solution/suspension with about 100 ⁇ g of QS-21 , or as an emulsion with about 1 ml of Montanide ISA-51 adjuvant.
  • Patients are immunized e.g. at day 0 and months 1 , 2, 3, 6, 9, and 12, with the peptides plus adjuvant, for a total of seven immunizations. With rare exceptions, the vaccinations are administered to the same arm with each vaccine. The peptides were administered s.c.
  • preferred TRAG-3 fragments are frag- ments capable of associating with an MHC molecule. Because different MHC molecules have different affinities for a given peptide, different TRAG-3 fragments may be preferred with different embodiments of the invention.
  • the invention also relates to compositions comprising different TRAG-3 fragments, which preferably have affinity for different MHC molecules.
  • MHC molecules according to the present invention are MHC class I molecules and MHC class II molecules, more preferably MHC class I molecules.
  • Preferred MHC class I molecules are the most commonly occurring MHC class I molecules.
  • the preferred MHC class I molecules may be selected from the group consisting of HLA-A1 , HLA-A2, HLA-A3, HLA-A11 , HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B27, HLA-B35, HLA-B60, HLA-B61 , and HLA-B62.
  • compositions according to the present invention comprises at least 1 , more preferably at least 2, even more preferably at least 3, yet more preferably at least 4, for example at least 5, such as at least 6, for example 7 different TRAG-3 fragments each capable of associating with a different HLA molecule selected from the group consisting of HLA-A1 , HLA-A2, HLA-A3, HLA-A11 , HLA-A24, HLA-A68,
  • a preferred composition may comprise one TRAG-3 peptide capable of associating with HLA-A2 and a TRAG-3 peptide capable of associating with HLA-A1 and a TRAG-3 peptide capable of associating with HLA-A3 and a TRAG-3 peptide capable of associating with HLA-A24.
  • the present invention relates to a method for activating and expanding T cells specific for TRAG-3 or fragments thereof as well as to T cells obtained by such methods.
  • the methods relate to cytotoxic T lymphocytes and/or helper T cells specific for TRAG-3 fragments, more preferably the methods relate to cytotoxic T lymphocytes specific for TRAG-3 fragments.
  • the methods preferably comprise the steps of co-cultivating T cells and TRAG-3, or at least one fragment thereof, thereby activating the T cells, and optionally isolating activated TRAG-3-specific T cells or TRAG-3 fragment specific T cells.
  • Co-cultivating T cells and TRAG-3 or fragments thereof may be done by any conventional method.
  • methods involving antigen presenting cells, such as dendritic cells (DC) may be used.
  • the method may thus comprise generating and loading monocyte-derived DC with TRAG-3 fragment(s) and co-cultivating said DC and peripheral blood monocytes (PBMC) comprising T cells or T cells purified from PBMC.
  • PBMC peripheral blood monocytes
  • the TRAG-3-specific T cells may then be isolated.
  • the TRAG-3-specific T cells may then be isolated.
  • the PBMC peripheral blood monocytes
  • TRAG-3-specific T cells are cytotoxic T lymphocytes (CTL).
  • CTL cytotoxic T lymphocytes
  • the method may comprise generating Drosophila melanogaster cells expressing one or more different HLA molecules, loading said Drosophila melanogaster cells with TRAG-3 fragment(s) and co-cultivating said Drosophila cells with peripheral blood monocytes (PBMC) comprising T cells or T cells purified from PBMC.
  • PBMC peripheral blood monocytes
  • TRAG-3-specific T cells may be generated.
  • said T cells are cytotoxic T lymphocytes.
  • T cells may subsequently be isolated.
  • Drosophila melanogaster cells is that they are non-viable at 37°C.
  • Drosophila melanogaster cells were used as APCs 41 ' 42 . These cells are efficient vehicles for the presentation of peptides in the context of HLA class I, especially for de novo immunization of CD8 + CTL.
  • the Schneider S2 Drosophila cell line (American Type Culture Collection CRL 10974, Rockville, MD) was transduced with HLA- A*0201 , CD80 (B7-1) and CD54 (intracellular adhesion molecule-1) with a pRmHa-3 plasmid vector. Drosophila cells were grown in Schneider's medium (10 6 cells/mL) with 10% fetal bovine serum and CuS0 4 at 27°C, the optimal temperature for these insect cells. They were harvested, washed, and resuspended in X-press medium (Bio Whittaker, Walkersville, MD) containing 100 ⁇ g/mL of the HLA-restricted peptide epitope.
  • CD8 + T cells were obtained from peripheral blood mononuclear cells (PBMCs) by positive selection with a novel anti-CD8 monoclonal antibody (mAb) captured with a sheep anti-mouse magnetic bead (Dynal, Lake Success, NY) 43 .
  • PBMCs peripheral blood mononuclear cells
  • mAb novel anti-CD8 monoclonal antibody
  • mAb sheep anti-mouse magnetic bead
  • Drosophila cells were incubated with the CD8 + T cells at 37°C at a ratio of 1 :10 in RPMI 1640 medium containing 10% autologous serum. Two days later, 20 IU of IL-2 and 30 IU of IL-7 were added to the growth medium. Incubation was continued for 1 week, after when the Drosophila cells were replaced with autologous irradiated PBMCs (30 Gy) and the HLA-restricted peptide. This was repeated for one addi- tional round of stimulation, after when the CD8 + T cells were tested for cytotoxicity by a 4-hour [ 51 Cr]-release assay. The final preparation contained at least 92% CD8 + T cells, with 4% or less CD16 + (natural killer) cells and 4% or less CD4 + T cells.
  • the present invention also relates to methods of treating a clinical condition in an individual in need thereof, comprising (re)infusing TRAG-3-specific T cells into said individual. Furthermore the invention relates to use of TRAG-3-specific T cells for the preparation of a medicament for treatment of a clinical condition in an individual in need thereof and to medicaments for treating a clinical condition comprising TRAG-3 specific T cells as active ingredient.
  • a method for obtaining T cells from an individual and reinfusing the T cells after immunization ex vivo Leukapheresis was performed to obtain approximately 1 x 10 10 peripheral-blood mononuclear cells (PBMCs). After three rounds (3 weeks) of in vitro immunization, formal mycologic and bacteriologic testing was performed to verify sterility before the cells were administered. No Drosophila cells remained in the CTL preparation after the immunization procedure. Drosophila cells are viable at 27°C but are nonviable at 37°C. Furthermore, two rounds of immunization with changes of medium each time were performed subsequent to the initial immunization with the fly cells. Finally, the polymerase chain reaction was used in order to detect residual Drosophila DNA in the final preparation of CTL before reinfusion. Drosophila DNA was uniformly absent by this sensitive method.
  • PBMCs peripheral-blood mononuclear cells
  • the CTLs were resuspended in 200 mL of 0.9% saline with 5% human serum albumin in a transfer pack (Baxter [McGaw Park, IL] catalog no. 4R-2014 plastic blood cell infusion bag) and were administered intravenously over a period of 1 hour on the stem-cell transplantation unit.
  • a transfer pack Baxter [McGaw Park, IL] catalog no. 4R-2014 plastic blood cell infusion bag
  • experienced nurses took vital signs every 15 minutes and monitored the patients for signs of toxicity or immediate hypersensitivity reactions.
  • the present invention furthermore relates to phar- maceutical compositions and kit of parts for use in combination therapy.
  • Combination therapy denotes treatment of an individual in need thereof with more than one different method.
  • combination therapy may in one aspect involve administration of a pharmaceutical composition or a kit of parts com- prising a vaccine composition as described herein above and an anti-cancer medicament.
  • Anti-cancer medicaments may be any of the pharmaceutical compositions described herein below, for example a chemotherapeutic agent or a immunotherapeutic agent.
  • combination therapy may involve administration to an individual of a chemotherapeutic agent and/or an immunotherapeutic agent in combination with one or more of i) TRAG-3, or a fragment thereof, ii) an antigen presenting cell presenting TRAG-3, and iii) an activated, TRAG-3 peptide-specific T cell.
  • combination therapy may also involve radiation therapy, gene therapy and/or sur- gery.
  • “In combination” herein means administration or treatment simultanously or sequentially in any order in such a way as to achieve a therapeutic effect.
  • Combination therapy thus may include administration, simultaneously, or sequentially in any order, of e.g.:
  • TRAG-3 and/or fragments thereof + at least one chemotherapeutic agent ii) TRAG-3 and/or fragments thereof + at least one immunotherapeutic agent iii) Antigen presenting cell presenting TRAG-3 and/or fragments thereof + at least one chemotherapeutic agent iv) Antigen presenting cell presenting TRAG-3 and/or fragments thereof + at least one immunotherapeutic agent v) Activated T cells + at least one chemotherapeutic agent vi) Activated T cells + at least one immunotherapeutic agent vii) TRAG-3 and/or fragments thereof+another protein and/or fragments thereof
  • the chemotherapeutic agent can be e.g. methotrexate, vincristine, adriamycin, cis- platin, non-sugar containing chloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin, carmustaine and poliferposan, MM1270, BAY 12-9566, RAS famesyl transferase inhibitor, famesyl transferase inhibitor, MMP, MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470, Hycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone, Metaret/Suramin, Batimastat, E7070, BCH-4556, CS- 682
  • chemo- theraputic agent may be any of the chemotherapeutic agents mentioned in table 3 of US 6,482,843 column 13 to 18.
  • the immunotherapeutic agent can be e.g. Ributaxin, Herceptin, Quadramet,
  • the immunotherapeutic agent may be any cytokine or interferon.
  • the therapeutic compositions or vaccine compositions of the invention can also be used in combination with other anti-cancer strategies, and such combination thera- pies are effective in inhibiting and/or eliminating tumor growth and metastasis.
  • the methods of the present invention can advantageously be used with other treatment modalities, including, without limitation, radiation, surgery, gene therapy and chemotherapy.
  • “Combination therapy” can include the introduction of heterologous nucleic acids into suitable cells, generally known as gene therapy.
  • gene therapy may involve introduction of tumor suppressor genes or apoptosis promoting genes into tumor cells.
  • nucleic acid sequences inhibiting expression of oncogenes or apoptosis inhibiting genes may be introduced to tumor cells.
  • genes that encode enzymes capable of conferring to tumor cells sensitivity to chemotherapeutic agents may be introduced.
  • the present invention in one embodiment provides a method comprising the step of treating cancer by introducing a gene vector, encoding a protein capable of enzymatically converting a prodrug, i.e., a non-toxic compound, into a toxic compound.
  • the therapeutic nucleic acid sequence is a nucleic acid coding for a product, wherein the product causes cell death by itself or in the presence of other drugs.
  • a representative example of such a therapeutic nucleic acid is one, which codes for thymidine kinase of herpes simplex virus. Additional examples are thymidine kinase of varicella zoster virus and the bacterial gene cytosine deaminase, which can convert 5-fluorocytosine to the highly toxic compound 5-fluorouracil.
  • the other protein is preferably a protein, which is expressed in cancer cells, preferably a protein which is expressed at higher levels in cancer cells compared with non-malignant cells.
  • the other protein is preferably a protein involved in the regulation of apoptosis, more preferably a protein selected from the group consisting of ML-IAP, survivin, Bcl-2, Bcl-X L and Mcl-1.
  • the vaccine compositions or the therapeutic/medicaments dislcosed herein may be used to treat a number of different clinical conditions. Furthermore, the present invention relates to methods of treatment of said clinical conditions in an individual in need thereof, methods of diagnosing said clinical conditions and use of TRAG-3 or fragments thereof for preparation of a pharmaceutical composition for treatment of a clinical condition in an individual in need thereof as well as to pharmaceutical compositions for treating a clinical condition comprising TRAG-3 or fragments thereof as active ingredient.
  • the clinical condition is a cancer.
  • cancer as used herein is meant to encompass any cancer, neoplastic and preneoplastic disease.
  • Said cancer may for example be selected from the group consisting of colon carcinoma, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangeosarcoma, lymphangeoendothelia sarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas,
  • the clinical condition is a type of cancer frequently expressing TRAG-3 or a type of cancer wherein cell lines derived from said type of cancer frequently expresses TRAG-3.
  • TRAG-3 is found to be expressed in cancers of the colon, CNS, ovary, kidney, prostate, and breast, as well as non-small-cell lung cancers, melanomas, chondrosarcomas, and leukemias, but not in normal, non-testis tissue 25"27,44 . It is very much preferred however, that the clinical condition is a cancer expressing TRAG-3.
  • the clinical condition is comprised in the group of malig- nant melanoma, chondrosarcoma, non-small cell lung carcinoma, especially lung adenocarcinoma, leukemia, colon cancer, ovary cancer, kidney cancer, prostate cancer, or breast cancer.
  • the clinical condition is an auto-immune dis- ease.
  • Autoimmune diseases may be loosely grouped into those primarily restricted to specific organs or tissues and those that affect the entire body.
  • organ- specific disorders include multiple sclerosis (myelin coating on nerve processes), type I diabetes mellitus (pancreas), Hashimotos thyroiditis (thyroid gland), pernicious anemia (stomach), Addison's disease (adrenal glands), myasthenia gravis (acetylcholine receptors at neuromuscular junction), rheumatoid arthritis (joint lining), uveitis (eye), psoriasis (skin), Guillain-Barre Syndrome (nerve cells) and Grave's disease (thyroid).
  • Systemic autoimmune diseases include sys- temic lupus erythematosus and dermatomyositis.
  • hypersensitivity disorders include asthma, eczema, atopical dermatitis, contact dermatitis, other eczematous dermatitides, seborrheic dermatitis, rhinitis, Lichen planus, Pemplugus, bullous Pemphigoid, Epidermolysis bullosa, urit- caris, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Alopecia areata, atherosclerosis, primary biliary cirrhosis and nephrotic syndrome.
  • a vaccine composition or a medicament to be administered to a given individual will comprise at least one peptide capable of associating with HLA molecules of that particular individual.
  • the methods according to the present invention allows vaccination even of immu- nologically naive individuals, because the vaccine compositions according to the invention preferably comprises immunologically dominant TRAG-3 fragments.
  • the individual in need of treatment has not previously been subjected to immune therapy against a neoplastic disease.
  • the individual has not previously been sub- jected to an immune therapy that comprised immunisation with a component comprising TRAG-3 or a fragment thereof.
  • said individual has not been immunised with a tumor cell expressing TRAG-3.
  • the invention in preferred embodiments relates to pharmaceutical compositions which comprise TRAG-3 as represented by SEQ ID NO:1 and/or TRAG-3L as represented by SEQ ID NO:2 , and/or variants or fragments of these molecules as defined herein above for the treatment of pathological disorders related to or me- diated by TRAG-3.
  • compositions according to the invention can be formulated according to known methods such as by the admixture of one or more pharmaceutically or veterinary acceptable excipients or carriers. Ex- amples of such excipients, carriers and methods of formulation may be found e.g. in Remington's Pharmaceutical Sciences (Maack Publishing Co, Easton, PA).
  • a pharmaceutically or veterinary acceptable composition suitable for effective administration such compositions will contain an effective amount of a polypeptide, nucleic acid, antibody or compound modulator.
  • Therapeutic or diagnostic compositions of the invention are administered to an individual. Said individual is a vertebrate, preferably a mammal, more preferably a human. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of ad- ministration.
  • the term functional derivative includes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, eg, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans and other animals.
  • a therapeutically effective dose refers to that amount of compound, peptide, antibody or nucleic acid which ameliorate or prevent a dysfunctional apoptotic condition. The exact dosage is chosen by the individual physician in view of the patient to be treated.
  • compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular. Admini- stration of medicaments is accomplished orally or parenterally. Methods of parental delivery include topical, intra-arterial (directly to the tissue), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
  • the present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention.
  • compositions containing compounds identified according to this invention as the active ingredient for use in the modulation of a protein which is associated with resistance to chemotherapy in cancer can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration.
  • the compounds can be adminis- tered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.
  • they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
  • An effective but non-toxic amount of the compound, nucleic acid, or peptide desired can be employed as a modulating agent for resistance to chemotherapy in cancer.
  • the daily dosage of the products may be varied over a wide range from 0.001 to 1 ,000 mg per adult human/per day.
  • the compositions are preferably provided in the form of scored or unscored tablets containing 0.01 , 0.05, 0.1 , 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 100 mg/kg of body weight per day.
  • the range is more particularly from about 0.001 mg/kg to 10 mg/kg of body weight per day. Even more particularly, the range varies from about 0.05 mg/kg to about 1 mg/kg.
  • the dosage level will vary depending upon the potency of the particular compound. Certain compounds will be more potent than others. In addition, the dosage level will vary depending upon the bioavailability of the compound. The more bioavailable and potent the compound, the less compound will need to be administered through any delivery route, including but not limited to oral delivery.
  • the dosages of TRAG-3 modulators are adjusted when combined to achieve desired effects. On the other hand, dosages of these various agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors.
  • combination therapies comprising the step of administering the vaccine compositions according to the invention in combination with a chemotherapeutic agent, such as a taxane, for example taxol, and/or an immunotherapeutic agent and/or a cancer vaccine.
  • a chemotherapeutic agent such as a taxane, for example taxol
  • an immunotherapeutic agent and/or a cancer vaccine comprising the step of administering the vaccine compositions according to the invention in combination with a chemotherapeutic agent, such as a taxane, for example taxol, and/or an immunotherapeutic agent and/or a cancer vaccine.
  • the present invention is also directed to variants and functional equivalents of the above-listed fragments of TRAG-3.
  • TRAG-3 peptides with high affinity to a particular HLA molecule are listed herein below with an indication of the position in which a substitution preferably has occurred for each of the above-listed fragments.
  • the preferred amino acid residue in the respective position of the variant is indicated in the table.
  • a preferred TRAG-3 peptide variant capable of binding to HLA-B54 has a proline at the second position.
  • variants are determined on the basis of their degree of identity or their degree of homology with any predetermined sequence of consecutive amino acid sequences of a fragment of TRAG-3, such as e.g. SEQ ID ⁇ O:3 - SEQ ID NO:311.
  • variants preferably have at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85% sequence identity, for example at least 90% sequence identity, such as at least 91 % sequence identity, for example at least 91 % sequence identity, such as at least 92% sequence identity, for example at least 93% sequence identity, such as at least 94% sequence identity, for example at least 95% sequence identity, such as at least 96% sequence identity, for example at least 97% sequence identity, such as at least 98% sequence identity, for example 99% sequence identity with the predetermined TRAG-3 sequence of consecutive amino acid residues.
  • Sequence identity is determined in one embodiment by utilising fragments of peptides comprising at least 9 contiguous amino acids and having an amino acid sequence which is at least 80%, such as 85%, for example 90%, such as 95%, for example 99% identical to the amino acid sequence of any of SEQ ID NO: 3 - SEQ
  • predetermined sequence is a defined sequence used as a basis for a sequence compari- sion; a predetermined sequence may be a subset of a larger sequence.
  • Optimal alignment of sequences for aligning a comparison window may be con- ducted by the local homology algorithm of Smith and Waterman (1981 ) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lip- man (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science
  • sequence identity means that two amino acid sequences are identical over the window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of posi- tions at which identical amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparision (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • a degree of identity of amino acid sequences is a function of the number of identical amino acids at positions shared by the amino acid sequences.
  • a degree of homology or similarity of amino acid sequences is a function of the number of amino acids, i.e. structurally related, at positions shared by the amino acid sequences.
  • substantially identical means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 75 percent sequence identity, such as at least 80 percent sequence identity, for example at least 85 percent sequence identity, such as e.g.
  • amino acid residues such as 10 amino acid residues, for example 11 amino acid residues, such as 12 amino acid residues, for example 13 amino acid residues, such as 14 amino acid residues, for example 15 amino acid residues, such as 20 amino acid residues, for example 30 amino acid residues, such as 40 amino acid residues, for example 50 amino acid residues, such as 60 amino acid residues, for example 70 amino acid residues, such as 80 amino acid residues, for example 90 amino acid residues, such as 100 amino acid residues, for example 110 amino acid residues, such as 120 amino acid residues, for example 127 amino acid residues.
  • residue positions which are not identical differ by conservative amino acid substitutions.
  • Conservative amino acid substitutions refer in one embodiment to the interchange- ability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine, a group of amino acids having amide-containing side chains is asparagine and gluta- mine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine-glutamine.
  • variants are also determined based on a predetermined number of conservative amino acid substitutions as defined herein below.
  • Conservative amino acid substitution as used herein relates to the substitution of one amino acid (within a predetermined group of amino acids) for another amino acid (within the same group), wherein the amino acids exhibit similar or substantially similar characteris- tics.
  • conservative amino acid substitution as applied herein, one amino acid may be substituted for another within the groups of amino acids indicated herein below:
  • Amino acids having polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys,)
  • Amino acids having non-polar side chains (Gly, Ala, Val, Leu, lie, Phe, Trp, Pro, and Met)
  • Amino acids having aliphatic side chains (Gly, Ala Val, Leu, lie)
  • Amino acids having acidic side chains (Asp, Glu)
  • Amino acids having basic side chains (Lys, Arg, His) Amino acids having amide side chains (Asn, Gin)
  • a variant or a fragment thereof according to the invention may comprise at least one substitution, such as a plurality of substitutions introduced independ- ently of one another. It is clear from the above outline that the same variant or frag- ment thereof may comprise more than one conservative amino acid substitution from more than one group of conservative amino acids as defined herein above.
  • the addition or deletion of at least one amino acid may be an addition or deletion of from preferably 2 to 250 amino acids, such as from 10 to 20 amino acids, for example from 20 to 30 amino acids, such as from 40 to 50 amino acids.
  • additions or deletions of more than 50 amino acids such as additions from 50 to 100 amino acids, addition of 100 to 150 amino acids, addition of 150-250 amino acids, are also comprised within the present invention.
  • the deletion and/or the addition may - independently of one another - be a deletion and/or an addition within a sequence and/or at the end of a sequence.
  • polypeptide fragments according to the present invention may in one embodiment comprise a sequence of con- secutive TRAG-3 amino acid residues of less than 127 amino acid residues, such as less than 120 amino acid residues, for example less than 110 amino acid residues, such as less than 100 amino acid residues, for example less than 90 amino acid residues, such as less than 85 amino acid residues, for example less than 80 amino acid residues, such as less than 75 amino acid residues, for example less than 70 amino acid residues, such as less than 65 amino acid residues, for example less than 60 amino acid residues, such as less than 55 amino acid residues, for example less than 50 amino acid residues, such as less than 45 amino acid residues, for example less than 30 amino acid residues, such as less than 25 amino acid residues, for example less than 20 amino acid residues, such as less than 15 amino acid resi- dues, for example 14 consecutive amino acid residues, such as 13 consecutive amino acid residues, for example 12 consecutive amino acid residues, such as
  • Frctional equivalency as used in the present invention is according to one preferred embodiment established by means of reference to the corresponding functionality of a predetermined fragment of the sequence.
  • Functional equivalency can be established by e.g. similar binding affinities to HLA class I molecules, or similar potency demonstrated by ELISPOT assays.
  • Functional equivalents or variants of a TRAG-3 fragment as described herein will be understood to exhibit amino acid sequences gradually differing from preferred, predetermined sequences, as the number and scope of insertions, deletions and substi- tutions including conservative substitutions, increases. This difference is measured as a reduction in homology between a preferred, predetermined sequence and the TRAG-3 variant fragment or TRAG-3 functional equivalent.
  • TRAG-3 fragments comprising or consisting of consecutive TRAG-3 amino acid residues as well as variants and functional equivalents thereof are included within the scope of this invention, regardless of the degree of homology they show to a predetermined sequence. The reason for this is that some regions of the TRAG-3 fragments are most likely readily mutatable, or capable of being completely deleted, without any significant effect on e.g. the binding activity of the resulting fragment.
  • a functional variant obtained by substitution may well exhibit some form or degree of native binding activity, and yet be less homologous, if residues containing functionally similar amino acid side chains are substituted.
  • Functionally similar in this respect refers to dominant characteristics of the side chains such as hydrophobic, basic, neutral or acidic, or the presence or absence of steric bulk. Accordingly, in one embodiment of the invention, the degree of identity is not a principal measure of a fragment being a variant or functional equivalent of a preferred predetermined fragment according to the present invention.
  • the homology between amino acid sequences may be calculated using well known algorithms such as any one of BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, and BLOSUM 90.
  • Fragments sharing homology with fragments comprising or consisting of consecutive TRAG-3 amino acid residues are to be considered as falling within the scope of the present invention when they are preferably at least about 90 percent homologous, for example at least 92 percent homologous, such as at least 94 percent homologous, for example at least 95 percent homologous, such as at least 96 percent homologous, for example at least 97 percent homologous, such as at least 98 per- cent homologous, for example at least 99 percent homologous with a predetermined TRAG-3 fragment.
  • the homology percentages indicated above are identity percentages.
  • Additional factors that may be taken into consideration when determining functional equivalence according to the meaning used herein are i) the ability of antisera to detect a TRAG-3 fragment according to the present invention, or ii) the ability of a functionally equivalent TRAG-3 fragment to compete with a predetermined TRAG-3 fragment in an assay.
  • One method for determining a sequence of immunogenically active amino acids within a known amino acid sequence has been described by Geysen in US 5,595,915 and is incorporated herein by reference.
  • a non-conservative substitution leading to the formation of a functionally equivalent fragment would for example i) differ substantially in polarity, for example a residue with a non-polar side chain (Ala, Leu, Pro, Trp, Val, He, Leu, Phe or Met) substituted for a residue with a polar side chain such as Gly, Ser, Thr, Cys, Tyr, Asn, or Gin or a charged amino acid such as Asp, Glu, Arg, or Lys, or substituting a charged or a polar residue for a non- polar one; and/or ii) differ substantially in its effect on polypeptide backbone orientation such as substitution of or for Pro or Gly by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such as Glu or Asp for a
  • Variants obtained by substitution of amino acids may in one preferred embodiment be made based upon the hydrophobicity and hydrophilicity values and the relative similarity of the amino acid side-chain substituents, including charge, size, and the like.
  • Exemplary amino acid substitutions which take various of the foregoing charac- teristics into consideration are well known to those of skill in the art and include: ar- ginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • the present invention relates to functional variants compris- ing substituted amino acids having hydrophilic values or hydropathic indices that are within +/-4.9, for example within +/-4.7, such as within +/-4.5, for example within +/- 4.3, such as within +/-4.1 , for example within +/-3.9, such as within +/-3.7, for example within +/- 3.5, such as within +/-3.3, for example within +/- 3.1 , such as within +/- 2.9, for example within +/- 2.7, such as within +/-2.5, for example within +/- 2.3, such as within +/- 2.1 , for example within +/- 2.0, such as within +/- 1.8, for example within +/- 1.6, such as within +/- 1.5, for example within +/- 1.4, such as within +/- 1.3 for example within +/- 1.2, such as within +/- 1.1 , for example within +/- 1.0, such as within +/- 0.9, for example within within within +/
  • hydrophilic and hydropathic amino acid indices in conferring interactive biologic function on a protein is well understood in the art (Kyte & Doolit- tie, 1982 and Hopp, U.S. Pat. No. 4,554,101 , each incorporated herein by reference).
  • amino acid hydropathic index values as used herein are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methion- ine (+1.9); alanine (+1.8); glycine (-0.4 ); threonine (-0.7 ); serine (-0.8 ); tryptophan
  • amino acid hydrophilicity values are: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); praline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cys- teine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4) (U.S. 4,554,101).
  • sterically similar compounds may be formulated to mimic the key portions of the peptide structure and that such compounds may also be used in the same manner as the peptides of the invention. This may be achieved by techniques of modelling and chemical designing known to those of skill in the art. For example, esterification and other alkylations may be employed to modify the amino terminus of, e.g., a di-arginine peptide backbone, to mimic a tetra peptide structure. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
  • variants and functional equivalents of TRAG-3 also includes derivatives of TRAG-3 or fragments thereof, for example TRAG-3 or TRAG-3 fragments substituted with one or more chemical moieties.
  • Functional equivalents also comprise glycosy- lated and covalent or aggregative conjugates formed with the same or other TRAG- 3 fragments, including dimers or unrelated chemical moieties. Such functional equivalents are prepared by linkage of functionalities to groups which are found in fragment including at any one or both of the N- and C-termini, by means known in the art.
  • Functional equivalents may thus comprise TRAG-3 fragments conjugated to aliphatic or acyl esters or amides of the carboxyl terminus, alkylamines or residues containing carboxyl side chains, e.g., conjugates to alkylamines at aspartic acid residues; O-acyl derivatives of hydroxyl group-containing residues and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues, e.g. conjugates with fMet-Leu-Phe or immunogenic proteins.
  • Derivatives of the acyl groups are selected from the group of alkyl-moieties (including C3 to C10 normal alkyl), thereby forming alkanoyl species, and carbocyclic or heterocyclic compounds, thereby forming aroyl species.
  • the reactive groups preferably are difunctional compounds known per se for use in cross-linking proteins to insoluble matrices through reactive side groups.
  • Covalent or aggregative functional equivalents and derivatives thereof are useful as reagents in immunoassays or for affinity purification procedures.
  • a TRAG-3 fragment according to the present invention may be insolubilized by covalent bonding to cyanogen bromide-activated Sepharose by methods known per se or adsorbed to polyolefin surfaces, either with or without glutaraldehyde cross-linking, for use in an assay or purification of anti-TRAG-3 fragment antibodies or cell surface receptors. Fragments may also be labelled with a detectable group, e.g., radioiodi- nated by the chloramine T procedure, covalently bound to rare earth chelates or conjugated to another fluorescent moiety for use in e.g. diagnostic assays.
  • a detectable group e.g., radioiodi- nated by the chloramine T procedure, covalently bound to rare earth chelates or conjugated to another fluorescent moiety for use in e.g. diagnostic assay
  • TRAG-3 fragments according to the invention may be synthesised both in vitro and in vivo.
  • the TRAG-3 fragments of the invention are synthesised by automated synthesis. Any of the commercially available solid-phase techniques may be employed, such as the Merrifield solid phase synthesis method, in which amino acids are sequentially added to a growing amino acid chain. (See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963).
  • Solid phase synthesis will enable the incorporation of desirable amino acid substitutions into any TRAG-3 fragment according to the present invention. It will be understood that substitutions, deletions, insertions or any subcombination thereof may be combined to arrive at a final sequence of a functional equivalent. Insertions shall be understood to include amino-terminal and/or carboxyl-terminal fusions, e.g. with a hydrophobic or immu- nogenic protein or a carrier such as any polypeptide or scaffold structure capable as serving as a carrier.
  • Oligomers including dimers including homodimers and heterodimers of TRAG-3 fragments according to the invention are also provided and fall under the scope of the invention. Functional equivalents and variants of TRAG-3 fragments can be pro- prised as homodimers or heterodimers with other amino acid sequences or with native TRAG-3 sequences. Heterodimers include dimers containing immunoreactive TRAG-3 fragments as well as TRAG-3 fragments that need not have or exert any biological activity.
  • the invention also relates to nucleic acids encoding any of the polypeptide fragments described herein, as well as to said nucleic acids for use as medicaments, pharmaceutical compositions comprising said nucleic acids, vaccine compositions comprising the nucleic acids and use of the nucleic acids for the preparation of a medicament for the treatment of cancer.
  • Vaccine compositions and pharmaceutical compositions may be any of the compositions disclosed herein comprising nucleic acids in stead of or in addition to TRAG-3 polypeptide fragments.
  • the nucleic acid may be any nucleic acid, such as DNA or RNA.
  • the nucleic acids may be comprised in a suitable vector, such as an expression vector. Numerous vectors are available and the skilled person will be able to select a useful vector for the specific purpose.
  • the vector may, for example, be in the form of a plasmid, cosmid, viral particle or artificial chromosome.
  • Assembly assays for binding of the synthetic peptides to class I MHC molecules metabolically labeled with [ 35 S]-methionine were carried out as described 29 ' 45 .
  • the assembly assay is based on stabilization of the class I molecule after loading of peptide to the peptide transporter deficient cell line T2. Subsequently correctly folded stable MHC heavy chains are immunoprecipitated using conformation-dependent antibodies. After IEF electrophoresis, gels were exposed to phosphorlmager screens, and peptide binding was quantitated using the Imagequant Phosphorlmager program (Molecular Dynamics, Sunnyvale, CA).
  • PBL were stimulated once in vitro prior to analysis 33 ' 34 .
  • PBL or crushed lymph nodes were thawed and plated in 2 ml/well at a concentration of 2 x 10 6 cells in 24-well plates (Nunc, Denmark) in X-vivo medium (Bio Whittaker, Walkersville, Maryland), 5% heat-inactivated human serum, and 2 mM of L-glutamine in the presence of 10 ⁇ M of peptide.
  • IL-2 interleukin-2
  • the ELISPOT assay used to quantify peptide epitope-specific interferon- ⁇ releasing effector cells was performed as described previously 46 . Briefly, nitrocellulose bottomed 96-well plates (Multiscreen MAIP N45, Millipore, Hedehusene, Denmark) were coated with anti-IFN- ⁇ antibody (1-D1K, Mabtech, Nacka, Sweden). The wells were washed, blocked by X-vivo medium, and cells were added in duplicates at dif- ferent cell concentrations. Peptides or T2 stimulator cells were then added to each well and the plates were incubated overnight.
  • the spots were quantitated using a computerized ELISPOT counter (Immu- nospot, CTL inc., CA, USA) and the peptide specific CTL frequency could be calculated from the numbers of spot-forming cells.
  • the assays were all performed in duplicates for each peptide antigen.
  • TRAG-3 is recognized as a tumor antigen in cancer patients as TRAG-3 is subjected to T-cell responses.
  • the amino acid sequence of the TRAG-3 polypeptide was screened for conceivable HLA-A2 nonamer and HLA-A2 decamer peptide epitopes by using the main HLA-A2 specific anchor residues 47 .
  • TRAG-3 deduced peptides were synthesized and examined for binding to HLA-A2 by comparison with the HLA-A2 high affinity positive control peptide C2 (GLCTLVAML) from EBV (Table 1 ).
  • Two of the peptides addressd the insert region in the splice variant TRAG-3L (Feller et al., 2000), either completely (TJ.95 (SEQ ID NO:119)) or partially (TJ.102 (SEQ ID NO:126)).
  • the peptide concentration required for half maximal recovery of class I MHC were 0.7 ⁇ M for the positive control.
  • T_4 SEQ ID NO:6
  • T_45 SEQ ID NO:232 or 309
  • the high sensitivity of this assay allows reliable detection of as few as 10-100 specific T cells/1 million.
  • the T cells were stimulated once in vitro to extend the sensitivity. This method has previously been shown to be highly effective to identify peptide epitopes recognized by CTL in cancer patients 48"50 .
  • other ex vivo assays such as intracellular IFN- ⁇ staining or tetramer analysis by flow cytometry require approximately 10-fold higher specific T-cell frequencies for their detection.
  • TJ.102 (126), the latter overlapping the insert region of the splice variant TRAG_3L.
  • lymphocytes in this group originated from blood samples from patients with chronic lymphatic leukemia (CLL), lymphoma (LYM) or myeloma- tosis (MYE). The responding samples originated from one CLL, two LYM, and two MYE patients.
  • CLL chronic lymphatic leukemia
  • LYM lymphoma
  • MYE myeloma- tosis
  • the non-responding lymphocytes originated from two CLL, three LYM, and three MYE patients. Due to limited patient material available from the melanoma patients and the patients suffering from hematological malignancies, it was not possible to analyze for reactivity against the full panel of HLA-A*0201 bind- ing peptides in these patients.
  • the responding melanoma patient material originated from tumor infiltration lymphocytes (TIL) from lymph node metastases, in addition to PBL.
  • TIL tumor infiltration lymphocytes
  • the non-responding lymphocytes were in three cases TIL from lymph node metastases, one subcutaneous me- tastasis, one from primary tumor, and one blood sample.
  • peptide binding to class I is one in a number of different factors which determine the immunogenicity of a given peptide.
  • tolerance seems to be induced, and reactive T cells are eliminated or inactivated 54 .
  • T_58 SEQ ID NO:60
  • CTL specific for this peptide were cytolytic against HLA matched tumor targets expressing the TRAG-3 protein 28 .
  • these data support the notion of TRAG-3 derived peptides as targets for cytolytic T cells, strengthened and extended in the present study.
  • T_58 (SEQ ID NO:60) peptide is not the prominent natural target for anti-TRAG- 3 CTL responses, although it could be speculated that the CTL recognizing one of peptides T_57.9 (SEQ ID NO:59), T_57.10 (SEQ ID NO:310), and T_58 (SEQ ID NO:60) may in fact react against all three peptides. In some patients, responses were detected against two or all three of these peptides, supporting this notion ( Figure 2, patient B1 , Figure 2, patients H2-MYE, H3-LYM, and H5-LYM, and Figure 4, patients M3-PBL, and M5-PBL).
  • TRAG-3 As elevated expression of TRAG-3 in cells is associated with resistance to cancer chemotherapy and most likely also correlates with tumor progression in cancer patients, the combination of a TRAG-3-based immunotherapy with conventional cancer chemotherapy is believed to be an effective way to improve current cancer treat- ments.
  • Phosphorylated Peptides Can Be Transported by TAP Molecules, Presented by Class I MHC Molecules, and Recognized by Phosphopeptide-Specific CTL. J.Immunol., 763: 3812-3818, 1999. 46. Berke, Z., Andersen, M. H., Pedersen, M., Fugger, L., Zeuthen, J., and Haurum, J. S. Peptides spanning the junctional region of both the abl/bcr and the bcr/abl fusion polypeptides bind common HLA class I molecules. Leukemia, 14: 419- 426, 2000.
  • Keogh E., Fikes, J., Southwood, S., Celis, E., Chesnut, R., and Sette, A. Identification of new epitopes from four different tumor-associated antigens: recognition of naturally processed epitopes correlates with HLA-A*0201 -binding affinity. J.lmmunol., 767: 787-796, 2001.

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Abstract

The present invention relates to TRAG-3 peptide fragments and their use in the treatment of cancer. In particular, the disclosed fragments are useful in the preparation of vaccine compositions, which can be employed in cancer treatment. The invention also relates to TRAG-3 specific T-cells and their use in cancer treatment.

Description

Therapeutic Cancer Vaccine with Fragments of TRAG-3
All patent and non-patent references cited in the application, are hereby incorporated by reference in their entirety.
Field of the Invention
The present invention relates to polypeptide fragments of Taxol Resistance Associ- ated Gene-3 (TRAG-3) (SEQ ID NO:1) and its splice variant TRAG-3L (SEQ ID
NO:2). The present invention also relates to a therapeutic vaccine comprising one or more polypeptide fragments of TRAG-3 and/or TRAG-3L. The vaccine can be used for prophylactic, ameliorating and/or curative treatment of e.g. cancers and autoimmune diseases.
Background of the Invention
It is well established that peptide epitopes derived from human tumor-associated antigens (TAA) can be recognized by cytotoxic T lymphocytes (CTLs) in the context of MHC molecules and that most - if not all - tumors express such antigens. Consequently, exciting clinical efforts are ongoing to target these TAA in strategies such as vaccination and adoptive T-cell therapy in order to generate effective anti-tumor CTL responses in patients1.
Numerous TAA have been characterized and the peptide epitopes defined2. Some of these defined peptides have been used in peptide based vaccination trials, in particular peptides derived from melanocyte differentiation antigens have been used in vaccinations against melanoma, in some cases demonstrating remarkable re- sponse rates3"6.
Furthermore, immunoselection of antigen loss variants can be a serious obstacle for the curative potential of most of the known CTL epitopes in clinical oncology, and the selection of antigen deficient mutant tumors is a well-recognized limitation in the therapeutic strategies when targeting antigens that do not have a role in cancer growth6"8. The reason is that most characterized peptides are derived from polypep- tides, which are not essential for the survival of the tumor cell. Thus, if powerful CTL responses are induced against these peptide antigens by therapeutical measures such as vaccinations, tumor cells lacking the expression of the targeted antigen are very likely to escape the raised immune responses9"10.
There is a need for more efficient therapeutical vaccines and improved methods of treatment of cancer and autoimmune diseases.
Summary of the Invention
The application of tumor antigens the expression of which is associated with resistance to chemotherapy in cancer represents a strategy for succesful treatment of cancers, particularly combined with chemotherapy.
However, the identification of fragments of a specific length which comprise good antigenic properties is difficult.
The cancer/testis (CT) antigen family of TAA are more broadly expressed among cancers, and therapeutic vaccination against cancer with CT derived peptides have been shown to induce clinically relevant anti-tumor responses11"13.
CT antigens is a group of proteins expressed in male germline cells and certain tumor types, but not, or at least a 1000 fold less, in other healthy tissues. Because of the blood-testis barrier and the immune-privileged status of germline cells, these antigens can be considered functionally tumor-specific, and hence, represent attractive targets for immunotherapy14. The main additional characteristics of this group are mapping to the X chromosome, the presence of multigene families, and immunogenicity in cancer patients15. Subsequent studies have identified additional characteristics, like heterogenous antigen expression in cancer, and association with tumor progression and the metastatic potential of cancer cells, but also exceptions to all of these characteristics16"18.
The Taxol Resistance Associated Gene-3 (TRAG-3) represents one example of a CT antigen the expression of which is essential for the survival of tumor cells. Taxol is a natural product derived form the bark of Taxus brevafolio, and taxanes represent one of the most important anti-tumor agents introduced in cancer chemotherapy over the past decade19. Taxanes inhibits microtubule depolymerization and demonstrate a broad spectrum of anti-tumor activity in various cancers including cancers of the breast, ovary, and lung20"22. However, despite effective initial responses for these malignancies, taxol treatment is rarely a curative intervention for the treatment of metastatic disease due to the development of taxol- resistance23. In fact drug resistance represents a general characteristic observed in virtually every type of tumor (with few exceptions), with every known type of anticancer chemotherapeutic drug24. Several lines of research suggests that the development of drug resistance is associated with the expression of proteins not expressed in sensitive cells, underlining the potential of immunological targeting of proteins associated with drug resistance.
TRAG-3 was identified by comparing mRNA expression profiles of the taxol- sensitive ovarian cancer line SKOV3, to a taxol-resistant daughter line25"26. Thus, TRAG-3 was identified as a transcript associated with taxol resistance in cancer.
At least two alternatively spliced forms of TRAG-3 exist. The TRAG-3 cDNA encodes a 110 amino acid protein (SEQ ID NO:1) whereas the cDNA of an identified splice variant TRAG-3L encodes a protein of 127 amino acids (SEQ ID NO:2) with an insert between position 88 and 89 in the TRAG-3 amino acid sequence.
Additional TRAG-3 or TRAG-3L sequences with only a few amino acid substitutions to SEQ ID NO:1 and SEQ ID NO:2 are described in US 6,362,321 and in WO 2003004615.
As used herein and unless something else is stated, the term TRAG-3 refers to all TRAG-3 gene products, regardless of splice variants and hence, includes TRAG-3 sequences such as set out in SEQ ID NO:1 and SEQ ID NO:2 as well as TRAG-3 sequences such as described in US 6,362,321 and in WO 2003004615
Although there is no evidence for a direct role in the acquisition of taxol resistance, TRAG-3 are expressed in cancers of the colon, CNS, ovary, kidney, prostate, and breast, as well as non-small-cell lung cancers, melanomas and leukemias, but not in normal non-testis tissue25"27.
The broad expression among tumors suggests that exposure to taxol is not a requisite for TRAG-3 expression but probably that the expression frequencies increase with tumor progression as observed with other CT antigens24.
TRAG-3-derived peptides have previously been characterized by means of establishing specific CTL by repeated in vitro peptide stimulation28.
The present invention demonstrates that T cells infiltrating the tumor environment or circulating in the peripheral blood of cancer patients specifically recognize TRAG-3- derived peptides. Thus TRAG-3 is on the one hand important for the resistance of the cancer cell against taxol and on the other hand a target for immunological effector cells.
The present invention is in one aspect directed to fragments of TRAG-3 and of
TRAG-3L capable of eliciting a specific T-cell response.
In a further aspect the present invention is directed to functional equivalents and variants of TRAG-3 and TRAG-3L.
In another preferred aspect the present invention is directed to a therapeutic vaccine comprising TRAG-3 and/or TRAG-3L, and/or one or more fragments of TRAG-3 and/or TRAG-3L capable of eliciting a specific T-cell response, including a response involving the activation of CTL and/or T helper (Th) cells. The vaccine composition preferably further comprises an adjuvant and/or a pharmaceutically acceptable carrier.
In yet another aspect there is provided a pharmaceutical composition comprising TRAG-3 and/or TRAG-3L, and/or one or more fragments of of TRAG-3 and/or TRAG-3L capable of eliciting a specific T-cell response, including a response involving the activation of CTL and/or Th cells, and a bioactive compound selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent, and a second cancer vaccine composition. The pharmaceutical composition can further comprise an adjuvant and/or a pharmaceutically acceptable carrier. In a still further aspect of the invention there is provided a kit of parts comprising TRAG-3 and/or TRAG-3L, and/or one or more fragments of TRAG-3 and/or TRAG- 3L capable of eliciting a specific T-cell response, including a response involving the activation of CTL and/or Th cells, and a bioactive compound selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent, and a second cancer vaccine composition, wherein the one or more fragments of TRAG-3 and/or TRAG-3L and the bioactive compound can be administered simultaneously, or sequentially in any order. The kit of parts can optionally comprise a manual comprising information on the dosage regime or the administration of the TRAG-3 fragment and the bioactive compound.
In yet another aspect of the invention there is provided a method for treatment of a human or animal, wherein said method comprises the step of administering to an individual in need of said treatment the pharmaceutical composition or the components of the kit of parts according to the invention. Said treatment can be a curative, ameliorating or prophylactic treatment.
In a still further aspect of the invention there is provided the use of a fragment of TRAG-3 in combination with a bioactive agent in the manufacture of a pharmaceutical composition for treatment of a cancer and/or an auto-immune disease in an individual in need of said treatment. The treatment can be a curative, ameliorating or prophylactic treatment.
In yet another aspect of the invention there is provided antibodies being capable of specific recognition of TRAG-3 fragments.
In a still further aspect of the invention there is provided a method for activating and expanding T-cells specific for TRAG-3 or fragments thereof, said method comprising the steps of co-cultivating T cells and TRAG-3, and/or at least one fragment thereof, thereby activating the T cells, and isolating activated TRAG-3-specific T cells and/or TRAG-3 fragment-specific T cells.
In yet another aspect of the invention there is provided a method for treating an individual diagnosed with a cancer, or a patient at risk of developing a cancer, said method comprising the steps of administering to said individual at least one isolated and activated TRAG-3-specific T cell, and/or at least one isolated and activated TRAG-3 fragment-specific T cell.
Brief Description of the Figures
Figure 1
Stabilization of HLA-A*0201 by TRAG-3 derived peptides. Examples of quantification of binding affinity for the TRAG-3 derived peptides. X-axis represents the pep- tide concentration assayed, Y-axis the arbitrary value of number of decays for the given peptide's band.
Figure 2
Spontaneous immune responses against derived peptides in HLA-A2 positive breast cancer patients. Response was measured in ELISPOT, where IFN-γ released by T cells upon TCR stimulation is captured by well-coated primary anti-IFN-γ mAB.
Addition of secondary mAB and substrate leads to formation of a small spot for each
T cell that has released IFN-γ. Due to limited patient material, it was not possible to analyze for reactivity against the full panel of peptides in all patients. The number of antigen specific spots formed per 105 lymphocytes is shown, with error bars marking the range. In the empty slots, no response was detected.
Figure 3
Spontaneous immune responses against derived peptides in HLA-A2 positive hema- topoietic patients. The values represent the number of antigen specific spots per 105 lymphocytes, with error bars marking the range. In the empty slots, no response was detected. CLL: Chronical lymphomo leukemia, MYE: Myelomatose, LYM: Lym- phoma.
Figure 4
Spontaneous immune responses against TRAG-3-derived peptides in HLA-A2 positive melanoma patients. The values represent the number of antigen specific spots per 105 lymphocytes, with error bars marking the range. In the empty slots, no response was detected. Antigen-specific responses for patient M3 were tested in singlets against negative controls in dublets. LN: Tumor infiltration lymphocytes (TIL) from lymph node invading tumor, ME: TIL from metastasis, PBL: Peripheral blood lymphocytes from blood samples.
Detailed description of the invention
Amino acids
Throughout the description and claims the one letter code for natural amino acids are used. Where the L or D form has not been specified it is to be understood that the amino acid in question has the natural L form, cf. Pure & Appl. Chem. Vol. (56(5) pp 595-624 (1984) or the D form, so that the peptides formed may be constituted of amino acids of L form, D form, or a sequence of mixed L forms and D forms.
Where nothing is specified it is to be understood that the C-terminal amino acid of a polypeptide of the invention exists as the free carboxylic acid, this may also be specified as "-OH". The N-terminal amino acid of a polypeptide comprises a free amino-group, this may also be specified as "H-".
Accordingly, the group comprises: Alanine (a), Cysteine (c), Aspartic acid (d), Glu- tamic acid (e), Phenylalanine (f), Glycine (g), Histidine (h), Isoleucine (i), Lysine (k), Leucine (I), Methionine (m), Asparagine (n), Proline (p), Glutamine (q), Arginine (r), Serine (s), Threonine (t), Valine (v), Tryptophan (w), and Tyrosine (y).
The term "nucleic acid" is meant to encompass DNA and RNA as well as derivatives thereof such as peptide nucleic acids (PNA) or locked nucleic acids (LNA) throughout the description.
Preferred embodiments of the present invention is disclosed herein below.
Fragments of TRAG-3 and therapeutical vaccine compositions comprising such fragments or full length TRAG-3
In preferred embodiments the present invention is directed to fragments of TRAG-3 and therapeutical vaccine compositions comprising one or more fragments of TRAG-3, and a pharmaceutically acceptable carrier. The vaccine composition can further comprise a carrier and/or an adjuvant compound. A vaccine composition comprising full length TRAG-3 is also provided.
Preferred fragments of consecutive TRAG-3 amino acid residues according to the present invention can be selected from the sequence of amino acid residues of TRAG-3 as listed herein below as SEQ ID NO:1 :
mwmgliqlve gvkrkdqgfl ekefyhktni kmhcefhacw paftvlgeaw rdqvdwsill rdaglvkmsr kprassplsn nhpptpkrfp rqlgrekgpi eevpgtkgsp (SEQ ID NO:1).
and from the sequences of amino acid residues of the splice variant TRAG-3L as listed herein below as SEQ ID NO:2:
mwmgliqlve gvkrkdqgfl ekefyhktni kmhcefhacw paftvlgeaw rdqvdwsill rdaglvkmsr kprassplsn nhpptpkrrg sgrhplnpgp ealskfprql grekgpieev pgtkgsp (SEQ ID NO:2).
According to one preferred embodiment of the present invention fragments of consecutive TRAG-3 amino acid residues can also be selected from the sequences of amino acid residues of full lengthTRAG-3 as listed in SEQ ID NO:2 and SEQ ID
NO:4 of patent application US 6,362,321 and SEQ ID NO:25 of patent application WO2003004615.
As used herein and unless something else is stated, the term TRAG-3 refers to all TRAG-3 gene products mentioned above, regardless of splice variants.
It is not to be readily predicted which fragments of TRAG-3 will constitute good antigens or efficient targets for spontaneous CTL responses. Thus, it is critical to, and within the scope of, the present invention that the fragments of TRAG-3 are tested for binding affinity to HLA molecules, elicited CTL response and/or any other antigenic properties to identify those fragments of TRAG-3 which constitute good antigens. Fragments comprising or consisting of a seguence of 9 consecutive amino acid residues of TRAG-3
The present invention in one preferred embodiment is directed to fragments of TRAG-3 comprising or consisting of at least 9 consecutive amino acid residues and vaccine compositions comprising fragments of TRAG-3 comprising or consisting of at least 9 consecutive amino acid residues, including the fragments of TRAG-3 as represented by SEQ ID NO:1 listed below:
mwmgliqlv (SEQ ID NO:3); wmgliqlve (SEQ ID NO:4); mgliqlveg (SEQ ID NO:5); gliqlvegv (SEQ ID NO:6); liqlvegvk (SEQ ID NO:7); iqlvegvkr (SEQ ID NO:8); qlvegvkrk (SEQ ID NO:9); Ivegvkrkd (SEQ ID NO:10); vegvkrkdq (SEQ ID NO:11); egvkrkdqg (SEQ ID NO:12); gvkrkdqgf (SEQ ID NO:13); vkrkdqgfl (SEQ ID NO:14); krkdqgfle (SEQ ID NO: 15); rkdqgflek (SEQ ID NO: 16); kdqgfleke (SEQ ID NO: 17); dqgflekef (SEQ ID NO:18); qgflekefy (SEQ ID NO:19); gflekefyh (SEQ ID NO:20); flekefyhk (SEQ ID NO:21); lekefyhkt (SEQ ID NO:22); ekefyhktn (SEQ ID NO:23); kefyhktni (SEQ ID NO:24); efyhktnik (SEQ ID NO:25); fyhktnikm (SEQ ID NO:26); yhktnikmh (SEQ ID NO:27); hktnikmhc (SEQ ID NO:28); ktnikmhce (SEQ ID NO:29)
; tnikmhcef (SEQ ID NO:30); nikmhcefh (SEQ ID NO:31); ikmhcefha (SEQ ID NO: 32); kmhcefhac (SEQ ID NO:33); mhcefhacw (SEQ ID NO:34); hcefhacwp (SEQ ID
NO:35); cefhacwpa (SEQ ID NO:36); efhacwpaf (SEQ ID NO:37); fhacwpaft (SEQ
ID NO:38); hacwpaftv (SEQ ID NO:39); acwpaftvl (SEQ ID NO:40); cwpaftvlg (SEQ
ID NO:41); wpaftvlge (SEQ ID NO:42); paftvlgea (SEQ ID NO:43); aftvlgeaw (SEQ
ID NO:44); ftvlgeawr (SEQ ID NO:45); tvlgeawrd (SEQ ID NO:46); vlgeawrdq (SEQ ID NO:47); Igeawrdqv (SEQ ID NO:48); geawrdqvd (SEQ ID NO:49); eawrdqvdw
(SEQ ID NO:50); awrdqvdws (SEQ ID NO:51); wrdqvdwsr (SEQ ID NO:52); rdqvdwsrl (SEQ ID NO:53); dqvdwsill (SEQ ID NO:54); qvdwsillr (SEQ ID NO:55); vdwsillrd (SEQ ID NO:56); dwsillrda (SEQ ID NO:57); wsillrdag (SEQ ID NO:58); sillrdagl (SEQ ID NO:59); illrdaglv (SEQ ID NO:60); llrdaglvk (SEQ ID NO:61); Irdaglvkm (SEQ ID NO:62); rdaglvkms (SEQ ID NO:63); daglvkmsr (SEQ ID NO:64); aglvkmsrk (SEQ ID NO:65); glvkmsrkp (SEQ ID NO:66); Ivkmsrkpr (SEQ ID NO:67); vkmsrkpra (SEQ ID NO:68); kmsrkpras (SEQ ID NO:69); msrkprass (SEQ ID NO:
70); srkprassp (SEQ ID NO:71); rkprasspl (SEQ ID NO:72); kprasspls (SEQ ID NO:
73); prassplsn (SEQ ID NO:74); rassplsnn (SEQ ID NO:75); assplsnnh (SEQ ID NO: 76); ssplsnnhp (SEQ ID NO:77); splsnnhpp (SEQ ID NO:78); plsnnhppt (SEQ ID NO:79); Isnnhpptp (SEQ ID NO:80); snnhpptpk (SEQ ID NO:81); nnhpptpkr (SEQ ID NO:82); nhpptpkrf (SEQ ID NO:83); hpptpkrfp (SEQ ID NO:84); pptpkrfpr (SEQ ID NO:85); ptpkrfprq (SEQ ID NO:86); tpkrfprql (SEQ ID NO:87); pkrfprqlg (SEQ ID NO:88); krfprqlgr (SEQ ID NO:89); rfprqlgre (SEQ ID NO:90); fprqlgrek (SEQ ID NO: 91); prqlgrekg (SEQ ID NO:92); rqlgrekgp (SEQ ID NO:93); qlgrekgpi (SEQ ID NO:
94); Igrekgpie (SEQ ID NO:95); grekgpiee (SEQ ID NO:96); rekgpieev (SEQ ID NO: 97); ekgpieevp (SEQ ID NO:98); kgpieevpg (SEQ ID NO:99); gpieevpgt (SEQ ID NO:100); pieevpgtk (SEQ ID NO:101); ieevpgtkg (SEQ ID NO:102); eevpgtkgs (SEQ ID NO:103); evpgtkgsp (SEQ ID NO:104).
In one embodiment of the present invention the fragment is preferably not SEQ ID NO:6, SEQ ID NO:39, SEQ ID NO:59, or SEQ ID NO:60.
The present invention is also directed to fragments of TRAG-3L comprising or consisting of at least 9 consecutive amino acid residues and vaccine compositions comprising of fragments of TRAG-3L comprising or consisting of at least 9 consecutive amino acid residues, including the fragments of TRAG-3L as represented by SEQ ID NO:2, which are not identical to the above, listed below:
nhpptpkrr (SEQ ID NO:105); hpptpkrrg (SEQ ID NO:106); pptpkrrgs (SEQ ID NO:
107); ptpkrrgsg (SEQ ID NO:108); tpkrrgsgr (SEQ ID NO:109); pkrrgsgrh (SEQ ID NO:110); krrgsgrhp (SEQ ID NO:111); rrgsgrhpl (SEQ ID NO:112); rgsgrhpln (SEQ ID NO:113); gsgrhplnp (SEQ ID NO:114); sgrhplnpg (SEQ ID NO:115); grhplnpgp (SEQ ID NO:116); rhplnpgpe (SEQ ID NO:117); hplnpgpea (SEQ ID NO:118); plnpgpeal (SEQ ID NO:119); Inpgpeals (SEQ ID NO:120); npgpealsk (SEQ ID NO:
121); pgpealskf (SEQ ID NO:122); gpealskfp (SEQ ID NO:123); pealskfpr (SEQ ID NO:124); ealskfprq (SEQ ID NO:125); alskfprql (SEQ ID NO: 126); Iskfprqlg (SEQ ID NO:127); skfprqlgr (SEQ ID NO:128); kfprqlgre (SEQ ID NO:129).
Fragments comprising or consisting of a seguence of 10 consecutive amino acid residues of TRAG-3
The present invention also relates to fragments of TRAG-3 comprising or consisting of at least 10 consecutive amino acid residues and vaccine compositions comprising fragments of TRAG-3 comprising or consisting of at least 10 consecutive amino acid residues.
The present invention is directed to fragments comprising or consisting of 10 consecutive amino acid residues of TRAG-3 as represented by SEQ ID NO:1 , such as e.g. the 10 most N-terminal amino acid residues of SEQ ID NO:1, i.e. mwmgliqlve (SEQ ID NO:130), as well as vaccine compositions comprising such fragments. Additional fragments comprising or consisting of 10 consecutive amino acid residues of TRAG-3 represented by SEQ ID NO:1 can thus, by way of example, be obtained in the following way:
i) selecting any one of the above fragments defined by SEQ ID NO:4 to SEQ ID NO:103 (i.e. a fragment listed above defined by SEQ ID NO:N, wherein N is an integer larger than 3 and smaller than 104), and
ii) adding the C-terminal amino acid residue of SEQ ID NO:N+1 to the C- terminal amino acid residue of the fragment selected in step i).
In a similar way, fragments comprising or consisting of 10 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2 can be obtained.
The present invention is directed to such fragments comprising or consisting of 10 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2, as well as vaccine compositions comprising such fragments, including the fragments of TRAG-3L as represented by SEQ ID NO:2, which are not identical to the ones obtained above, listed below:
nnhpptpkrr (SEQ ID NO:131); nhpptpkrrg (SEQ ID NO:132); hpptpkrrgs (SEQ ID NO:133); pptpkrrgsg (SEQ ID NO:134); ptpkrrgsgr (SEQ ID NO:135); tpkrrgsgrh (SEQ ID NO:136); pkrrgsgrhp (SEQ ID NO:137); krrgsgrhpl (SEQ ID NO:138); rrgsgrhpln (SEQ ID NO:139); rgsgrhplnp (SEQ ID NO:140); gsgrhplnpg (SEQ ID NO:141); sgrhplnpgp (SEQ ID NO:142); grhplnpgpe (SEQ ID NO:143); rhplnpgpea (SEQ ID NO:144); hplnpgpeal (SEQ ID NO:145); plnpgpeals (SEQ ID NO:146); Inpgpealsk (SEQ ID NO:147); npgpealskf (SEQ ID NO:148); pgpealskfp (SEQ ID NO: 149); gpealskfpr (SEQ ID NO: 150); pealskfprq (SEQ ID NO: 151); ealskfprql (SEQ ID NO:152); alskfprqlg (SEQ ID NO:153); Iskfprqlgr (SEQ ID NO:154); skfprqlgre (SEQ ID NO:155); kfprqlgrek (SEQ ID NO:156).
Fragments comprising or consisting of a seguence of 11 consecutive amino acid residues of TRAG-3
The present invention also relates to fragments of TRAG-3 comprising or consisting of at least 11 consecutive amino acid residues and vaccine compositions comprising fragments of TRAG-3 comprising or consisting of at least 11 consecutive amino acid residues.
The present invention is directed to fragments comprising or consisting of 11 consecutive amino acid residues of TRAG-3 as represented by SEQ ID NO:1 , such as e.g. the 11 most N-terminal amino acid residues of SEQ ID NO:1 , i.e. mwmgliqlveg (SEQ ID NO: 157), as well as vaccine compositions comprising such fragments. Additional fragments comprising or consisting of 11 consecutive amino acid residues of TRAG-3 represented by SEQ ID NO:1 can thus be obtained in the following way:
i) selecting any one of the above fragments defined by SEQ ID NO:4 to SEQ ID NO:102 (i.e. a fragment listed above defined by SEQ ID NO:N, wherein N is an integer larger than 3 and smaller than 103), and
ii) adding the 2 most C-terminal amino acid residue of SEQ ID NO:N+2 to the C-terminal amino acid residue of the fragment selected in step i).
In a similar way, fragments comprising or consisting of 11 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2 can be obtained.
The present invention is directed to such fragments comprising or consisting of 11 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2, as well as vaccine compositions comprising such fragments, including the fragments of TRAG-3L as represented by SEQ ID NO:2, which are not identical to the ones obtained above, listed below: snnhpptpkrr (SEQ ID NO: 158); nnhpptpkrrg (SEQ ID NO: 159); nhpptpkrrgs (SEQ ID NO:160); hpptpkrrgsg (SEQ ID NO:161); pptpkrrgsgr (SEQ ID NO:162); ptpkrrgsgrh (SEQ ID NO:163); tpkrrgsgrhp (SEQ ID NO:164); pkrrgsgrhpl (SEQ ID NO:165); krrgsgrhpln (SEQ ID NO:166); rrgsgrhplnp (SEQ ID NO:167); rgsgrhplnpg (SEQ ID
NO:168); gsgrhplnpgp (SEQ ID NO:169); sgrhplnpgpe (SEQ ID NO:170); grhplnpgpea (SEQ ID NO:171); rhplnpgpeal (SEQ ID NO:172); hplnpgpeals (SEQ ID NO:173); plnpgpealsk (SEQ ID NO:174); Inpgpealskf (SEQ ID NO:175); npgpealskfp (SEQ ID NO:176); pgpealskfpr (SEQ ID NO:177); gpealskfprq (SEQ ID NO:178); pealskfprql (SEQ ID NO:179); ealskfprqlg (SEQ ID NO:180); alskfprqlgr
(SEQ ID NO:181); Iskfprqlgre (SEQ ID NO:182); skfprqlgrek (SEQ ID NO:183); kfprqlgrekg (SEQ ID NO:184).
Fragments comprising or consisting of a seguence of 12 consecutive amino acid residues of TRAG-3
The present invention also relates to fragments of TRAG-3 comprising or consisting of at least 12 consecutive amino acid residues and vaccine compositions comprising fragments of TRAG-3 comprising or consisting of at least 12 consecutive amino acid residues.
The present invention is directed to fragments comprising or consisting of 12 consecutive amino acid residues of TRAG-3 as represented by SEQ ID NO:1 , such as e.g. the 12 most N-terminal amino acid residues of SEQ ID NO:1 , i.e. mwmgliqlvegv (SEQ ID NO:185), as well as vaccine compositions comprising such fragments. Additional fragments comprising or consisting of 12 consecutive amino acid residues of TRAG-3 represented by SEQ ID NO:1 can thus be obtained in the following way: selecting any one of the above fragments defined by SEQ ID NO:4 to SEQ ID NO:101 (i.e. a fragment listed above defined by SEQ ID NO:N, wherein N is an integer larger than 3 and smaller than 102), and ii) adding the 3 most C-terminal amino acid residue of SEQ ID NO:N+3 to the C-terminal amino acid residue of the fragment selected in step i).
In a similar way, fragments comprising or consisting of 12 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2 can be obtained.
A preferred embodiment of the present invention is directed to such fragments comprising or consisting of 12 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2, as well as vaccine compositions comprising such fragments, including the fragments of TRAG-3L as represented by SEQ ID NO:2, which are not identical to the ones obtained above, listed below:
Isnnhpptpkrr (SEQ ID NO:186); snnhpptpkrrg (SEQ ID NO:187); nnhpptpkrrgs (SEQ
ID NO:188); nhpptpkrrgsg (SEQ ID NO:189); hpptpkrrgsgr (SEQ ID NO:190); pptpkrrgsgrh (SEQ ID NO:191); ptpkrrgsgrhp (SEQ ID NO:192); tpkrrgsgrhpl (SEQ
ID NO:193); pkrrgsgrhpln (SEQ ID NO:194); krrgsgrhplnp (SEQ ID NO:195); rrgsgrhplnpg (SEQ ID NO:196); rgsgrhplnpgp (SEQ ID NO:197); gsgrhplnpgpe
(SEQ ID NO:198); sgrhplnpgpea (SEQ ID NO:199); grhplnpgpeal (SEQ ID NO:200); rhplnpgpeals (SEQ ID NO:201); hplnpgpealsk (SEQ ID NO:202); plnpgpealskf (SEQ ID NO:203); Inpgpealskfp (SEQ ID NO:204); npgpealskfpr (SEQ ID NO:205); pgpealskfprq (SEQ ID NO:206); gpealskfprql (SEQ ID NO:207); pealskfprqlg (SEQ
ID NO:208); ealskfprqlgr (SEQ ID NO:209); alskfprqlgre (SEQ ID NO:210);
Iskfprqlgrek (SEQ ID NO:211); skfprqlgrekg (SEQ ID NO:212); kfprqlgrekgp (SEQ ID
NO:213).
Fragments comprising or consisting of a seguence of 13 consecutive amino acid residues of TRAG-3
The present invention also relates to fragments of TRAG-3 comprising or consisting of at least 13 consecutive amino acid residues and vaccine compositions comprising fragments of TRAG-3 comprising or consisting of at least 13 consecutive amino acid residues.
The present invention is directed to fragments comprising or consisting of 13 consecutive amino acid residues of TRAG-3 as represented by SEQ ID NO:1 , such as e.g. the 13 most N-terminal amino acid residues of SEQ ID N0:1 , i.e. mwmgliqlvegvk (SEQ ID NO:214), as well as vaccine compositions comprising such fragments. Additional fragments comprising or consisting of 13 consecutive amino acid residues of TRAG-3 represented by SEQ ID NO:1 can thus be obtained in the following way:
i) selecting any one of the above fragments defined by SEQ ID NO:4 to SEQ ID NO: 100 (i.e. a fragment listed above defined by SEQ ID NO:N, wherein N is an integer larger than 3 and smaller than 101), and
ii) adding the 4 most C-terminal amino acid residue of SEQ ID NO:N+4 to the C-terminal amino acid residue of the fragment selected in step i).
In a similar way, fragments comprising or consisting of 13 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2 can be obtained.
The present invention is also directed to such fragments comprising or consisting of 13 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2, as well as vaccine compositions comprising such fragments, including the fragments of TRAG-3L as represented by SEQ ID NO:2, which are not identical to the ones obtained above, listed below:
plsnnhpptpkrr (SEQ ID NO:215); Isnnhpptpkrrg (SEQ ID NO:216); snnhpptpkrrgs (SEQ ID NO:217); nnhpptpkrrgsg (SEQ ID NO:218); nhpptpkrrgsgr (SEQ ID NO:219); hpptpkrrgsgrh (SEQ ID NO:220); pptpkrrgsgrhp (SEQ ID NO:221); ptpkrrgsgrhpl (SEQ ID NO:222); tpkrrgsgrhpln (SEQ ID NO:223); pkrrgsgrhplnp (SEQ ID NO:224); krrgsgrhplnpg (SEQ ID NO:225); rrgsgrhplnpgp (SEQ ID NO:226); rgsgrhplnpgpe (SEQ ID NO:227); gsgrhplnpgpea (SEQ ID NO:228); sgrhplnpgpeal (SEQ ID NO:229); grhplnpgpeals (SEQ ID NO:230); rhplnpgpealsk (SEQ ID NO:231); hplnpgpealskf (SEQ ID NO:232); plnpgpealskfp (SEQ ID
NO:233); Inpgpealskfpr (SEQ ID NO:234); npgpealskfprq (SEQ ID NO:235); pgpealskfprql (SEQ ID NO:236); gpealskfprqlg (SEQ ID NO:237); pealskfprqlgr (SEQ ID NO:238); ealskfprqlgre (SEQ ID NO:239); alskfprqlgrek (SEQ ID NO:240); Iskfprqlgrekg (SEQ ID NO:241); skfprqlgrekgp (SEQ ID NO:242); kfprqlgrekgpi (SEQ ID NO:243). Fragments comprising or consisting of a sequence of 14 consecutive amino acid residues of TRAG-3
The present invention also relates to fragments of TRAG-3 comprising or consisting of at least 14 consecutive amino acid residues and vaccine compositions comprising fragments of TRAG-3 comprising or consisting of at least 14 consecutive amino acid residues.
The present invention is directed to fragments comprising or consisting of 14 consecutive amino acid residues of TRAG-3 as represented by SEQ ID NO:1 , such as e.g. the 14 most N-terminal amino acid residues of SEQ ID NO:1 , i.e. mwmgliqlvegvkr (SEQ ID NO:244), as well as vaccine compositions comprising such fragments. Additional fragments comprising or consisting of 14 consecutive amino acid residues of TRAG-3 represented by SEQ ID NO:1 can thus be obtained in the following way:
i) selecting any one of the above fragments defined by SEQ ID NO:4 to SEQ ID NO:99 (i.e. a fragment listed above defined by SEQ ID NO:N, wherein N is an integer larger than 3 and smaller than 100), and
ii) adding the 5 most C-terminal amino acid residue of SEQ ID NO:N+5 to the C-terminal amino acid residue of the fragment selected in step i).
In a similar way, fragments comprising or consisting of 14 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2 can be obtained.
The present invention is directed to such fragments comprising or consisting of 14 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2, as well as vaccine compositions comprising such fragments, including the fragments of TRAG-3L as represented by SEQ ID NO:2, which are not identical to the ones obtained above, listed below: splsnnhpptpkrr (SEQ ID NO:245); plsnnhpptpkrrg (SEQ ID NO:246); Isnnhpptpkrrgs (SEQ ID NO:247); snnhpptpkrrgsg (SEQ ID NO:248); nnhpptpkrrgsgr (SEQ ID NO:249); nhpptpkrrgsgrh (SEQ ID NO:250); hpptpkrrgsgrhp (SEQ ID NO:251); pptpkrrgsgrhpl (SEQ ID NO:252); ptpkrrgsgrhpln (SEQ ID NO:253); tpkrrgsgrhplnp (SEQ ID NO:254); pkrrgsgrhplnpg (SEQ ID NO:255); krrgsgrhplnpgp (SEQ ID
NO:256); rrgsgrhplnpgpe (SEQ ID NO:257); rgsgrhplnpgpea (SEQ ID NO:258); gsgrhplnpgpeal (SEQ ID NO:259); sgrhplnpgpeals (SEQ ID NO:260); grhplnpgpealsk (SEQ ID NO:261); rhplnpgpealskf (SEQ ID NO:262); hplnpgpealskfp (SEQ ID NO:263); plnpgpealskfpr (SEQ ID NO:264); Inpgpealskfprq (SEQ ID NO:265); npgpealskfprql (SEQ ID NO:266); pgpealskfprqlg (SEQ ID NO:267); gpealskfprqlgr (SEQ ID NO:268); pealskfprqlgre (SEQ ID NO:269); ealskfprqlgrek (SEQ ID NO:270); alskfprqlgrekg (SEQ ID NO:271); Iskfprqlgrekgp (SEQ ID NO:272); skfprqlgrekgpi (SEQ ID NO:273); kfprqlgrekgpie (SEQ ID NO:274).
Fragments comprising or consisting of a seguence of 15 consecutive amino acid residues of TRAG-3
The present invention also relates to fragments of TRAG-3 comprising or consisting of at least 15 consecutive amino acid residues and vaccine compositions comprising fragments of TRAG-3 comprising or consisting of at least 15 consecutive amino acid residues.
The present invention is directed to fragments comprising or consisting of 15 consecutive amino acid residues of TRAG-3 as represented by SEQ ID NO:1 , such as e.g. the 15 most N-terminal amino acid residues of SEQ ID NO:1 , i.e. mwmgliqlvegvkrk (SEQ ID NO:275), as well as vaccine compositions comprising such fragments. Additional fragments comprising or consisting of 15 consecutive amino acid residues of TRAG-3 represented by SEQ ID NO:1 can thus be obtained in the following way:
i) selecting any one of the above fragments defined by SEQ ID NO:4 to SEQ ID NO:98 (i.e. a fragment listed above defined by SEQ ID NO:N, wherein N is an integer larger than 3 and smaller than 99), and ii) adding the 6 most C-terminal amino acid residue of SEQ ID NO:N+6 to the C-terminal amino acid residue of the fragment selected in step i).
In a similar way, fragments comprising or consisting of 15 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2 can be obtained.
The present invention is directed to such fragments comprising or consisting of 15 consecutive amino acid residues of TRAG-3L as represented by SEQ ID NO:2, as well as vaccine compositions comprising such fragments, including the fragments of TRAG-3L as represented by SEQ ID NO:2, which are not identical to the ones obtained above, listed below:
ssplsnnhpptpkrr (SEQ ID NO:276); splsnnhpptpkrrg (SEQ ID NO:277); plsnnhpptpkrrgs (SEQ ID NO:278); Isnnhpptpkrrgsg (SEQ ID NO:279); snnhpptpkrrgsgr (SEQ ID NO:280); nnhpptpkrrgsgrh (SEQ ID NO:281 ); nhpptpkrrgsgrhp (SEQ ID NO:282); hpptpkrrgsgrhpl (SEQ ID NO:283); pptpkrrgsgrhpln (SEQ ID NO:284); ptpkrrgsgrhplnp (SEQ ID NO:285); tpkrrgsgrhplnpg (SEQ ID NO:286); pkrrgsgrhplnpgp (SEQ ID NO:287); krrgsgrhplnpgpe (SEQ ID NO:288); rrgsgrhplnpgpea (SEQ ID NO:289); rgsgrhplnpgpeal (SEQ ID NO:290); gsgrhplnpgpeals (SEQ ID NO:291); sgrhplnpgpealsk (SEQ ID NO:292); grhplnpgpealskf (SEQ ID NO:293); rhplnpgpealskfp (SEQ ID NO:294); hplnpgpealskfpr (SEQ ID NO:295); plnpgpealskfprq (SEQ ID NO:296); Inpgpealskfprql (SEQ ID NO:297); npgpealskfprqlg (SEQ ID NO:298); pgpealskfprqlgr (SEQ ID NO:299); gpealskfprqlgre (SEQ ID NO:300); pealskfprqlgrek (SEQ ID NO:301); ealskfprqlgrekg (SEQ ID NO:302); alskfprqlgrekgp (SEQ ID NO:303); Iskfprqlgrekgpi
(SEQ ID NO:304); skfprqlgrekgpie (SEQ ID NO:305); kfprqlgrekgpiee (SEQ ID
NO:306).
Accordingly, for any of the above fragments of TRAG-3 as represented by SEQ ID
NO:1 comprising or consisting of from 10 to 15 consecutive amino acid residues of TRAG-3, N is an integer of from 4 to preferably less than 104, such as 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 (fragments comprising or consisting of 15 or less consecutive amino acid residues of TRAG-3), 100 (fragments comprising or consisting of 14 or less consecutive amino acid residues of TRAG-3), 101 (fragments comprising or consisting of 13 or less consecutive amino acid residues of TRAG-3), 102 (fragments comprising or consisting of 12 or less consecutive amino acid residues of TRAG-3), 103 (fragments comprising or consisting of 11 or less consecutive amino acid residues of TRAG-3), 104 (fragments comprising or consisting of 10 or less consecutive amino acid residues of TRAG-3).
Accordingly, in preferred embodiments the present invention is directed to fragments of TRAG-3 as represented by SEQ ID NO:1 comprising or consisting of more than 9 consecutive amino acid residues. Such fragments can comprise for example 10 amino acid residues, such as 11 amino acid residues, for example 12 amino acid residues, such as 13 amino acid residues, for example 14 amino acid residues, such as 15 amino acid residues, for example 16 amino acid residues, such as 17 amino acid residues, for example 18 amino acid residues, such as 19 amino acid residues, for example 20 amino acid residues, such as 21 amino acid residues, for example 22 amino acid residues, such as 23 amino acid residues, for example 24 amino acid residues, such as 25 amino acid residues, for example 26 amino acid residues, such as 27 amino acid residues, for example 28 amino acid residues, such as 29 amino acid residues, for example 30 amino acid residues, such as 31 amino acid residues, for example 32 amino acid residues, such as 33 amino acid residues, for example 34 amino acid residues, such as 35 amino acid residues, for example 36 amino acid residues, such as 37 amino acid residues, for example 38 amino acid residues, such as 39 amino acid residues, for example 40 amino acid residues, such as 41 amino acid residues, for example 42 amino acid residues, such as 43 amino acid residues, for example 44 amino acid residues, such as 45 amino acid residues, for example 46 amino acid residues, such as 47 amino acid residues, for example 48 amino acid residues, such as 49 amino acid residues, for example 50 amino acid residues, such as 51 amino acid residues, for example 52 amino acid residues, such as 53 amino acid residues, for example 54 amino acid residues, such as 55 amino acid residues, for example 56 amino acid residues, such as 57 amino acid residues, for example 58 amino acid residues, such as 59 amino acid residues, for example 60 amino acid residues, such as 61 amino acid residues, for example 62 amino acid residues, such as 63 amino acid residues, for example 64 amino acid residues, such as 65 amino acid residues, for example 66 amino acid residues, such as 67 amino acid residues, for example 68 amino acid residues, such as 69 amino acid residues, for example 70 amino acid residues, such as 71 amino acid residues, for example 72 amino acid residues, such as 73 amino acid residues, for example 74 amino acid residues, such as 75 amino acid residues, for example 76 amino acid residues, such as 77 amino acid residues, for example 78 amino acid residues, such as 79 amino acid residues, for example 80 amino acid residues, such as 81 amino acid residues, for example 82 amino acid residues, such as 83 amino acid residues, for example 84 amino acid residues, such as 85 amino acid residues, for example 86 amino acid residues, such as 87 amino acid residues, for example 88 amino acid residues, such as 89 amino acid residues, for example 90 amino acid residues, such as 91 amino acid residues, for example 92 amino acid residues, such as 93 amino acid residues, for example 94 amino acid residues, such as 95 amino acid residues, for example 96 amino acid residues, such as 97 amino acid residues, for example 98 amino acid residues, such as 99 amino acid residues, for example 100 amino acid residues, such as 101 amino acid residues, for example 102 amino acid residues, such as 103 amino acid residues, for example 104 amino acid residues, such as 105 amino acid residues, for example 106 amino acid residues, such as 107 amino acid residues, for example 108 amino acid residues, such as 109 amino acid residues, for example 110 amino acid residues,
wherein the most N-terminal amino acid residue of each of the above fragments is for example residue 1 of SEQ ID NO:1 , such as residue 2 of SEQ ID NO:1 , for ex- ample residue 3 of SEQ ID NO:1 , such as residue 4 of SEQ ID NO:1 , for example residue 5 of SEQ ID NO:1 , such as residue 6 of SEQ ID NO:1 , for example residue 7 of SEQ ID NO:1, such as residue 8 of SEQ ID NO: 1, for example residue 9 of SEQ ID NO:1 , such as residue 10 of SEQ ID NO:1 , for example residue 11 of SEQ ID NO:1 , such as residue 12 of SEQ ID NO:1 , for example residue 13 of SEQ ID NO:1 , such as residue 14 of SEQ ID NO:1 , for example residue 15 of SEQ ID NO:1 , such as residue 16 of SEQ ID NO: 1 , for example residue 17 of SEQ ID NO:1 , such as residue 18 of SEQ ID NO:1 , for example residue 19 of SEQ ID NO:1 , such as residue 20 of SEQ ID NO:1 , for example residue 21 of SEQ ID NO:1 , such as residue 22 of SEQ ID NO:1 , for example residue 23 of SEQ ID NO:1 , such as residue 24 of SEQ ID NO:1 , for example residue 25 of SEQ ID NO:1 , such as residue 26 of SEQ ID NO:1 , for example residue 27 of SEQ ID NO:1 , such as residue 28 of SEQ ID NO:1 , for example residue 29 of SEQ ID NO:1 , such as residue 30 of SEQ ID NO:1 , for example residue 31 of SEQ ID NO:1 , such as residue 32 of SEQ ID NO:1 , for example residue 33 of SEQ ID NO:1 , such as residue 34 of SEQ ID NO:1 , for example residue 35 of SEQ ID NO:1 , such as residue 36 of SEQ ID NO:1 , for example residue 37 of SEQ ID NO:1 , such as residue 38 of SEQ ID NO:1 , for example residue 39 of SEQ ID NO:1 , such as residue 40 of SEQ ID NO:1 , for example residue 41 of SEQ ID NO:1 , such as residue 42 of SEQ ID NO:1 , for example residue 43 of SEQ ID NO:1 , such as residue 44 of SEQ ID NO:1 , for example residue 45 of SEQ ID NO:1 , such as residue 46 of SEQ ID NO:1 , for example residue 47 of SEQ
ID NO:1 , such as residue 48 of SEQ ID NO:1 , for example residue 49 of SEQ ID NO:1 , such as residue 50 of SEQ ID NO: 1 , for example residue 51 of SEQ ID NO:1 , such as residue 52 of SEQ ID NO:1 , for example residue 53 of SEQ ID NO:1 , such as residue 54 of SEQ ID NO:1 , for example residue 55 of SEQ ID NO:1 , such as residue 56 of SEQ ID NO:1 , for example residue 57 of SEQ ID NO:1 , such as residue 58 of SEQ ID NO:1 , for example residue 59 of SEQ ID NO:1 , such as residue 60 of SEQ ID NO:1 , for example residue 61 of SEQ ID NO:1 , such as residue 62 of SEQ ID NO:1 , for example residue 63 of SEQ ID NO:1 , such as residue 64 of SEQ ID NO:1 , for example residue 65 of SEQ ID NO:1 , such as residue 66 of SEQ ID NO:1 , for example residue 67 of SEQ ID NO:1 , such as residue 68 of SEQ ID NO:1 , for example residue 69 of SEQ ID NO:1 , such as residue 70 of SEQ ID NO:1 , for example residue 71 of SEQ ID NO:1 , such as residue 72 of SEQ ID NO:1 , for example residue 73 of SEQ ID NO:1 , such as residue 74 of SEQ ID NO:1 , for example residue 75 of SEQ ID NO:1 , such as residue 76 of SEQ ID NO:1 , for example resi- due 77 of SEQ ID NO:1 , such as residue 78 of SEQ ID NO:1 , for example residue
79 of SEQ ID NO:1 , such as residue 80 of SEQ ID NO:1 , for example residue 81 of SEQ ID NO:1 , such as residue 82 of SEQ ID NO:1 , for example residue 83 of SEQ ID NO:1 , such as residue 84 of SEQ ID NO: 1 , for example residue 85 of SEQ ID NO:1 , such as residue 86 of SEQ ID NO:1 , for example residue 87 of SEQ ID NO:1 , such as residue 88 of SEQ ID NO:1 , for example residue 89 of SEQ ID NO:1 , such as residue 90 of SEQ ID NO:1 , for example residue 91 of SEQ ID NO:1 , such as residue 92 of SEQ ID NO:1 , for example residue 93 of SEQ ID NO:1 , such as residue 94 of SEQ ID NO:1 , for example residue 95 of SEQ ID NO:1 , such as residue 96 of SEQ ID NO:1 , for example residue 97 of SEQ ID NO:1 , such as residue 98 of SEQ ID NO:1 , for example residue 99 of SEQ ID NO:1 , such as residue 100 of SEQ ID NO: 1, for example residue 101 of SEQ ID NO:1, such as residue 102 of SEQ ID NO: 1 , for example residue 103 of SEQ ID NO:1 , such as residue 104 of SEQ ID NO:1 , for example residue 105 of SEQ ID NO:1 , such as residue 106 of SEQ ID NO: 1, for example residue 107 of SEQ ID NO: 1 , such as residue 108 of SEQ ID NO: 1 , for example residue 109 of SEQ ID NO:1 , such as residue 110 of SEQ ID
NO:1 , with the proviso that the length of a given fragment, measured as the number of amino acid residues, is shorter than or equal to the result obtained by substracting from 110 (the number of residues in the full length TRAG-3 sequence) the position (number) in SEQ ID NO:1 of the N-terminal amino acid residue of the fragment in question.
In other preferred embodiments, the present invention is directed to fragments of TRAG-3L as represented by SEQ ID NO:2 comprising or consisting of more than 9 consecutive amino acid residues. Such fragments can comprise for example 10 amino acid residues, such as 11 amino acid residues, for example 12 amino acid residues, such as 13 amino acid residues, for example 14 amino acid residues, such as 15 amino acid residues, for example 16 amino acid residues, such as 17 amino acid residues, for example 18 amino acid residues, such as 19 amino acid residues, for example 20 amino acid residues, such as 21 amino acid residues, for example 22 amino acid residues, such as 23 amino acid residues, for example 24 amino acid residues, such as 25 amino acid residues, for example 26 amino acid residues, such as 27 amino acid residues, for example 28 amino acid residues, such as 29 amino acid residues, for example 30 amino acid residues, such as 31 amino acid residues, for example 32 amino acid residues, such as 33 amino acid residues, for example 34 amino acid residues, such as 35 amino acid residues, for example 36 amino acid residues, such as 37 amino acid residues, for example 38 amino acid residues, such as 39 amino acid residues, for example 40 amino acid residues, such as 41 amino acid residues, for example 42 amino acid residues, such as 43 amino acid residues, for example 44 amino acid residues, such as 45 amino acid residues, for example 46 amino acid residues, such as 47 amino acid residues, for example 48 amino acid residues, such as 49 amino acid residues, for example 50 amino acid residues, such as 51 amino acid residues, for example 52 amino acid residues, such as 53 amino acid residues, for example 54 amino acid residues, such as 55 amino acid residues, for example 56 amino acid residues, such as 57 amino acid residues, for example 58 amino acid residues, such as 59 amino acid residues, for example 60 amino acid residues, such as 61 amino acid residues, for example 62 amino acid residues, such as 63 amino acid residues, for example 64 amino acid residues, such as 65 amino acid residues, for example 66 amino acid residues, such as 67 amino acid residues, for example 68 amino acid residues, such as 69 amino acid residues, for example 70 amino acid residues, such as 71 amino acid residues, for example 72 amino acid residues, such as 73 amino acid residues, for example 74 amino acid residues, such as 75 amino acid residues, for example 76 amino acid residues, such as 77 amino acid residues, for example 78 amino acid residues, such as 79 amino acid residues, for example 80 amino acid residues, such as 81 amino acid residues, for example
82 amino acid residues, such as 83 amino acid residues, for example 84 amino acid residues, such as 85 amino acid residues, for example 86 amino acid residues, such as 87 amino acid residues, for example 88 amino acid residues, such as 89 amino acid residues, for example 90 amino acid residues, such as 91 amino acid residues, for example 92 amino acid residues, such as 93 amino acid residues, for example
94 amino acid residues, such as 95 amino acid residues, for example 96 amino acid residues, such as 97 amino acid residues, for example 98 amino acid residues, such as 99 amino acid residues, for example 100 amino acid residues, such as 101 amino acid residues, for example 102 amino acid residues, such as 103 amino acid residues, for example 104 amino acid residues, such as 105 amino acid residues, for example 106 amino acid residues, such as 107 amino acid residues, for example 108 amino acid residues, such as 109 amino acid residues, for example 110 amino acid residues, such as 111 amino acid residues, for example 112 amino acid residues, such as 113 amino acid residues, for example 114 amino acid residues, such as 115 amino acid residues, for example 116 amino acid residues, such as 117 amino acid residues, for example 118 amino acid residues, such as 119 amino acid residues, for example 120 amino acid residues, such as 121 amino acid residues, for example 122 amino acid residues, such as 123 amino acid residues, for example 124 amino acid residues, such as 125 amino acid residues, for example 126 amino acid residues, such as 127 amino acid residues,
wherein the most N-terminal amino acid residue of each of the above fragments is for example residue 1 of SEQ ID NO:2, such as residue 2 of SEQ ID NO:2, for example residue 3 of SEQ ID NO:2, such as residue 4 of SEQ ID NO:2, for example residue 5 of SEQ ID NO:2, such as residue 6 of SEQ ID NO:2, for example residue 7 of SEQ ID NO:2, such as residue 8 of SEQ ID NO:2, for example residue 9 of SEQ ID NO:2, such as residue 10 of SEQ ID NO:2, for example residue 1 1 of SEQ ID NO:2, such as residue 12 of SEQ ID NO:2, for example residue 13 of SEQ ID NO:2, such as residue 14 of SEQ ID NO:2, for example residue 15 of SEQ ID NO:2, such as residue 16 of SEQ ID NO:2, for example residue 17 of SEQ ID NO:2, such as residue 18 of SEQ ID NO:2, for example residue 19 of SEQ ID NO:2, such as residue 20 of SEQ ID NO:2, for example residue 21 of SEQ ID NO:2, such as residue 22 of SEQ ID NO:2, for example residue 23 of SEQ ID NO:2, such as residue 24 of SEQ ID NO:2, for example residue 25 of SEQ ID NO:2, such as residue 26 of SEQ ID NO:2, for example residue 27 of SEQ ID NO:2, such as residue 28 of SEQ
ID NO:2, for example residue 29 of SEQ ID NO:2, such as residue 30 of SEQ ID NO:2, for example residue 31 of SEQ ID NO:2, such as residue 32 of SEQ ID NO:2, for example residue 33 of SEQ ID NO:2, such as residue 34 of SEQ ID NO:2, for example residue 35 of SEQ ID NO:2, such as residue 36 of SEQ ID NO:2, for ex- ample residue 37 of SEQ ID NO:2, such as residue 38 of SEQ ID NO:2, for example residue 39 of SEQ ID NO:2, such as residue 40 of SEQ ID NO:2, for example residue 41 of SEQ ID NO:2, such as residue 42 of SEQ ID NO:2, for example residue 43 of SEQ ID NO:2, such as residue 44 of SEQ ID NO:2, for example residue 45 of SEQ ID NO:2, such as residue 46 of SEQ ID NO:2, for example residue 47 of SEQ ID NO:2, such as residue 48 of SEQ ID NO:2, for example residue 49 of SEQ ID
NO:2, such as residue 50 of SEQ ID NO:2, for example residue 51 of SEQ ID NO:2, such as residue 52 of SEQ ID NO:2, for example residue 53 of SEQ ID NO:2, such as residue 54 of SEQ ID NO:2, for example residue 55 of SEQ ID NO:2, such as residue 56 of SEQ ID NO:2, for example residue 57 of SEQ ID NO:2, such as resi- due 58 of SEQ ID NO:2, for example residue 59 of SEQ ID NO:2, such as residue
60 of SEQ ID NO:2, for example residue 61 of SEQ ID NO:2, such as residue 62 of SEQ ID NO:2, for example residue 63 of SEQ ID NO:2, such as residue 64 of SEQ ID NO:2, for example residue 65 of SEQ ID NO:2, such as residue 66 of SEQ ID NO:2, for example residue 67 of SEQ ID NO:2, such as residue 68 of SEQ ID NO:2, for example residue 69 of SEQ ID NO:2, such as residue 70 of SEQ ID NO:2, for example residue 71 of SEQ ID NO:2, such as residue 72 of SEQ ID NO:2, for example residue 73 of SEQ ID NO:2, such as residue 74 of SEQ ID NO:2, for example residue 75 of SEQ ID NO:2, such as residue 76 of SEQ ID NO:2, for example residue 77 of SEQ ID NO:2, such as residue 78 of SEQ ID NO:2, for example residue 79 of SEQ ID NO:2, such as residue 80 of SEQ ID NO:2, for example residue 81 of SEQ ID NO:2, such as residue 82 of SEQ ID NO:2, for example residue 83 of SEQ ID NO:2, such as residue 84 of SEQ ID NO:2, for example residue 85 of SEQ ID NO:2, such as residue 86 of SEQ ID NO:2, for example residue 87 of SEQ ID NO:2, such as residue 88 of SEQ ID NO:2, for example residue 89 of SEQ ID NO:2, such as residue 90 of SEQ ID NO:2, for example residue 91 of SEQ ID NO:2, such as residue 92 of SEQ ID NO:2, for example residue 93 of SEQ ID NO:2, such as residue 94 of SEQ ID NO:2, for example residue 95 of SEQ ID NO:2, such as residue 96 of SEQ ID NO:2, for example residue 97 of SEQ ID NO:2, such as residue 98 of SEQ ID NO:2, for example residue 99 of SEQ ID NO:2, such as residue 100 of SEQ ID NO:2, for example residue 101 of SEQ ID NO:2, such as residue 102 of SEQ ID
NO:2, for example residue 103 of SEQ ID NO:2, such as residue 104 of SEQ ID NO:2, for example residue 105 of SEQ ID NO:2, such as residue 106 of SEQ ID NO:2, for example residue 107 of SEQ ID NO:2, such as residue 108 of SEQ ID NO:2, for example residue 109 of SEQ ID NO:2, such as residue 110 of SEQ ID NO:2, for example residue 111 of SEQ ID NO:2, such as residue 112 of SEQ ID
NO:2, for example residue 113 of SEQ ID NO:2, such as residue 114 of SEQ ID NO:2, for example residue 115 of SEQ ID NO:2, such as residue 116 of SEQ ID NO:2, for example residue 117 of SEQ ID NO:2, such as residue 118 of SEQ ID NO:2, for example residue 119 of SEQ ID NO:2, such as residue 120 of SEQ ID NO:2, for example residue 121 of SEQ ID NO:2, such as residue 122 of SEQ ID
NO:2, for example residue 123 of SEQ ID NO:2, such as residue 124 of SEQ ID NO:2, for example residue 125 of SEQ ID NO:2, such as residue 126 of SEQ ID NO:2, for example residue 127 of SEQ ID NO:2, with the proviso that the length of a given fragment, measured as the number of amino acid residues, is shorter than or equal to the result obtained by substracting from 127 (the number of residues in the full length splice variant of TRAG-3, TRAG- 3L as represented in SEQ ID NO:2) the position (number) in SEQ ID NO:2 of the N- terminal amino acid residue of the fragment in question.
Further preferred embodiments of the present invention are directed to polypeptides comprising a sequence of 9 or more consecutive amino acid residues of TRAG-3, such as the polypeptide fragments mentioned above, wherein the polypeptide comprises more than 9 amino acid residues. Such polypeptides can comprise for example no more than 10 amino acid residues, such as no more than 11 amino acid resi- dues, for example no more than 12 amino acid residues, such as no more than 13 amino acid residues, for example no more than 14 amino acid residues, such as no more than 15 amino acid residues, for example no more than 16 amino acid residues, such as no more than 17 amino acid residues, for example no more than 18 amino acid residues, such as no more than 19 amino acid residues, for example no more than 20 amino acid residues, such as no more than 21 amino acid residues, for example no more than 22 amino acid residues, such as no more than 23 amino acid residues, for example no more than 24 amino acid residues, such as no more than 25 amino acid residues, for example no more than 30 amino acid residues, such as no more than 40 amino acid residues, for example no more than 50 amino acid residues, such as no more than 60 amino acid residues, for example no more than 70 amino acid residues, such as no more than 80 amino acid residues, for example no more than 90 amino acid residues, such as no more than 100 amino acid residues, for example no more than 125 amino acid residues, such as no more than 150 amino acid residues, for example no more than 175 amino acid residues, such as no more than 200 amino acid residues, for example more than 200 amino acid residues.
Much preferred fragments of TRAG-3 are listed herein below in Table 1 and characterised by their C50 value (μM) as the concentration of the peptide required for half maximal binding to HLA-A2. The value listed after the designation T_ (TRAG-3) or
T_L (TRAG-3L) indicates the position of the first amino acid in the sequence. The value listed after the . indicates the number of consecutive amino acid residues in the peptide fragments. C2 is a high affinity, positive control peptide from Epstein Barr Virus (EBV).
Table 1
TRAG-3 fragment Sequence C50 (μM) T_4 GLIQLVEGV (SEQ ID NO: 6) 0.3 T_29 NIKMHCEFHA (SEQ ID NO: 230 >100 or 307) T_37.9 HACWPAFTV (SEQ ID NO: 39) >100 T_37.10 HACWPAFTVL (SEQ ID NO: 308) Not binding T_45 VLGEAWRDQV (SEQ ID NO: 232 0.7 or 309) T_57.9 SILLRDAGL (SEQ ID NO: 59) >100 T_57.10 SILLRDAGLV (SEQ ID NO: 233 35 or 310) T_58 ILLRDAGLV (SEQ ID NO: 60) 0.7 T_59 LLRDAGLVKM (SEQ ID NO: 234 5 or 311) T_92 QLGREKGPI (SEQ ID NO: 94) Not binding T_L95 PLNPGPEAL (SEQ ID NO: 119) Not binding T_L102 ALSKFPRQL (SEQ ID NO: 126) 16 C2 GLCTLVAML 0.7
Preferred TRAG-3 fragments
In one aspect the present invention thus relates to a polypeptide fragment capable of raising a specific T-cell response, said fragment comprising a peptide consisting of at least 9 consecutive amino acid residues of TRAG-3, preferably of SEQ ID NO:1 or SEQ ID NO:2, wherein said peptide is selected from the group consisting
NIKMHCEFHA (SEQ ID NO:230), VLGEAWRDQV (SEQ ID NO:232), SILLRDAGLV (SEQ ID NO:233), ALSKFPRQL (SEQ ID NO:126)and LLRDAGLVKM (SEQ ID NO:23), preferably from the group consisting of NIKMHCEFHA (SEQ ID NO:230), VLGEAWRDQV (SEQ ID NO:232), SILLRDAGLV (SEQ ID NO:233), ALSKFPRQL (SEQ ID NO:126); and wherein said polypeptide fragment consists of at the most 100 amino acids.
Preferably the polypeptide fragment consists of at the most 90, more preferably at the most 80, even more preferably at the most 70, yet more preferably at the most 60 even more preferably at the most 70, yet more preferably at the most 60, even more preferably at the most 50, yet more preferably at the most 40, even more preferably at the most 30, yet more preferably at the most 20, even more preferably at the most 18, yet more preferably at the most 16, even more preferably at the most 15, yet more preferably at the most 14, even more preferably at the most 13, yet more preferably at the most 12, such as at the most 11 , for example at the most 10, such as at the most 9 amino acids.
It is preferred that the polypeptide fragment comprises at least 9, such as at least 10, for example at least 11 , such as at least 12, for example at least 13, such as at least 14, for example at least 15 consecutive amino acids of TRAG-3, preferably of SEQ ID NO:1 or SEQ ID NO:2. Thus the polypeptide fragment may consist of 9, such as 10, for example 11 , such as 12, for example 13, such as 14, for example 15 consecutive amino acids of SEQ ID:1 , comprising (SEQ ID NO:230), (SEQ ID NO:232), (SEQ ID NO:233), (SEQ ID NO:126) or (SEQ ID NO:234), preferably,
(SEQ ID NO:230), (SEQ ID NO:232), (SEQ ID NO:233) or (SEQ ID NO:126).
Functional characterisation of preferred TRAG-3 fragments
Very preferred fragments of TRAG-3 according to the present invention are fragments of TRAG-3 capable of eliciting a specific T-cell response, which may be either a cytotoxic T-lymphocyte response or a helper T-cell response or both, but which preferably is a cytotoxic T-lymphocyte response. Several different state-of- the-art methods may be used to identify peptide fragments capable of eliciting a specific T-cell response. However it is difficult to predict whether a given peptide will be capable of eliciting a specific T-cell response. Even though computer models may predict functional epitopes, experimental evidence is usually required in order to verify whether a predicted epitope indeed is a functional epitope. Even fragments that associate with MHC molecules with high affinity will not necessarily give rise to a T-cell response as determined by for example ELISPOT assay (see herein below) or in an individual.
Hence, in one embodiment of the present invention it is preferred that the TRAG-3 fragments are capable of association with an MHC molecule, even more preferred that the fragment is capable of association with a class I MHC molecule. Preferred MHC class I molecules are frequently occuring MHC class I molecules, such as for example the MHC class I molecules described herein below. Association between TRAG-3 and an MHC molecule may for example be determined using the assembly assay described herein below.
Preferred TRAG-3 fragments according to the invention may be characterised by having a C50 value, measured as the concentration (μM) of the polypeptide fragment required for half maximal binding to an MHC molecule, preferably to an MHC class I molecule, in the range of from 500 to 1000, such as in the range of 200 to 500, for example in the range of 100 to 200, such as in the range of 50 to 100, for example in the range of 25 to 50, such as in the range of 10 to 25, for example in the range of 5 to 10 such as in the range of 1 to 5, for example in range of 0,1 to 1 such as in the range of 0,05 to 0,1 , for example less than 0,05. Said MHC class I molecule may be any MHC class I molecule, for example an HLA-A molecule, such as HLA-A2, for example the subtype HLA-A*0201. Preferred MHC class I molecules are frequently occuring MHC class I molecules, for example the MHC class I molecules described herein below.
More preferred fragments of TRAG-3 includes fragments of TRAG-3 wherein the C50 value is less than 1000 μM, even more preferably less than 200 μM, yet more preferably less than 100 μM, even more preferably less than 75 μM, yet more preferably less than 50 μM, even more preferably less than 40 μM, yet more preferably less than 31 μM, even more preferably less than 25 μM, yet more preferably less than 10 μM, even more preferably less than 5 μM, yet more preferably less than 1 μM, even more preferably less than 0,5 μM, yet more preferably less than 0,2 μM, even more preferably less than 0,1 μM, yet more preferably less than 0,05 μM, wherein the C50 value is the concentration of the peptide required for half maximal binding to an MHC molecule, preferably to an MHC class I molecule. Said MHC class I molecule may be any MHC class I molecule, such as an HLA-A molecule, for example HLA-A2, such as the subtype HLA-A*0201. Preferred MHC class I molecules are frequently occuring MHC class I molecules, for example the MHC class I molecules described herein below. In preferred embodiments of this invention the MHC class I molecules are selected from a group comprising the highly prevalent MHC class I alleles HLA-A2, HLA-A3, HLA-A1 , HLA-A11 , HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61 , and HLA-B62. These MHC class I alleles have been reported to cover more than 95% of the Caucasian population58. It is preferred that the C50 value is determined according to the assembly assay described herein below.
Accordingly, in one embodiment of the present invention preferred fragments of TRAG-3 may be selected from the group consisting of T_57.10 (SEQ ID NO:310), TJ.102 (SEQ ID NO: 126), T_59 (SEQ ID NO:311) , and T_45 (SEQ ID NO:309). More preferred fragments of TRAG-3 may be selected from the group consisting of
T_59 (SEQ ID NO:311) and T_45 (SEQ ID NO:309).
Assembly assay
Assembly assays for binding of the synthetic peptides to class I MHC molecules metabolically labeled with [35S]-methionine can be carried out as described below. It will be appreciated by the person skilled in the art that the protocol may be adoptes for any peptide and any class I MHC molecule. Previously, it has been demonstrated that the peptide concentration resulting in half-maximal binding in an assembly assay for peptide binding to class I MHC is a good approximation of Ka 29.
Briefly, Transporter Associated with antigen Processing (TAP) deficient cells (T2) were metabolically labeled with [35S]-methionine (Amersham, Freiburg, Germany). The cells were lyzed in 0.5 ml lysis buffer (150 mM NaCI, 50 mM TrisHCI, 0.5% NP-
40 (Fluka, Buchs, Switzerland), 5 mM EDTA, pH 7.5) with 0.5% Mega-9 (Sigma, St. Louis, USA) in the presence of protease inhibitors (2 mM PMSF, 5 mM io- doacetamide, 2 μg/ml pepstatin, 2 μg/ml leupeptin) with or without synthetic peptide. For assembly assays for binding to HLA-A2 the positive control peptide C2 (glctlvaml) from EBV can be used. After 20 min of incubation, the cell nuclei were removed by centrifugation (5 min, 10,000 g) and 50 μl (10% v/v) freshly washed Staphylococcus aureus organisms (Pansorbin, Calbiochem, Nottingham, UK) were added to the samples. The next day Pansorbin was removed by centrifugation (12 min, 16.000 g) and monoclonal antibody (for immunoprecipitation of HLA class I peptide complexes the specific, conformation-dependent antibody W6/32 (DakoCytomation, Code no. M0736) was added at a final concentration of 10 μg/ml, incubated for 90 min, followed by addition of protein A-Sepharose (75 μl, 10% v/v) and incubation for 1 h. The beads were washed 4 times and stored at -20°C until analysis by electrophoresis.
For peptide binding to HLA-B*2705 and H-2Kk a modified assembly can be used as described previously by us30. When transfected into T2 cells B*2705 and K are unusually stable in the absence of added peptide. To circumvent this, a mild heating step was introduced in order to preferentially destabilize MHC molecules which remain empty after peptide incubation. Briefly, the cell lysates are incubated with peptide for 2 hours at 4°C, allowing the binding of peptide to empty class I molecules. Next, the cell lysates were heated (60°C for 5 min. for T2-B*2705 or 55°C for 2 min. for T2-2Kk). Pansorbin was then added to the samples as in the conventional as- sembly assay.
Electrophoresis
In the case of an assembly assay with the HLA class l-specific conformation- dependent monoclonal antibody W6/32 (DakoCytomation, Code no. M0736), samples were eluted in reducing buffer (9.5 M urea, 2% NP-40, 5% 2-mercaptoethanol, 2% Ampholines, pH range 3.5-9.5 (Pharmacia Biotech, Uppsala, Sweden) and focused for 16 hours at 880 V on 5.5% polyacrylamide isoelectric focusing (IEF) gels30"31.
Samples from assembly assays with allele-specific antibodies can be eluted by boiling (5 min.) in SDS reducing buffer (50 mM Tris-HCI pH 6.8, 2% SDS, 5% 2- mercaptoethanol, 10% blycerol, 2.5% bromophenol blue) and electrophorezed on 12% SDS-PAGE gels (1 hour at 200V). All gels are fixed in 10% acetic acid with 5% methanol and dried onto 3 MM paper (Whatman, Maidstone, UK). MHC heavy chain bands were quantified using the Im- agequant Phosphorimager program (Molecular Dynamics, Sunnyvale, CA, USA). The intensity of the band is directly related to the amount of peptide-bound class I MHC complex recovered during the assay.
C50 value (μM) as the concentration of the peptide required for half maximal binding to MHC may thus be determined. The binding affinities of the analysed peptides are determined according to their efficiency of stabilising the HLA class I molecules. The binding affinity is represented as the peptide concentration required to reach half- maximal stabilistation of appropriate HLA-molecule. Previous analyses have shown that the C50 values measured in this assay fits well with the dissociation constant (Kd) of the complex32.
Epitope prediction
In another embodiment of the present invention preferred TRAG-3 fragments are TRAG-3 fragments predicted to be epitopes, in particular HLA-restricted epitopes.
Epitope prediction may be performed using any suitable algorithm, preferably an algorithmus based on the book "MHC Ligands and Peptide Motifs" by H.G. Ram- mensee, J.Bachmanna and S.Stevanovic. However, such epitope prediction is not reliable, and thus it is preferred within the present invention that TRAG-3 fragments are identified using ELISPOT assays as described herein elsewhere. One preferred method of predicting epitopes is using the SYFPEITHI epitope prediction algorithm. Further explanations on the algorithm can be found in HG Rammensee, J Bach- mann, NPN Emmerich, OA Bachor and S Stevanovic (1999) SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50: 213-219. SYFPEITHI da- tabase can be found on the internet site: http://syfpeithi.bmi- heidelberg.com/scripts/MHCServer.dll/home.htm.
The algorithm calculates the predicted ligation strength to a defined HLA type for a sequence of amino acids. Preferred TRAG-3 fragments has a predicted ligation strength to a given HLA molecule as calculated using said algorithm of at least 10, preferably at least 12, more preferably at least 15, even more preferably at least 16, such as at least 18, for example at least 20.
Specific T-cell response
However, more preferred fragments according to the present invention are TRAG-3 fragments capable of raising a specific T-cell response as determined by an ELISPOT assay, for example the ELISPOT assay described herein below. Some fragments of TRAG-3 although not binding MHC with high affinity still may give rise to a T-cell response as determined by ELISPOT. Other fragments of TRAG-3 may be capable of binding MHC with high affinity and gives rise to a T-cell response as determined by ELISPOT. Both kinds of fragments are preferred fragments according to the invention.
Hence, preferred fragments according to the present invention are TRAG-3 fragments capable of raising a specific T-cell response as measured by an ELISPOT assay, wherein more than 50 fragment specific spots per 108 cells, more preferably per 107 cells, even more preferably per 106 cells, yet more preferably per 105 cells, for example per 104 cells are measured
Hence, in one embodiment of the present invention preferred TRAG-3 fragments may be selected from the group consisting of T_29 (SEQ ID NO:307), T_45 (SEQ ID NO:309), T_57.10 (SEQ ID NO:310), and TJ.102 (SEQ ID NO:126), or more preferably from the group consisting of T_45 (SEQ ID NO:309), T_57.10 (SEQ ID NO:310), and TJ.102 (SEQ ID NO:126).
Antigen stimulation of peripheral blood lymphocytes (PBL).
Peripheral blood was obtained from patients suffering from breast cancer, melanoma, and hematopoetic malignancies before vaccination and subsequent to a series of vaccinations. In order to identify peptide-specific T-cell precursors, peripheral blood lymphocytes (PBL) were used directly in the ELISPOT (designated direct ELISPOT). However, to extend the sensitivity of the ELISPOT assay, PBL were stimulated once in vitro prior to analysis33"34. At day 0, PBL or crushed lymph nodes were thawed and plated in 2 ml/well at a concentration of 2 x 106 cells in 24-well plates (Nunc, Denmark) in X-vivo medium (Bio Whittaker, Walkersville, Maryland), 5% heat-inactivated human serum, and 2 mM of L-glutamine in the presence of 10 μM of peptide. Two days later 20 lU/ml recombinant interleukin-2 (IL-2) (Chiron, Rat- ingen, Germany) was added to the cultures. The cultured cells were tested for reactivity in the ELISPOT on day 10.
ELISPOT assay
The ELISPOT assay used to quantify peptide epitope specific interferon-γ releasing effector cells was adapted from Lalvani et al.35, and Scheibenbogen et al.36. Briefly, nitrocellulose bottomed 96-well plates (Multiscreen MAIP N45, Millipore) were coated overnight at room temperature with 7.5 μg/ml of anti-IFN-γ antibody (1-D1 K, Mabtech, Sweden) in 75 μl sterile PBS. Subsequently, the wells were washed six times with PBS, and non-specific binding were blocked by X-vivo medium for 2 hours at 37°C. Freshly isolated PBL or cells, which had been stimulated once in vitro, were added in duplicates at different cell concentration (from 106 to 105 cells per well for non-stimulated PBL, and from 105 to 104 cells per well for PBL which had been stimulated once in vitro), in 100 μl X-vivo medium. 104 T2 stimulator cells
(unpulsed or pulsed with 1 μg peptide) or peptides to a final concentration of 2 μM where then added to each well and incubated overnight at 37°C.
The following day, media was discarded and the wells were washed (six times) with PBS containing 0.05% Tween (PBS/Tw) before the addition of biotinylated secondary antibody (7-B6-1-Biotin, Mabtech) at 0.5 μg/ml in 75 μl PBS containing 1 % BSA and 0.02% NaN3 (PBS/BSA). The plates were incubated at room temperature for 2 hours. The wells were washed (six times) with PBS/Tw. Then 75 μl of Avidin- enzyme conjugate (AP-Avidin, Calbiochem) diluted 1 :2000 in PBS/BSA were added to each well and incubated for 1 hour at room temperature. The plates were washed twice and enzyme substrate (DAKO, Carpinteria; CA) was added to each well, and incubated for 5-10 min. at room temperature. The reaction was terminated by washing with tap-water upon the emergency of dark purple spots. Spots were quantitated using a computerized ELISPOT counter (Immunospot, CTL inc., CA, USA) and the peptide specific CTL frequency could be calculated as the number of spot-forming cells.
It is preferred that the ELISPOT assay is performed using PBL derived from an indi- vidual that has not previously been immunised with TRAG-3 or a fragment thereof. More preferably, said individual has not been subjected to any kind of immune therapy against a neoplastic disease. Hence it is for example preferred that the individual has not been immunised with tumor cells previously. PBL from individuals that have been subjected to immune therapy, in particular immune therapy comprising TRAG-3, may give a positive result against a given peptide in an ELISPOT assay, even though PBL from a naive person would not have given a positive result.
Activation of T-cell growth
In yet another embodiment of the present invention, preferred TRAG-3 fragments are fragments capable of activating T-cell growth in vitro. In particular, preferred TRAG-3 fragments induce expansion of antigen-specific CTL using DC loaded with said fragments. A method of expanding antigen-specific CTLs is described herein below. Accordingly, very preferred TRAG-3 fragments include fragments, wherein more than 105 antigen specific CTLs, more preferably 106, even more preferably 107 antigen specific CTLs may be harvested after 4 stimulation cycles starting with 104 PBMC as described herein below.
Expansion of antigen-specific CTL using antigen-loaded DC
The generation of dendritic cells (DC) can be performed as described37. PBMC were plated in 85 mm dishes (either bacteriological, Primaria or Tissue culture dishes, Falcon, Cat. No. 1005, 3038 or 3003; Becton Dickinson, Hershey, USA) at a density of 50x106 cells per dish in 10 ml of complete culture medium and incubated at 37°C and 5% C02 for 1 h. After a microscopic control of adherence, the non-adherent fraction was removed and 10 ml of fresh, warm complete medium (RPMI 1640 (Prod. Nr. 12-167, Bio Whittaker, Walkersville, USA) supplemented with gentamicin (Refobacin 10, Merck, Darmstadt, Germany) at 20 μg/ml final concentration, gluta- mine at 2 mM final concentration (Prod. Nr. 17-605, Bio Whittaker) and 1 % heat inactivated (56° for 30 min) human plasma were added (day 0). The non-adherent fractions were centrifuged and plated once more in new 85 mm tissue-culture-dishes for readherence. The non-adherent fraction from these 'replate' dishes was dis- carded after 1 hour adherence. All adherent fractions were cultured until day 1 , then culture medium was taken off carefully so that loosely adherent cells were not removed, and new culture medium containing GM-CSF (800 U/ml final concentration) and IL-4 (1000 U/ml final concentration) is added. Cytokines were added again on day 3 in 3 ml fresh medium (containing 8000 U GM-CSF and 10,000 U IL-4) per dish. On day 5 all non-adherent cells were harvested, counted and replated in fresh complete medium (containing cytokines in the same dosage as described above) in 6 well plates at a density of 5χ105 cells/well in 3 ml medium. On day 6 750 μl mono- cyte conditioned medium (MCM) were added to induce maturation of DC, and on day 7 or 8 cells were harvested. All DC preparations were highly enriched in mature DC with > 90% showing a characteristic phenotype by flow cytometry (HLA-DR+++, CD86+++, CD40+, CD25+, CD14-). More than 80% of the cells expressed the CD83 antigen as marker for mature DC.
MCM was prepared in the following way: Ig coated bacteriological plates (85 mm,
Falcon 1005) are prepared immediately prior to use. As immunoglobulin we used Sandoglobin(TM) (Novartis). Coating was performed with 10 ml of diluted (with PBS without calcium or magnesium, Bio Whittaker) immunoglobulin (10 μg/ml) for 10 min at room temperature. After the coating procedure plates were rinsed twice with PBS without calcium or magnesium (Bio Whittaker). 50χ106 PBMC are plated on these dishes in complete medium without cytokines and incubated at 37°C, 5% C02 for 20 h. Then the MCM is harvested, centrifuged at 1360 g for 10 min (22°C), sterile filtered (0.22 μm filters, Millipore, Molsheim, France) and frozen down in aliquots at - 20°C.
5 x 106 cells/well DC were pulsed with 50 μg/ml HLA-restricted peptide, for example an TRAG-3 fragment, for 2 hours at 37° C peptide. About 104 PBMC/well and 5 103 antigen-loaded autologous DC/well were co-cultured in a 96-well round-bottom plate in 200 μl MCM medium/well supplemented with 5% autologous plasma. On day 7, PBMC were restimulated either with antigen-loaded DC. After a total of 4 to 5 stimu- lation cycles CD8+ T lymphocytes were enriched from PBMC by depletion of CD4+, CD11 b+, CD16+, CD19+, CD36+ and CD56+ cells with magnetic cell sorting using a midiMACS device (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The resulting population consisted of >90% CD8+ T cells.
Conventional [51Cr]-release assays for CTL-mediated cytotoxicity can be used to test the specificity of CTL lines against relevant target cells, e.g. autologous EBV- transformed B cell lines or cancer cell lines. This procedure is performed according to Brunner et al.59.
For expansion of specific CTLs, such as tumor specific CTLs, the CD8-enriched T cells were transferred to 25-cm2 flasks coated with anti-CD3/anti-CD28 mAbs, as described previously38. Briefly, 25-cm2 flasks (Falcon, Heidelberg, Germany) were coated with anti-human CD3 mAb (OKT3, Ortho Pharmaceutical Corp., Raritan, NJ) and anti-human CD28 mAb (L293, Becton Dickinson) at a concentration of 1 μg/ml in PBS/100 mM HEPES buffer (pH 9). After incubation overnight at 4°C, coated flasks were washed twice with PBS. CD8+ T cells were placed on the precoated and washed flasks at 5 * 105 cells/ml in 10 ml MCM medium supplemented with 10% human AB serum (PAA Laboratories GmbH, Coelbe, Germany) and 100 IU IL-2/ml (EuroCetus, Amsterdam, Netherlands). Cells were restimulated with anti-CD3/anti-
CD28 mAbs once a week, culture medium and IL-2 (100 lU/ml) was changed twice a week. Part of CD8-purified CTL were expanded by weekly restimulation with antigen-loaded DC or PBL.
In some embodiments of the present invention preferred TRAG-3 fragments are fragments capable of being presented by a specific MHC molecule. Hence, very preferred TRAG-3 fragments may be fragments capable of raising a specific T-cell response in an individual with a specific tissue type.
In another embodiment of the present invention preferred fragments of TRAG-3 are fragments which may give rise to a specific T-cell response without leading to antibody production. Epitopes only leading to a T-cell response, but not an IgG response have been described in the prior art39"40. Methods of identifying fragments
In one aspect the present invention also relates to methods of selecting a peptide comprising a fragment of TRAG-3 for use in a vaccine composition comprising the steps of
i) providing an individual who has not been subjected to immune therapy ii) providing fragments of TRAG-3 iii) testing specific T-cell responses against fragments of TRAG-3 in said individ- ual iv) selecting fragments of TRAG-3 wherein said T-cell response corresponds to or is better than a predetermined selection criterium.
The T-cell response may be tested according to any suitable method, it is however preferred that testing said T-cell response comprises an ELISPOT assay. The
ELISPOT assay is preferably the assay described herein above.
Preferably, fragments giving rise to more than 50 peptide specific spots per 106 cells in an ELISPOT assay, preferably the ELISPOT assay described herein above, are selected.
Vaccine compositions and uses thereof
The present invention in one embodiment is directed to an immunogenic composition such as a vaccine composition capable of raising e.g. a specific T-cell response. The vaccine composition comprises full length TRAG-3 and/or one or more fragments thereof. Preferably, the vaccine compositions comprise isolated TRAG-3 and/or one or more isolated fragments thereof. The terms "TRAG-3 peptide" and "TRAG-3 fragments" as used herein below refers to full length TRAG-3 and fragments thereof and to full length TRAG-3L and fragments thereof as described herein elsewhere. Any of the TRAG-3 fragments described herein above may be comprised within said vaccine, in particular the preferred TRAG-3 fragments described above may be comprised within a vaccine. Hence, the vaccine compositions according to the invention may comprise more than one different TRAG-3 fragment, such as 2, for example 3, such as 4, for example 5, such as 6, for example 7, such as 8, for example 9, such as 10, such as a number of fragments in the range of from 5 to 10, for example in the range of from 10 to 15, such as in the range of from 15 to 20, for example in the range of from 20 to 30, such as in the range of from 30 to 40, for example in the range of from 40 to 60, such as in the range of from 60 to 100, for example in the range of from 100 to 200.
In one embodiment of the present invention the different TRAG-3 fragments are selected so that one vaccine composition comprises fragments capable of associating with different MHC molecules, such as different MHC class I molecule, i.e. the TRAG-3 fragments are restricted to specific HLA's. Preferably, one vaccine composition comprises fragments capable of associating with the most frequently occurring MHC class I molecules. Hence preferred vaccine compositions comprises different fragments capable of associating with at least 2 preferred, more preferably at least 3 preferred, even more preferably at least 4 preferred MHC class I molecules.
In another embodiment of the invention, the vaccine composition comprises one or more fragments capable of associating to an MHC class I molecule and one or more fragments capable of associating with an MHC class II molecule. Peptide vaccine preparations capable of being used in accordance with the present invention may thus comprise a class l-restricted TRAG-3 peptide and/or a class II-TRAG-3 peptide and/or fusion peptides comprising both peptides. Hence, such a vaccine composi- tion is preferably capable of raising a specific cytotoxic T-lymphocyte response and/or a specific helper T-cell response.
The vaccine composition can further comprise an adjuvant and/or a carrier. Examples of useful adjuvants and carriers are given herein below. Thus TRAG-3, or the fragment thereof, present in the composition can be associated with a carrier such as e.g. a protein or an antigen-presenting cell such as e.g. a dendritic cell (DC) capable of presenting TRAG-3 or a fragment thereof to a T cell.
Adjuvants are any substance whose admixture into the vaccine composition in- creases or otherwise modifies the immune response to TRAG-3 or a TRAG-3 frag- ment. Carriers are scaffold structures, for example a polypeptide or a polysaccha- ride, to which TRAG-3, or the fragment thereof is capable of being associated. Adjuvants could for example be selected from the group consisting of: AIK(S04)2, AINa(S04)2, AINH4 (S0 ), silica, alum, AI(OH)3, Ca3 (P04)2, kaolin, carbon, alumi- num hydroxide, muramyl dipeptides, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'- dipalmitoyl-sn -glycero-3-hydroxphosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-PE), RIBI (MPL+TDM+CWS) in a 2% squalene/Tween-80.RTM. emulsion, lipopolysaccharides and its various derivatives, including lipid A, Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (for example, poly IC and poly AU acids), wax D from Mycobacte- rium, tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, liposomes or other lipid emulsions, Titermax, ISCOMS, Quil A, ALUN (see US 58767 and 5,554,372), Lipid A derivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrixes or GMDP, Interleukin 1 , Interleukin 2, Montanide ISA-51 and QS-21. Preferred adjuvants to be used with the invention include Montanide ISA-51 and QS-21. The most preferred adjuvants to be used with the present invention are adjuvants suitable for human use.
Montanide ISA-51 (Seppic, Inc.) is a mineral oil-based adjuvant analogous to incomplete Freund's adjuvant, which must be administered as an emulsion. QS-21 (Anti- genics; Aquila Biopharmaceuticals, Framingham, MA) is a highly purified, water- soluble saponin that handles as an aqueous solution. QS-21 and Montanide ISA-51 adjuvants can be provided in sterile, single-use vials.
In a preferred embodiment of the invention the adjuvant is a Montanide adjuvant (all available from Seppic Inc., Belgium), which may be selected from the group consist- ing of Montanide ISA-51 , Montanide ISA-50, Montanide ISA-70, Montanide ISA-206,
Montanide ISA-25, Montanide ISA-720, Montanide ISA-708, Montanide ISA-763A, Montanide ISA-207, Montanide ISA-264, Montanide ISA-27, Montanide ISA-35, Montanide ISA 51 F, Montanide ISA 016D and Montanide IMS, preferably from the group consisting of Montanide ISA-51 , Montanide IMS and Montanide ISA-720, more preferably from the group consisting of Montanide ISA-51. Additional preferred adjuvants capable of being used in vaccine compositions comprising TRAG-3, and/or one or more fragments thereof, are e.g. any substance which promote an immune response. Frequently, the adjuvant of choice is Freund's complete or incomplete adjuvant, or killed B. pertussis organisms, used e.g. in com- bination with alum precipitated antigen. A general discussion of adjuvants is provided in Goding, Monoclonal Antibodies: Principles & Practice (2nd edition, 1986) at pages 61-63. Goding notes, however, that when the antigen of interest is of low molecular weight, or is poorly immunogenic, coupling to an immunogenic carrier is recommended. Examples of such carrier molecules include keyhole limpet haemocya- nin, bovine serum albumin, ovalbumin and fowl immunoglobulin. Various saponin extracts have also been suggested to be useful as adjuvants in immunogenic compositions. Recently, it has been proposed to use granulocyte-macrophage colony stimulating factor (GM-CSF), a well known cytokine, as an adjuvant (WO 97/28816).
Desirable functionalities of adjuvants capable of being used in accordance with the present invention are listed in the below table.
Table 1 o ss of adjuvant action Action Adjuvant type Benefit 1. Immunomodulafion Generally small molecules or proteins which modify the Upregulatioπ of immune response. Selection of Th1 or cytokine network Th2 2. Presentation Generally amphipathic molecules or complexes which Increased neutralizing antibody response. Greater interact with immunogen in its native conformation duration of response 3. CTL induction , Particles which can bind or enclose immunogen and Cytosotic processing of protein yielding correct c'ass which can fuse with or disrupt cell membranes 1 restricted peplides , /o emulsions for direct attachment of peptide to cell Simple process if promiscuous peptidefs) known surface HC-1 4. Targeting . Particulatβ adjuvants which bind Immunogen. Adjuvants Efficient uso of adjuvant and immunogen which saturate Kupffer cells . Carbohydrate adjuvants which target leclin receptors on As above. May also determine type of response if macrophages and DCs targeting selective 5. Depot generation . w/o emulsion for short term Efficiency . Microspheres or nanospheres lor long term Potential for single-dose vaccine
Source: John C. Cox and Alan R. Coulter,. Vaccine 1997 Feb;15(3):248-56)
A vaccine composition according to the present invention may comprise more than one different adjuvant. Furthermore, the invention encompasses a therapeutic composition further comprising any adjuvant substance including any of the above or combinations thereof. It is also contemplated that TRAG-3, or one or more fragments thereof, and the adjuvant can be administered separately in any appropriate sequence. A carrier may be present independently of an adjuvant. The function of a carrier can for example be to increase the molecular weight of in particular TRAG-3 fragments in order to increase their activity or immunogenicity, to confer stability, to increase the biological activity, or to increase serum half-life. Furthermore, a carrier may aid presenting TRAG-3, or the fragments thereof to T cells. The carrier may be any suitable carrier known to the person skilled in the art, for example a protein or an antigen presenting cell. A carrier protein could be but is not limited to keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid. For immunization of humans, the carrier must be a physiologically acceptable carrier acceptable to humans and safe. However, tetanus toxoid and/or diptheria toxoid are suitable carriers in one embodiment of the invention. Alternatively, the carrier may be dextrans for example sepharose.
DC-based vaccines and therapeutic procedures using DC
In one embodiment, the vaccine composition may comprise dendritic cells. The dendritic cells (DC) may be prepared and used in therapeutic procedure according to any suitable protocol, for example as described herein below.
It will be appreciated by the person skilled in the art that the protocol may be adopted to use with patients with different HLA type and different diseases. The peptide in the procedure described in example 3 can be any fragment of TRAG-3 such as e.g. fragments of TRAG-3 described herein, in particular preferred fragments of TRAG-3 described herein.
Cancer patients such as e.g. stage IV metastatic melanoma patients with progressive disease were entered into a DC-based vaccination trial e.g. after having failed to respond to chemotherapy. All patients provided informed consent to participate in the experimental vaccination and to donate blood for immunological monitoring.
Serological HLA typing revealed that the patients were HLA-A2. Dendritic cells (DC) were pulsed with 50 μg/ml HLA-restricted peptide for 1hour at 37°C peptide and 5 x 106 cells were administered subcutaneously at day 1 and 14, subsequently every 4 weeks, additional leukapheresis after 5 vaccinations.
All vaccine preparations were highly enriched in mature DC with > 90% showing a characteristic phenotype by flow cytometry (HLA-DR+++, CD86+++, CD40+,
CD25+, CD14-). More than 80% of the cells expressed the CD83 antigen as marker for mature DC. The peptide used in the vaccination trial were synthesized at a GMP quality by Clinalfa (purity >98%).
The generation of DC for clinical use and quality control was performed as described37. PBMC were plated in 85 mm dishes (either bacteriological, Primaria or Tissue culture dishes, Falcon, Cat. No. 1005, 3038 or 3003; Becton Dickinson, Hershey, USA) at a density of 50x106 cells per dish in 10 ml of complete culture medium and incubated at 37°C and 5% C02 for 1 h. After a microscopic control of ad- herence, the non-adherent fraction was removed and 10 ml of fresh, warm complete medium (RPMI 1640 (Prod. Nr. 12-167, Bio Whittaker, Walkersville, USA) supplemented with gentamicin (Refobacin 10, Merck, Darmstadt, Germany) at 20 μg/ml final concentration, glutamine at 2 mM final concentration (Prod. Nr. 17-605, Bio Whittaker) and 1 % heat inactivated (56° for 30 min) human plasma) were added (day 0).
The non-adherent fractions were centrifuged and plated once more in new 85 mm tissue-culture-dishes for readherence. The non-adherent fraction from these 'replate' dishes was discarded after 1hour adherence. All adherent fractions were cultured until day 1 , then culture medium was taken off carefully so that loosely adherent cells were not removed, and new culture medium containing GM-CSF (800 U/ml final concentration) and IL-4 (1000 U/ml final concentration) was added. Cytokines were added again on day 3 in 3 ml fresh medium (containing 8000 U GM-CSF and 10,000 U IL-4) per dish. On day 5 all non-adherent cells were harvested, counted and replated in fresh complete medium (containing cytokines in the same dosage as described above) in 6 well plates at a density of 5χ105 cells/well in 3 ml medium. On day 6 750 μl monocyte conditioned medium (MCM) were added to induce maturation of DC, and on day 7 or 8 cells were harvested. MCM (monocyte conditioned medium) was prepared in the following way: Ig coated bacteriological plates (85 mm, Falcon 1005) were prepared immediately prior to use. As immunoglobulin we used Sandoglobin(TM> (Novartis). Coating was performed with 10 ml of diluted (with PBS without calcium or magnesium, Bio Whittaker) immu- noglobulin (10 μg/ml) for 10 min at room temperature. After the coating procedure plates were rinsed twice with PBS without calcium or magnesium (Bio Whittaker). 50χ106 PBMC were plated on these dishes in complete medium without cytokines and incubated at 37°C, 5% C02 for 20 h. Then the monocyte conditioned medium was harvested, centrifuged at 1360 g for 10 min (22°C), sterile filtered (0.22 μm fil- ters, Millipore, Molsheim, France) and frozen down in aliquots at -20°C.
Peptide vaccines
Vaccine compositions may be prepared and administered using any conventional protocol known by a person skilled in the art. Below a non-limiting example of preparation of a vaccine composition according to the invention is given as well as a non- limiting example of adminstration of such as a vaccine. It will be appreciated by the person skilled in the art that the protocol may be easily adapted to any of the vac- cine compositions described herein.
TRAG-3 peptides can e.g. be synthesized e.g. at the UVA Biomolecular Core Facility with a free amide NH2 terminus and free acid COOH terminus. Each was provided as a lyophilized peptide, which was then reconstituted in sterile water and diluted with Lactated Ringer's solution (LR, Baxter Healthcare, Deerfield, IL) as a buffer for a final concentration of 67-80% Lactated Ringer's in water. These solutions were then sterile-filtered, placed in borosilicate glass vials, and submitted to a series of quality assurance studies including confirmation of identity, sterility, general safety, and purity, in accordance with FDA guidelines, as defined in IND 6453. Tests of pep- tide stability demonstrated no decrease in purity or in the peptide concentration, when these peptide solutions were stored at -20°C for 3 years.
In practical circumstances, patients will receive a vaccine comprising about 100 μg of a class I HLA-restricted TRAG-3 peptide with or without a class II HLA-restricted TRAG-3 helper peptide. The patients are vaccinated with e.g. about 100 μg of the class I HLA peptide in adjuvant alone, or were vaccinated with e.g. about 100 μg of the HLA class l-restricted peptide plus 190 μg of the class ll-restricted helper peptide. The higher dose of the helper peptide was calculated to provide equimolar quantities of the helper and cytotoxic epitopes. Additionally, patients can be vacci- nated with a longer peptide comprising the amino acid sequences of both peptides.
The above peptides, in 1-ml aqueous solution, can be administered either as a solution/suspension with about 100 μg of QS-21 , or as an emulsion with about 1 ml of Montanide ISA-51 adjuvant.
Patients are immunized e.g. at day 0 and months 1 , 2, 3, 6, 9, and 12, with the peptides plus adjuvant, for a total of seven immunizations. With rare exceptions, the vaccinations are administered to the same arm with each vaccine. The peptides were administered s.c.
MHC molecules
In one embodiment of the present invention preferred TRAG-3 fragments are frag- ments capable of associating with an MHC molecule. Because different MHC molecules have different affinities for a given peptide, different TRAG-3 fragments may be preferred with different embodiments of the invention. The invention also relates to compositions comprising different TRAG-3 fragments, which preferably have affinity for different MHC molecules.
Preferred MHC molecules according to the present invention are MHC class I molecules and MHC class II molecules, more preferably MHC class I molecules.
Preferred MHC class I molecules are the most commonly occurring MHC class I molecules. In one embodiment of the present invention the preferred MHC class I molecules may be selected from the group consisting of HLA-A1 , HLA-A2, HLA-A3, HLA-A11 , HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B27, HLA-B35, HLA-B60, HLA-B61 , and HLA-B62. Preferred compositions according to the present invention comprises at least 1 , more preferably at least 2, even more preferably at least 3, yet more preferably at least 4, for example at least 5, such as at least 6, for example 7 different TRAG-3 fragments each capable of associating with a different HLA molecule selected from the group consisting of HLA-A1 , HLA-A2, HLA-A3, HLA-A11 , HLA-A24, HLA-A68,
HLA-B7, HLA-B8, HLA-B14, HLA-B27, HLA-B35, HLA-B60, HLA-B61 , and HLA- B62.
By way of example, a preferred composition may comprise one TRAG-3 peptide capable of associating with HLA-A2 and a TRAG-3 peptide capable of associating with HLA-A1 and a TRAG-3 peptide capable of associating with HLA-A3 and a TRAG-3 peptide capable of associating with HLA-A24.
Ex vivo methods for obtaining and cultivating T cells
In one embodiment, the present invention relates to a method for activating and expanding T cells specific for TRAG-3 or fragments thereof as well as to T cells obtained by such methods. Preferably, the methods relate to cytotoxic T lymphocytes and/or helper T cells specific for TRAG-3 fragments, more preferably the methods relate to cytotoxic T lymphocytes specific for TRAG-3 fragments. The methods preferably comprise the steps of co-cultivating T cells and TRAG-3, or at least one fragment thereof, thereby activating the T cells, and optionally isolating activated TRAG-3-specific T cells or TRAG-3 fragment specific T cells.
Co-cultivating T cells and TRAG-3 or fragments thereof may be done by any conventional method. For example methods involving antigen presenting cells, such as dendritic cells (DC) may be used. The method may thus comprise generating and loading monocyte-derived DC with TRAG-3 fragment(s) and co-cultivating said DC and peripheral blood monocytes (PBMC) comprising T cells or T cells purified from PBMC. Optionally the TRAG-3-specific T cells may then be isolated. Preferably, the
TRAG-3-specific T cells are cytotoxic T lymphocytes (CTL).
One preferred method for generating and loading of monocyte-derived DC and for co-cultivating DC and peripheral blood monocytes (PBMC) is described herein above in the section "Expansion of antigen specific CTL using antigen-loaded CD": However, different kinds of antigen presenting cells (APC) may be used with the invention.
In one example, there is provided a method for in vitro immunization with Drosophila cells as APCs. Hence, the method may comprise generating Drosophila melanogaster cells expressing one or more different HLA molecules, loading said Drosophila melanogaster cells with TRAG-3 fragment(s) and co-cultivating said Drosophila cells with peripheral blood monocytes (PBMC) comprising T cells or T cells purified from PBMC. Thereby, TRAG-3-specific T cells may be generated. Preferably, said T cells are cytotoxic T lymphocytes. Optionally, T cells may subsequently be isolated. One advantage of using Drosophila melanogaster cells is that they are non-viable at 37°C.
Drosophila melanogaster cells were used as APCs41'42. These cells are efficient vehicles for the presentation of peptides in the context of HLA class I, especially for de novo immunization of CD8+ CTL. The Schneider S2 Drosophila cell line (American Type Culture Collection CRL 10974, Rockville, MD) was transduced with HLA- A*0201 , CD80 (B7-1) and CD54 (intracellular adhesion molecule-1) with a pRmHa-3 plasmid vector. Drosophila cells were grown in Schneider's medium (106 cells/mL) with 10% fetal bovine serum and CuS04 at 27°C, the optimal temperature for these insect cells. They were harvested, washed, and resuspended in X-press medium (Bio Whittaker, Walkersville, MD) containing 100 μg/mL of the HLA-restricted peptide epitope.
CD8+ T cells were obtained from peripheral blood mononuclear cells (PBMCs) by positive selection with a novel anti-CD8 monoclonal antibody (mAb) captured with a sheep anti-mouse magnetic bead (Dynal, Lake Success, NY)43.
After incubation at 27°C with the HLA-restricted peptide epitope for 3 hours, the
Drosophila cells were incubated with the CD8+ T cells at 37°C at a ratio of 1 :10 in RPMI 1640 medium containing 10% autologous serum. Two days later, 20 IU of IL-2 and 30 IU of IL-7 were added to the growth medium. Incubation was continued for 1 week, after when the Drosophila cells were replaced with autologous irradiated PBMCs (30 Gy) and the HLA-restricted peptide. This was repeated for one addi- tional round of stimulation, after when the CD8+ T cells were tested for cytotoxicity by a 4-hour [51Cr]-release assay. The final preparation contained at least 92% CD8+ T cells, with 4% or less CD16+ (natural killer) cells and 4% or less CD4+ T cells.
For the [51Cr] release assay, 106 target cells were labeled in 50 μl R10 medium with
[51Cr] (100 μCi) in a round bottomed well of a 96-well plate at 37°C for 60 min. If necessary, 4 μg of peptide was added. The target cells were washed 4 times and plated out in 96-well plates in 100 μl R10 medium. T-cells were added at various effector:target ratios in another 100 μl R10 medium and incubated at 37°C for 4 hrs. 100 μl of medium was aspirated and [51Cr] release was counted in a Gamma- counter.
The maximum [51Cr] release was determined in separate wells by addition of 100 μl 10% Triton X-100, and the spontaneous release was determined by the addition of 100 μl R10 only to target cells. The specific lysis was calculated using the following formula: ((experimental release
- spontaneous release)/(maximum release - spontaneous release)) x 100
The present invention also relates to methods of treating a clinical condition in an individual in need thereof, comprising (re)infusing TRAG-3-specific T cells into said individual. Furthermore the invention relates to use of TRAG-3-specific T cells for the preparation of a medicament for treatment of a clinical condition in an individual in need thereof and to medicaments for treating a clinical condition comprising TRAG-3 specific T cells as active ingredient.
Methods of (re)infusing T cells to an individual are known in the art. One example of a suitable method is outlined herein below. Other methods are described in Rosenberg SA and Dudley ME (2004) PNAS 101:14639-14645.
In yet another embodiment there is provided a method for obtaining T cells from an individual and reinfusing the T cells after immunization ex vivo. Leukapheresis was performed to obtain approximately 1 x 1010 peripheral-blood mononuclear cells (PBMCs). After three rounds (3 weeks) of in vitro immunization, formal mycologic and bacteriologic testing was performed to verify sterility before the cells were administered. No Drosophila cells remained in the CTL preparation after the immunization procedure. Drosophila cells are viable at 27°C but are nonviable at 37°C. Furthermore, two rounds of immunization with changes of medium each time were performed subsequent to the initial immunization with the fly cells. Finally, the polymerase chain reaction was used in order to detect residual Drosophila DNA in the final preparation of CTL before reinfusion. Drosophila DNA was uniformly absent by this sensitive method.
For infusion into the patient, the CTLs were resuspended in 200 mL of 0.9% saline with 5% human serum albumin in a transfer pack (Baxter [McGaw Park, IL] catalog no. 4R-2014 plastic blood cell infusion bag) and were administered intravenously over a period of 1 hour on the stem-cell transplantation unit. Experienced nurses took vital signs every 15 minutes and monitored the patients for signs of toxicity or immediate hypersensitivity reactions.
Combination therapy
In a very preferred embodiment, the present invention furthermore relates to phar- maceutical compositions and kit of parts for use in combination therapy.
Combination therapy as used herein denotes treatment of an individual in need thereof with more than one different method. Hence combination therapy may in one aspect involve administration of a pharmaceutical composition or a kit of parts com- prising a vaccine composition as described herein above and an anti-cancer medicament. Anti-cancer medicaments may be any of the pharmaceutical compositions described herein below, for example a chemotherapeutic agent or a immunotherapeutic agent.
In particular combination therapy may involve administration to an individual of a chemotherapeutic agent and/or an immunotherapeutic agent in combination with one or more of i) TRAG-3, or a fragment thereof, ii) an antigen presenting cell presenting TRAG-3, and iii) an activated, TRAG-3 peptide-specific T cell. However, combination therapy may also involve radiation therapy, gene therapy and/or sur- gery. "In combination" herein means administration or treatment simultanously or sequentially in any order in such a way as to achieve a therapeutic effect.
Combination therapy thus may include administration, simultaneously, or sequentially in any order, of e.g.:
i) TRAG-3 and/or fragments thereof + at least one chemotherapeutic agent ii) TRAG-3 and/or fragments thereof + at least one immunotherapeutic agent iii) Antigen presenting cell presenting TRAG-3 and/or fragments thereof + at least one chemotherapeutic agent iv) Antigen presenting cell presenting TRAG-3 and/or fragments thereof + at least one immunotherapeutic agent v) Activated T cells + at least one chemotherapeutic agent vi) Activated T cells + at least one immunotherapeutic agent vii) TRAG-3 and/or fragments thereof+another protein and/or fragments thereof
Further combinations include i) and ii); iii) and iv); v) and vi); i) and iii); i) and iv), i) and v); i) and vi); ii) and iii); ii) and iv); ii) and v); ii) and vi); iii) and v); iii) and vi); iv) and v); iv) and vi); i) and iv) and any of v) and vi).
The chemotherapeutic agent can be e.g. methotrexate, vincristine, adriamycin, cis- platin, non-sugar containing chloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin, carmustaine and poliferposan, MM1270, BAY 12-9566, RAS famesyl transferase inhibitor, famesyl transferase inhibitor, MMP, MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470, Hycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone, Metaret/Suramin, Batimastat, E7070, BCH-4556, CS- 682, 9-AC, AG3340, AG3433, lncel/VX-710, VX-853, ZD0101 , ISI641 , ODN 698, TA 2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951f, Lem- onal DP 2202, FK 317, Picibanil/OK-432, AD 32/Valrubicin, Metastron/strontium derivative, Temodal/Temozolomide, Evacet/liposomal doxorubicin, Yew- taxan/Placlitaxel, Taxol/Paclitaxel, Xeload/Capecitabine, Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid, SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609 (754)/RAS oncogene inhibitor, BMS-182751/oral platinum, UFT(Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776C85/5FU enhancer, Campto/Levamisole, Camptosar/lrinotecan, Tumodex/Ralitrexed, Leustatin/Cladribine, Paxex/Paclitaxel, Doxil/liposomal doxorubicin, Cae- lyx/liposomal doxorubicin, Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU 79553/Bis-Naphtalimide, LU 103793/Dolastain, Caetyx/liposomal doxorubicin, Gemzar/Gemcitabine, ZD 0473/Anormed, YM 116, Iodine seeds, CDK4 and CDK2 inhibitors, PARP inhibitors, D4809/Dexifosamide, Ifes/Mesnex/lfosamide, Vumon/Teniposide, Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD 9331 , Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane Analog, ni- trosoureas, alkylating agents such as melphelan and cyclophosphamide, Aminoglu- tethimide, Asparaginase, Busulfan, Carboplatin, Chlorombucil, Cytarabine HCI, Dactinomycin, Daunorubicin HCI, Estramustine phosphate sodium, Etoposide (VP16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea (hydroxycar- bamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolide acetate (LHRH-releasing factor analogue), Lomustine (CCNU), Mechlorethamine HCI (nitrogen mustard),
Mercaptopurine, Mesna, Mitotane (o.p'-DDD), Mitoxantrone HCI, Octreotide, Plica- mycin, Procarbazine HCI, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA), Azacitidine, Erthropoietin, Hexamethyl- melamine (HMM), Interleukin 2, Mitoguazone (methyl-GAG; methyl glyoxal bis- guanylhydrazone; MGBG), Pentostatin (2'deoxycoformycin), Semustine (methyl-
CCNU), Teniposide (VM-26) and Vindesine sulfate. Furthermore, the chemo- theraputic agent may be any of the chemotherapeutic agents mentioned in table 3 of US 6,482,843 column 13 to 18.
The immunotherapeutic agent can be e.g. Ributaxin, Herceptin, Quadramet,
Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART Ml 95, ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6, MDX-210, MDX-11 , MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pre- target, NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab,
SMART 1 D10 Ab, SMART ABL 364 Ab and ImmuRAIT-CEA. Furthermore the immunotherapeutic agent may be any cytokine or interferon.
The therapeutic compositions or vaccine compositions of the invention can also be used in combination with other anti-cancer strategies, and such combination thera- pies are effective in inhibiting and/or eliminating tumor growth and metastasis. The methods of the present invention can advantageously be used with other treatment modalities, including, without limitation, radiation, surgery, gene therapy and chemotherapy.
"Combination therapy" can include the introduction of heterologous nucleic acids into suitable cells, generally known as gene therapy. For example gene therapy may involve introduction of tumor suppressor genes or apoptosis promoting genes into tumor cells. Alternatively, nucleic acid sequences inhibiting expression of oncogenes or apoptosis inhibiting genes may be introduced to tumor cells. Furthermore, genes that encode enzymes capable of conferring to tumor cells sensitivity to chemotherapeutic agents may be introduced. Accordingly, the present invention in one embodiment provides a method comprising the step of treating cancer by introducing a gene vector, encoding a protein capable of enzymatically converting a prodrug, i.e., a non-toxic compound, into a toxic compound. In the method of the present invention, the therapeutic nucleic acid sequence is a nucleic acid coding for a product, wherein the product causes cell death by itself or in the presence of other drugs. A representative example of such a therapeutic nucleic acid is one, which codes for thymidine kinase of herpes simplex virus. Additional examples are thymidine kinase of varicella zoster virus and the bacterial gene cytosine deaminase, which can convert 5-fluorocytosine to the highly toxic compound 5-fluorouracil.
The other protein is preferably a protein, which is expressed in cancer cells, preferably a protein which is expressed at higher levels in cancer cells compared with non-malignant cells. The other protein is preferably a protein involved in the regulation of apoptosis, more preferably a protein selected from the group consisting of ML-IAP, survivin, Bcl-2, Bcl-XL and Mcl-1.
Clinical indications capable of being treated with the present invention
The vaccine compositions or the therapeutic/medicaments dislcosed herein may be used to treat a number of different clinical conditions. Furthermore, the present invention relates to methods of treatment of said clinical conditions in an individual in need thereof, methods of diagnosing said clinical conditions and use of TRAG-3 or fragments thereof for preparation of a pharmaceutical composition for treatment of a clinical condition in an individual in need thereof as well as to pharmaceutical compositions for treating a clinical condition comprising TRAG-3 or fragments thereof as active ingredient.
In a preferred embodiment of the invention, the clinical condition is a cancer. The term "cancer" as used herein is meant to encompass any cancer, neoplastic and preneoplastic disease. Said cancer may for example be selected from the group consisting of colon carcinoma, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangeosarcoma, lymphangeoendothelia sarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystandeocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioblastomas, neuronomas, craniopharingiomas, schwannomas, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroama, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemias and lymphomas, acute lymphocytic leukemia and acute myelocytic polycythemia vera, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease, acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's Disease, non-Hodgkin's lymphomas, rectum cancer, urinary cancers, uterine cancers, oral cancers, skin cancers, stomach cancer, brain tumors, liver cancer, laryngeal cancer, esophageal cancer, mammary tumors, childhood-null acute lymphoid leukemia (ALL), thymic ALL, B-cell ALL, acute myeloid leukemia, myelomonocytoid leukemia, acute megakaryocytoid leukemia, Burkitt's lymphoma, acute myeloid leukemia, chronic myeloid leukemia, and T-cell leukemia, small and large non-small cell lung carcinoma, acute granulocytic leukemia, germ cell tumors, endometrial cancer, gastric cancer, cancer of the head and neck, chronic lymphoid leukemia, hairy cell leukemia and thyroid cancer. In preferred embodiments of the invention the clinical condition is a type of cancer frequently expressing TRAG-3 or a type of cancer wherein cell lines derived from said type of cancer frequently expresses TRAG-3. For example TRAG-3 is found to be expressed in cancers of the colon, CNS, ovary, kidney, prostate, and breast, as well as non-small-cell lung cancers, melanomas, chondrosarcomas, and leukemias, but not in normal, non-testis tissue25"27,44. It is very much preferred however, that the clinical condition is a cancer expressing TRAG-3.
In preferred embodiments, the clinical condition is comprised in the group of malig- nant melanoma, chondrosarcoma, non-small cell lung carcinoma, especially lung adenocarcinoma, leukemia, colon cancer, ovary cancer, kidney cancer, prostate cancer, or breast cancer.
In another embodiment of the invention the clinical condition is an auto-immune dis- ease.
Autoimmune diseases may be loosely grouped into those primarily restricted to specific organs or tissues and those that affect the entire body. Examples of organ- specific disorders (with the organ affected) include multiple sclerosis (myelin coating on nerve processes), type I diabetes mellitus (pancreas), Hashimotos thyroiditis (thyroid gland), pernicious anemia (stomach), Addison's disease (adrenal glands), myasthenia gravis (acetylcholine receptors at neuromuscular junction), rheumatoid arthritis (joint lining), uveitis (eye), psoriasis (skin), Guillain-Barre Syndrome (nerve cells) and Grave's disease (thyroid). Systemic autoimmune diseases include sys- temic lupus erythematosus and dermatomyositis.
Other examples of hypersensitivity disorders include asthma, eczema, atopical dermatitis, contact dermatitis, other eczematous dermatitides, seborrheic dermatitis, rhinitis, Lichen planus, Pemplugus, bullous Pemphigoid, Epidermolysis bullosa, urit- caris, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Alopecia areata, atherosclerosis, primary biliary cirrhosis and nephrotic syndrome. Related diseases include intestinal inflammations, such as Coeliac disease, proctitis, eosi- nophilia gastroenteritis, mastocytosis, inflammatory bowel disease, Chrohn's disease and ulcerative colitis, as well as food-related allergies. The individual in need of treatment may be any individual, preferably a human being. Peptides will in general have different affinities to different HLA molecules. Hence, in the embodiments of the present invention wherein the vaccine composition or the medicament comprises TRAG-3 peptides, it is preferred that a vaccine composition or a medicament to be administered to a given individual will comprise at least one peptide capable of associating with HLA molecules of that particular individual.
The methods according to the present invention allows vaccination even of immu- nologically naive individuals, because the vaccine compositions according to the invention preferably comprises immunologically dominant TRAG-3 fragments. Hence, in one embodiment of the present invention the individual in need of treatment has not previously been subjected to immune therapy against a neoplastic disease. In particular it is preferred that the individual has not previously been sub- jected to an immune therapy that comprised immunisation with a component comprising TRAG-3 or a fragment thereof. Hence, for example it is preferred that said individual has not been immunised with a tumor cell expressing TRAG-3.
Pharmaceutical compositions
Accordingly, the invention in preferred embodiments relates to pharmaceutical compositions which comprise TRAG-3 as represented by SEQ ID NO:1 and/or TRAG-3L as represented by SEQ ID NO:2 , and/or variants or fragments of these molecules as defined herein above for the treatment of pathological disorders related to or me- diated by TRAG-3.
Pharmaceutically and/or veterinary useful therapeutic compositions according to the invention can be formulated according to known methods such as by the admixture of one or more pharmaceutically or veterinary acceptable excipients or carriers. Ex- amples of such excipients, carriers and methods of formulation may be found e.g. in Remington's Pharmaceutical Sciences (Maack Publishing Co, Easton, PA). To form a pharmaceutically or veterinary acceptable composition suitable for effective administration, such compositions will contain an effective amount of a polypeptide, nucleic acid, antibody or compound modulator. Therapeutic or diagnostic compositions of the invention are administered to an individual. Said individual is a vertebrate, preferably a mammal, more preferably a human. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of ad- ministration.
The term functional derivative includes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
Pharmaceutical and veterinary compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art. The therapeutically effective dose can be estimated initially either in cell culture assays, eg, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans and other animals. A therapeutically effective dose refers to that amount of compound, peptide, antibody or nucleic acid which ameliorate or prevent a dysfunctional apoptotic condition. The exact dosage is chosen by the individual physician in view of the patient to be treated.
Compounds identified according to the methods disclosed herein as well as, therapeutic antibodies, therapeutic nucleic acids and peptides contemplated herein may be used alone at appropriate dosages defined by routine testing in order to obtain optimal modulation of TRAG-3 activity. In addition, co-administration or sequential administration of these and other agents may be desirable.
The pharmaceutical or veterinary compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular. Admini- stration of medicaments is accomplished orally or parenterally. Methods of parental delivery include topical, intra-arterial (directly to the tissue), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compositions containing compounds identified according to this invention as the active ingredient for use in the modulation of a protein which is associated with resistance to chemotherapy in cancer can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the compounds can be adminis- tered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound, nucleic acid, or peptide desired can be employed as a modulating agent for resistance to chemotherapy in cancer.
The daily dosage of the products may be varied over a wide range from 0.001 to 1 ,000 mg per adult human/per day. For oral administration, the compositions are preferably provided in the form of scored or unscored tablets containing 0.01 , 0.05, 0.1 , 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 100 mg/kg of body weight per day. The range is more particularly from about 0.001 mg/kg to 10 mg/kg of body weight per day. Even more particularly, the range varies from about 0.05 mg/kg to about 1 mg/kg.
Of course the dosage level will vary depending upon the potency of the particular compound. Certain compounds will be more potent than others. In addition, the dosage level will vary depending upon the bioavailability of the compound. The more bioavailable and potent the compound, the less compound will need to be administered through any delivery route, including but not limited to oral delivery. The dosages of TRAG-3 modulators are adjusted when combined to achieve desired effects. On the other hand, dosages of these various agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors.
There is also provided combination therapies comprising the step of administering the vaccine compositions according to the invention in combination with a chemotherapeutic agent, such as a taxane, for example taxol, and/or an immunotherapeutic agent and/or a cancer vaccine.
Variants and functional equivalents of TRAG-3
The present invention is also directed to variants and functional equivalents of the above-listed fragments of TRAG-3.
The affinity of various HLA molecules towards a given peptide depends on the se- quence of said peptide. In Table 2 herein below the amino acids in a given position within a peptide that results in the highest affinity of said peptide to a given HLA molecule are described.
Hence, preferred variants of TRAG-3 peptides with high affinity to a particular HLA molecule are listed herein below with an indication of the position in which a substitution preferably has occurred for each of the above-listed fragments. The preferred amino acid residue in the respective position of the variant is indicated in the table.
Accordingly, by way of example, a preferred TRAG-3 peptide variant capable of binding to HLA-B54 has a proline at the second position.
Figure imgf000059_0001
Figure imgf000060_0001
HLA-Cw8 Y L,l,
HLA- A,L LN
Cw16
* In one embodiment there is no primary anchor residue, but in a preferred embodiment the primary anchor residue is R or A
Functional equivalents and variants are used interchangably herein. When being polypeptides, variants are determined on the basis of their degree of identity or their degree of homology with any predetermined sequence of consecutive amino acid sequences of a fragment of TRAG-3, such as e.g. SEQ ID ΝO:3 - SEQ ID NO:311.
One therefore initially defines a sequence of consecutive TRAG-3 amino acid residues and then defines variants and functional equivalents in relation thereto.
Accordingly, variants preferably have at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85% sequence identity, for example at least 90% sequence identity, such as at least 91 % sequence identity, for example at least 91 % sequence identity, such as at least 92% sequence identity, for example at least 93% sequence identity, such as at least 94% sequence identity, for example at least 95% sequence identity, such as at least 96% sequence identity, for example at least 97% sequence identity, such as at least 98% sequence identity, for example 99% sequence identity with the predetermined TRAG-3 sequence of consecutive amino acid residues.
Sequence identity is determined in one embodiment by utilising fragments of peptides comprising at least 9 contiguous amino acids and having an amino acid sequence which is at least 80%, such as 85%, for example 90%, such as 95%, for example 99% identical to the amino acid sequence of any of SEQ ID NO: 3 - SEQ
ID NO:311 , respectively, wherein the percent identity is determined with the algorithm GAP, BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release 7.0, using default gap weights.
The following terms are used to describe the sequence relationships between two or more polynucleotides: "predetermined sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A "predeter- mined sequence" is a defined sequence used as a basis for a sequence compari- sion; a predetermined sequence may be a subset of a larger sequence.
Optimal alignment of sequences for aligning a comparison window may be con- ducted by the local homology algorithm of Smith and Waterman (1981 ) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lip- man (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.
The term "sequence identity" means that two amino acid sequences are identical over the window of comparison.
The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of posi- tions at which identical amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparision (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
As applied to polypeptides, a degree of identity of amino acid sequences is a function of the number of identical amino acids at positions shared by the amino acid sequences. A degree of homology or similarity of amino acid sequences is a function of the number of amino acids, i.e. structurally related, at positions shared by the amino acid sequences.
The term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 75 percent sequence identity, such as at least 80 percent sequence identity, for example at least 85 percent sequence identity, such as e.g. at least 90 percent sequence identity, for example at least 95 percent sequence identity, such as at least 98 percent sequence identity, or even at least 99 percent sequence identity, compared to a predetermined sequence over a comparison window of at least 9 amino acid residues, such as 10 amino acid residues, for example 11 amino acid residues, such as 12 amino acid residues, for example 13 amino acid residues, such as 14 amino acid residues, for example 15 amino acid residues, such as 20 amino acid residues, for example 30 amino acid residues, such as 40 amino acid residues, for example 50 amino acid residues, such as 60 amino acid residues, for example 70 amino acid residues, such as 80 amino acid residues, for example 90 amino acid residues, such as 100 amino acid residues, for example 110 amino acid residues, such as 120 amino acid residues, for example 127 amino acid residues. Preferably, residue positions which are not identical differ by conservative amino acid substitutions.
An "unrelated" or "non-homologous" sequence shares less than 40% identity, though preferably less than 25% identity, with an TRAG-3 amino acid sequence of the present invention.
Conservative amino acid substitutions refer in one embodiment to the interchange- ability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine, a group of amino acids having amide-containing side chains is asparagine and gluta- mine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine-glutamine.
Additionally, variants are also determined based on a predetermined number of conservative amino acid substitutions as defined herein below. Conservative amino acid substitution as used herein relates to the substitution of one amino acid (within a predetermined group of amino acids) for another amino acid (within the same group), wherein the amino acids exhibit similar or substantially similar characteris- tics. Within the meaning of the term "conservative amino acid substitution" as applied herein, one amino acid may be substituted for another within the groups of amino acids indicated herein below:
Amino acids having polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys,)
Amino acids having non-polar side chains (Gly, Ala, Val, Leu, lie, Phe, Trp, Pro, and Met)
Amino acids having aliphatic side chains (Gly, Ala Val, Leu, lie)
Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro)
Amino acids having aromatic side chains (Phe, Tyr, Trp)
Amino acids having acidic side chains (Asp, Glu)
Amino acids having basic side chains (Lys, Arg, His) Amino acids having amide side chains (Asn, Gin)
Amino acids having hydroxy side chains (Ser, Thr)
Amino acids having sulphor-containing side chains (Cys, Met),
Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser, Thr)
Hydrophilic, acidic amino acids (Gin, Asn, Glu, Asp), and
Hydrophobic amino acids (Leu, lie, Val)
Accordingly, a variant or a fragment thereof according to the invention may comprise at least one substitution, such as a plurality of substitutions introduced independ- ently of one another. It is clear from the above outline that the same variant or frag- ment thereof may comprise more than one conservative amino acid substitution from more than one group of conservative amino acids as defined herein above.
The addition or deletion of at least one amino acid may be an addition or deletion of from preferably 2 to 250 amino acids, such as from 10 to 20 amino acids, for example from 20 to 30 amino acids, such as from 40 to 50 amino acids. However, additions or deletions of more than 50 amino acids, such as additions from 50 to 100 amino acids, addition of 100 to 150 amino acids, addition of 150-250 amino acids, are also comprised within the present invention. The deletion and/or the addition may - independently of one another - be a deletion and/or an addition within a sequence and/or at the end of a sequence.
The polypeptide fragments according to the present invention, including any functional equivalents thereof, may in one embodiment comprise a sequence of con- secutive TRAG-3 amino acid residues of less than 127 amino acid residues, such as less than 120 amino acid residues, for example less than 110 amino acid residues, such as less than 100 amino acid residues, for example less than 90 amino acid residues, such as less than 85 amino acid residues, for example less than 80 amino acid residues, such as less than 75 amino acid residues, for example less than 70 amino acid residues, such as less than 65 amino acid residues, for example less than 60 amino acid residues, such as less than 55 amino acid residues, for example less than 50 amino acid residues, such as less than 45 amino acid residues, for example less than 30 amino acid residues, such as less than 25 amino acid residues, for example less than 20 amino acid residues, such as less than 15 amino acid resi- dues, for example 14 consecutive amino acid residues, such as 13 consecutive amino acid residues, for example 12 consecutive amino acid residues, such as 11 consecutive amino acid residues, for example 10 consecutive amino acid residues, such as 9 consecutive amino acid residues of TRAG-3 (SEQ ID NO:1).
"Functional equivalency" as used in the present invention is according to one preferred embodiment established by means of reference to the corresponding functionality of a predetermined fragment of the sequence.
Functional equivalency can be established by e.g. similar binding affinities to HLA class I molecules, or similar potency demonstrated by ELISPOT assays. Functional equivalents or variants of a TRAG-3 fragment as described herein will be understood to exhibit amino acid sequences gradually differing from preferred, predetermined sequences, as the number and scope of insertions, deletions and substi- tutions including conservative substitutions, increases. This difference is measured as a reduction in homology between a preferred, predetermined sequence and the TRAG-3 variant fragment or TRAG-3 functional equivalent.
All TRAG-3 fragments comprising or consisting of consecutive TRAG-3 amino acid residues as well as variants and functional equivalents thereof are included within the scope of this invention, regardless of the degree of homology they show to a predetermined sequence. The reason for this is that some regions of the TRAG-3 fragments are most likely readily mutatable, or capable of being completely deleted, without any significant effect on e.g. the binding activity of the resulting fragment.
A functional variant obtained by substitution may well exhibit some form or degree of native binding activity, and yet be less homologous, if residues containing functionally similar amino acid side chains are substituted. Functionally similar in this respect refers to dominant characteristics of the side chains such as hydrophobic, basic, neutral or acidic, or the presence or absence of steric bulk. Accordingly, in one embodiment of the invention, the degree of identity is not a principal measure of a fragment being a variant or functional equivalent of a preferred predetermined fragment according to the present invention.
The homology between amino acid sequences may be calculated using well known algorithms such as any one of BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, and BLOSUM 90.
Fragments sharing homology with fragments comprising or consisting of consecutive TRAG-3 amino acid residues are to be considered as falling within the scope of the present invention when they are preferably at least about 90 percent homologous, for example at least 92 percent homologous, such as at least 94 percent homologous, for example at least 95 percent homologous, such as at least 96 percent homologous, for example at least 97 percent homologous, such as at least 98 per- cent homologous, for example at least 99 percent homologous with a predetermined TRAG-3 fragment. According to one embodiment of the invention the homology percentages indicated above are identity percentages.
Additional factors that may be taken into consideration when determining functional equivalence according to the meaning used herein are i) the ability of antisera to detect a TRAG-3 fragment according to the present invention, or ii) the ability of a functionally equivalent TRAG-3 fragment to compete with a predetermined TRAG-3 fragment in an assay. One method for determining a sequence of immunogenically active amino acids within a known amino acid sequence has been described by Geysen in US 5,595,915 and is incorporated herein by reference.
A further suitably adaptable method for determining structure and function relationships of peptide fragments is described by US 6,013,478, which is herein incorpo- rated by reference. Also, methods of assaying the binding of an amino acid sequence to a receptor moiety such as e.g. a T-cell receptor are known to the skilled artisan.
In addition to conservative substitutions introduced into any position of a preferred TRAG-3 fragment, it may also be desirable to introduce non-conservative substitutions in any one or more positions of such a fragment. A non-conservative substitution leading to the formation of a functionally equivalent fragment would for example i) differ substantially in polarity, for example a residue with a non-polar side chain (Ala, Leu, Pro, Trp, Val, He, Leu, Phe or Met) substituted for a residue with a polar side chain such as Gly, Ser, Thr, Cys, Tyr, Asn, or Gin or a charged amino acid such as Asp, Glu, Arg, or Lys, or substituting a charged or a polar residue for a non- polar one; and/or ii) differ substantially in its effect on polypeptide backbone orientation such as substitution of or for Pro or Gly by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such as Glu or Asp for a positively charged residue such as Lys, His or Arg (and vice versa); and/or iv) differ substantially in steric bulk, for example substitution of a bulky residue such as His, Trp, Phe or Tyr for one having a minor side chain, e.g. Ala, Gly or Ser (and vice versa). Variants obtained by substitution of amino acids may in one preferred embodiment be made based upon the hydrophobicity and hydrophilicity values and the relative similarity of the amino acid side-chain substituents, including charge, size, and the like. Exemplary amino acid substitutions which take various of the foregoing charac- teristics into consideration are well known to those of skill in the art and include: ar- ginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
In a further embodiment the present invention relates to functional variants compris- ing substituted amino acids having hydrophilic values or hydropathic indices that are within +/-4.9, for example within +/-4.7, such as within +/-4.5, for example within +/- 4.3, such as within +/-4.1 , for example within +/-3.9, such as within +/-3.7, for example within +/- 3.5, such as within +/-3.3, for example within +/- 3.1 , such as within +/- 2.9, for example within +/- 2.7, such as within +/-2.5, for example within +/- 2.3, such as within +/- 2.1 , for example within +/- 2.0, such as within +/- 1.8, for example within +/- 1.6, such as within +/- 1.5, for example within +/- 1.4, such as within +/- 1.3 for example within +/- 1.2, such as within +/- 1.1 , for example within +/- 1.0, such as within +/- 0.9, for example within +/- 0.8, such as within +/- 0.7, for example within +/- 0.6, such as within +/- 0.5, for example within +/- 0.4, such as within +/- 0.3, for example within +/- 0.25, such as within +/- 0.2 of the value of the amino acid it has substituted.
The importance of the hydrophilic and hydropathic amino acid indices in conferring interactive biologic function on a protein is well understood in the art (Kyte & Doolit- tie, 1982 and Hopp, U.S. Pat. No. 4,554,101 , each incorporated herein by reference).
The amino acid hydropathic index values as used herein are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methion- ine (+1.9); alanine (+1.8); glycine (-0.4 ); threonine (-0.7 ); serine (-0.8 ); tryptophan
(-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (- 3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5) (Kyte & Doolittle, 1982). The amino acid hydrophilicity values are: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); praline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cys- teine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4) (U.S. 4,554,101).
In addition to the peptidyl compounds described herein, sterically similar compounds may be formulated to mimic the key portions of the peptide structure and that such compounds may also be used in the same manner as the peptides of the invention. This may be achieved by techniques of modelling and chemical designing known to those of skill in the art. For example, esterification and other alkylations may be employed to modify the amino terminus of, e.g., a di-arginine peptide backbone, to mimic a tetra peptide structure. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
Variants and functional equivalents of TRAG-3 also includes derivatives of TRAG-3 or fragments thereof, for example TRAG-3 or TRAG-3 fragments substituted with one or more chemical moieties.
Peptides with N-terminal alkylations and C-terminal esterifications are also encompassed within the present invention. Functional equivalents also comprise glycosy- lated and covalent or aggregative conjugates formed with the same or other TRAG- 3 fragments, including dimers or unrelated chemical moieties. Such functional equivalents are prepared by linkage of functionalities to groups which are found in fragment including at any one or both of the N- and C-termini, by means known in the art.
Functional equivalents may thus comprise TRAG-3 fragments conjugated to aliphatic or acyl esters or amides of the carboxyl terminus, alkylamines or residues containing carboxyl side chains, e.g., conjugates to alkylamines at aspartic acid residues; O-acyl derivatives of hydroxyl group-containing residues and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues, e.g. conjugates with fMet-Leu-Phe or immunogenic proteins. Derivatives of the acyl groups are selected from the group of alkyl-moieties (including C3 to C10 normal alkyl), thereby forming alkanoyl species, and carbocyclic or heterocyclic compounds, thereby forming aroyl species. The reactive groups preferably are difunctional compounds known per se for use in cross-linking proteins to insoluble matrices through reactive side groups.
Covalent or aggregative functional equivalents and derivatives thereof are useful as reagents in immunoassays or for affinity purification procedures. For example, a TRAG-3 fragment according to the present invention may be insolubilized by covalent bonding to cyanogen bromide-activated Sepharose by methods known per se or adsorbed to polyolefin surfaces, either with or without glutaraldehyde cross-linking, for use in an assay or purification of anti-TRAG-3 fragment antibodies or cell surface receptors. Fragments may also be labelled with a detectable group, e.g., radioiodi- nated by the chloramine T procedure, covalently bound to rare earth chelates or conjugated to another fluorescent moiety for use in e.g. diagnostic assays.
TRAG-3 fragments according to the invention may be synthesised both in vitro and in vivo. In one embodiment the TRAG-3 fragments of the invention are synthesised by automated synthesis. Any of the commercially available solid-phase techniques may be employed, such as the Merrifield solid phase synthesis method, in which amino acids are sequentially added to a growing amino acid chain. (See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963).
Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied Biosystems, Inc. of Foster City, Calif., and may generally be operated according to the manufacturer's instructions. Solid phase synthesis will enable the incorporation of desirable amino acid substitutions into any TRAG-3 fragment according to the present invention. It will be understood that substitutions, deletions, insertions or any subcombination thereof may be combined to arrive at a final sequence of a functional equivalent. Insertions shall be understood to include amino-terminal and/or carboxyl-terminal fusions, e.g. with a hydrophobic or immu- nogenic protein or a carrier such as any polypeptide or scaffold structure capable as serving as a carrier.
Oligomers including dimers including homodimers and heterodimers of TRAG-3 fragments according to the invention are also provided and fall under the scope of the invention. Functional equivalents and variants of TRAG-3 fragments can be pro- duced as homodimers or heterodimers with other amino acid sequences or with native TRAG-3 sequences. Heterodimers include dimers containing immunoreactive TRAG-3 fragments as well as TRAG-3 fragments that need not have or exert any biological activity.
The invention also relates to nucleic acids encoding any of the polypeptide fragments described herein, as well as to said nucleic acids for use as medicaments, pharmaceutical compositions comprising said nucleic acids, vaccine compositions comprising the nucleic acids and use of the nucleic acids for the preparation of a medicament for the treatment of cancer. Vaccine compositions and pharmaceutical compositions may be any of the compositions disclosed herein comprising nucleic acids in stead of or in addition to TRAG-3 polypeptide fragments. The nucleic acid may be any nucleic acid, such as DNA or RNA. The nucleic acids may be comprised in a suitable vector, such as an expression vector. Numerous vectors are available and the skilled person will be able to select a useful vector for the specific purpose.
The vector may, for example, be in the form of a plasmid, cosmid, viral particle or artificial chromosome.
Examples
The following section serves to more fully describe the manner of using the above- described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes.
Example 1
Materials and Methods
Peptides
All peptides were purchased from Research Genetics (Huntsville, AL, USA) and provided at >80% purity as verified by HPLC and MS analysis. All peptides used are listed in Table 1.
Assembly assay for peptide binding to class I MHC molecules
Assembly assays for binding of the synthetic peptides to class I MHC molecules metabolically labeled with [35S]-methionine were carried out as described29'45. The assembly assay is based on stabilization of the class I molecule after loading of peptide to the peptide transporter deficient cell line T2. Subsequently correctly folded stable MHC heavy chains are immunoprecipitated using conformation-dependent antibodies. After IEF electrophoresis, gels were exposed to phosphorlmager screens, and peptide binding was quantitated using the Imagequant Phosphorlmager program (Molecular Dynamics, Sunnyvale, CA).
Antigen stimulation of PBL
To extend the sensitivity of the ELISPOT assay, PBL were stimulated once in vitro prior to analysis33'34. At day 0, PBL or crushed lymph nodes were thawed and plated in 2 ml/well at a concentration of 2 x 106 cells in 24-well plates (Nunc, Denmark) in X-vivo medium (Bio Whittaker, Walkersville, Maryland), 5% heat-inactivated human serum, and 2 mM of L-glutamine in the presence of 10 μM of peptide. Two days later 20 lU/ml recombinant interleukin-2 (IL-2) (Chiron, Ratingen, Germany) was added to the cultures. The cultured cells were tested for reactivity in the ELISPOT on day 10.
ELISPOT assay
The ELISPOT assay used to quantify peptide epitope-specific interferon-γ releasing effector cells was performed as described previously46. Briefly, nitrocellulose bottomed 96-well plates (Multiscreen MAIP N45, Millipore, Hedehusene, Denmark) were coated with anti-IFN-γ antibody (1-D1K, Mabtech, Nacka, Sweden). The wells were washed, blocked by X-vivo medium, and cells were added in duplicates at dif- ferent cell concentrations. Peptides or T2 stimulator cells were then added to each well and the plates were incubated overnight. The following day, media was discarded and the wells were washed prior to addition of biotinylated secondary antibody (7-B6-1-Biotin, Mabtech). The plates were incubated for 2 hours, washed and Avidin-enzyme conjugate (AP-Avidin, Calbiochem, Life Technologies) was added to each well. Plates were incubated at RT for 1 hour and the enzyme substrate (DAKO, Carpinteria; CA) was added to each well and incubated at RT for 5-10 min. The reaction was terminated by washing with tap-water upon the emergency of dark purple spots. The spots were quantitated using a computerized ELISPOT counter (Immu- nospot, CTL inc., CA, USA) and the peptide specific CTL frequency could be calculated from the numbers of spot-forming cells. The assays were all performed in duplicates for each peptide antigen.
Example 2
The present example demonstrates that TRAG-3 is recognized as a tumor antigen in cancer patients as TRAG-3 is subjected to T-cell responses. The amino acid sequence of the TRAG-3 polypeptide was screened for conceivable HLA-A2 nonamer and HLA-A2 decamer peptide epitopes by using the main HLA-A2 specific anchor residues47.
Twelve TRAG-3 deduced peptides were synthesized and examined for binding to HLA-A2 by comparison with the HLA-A2 high affinity positive control peptide C2 (GLCTLVAML) from EBV (Table 1 ). Two of the peptides adressed the insert region in the splice variant TRAG-3L (Feller et al., 2000), either completely (TJ.95 (SEQ ID NO:119)) or partially (TJ.102 (SEQ ID NO:126)). The peptide concentration required for half maximal recovery of class I MHC (C50 value) were 0.7 μM for the positive control.
Three TRAG-3 peptides T_4 (SEQ ID NO:6), T_45 (SEQ ID NO:232 or 309), and T_58 (SEQ ID NO:60) bound with similar high affinity as the positive control (C50 = 0.3, 0.7, and 0.7 μM, respectively) (Table 1). The peptides T_57.10 (SEQ ID NO:233 or 310), T_59 (SEQ ID NO:234 or 311), and T_L102 (SEQ ID NO: 126) bound in comparison with intermediate affinity (C50 = 35, 5, and 16 μM, respec- tively), whereas the peptides T_29 (SEQ ID NO:230 or 307), T_37.9 (SEQ ID NO:39), and T_57.9 (SEQ ID NO:59) bound only weakly to HLA-A2 (C50 >100 μM). The remaining three peptides had HLA-A2*0201 affinities below the detection limit of the assay (Not binding).
Using the ELISPOT IFN-γ secretion assay, we examined the presence of specific T- cell responses against the TRAG-3 deduced, HLA-A2 binding peptides in peripheral blood T cells (n = 7) or TIL (n = 22) from melanoma patients. The high sensitivity of this assay allows reliable detection of as few as 10-100 specific T cells/1 million. In addition, before analysis, the T cells were stimulated once in vitro to extend the sensitivity. This method has previously been shown to be highly effective to identify peptide epitopes recognized by CTL in cancer patients48"50. In contrast, other ex vivo assays such as intracellular IFN-γ staining or tetramer analysis by flow cytometry require approximately 10-fold higher specific T-cell frequencies for their detection.
We analyzed the T-cell responses in 9 breast cancer patients, 12 melanoma patients, and 13 patients suffering from the hematopoetic malignancies against the panel of TRAG-3 derived peptides capable of binding to the HLA-*0201 molecule.
In PBL from breast cancer patients, spontaneous immune responses were detected against one or several peptides in 7 of 9 patients (78%) (Figure 2). In this group, the majority of responses were directed against T_37.9 (SEQ ID NO:39), T_4 (SEQ ID NO:6), and T_57.10 (SEQ ID NO:310). However, single immune responses were detected against T_29 (SEQ ID NO:307), T_45 (SEQ ID NO:309), T_57.9 (SEQ ID NO:59), T_58 (SEQ ID NO:60), and
TJ.102 (126), the latter overlapping the insert region of the splice variant TRAG_3L.
In patients with various hematopoietic malignancies, immune responses against TRAG-3 derived peptides were detected in 5 of the 13 patients examined (38%) (Figure 3). In this group, immune responses against T_4 and T_58 tended to be slightly dominating, with dispersed responses against T_29, T_37.9, T_57.9, T_57.10, and TJ.102. Notably, no responses against the strong binder T_45 were observed in this group. The lymphocytes in this group originated from blood samples from patients with chronic lymphatic leukemia (CLL), lymphoma (LYM) or myeloma- tosis (MYE). The responding samples originated from one CLL, two LYM, and two MYE patients. The non-responding lymphocytes originated from two CLL, three LYM, and three MYE patients. Due to limited patient material available from the melanoma patients and the patients suffering from hematological malignancies, it was not possible to analyze for reactivity against the full panel of HLA-A*0201 bind- ing peptides in these patients.
In melanoma patients, TRAG-3 specific immune responses were detected in 7 of 12 patients (58%) (Figure 4). The immune responses against the strong binders T_4 (SEQ ID NO:6) and T_45 (SEQ ID NO:309) were prominent in this group, with dis- persed responses against T_29 (SEQ ID NO:307), T_37.9 (SEQ ID NO:39), T .57.9
(SEQ ID NO:59), T_57.10 (SEQ ID NO:310), and TJ.102 (SEQ ID NO:126). The responding melanoma patient material originated from tumor infiltration lymphocytes (TIL) from lymph node metastases, in addition to PBL. The non-responding lymphocytes were in three cases TIL from lymph node metastases, one subcutaneous me- tastasis, one from primary tumor, and one blood sample.
Two of nine breast cancer patients, five of twelve melanoma patients, and eight of thirteen patients suffering from hematopoietic malignancies did not display any TRAG-3-specific response (data not shown). Thus, spontaneous T-cell responses against TRAG-3 was detected in approximately half of the patients examined.
A recent study suggested that the induction of CTL responses correlate with binding, i.e., high affinity peptides are more frequently recognized by host T cells51. In the present study, responses against the high affinity binding peptide T_4 (SEQ ID NO:6) were detected in samples from 7 patients, however, responses against the intermediately binding peptide T_57.10 (SEQ ID NO:310) were almost as frequent (Figure 1). Surprisingly, the high affinity peptides T_45 (SEQ ID NO:309) and T_58 (SEQ ID NO:60) were only recognized in 3 and 4 patient samples, respectively. In this respect, it was recently shown that higher peptide binding affinity for HLA mole- cules does not necessarily equate to functional activity of the responding T cell52.
There are many factors, which determine a CTL response against a given peptide. These include expression level of the relevant source polypeptide, processing, TAP- transport, expression level of the class I MHC on the cell surface, TCR repertoire, CTL sensitivity, immuno-suppression and cytokines53. Thus, peptide binding to class I is one in a number of different factors which determine the immunogenicity of a given peptide. Additionally, in contrast to foreign peptides, when self-peptides are expressed on the cell surface at high density due to high MHC-binding affinity, tolerance seems to be induced, and reactive T cells are eliminated or inactivated54.
Subsequently, many immunodominant epitopes in CTL responses to self polypeptides may frequently be subdominant or cryptic, rather than dominant determinants. This may explain the observation that many epitopes from human melanoma antigens, which are non-mutated self-polypeptides, such as gp100 and MART-1 have relatively low binding affinities to class I MHC55. Given that the efficacy of tumor im- munotherapy most likely depends on the avidity of recruited CTL56, low affinity tumor epitopes might be important TAA, provided that they are able to mobilize their specific CTL repertoire and that they are presented by tumor cells efficiently enough to be recognized by CTL.
A recent study characterized the T_58 (SEQ ID NO:60) peptide, also described in the present study, and demonstrated that CTL specific for this peptide were cytolytic against HLA matched tumor targets expressing the TRAG-3 protein28. Clearly, these data support the notion of TRAG-3 derived peptides as targets for cytolytic T cells, strengthened and extended in the present study. Moreover, our data suggests that the T_58 (SEQ ID NO:60) peptide is not the prominent natural target for anti-TRAG- 3 CTL responses, although it could be speculated that the CTL recognizing one of peptides T_57.9 (SEQ ID NO:59), T_57.10 (SEQ ID NO:310), and T_58 (SEQ ID NO:60) may in fact react against all three peptides. In some patients, responses were detected against two or all three of these peptides, supporting this notion (Figure 2, patient B1 , Figure 2, patients H2-MYE, H3-LYM, and H5-LYM, and Figure 4, patients M3-PBL, and M5-PBL). However, in other patients responses were detected against one of the peptides only, suggesting that some T cells (in some patients) cross-react whereas others do not. Clearly, isolation and comparison of the clonotypic composition of cells responding against the respective peptides could provide the means to resolve this question57.
In conclusion, our data demonstrate that several peptides from the TRAG-3 protein are targets for spontaneous CTL responses in cancer patients. These peptides may be suited for future therapeutic vaccinations against cancer. The data presented herein above demonstrate that several peptides from the TRAG-3 protein, which is a tumor antigen of crucial importance for the resistance of cancer cells against taxol treatment, are targets for spontaneous, specific T-cell response in cancer patients.
As elevated expression of TRAG-3 in cells is associated with resistance to cancer chemotherapy and most likely also correlates with tumor progression in cancer patients, the combination of a TRAG-3-based immunotherapy with conventional cancer chemotherapy is believed to be an effective way to improve current cancer treat- ments.
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Claims

Claims
1. A polypeptide fragment capable of raising a specific T-cell response, said fragment comprising a peptide consisting of at least 9 consecutive amino acid residues of TRAG-3, wherein said peptide is selected from the group consisting NIKMHCEFHA (SEQ ID NO:230), VLGEAWRDQV (SEQ ID NO:232), SILLRDAGLV (SEQ ID NO:233), ALSKFPRQL (SEQ ID NO:126)and LLRDAGLVKM (SEQ ID NO:23), and wherein said polypeptide fragment consists of at the most 100 amino acids.
2. The polypeptide fragment according to claim 1 comprising or consisting of at least 10 consecutive amino acid residues of TRAG-3.
3. The polypeptide fragment according to claim 1 comprising or consisting of at least 11 consecutive amino acid residues of TRAG-3.
4. The polypeptide fragment according to claim 1 comprising or consisting of at least 12 consecutive amino acid residues of TRAG-3.
5. The polypeptide fragment according to claim 1 comprising or consisting of at least 13 consecutive amino acid residues of TRAG-3.
6. The polypeptide fragment according to claim 1 comprising or consisting of at least 14 consecutive amino acid residues of TRAG-3.
7. The polypeptide fragment according to claim 1 comprising or consisting of at least 15 consecutive amino acid residues of TRAG-3.
8. The polypeptide fragment according to claim 1 comprising sillrdaglv (SEQ ID NO:233) and consisting of no more than 15 amino acid residues.
9. The polypeptide fragment according to claim 1 comprising nikmhcefha (SEQ ID NO:230) and consisting of no more than 15 amino acid residues.
10. The polypeptide fragment according to claim 1 comprising vlgeawrdqv (SEQ ID NO:232) and consisting of no more than 15 amino acid residues.
11. The polypeptide fragment according to claim 1 comprising llrdaglvkm (SEQ ID NO:234) and consisting of no more than 15 amino acid residues.
12. The polypeptide fragment according to claim 1 comprising alskfprql (SEQ ID NO: 126) and consisting of no more than 15 amino acid residues.
13. The polypeptide fragment according to claim 1 , wherein the specific T-cell response is measured as more than 50 peptide specific spots per 108 cells in an ELISPOT assay.
14. The polypeptide fragment according to claim 13, wherein the specific T-cell response is measured as more than 50 peptide specific spots per 107 cells in an ELISPOT assay.
15. The polypeptide fragment according to claim 13, wherein the specific T-cell response is measured as more than 50 peptide specific spots per 106 cells in an ELISPOT assay.
16. The polypeptide fragment according to claim 13, wherein the specific T-cell response is measured as more than 50 peptide specific spots per 105 cells in an ELISPOT assay.
17. The polypeptide fragment according to claim 13, wherein the specific T-cell response is measured as more than 50 peptide specific spots per 104 cells in an ELISPOT assay.
18. The polypeptide fragment according to any of claims 13 to 17, wherein the specific T-cell response is measured in an ELISPOT assay performed using PBL from an individual that has not been subjected to immune therapy against a neoplastic disease.
19. The polypeptide fragment according to claim 1 , wherein the polypeptide fragment is characterized by having a C50 value, measured as the concentration (μM) of the polypeptide fragment required for half maximal binding to a MHC (Major Histocompatibility Complex) class I molecule, of less than 1000.
20. The polypeptide fragment according to claim 19, wherein the polypeptide fragment is characterized by having a C50 value, measured as the concentration (μM) of the polypeptide fragment required for half maximal binding to a MHC class I molecule, of less than 100.
21. The polypeptide fragment according to claim 19, wherein the polypeptide fragment is characterized by having a C50 value, measured as the concentration (μM) of the polypeptide fragment required for half maximal binding to a MHC class I molecule, of less than 31.
22. The polypeptide fragment according to claim 19, wherein the polypeptide fragment is characterized by having a C50 value, measured as the concentration (μM) of the polypeptide fragment required for half maximal binding to a MHC class I molecule, of less than 5.
23. The polypeptide fragment according to claim 19, wherein the polypeptide fragment is characterized by having a C50 value, measured as the concentration (μM) of the polypeptide fragment required for half maximal binding to a MHC class I molecule, of less than 1.
24. The polypeptide fragment according to any of claims 1 to 23, wherein the polypeptide fragment is capable of activating T-cell growth in vitro.
25. The polypeptide fragment according to claim 24, wherein the polypeptide fragment is capable of activating T-cell growth in vitro so that more than 10 antigen specific cytotoxic T lymphocytes (CTL) may be harvested after 4 stimulation cycles starting with 104 PBMC.
26. The polypeptide fragment according to claim 25, wherein the polypeptide fragment is capable of activating T-cell growth in vitro so that more than 106 antigen specific CTL may be harvested after 4 stimulation cycles starting with 104 PBMC.
27. The polypeptide fragment according to claim 25, wherein the polypeptide fragment is capable of activating T-cell growth in vitro so that more than 107 antigen specific CTL may be harvested after 4 stimulation cycles starting with 104 PBMC.
28. The polypeptide fragment according to any of claims 1 to 27, wherein the polypeptide fragment is capable of raising a specific cytotoxic T-cell response.
29. The polypeptide fragment according to claim 28, wherein the specific cytotoxic T-cell response is measured as more than 15% polypeptide- specific target lysis in a [51Cr] release assay.
30. A nucleic acid encoding the polypeptide fragment according to any of claims 1 to 29.
31. The nucleic acid according to claim 30 for use as a medicament.
32. A method of selecting a peptide comprising a fragment of TRAG-3 for use in a vaccine composition comprising the steps of (i) providing an individual who has not been subjected to immune therapy, (ii) providing fragments of TRAG-3, (iii) testing specific T-cell responses against the fragments of TRAG-3 in said individual, (iv) selecting fragments of TRAG-3, wherein said T-cell response corresponds to or is better than a predetermined selection criterium.
33. The method according to claim 32, wherein testing said T-cell response comprises an ELISPOT assay.
34. The method according to claim 33, wherein said predetermined selection criterium is more than 50 peptide specific spots per 108 cells in said ELISPOT assay.
35. The method according to claim 33, wherein said predetermined selection criterium is more than 50 peptide specific spots per 107 cells in said ELISPOT assay.
36. The method according to claim 33, wherein said predetermined selection criterium is more than 50 peptide specific spots per 106 cells in said ELISPOT assay.
37. The method according to claim 33, wherein said predetermined selection criterium is more than 50 peptide specific spots per 105 cells in said ELISPOT assay.
38. The method according to claim 33, wherein said predetermined selection criterium is more than 50 peptide specific spots per 104 cells in said ELISPOT assay.
39. A polypeptide fragment according to any of claims 1 to 29 for use as a medicament.
40. Use of one or more polypeptide fragments according to any of claims 1 to 29 or of a nucleic acid according to claim 30 in the manufacture of a pharmaceutical composition for treatment of a clinical condition in an individual in need thereof.
41. Use according to claim 40, wherein said clinical condition is cancer.
42. Use according to claim 41 , wherein the cancer is a malignant melanoma.
43. Use according to claim 41 , wherein the cancer is a lung cancer.
44. Use according to claim 43, wherein the lung cancer is a non-small cell lung cancer.
45. Use according to claim 41 , wherein the cancer is a leukemia.
46. Use according to claim 41 , wherein the cancer is a sarcoma.
47. Use according to claim 46, wherein the cancer is a chondrosarcoma.
48. Use according to claim 41 , wherein the cancer is a colon cancer.
49. Use according to claim 41 , wherein the cancer is an ovary cancer.
50. Use according to claim 41 , wherein the cancer is a kidney cancer.
51. Use according to claim 41 , wherein the cancer is a prostate cancer.
52. Use according to claim 41 , wherein the cancer is a breast cancer.
53. Use according to claim 41 , wherein the cancer is a cancer of the central nervous system.
54. Use according to claim 40, wherein said clinical condition is an autoimmune disease.
55. Use according to claim 40, wherein at least one of said polypeptide fragments is restricted to an HLA molecule present in said individual.
56. Use according to claim 55, wherein said HLA molecule is selected from the group consisting of HLA-A2, HLA-A3, HLA-A1 , HLA-A11 , HLA-A24, HLA- A68, HLA-B7, HLA-B8, HLA-B14, HLA-B27, HLA-B35, HLA-B60, HLA-B61 , and HLA-B62.
57. Use according to any of claims 55 and 56, wherein said HLA molecule is a HLA-A2 molecule.
58. Use according to claim 40, wherein said individual has not previously been subjected to immune therapy against a neoplastic disease.
59. A pharmaceutical composition for treating a clinical condition comprising a polypeptide according to any of claims 1 to 29 or the nucleic acid according to claim 30 as an active ingredient and a pharmaceutically acceptable carrier.
60. A vaccine composition comprising isolated TRAG-3 and/or at least one polypeptide fragment according to any of claims 1 to 29 or a nucleic acid according to claim 30; and a pharmaceutically acceptable carrier and/or adjuvant.
61. The vaccine composition according to claim 60, wherein the composition comprises said at least one polypeptide fragment in the form of at least one nucleic acid that encodes said at least one polypeptide fragment.
62. The vaccine composition according to any of claims 60 and 61 further comprising an adjuvant.
63. The vaccine composition according to claim 62, wherein the adjuvant is selected from the group consisting of Montanide ISA-51 and QS-21.
64. The vaccine composition according to any of claims 60 to 63, wherein the composition further comprises a pharmaceutically acceptable carrier.
65. The vaccine composition according to claim 64, wherein the carrier is a dendritic cell.
66. The vaccine composition according to claim 60, wherein said composition comprises more than one different polypeptide fragment according to any of claims 1 to 31.
67. The vaccine composition according to any of claims 66 and 67, wherein the composition comprises said more than one different polypeptide fragment in the form of at least one nucleic acid that encodes said more than one different polypeptide fragment.
68. The vaccine composition according to claim 66, wherein the composition comprises different TRAG-3 fragments, wherein said fragments are capable of associating with the most frequently occuring MHC class I molecules.
69. The vaccine composition according to any of claims 67 and 68, wherein the composition comprises at least 1 , more preferably at least 2, even more preferably at least 3, yet more preferably at least 4, for example at least 5, such as at least 6, for example 7 different TRAG-3 fragments each capable of associating with a different HLA molecule selected from the group consisting of HLA-A2, HLA-A3, HLA-A1 , HLA-A11 , HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B27, HLA-B35, HLA-B60, HLA-B61 , and HLA-B62.
70. A pharmaceutical composition comprising the vaccine composition according to any of claims 60 to 69 and an anti-cancer medicament.
71. The pharmaceutical composition according to claim 70, wherein the anti- cancer medicament is selected from the group consisting of chemo- therapeutic agents and immunotherapeutic agents.
72. The pharmaceutical composition according to claim 71 , wherein the anti- cancer medicament is selected from the group of taxanes.
73. The pharmaceutical composition according to claim 72, wherein the anticancer medicament is taxol.
74. A kit of parts comprising TRAG-3 and/or one or more fragments thereof and a bioactive compound selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent, and a second cancer vaccine composition.
75. The kit of parts according to claim 74, comprising one or more polypeptide fragments according to any of claims 1 to 31.
76. The kit of parts according to claim 75, wherein the the kit of parts comprises said one or more polypeptide fragments in the form of one or more nucleic acid that encode said one or more polypeptide fragment.
77. A method for treatment of an individual diagnosed with cancer, said method comprising the step of administering to the individual a polypeptide fragment according to any of claims 1 to 31 , or the vaccine composition according to any of claims 60 to 69, or the pharmaceutical composition according to any of claims 70 to 73, or the kit of parts according to any of claims 74 to 76.
78. The method according to claim 77, wherein said individual has not previously been subjected to immune therapy against a neoplastic disease.
79. The method according to claim 77, wherein the cancer is a malignant melanoma.
80. The method according to claim 77, wherein the cancer is a lung cancer.
81. The method according to claim 80, wherein the cancer is a non-small cell lung cancer.
82. The method according to claim 77, wherein the cancer is a leukemia.
83. The method according to claim 77, wherein the cancer is a sarcoma.
84. The method according to claim 83, wherein the cancer is a chondrosarcoma.
85. The method according to claim 77, wherein the cancer is a colon cancer.
86. The method according to claim 77, wherein the cancer is an ovary cancer.
87. The method according to claim 77, wherein the cancer is a kidney cancer.
88. The method according to claim 77, wherein the cancer is a prostate cancer.
89. The method according to claim 77, wherein the cancer is a breast cancer.
90. The method according to claim 77, wherein the cancer is a cancer of the central nervous system.
91. A method for prophylactic treatment of an individual at risk of developing a cancer, said method comprising the step of administering to the individual a polypeptide fragment according to any of claims 1 to 31 , or the vaccine composition according to any of claims 60 to 69, or the pharmaceutical composition according to any of claims 70 to 73, or the kit of parts according to any of claims 74 to 76.
92. A method for raising a specific T-cell response against an epitope of TRAG- 3 in an individual, said method comprising the steps of administering to the individual a polypeptide fragment according to any of claims 1 to 31 , and raising a specific T-cell response against an epitope of TRAG-3 in the individual.
93. A method according to any of claims 77 to 92, wherein the polypeptide fragment elicits a cytotoxic T-lymphocyte response.
94. A method according to any of claims 77 to 93, wherein the administration step comprises expressing a nucleic acid that encodes said polypeptide fragment.
95. A method according to any of claims 77 to 94, wherein the method comprises administering one or more polypeptide fragments according to any of claims 1 to 31 , and wherein at least one fragment is restricted to an HLA molecule present in said individual.
96. A method according to claim 95, wherein the administration step comprises expressing one or more nucleic acids that encode said one or more polypeptide fragments.
97. An antibody capable of specific recognition of a polypeptide fragment according to any of claims 1 to 31.
98. An antibody according to claim 97, wherein the antibody is a monoclonal antibody.
99. An antibody according to any of claims 97 and 98, wherein the antibody is a humanised antibody.
100. A method for activating and expanding T cells specific for TRAG-3 or fragments thereof comprising the steps of co-cultivating T cells and TRAG- 3, or one or more fragments thereof.
101. The method according to claim 100, wherein said fragments comprises one or more polypeptide fragments according to any of claims 1 to 31.
102. The methods according to any of claims 100 and 101 , wherein the method comprises generating and loading monocyte-derived dendritic cells (DC) with TRAG-3 fragment(s) and co-cultivating said DC and peripheral blood monocytes (PBMC) comprising T cells.
103. The methods according to any of claims 100 and 101 , wherein the method comprises generating Drosophila melanogaster cells expressing one or more different HLA molecules, loading said Drosophila melanogaster cells with TRAG-3 fragment(s) and co-cultivating said Drosophila cells with peripheral blood monocytes (PBMC) comprising T cells or T cells purified from PBMC.
104. TRAG-3-specific T cells obtained by the method according to any of claims 100 to 103.
105. T cells according to claim 104, wherein said TRAG-3-specific T cells are cytotoxic T lymphocytes.
106. Use of TRAG-3-specific T cells according to any of claims 104 and 105 for the preparation of a pharmaceutical composition for treatment of a clinical condition in an individual in need thereof.
107. Use of TRAG-3-specific T cells according to claim 106, wherein said clinical condition is cancer.
108. Use of TRAG-3-specific T cells according to claim 107, wherein the cancer is a malignant melanoma.
109. Use of TRAG-3-specific T cells according to claim 107, wherein the cancer is a lung cancer.
110. Use of TRAG-3-specific T cells according to claim 109, wherein the cancer is a non-small cell lung cancer.
111. Use of TRAG-3-specific T cells according to claim 107, wherein the cancer is a leukemia.
112. Use of TRAG-3-specific T cells according to claim 107, wherein the cancer is a sarcoma.
113. Use of TRAG-3-specific T cells according to claim 107, wherein the cancer is a chondrosarcoma.
114. Use of TRAG-3-specific T cells according to claim 107, wherein the cancer is a colon cancer.
115. Use of TRAG-3-specific T cells according to claim 107, wherein the cancer is an ovary cancer.
116. Use of TRAG-3-specific T cells according to claim 107, wherein the cancer is a kidney cancer.
117. Use of TRAG-3-specific T cells according to claim 107, wherein the cancer is a prostate cancer.
118. Use of TRAG-3-specific T cells according to claim 107, wherein the cancer is a breast cancer.
119. Use of TRAG-3-specific T cells according to claim 107, wherein the cancer is a cancer of the central nervous system.
120. Use of TRAG-3-specific T cells according to claim 107, wherein said clinical condition is an auto-immune disease.
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