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WO2009008713A1 - Inhibiteurs de tap à partir d'herpèsvirus 1 de primate d'europe et leur utilisation - Google Patents

Inhibiteurs de tap à partir d'herpèsvirus 1 de primate d'europe et leur utilisation Download PDF

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Publication number
WO2009008713A1
WO2009008713A1 PCT/NL2008/050440 NL2008050440W WO2009008713A1 WO 2009008713 A1 WO2009008713 A1 WO 2009008713A1 NL 2008050440 W NL2008050440 W NL 2008050440W WO 2009008713 A1 WO2009008713 A1 WO 2009008713A1
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Prior art keywords
tap
cell
cells
polypeptide
inhibitor
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Inventor
Maaike Elisabeth Ressing
Daphne Van Leeuwen
Daniëlle HORST
Emmanuel Jacques Henri Joseph Wiertz
Alan Bernard Rickinson
Victoria Anne Pudney
Andrew Douglas Hislop
Thorbald Van Hall
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Leids Universitair Medisch Centrum LUMC
Publiekrechtelijke Rechtspersoon Academisch Ziekenhuis Leiden HODN
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Leids Universitair Medisch Centrum LUMC
Publiekrechtelijke Rechtspersoon Academisch Ziekenhuis Leiden HODN
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • TAP-inhibitors from Old World primate ⁇ l -herpesviruses and their use
  • the present invention relates to the fields of medicine and molecular immunology.
  • the invention relates to TAP-inhibitors from Old World primate ⁇ l -herpesviruses compositions comprising such TAP-inhibitors and/or nucleotide sequences encoding the TAP-inhibitors as well as methods wherein the TAP-inhibitors are used for induction of tumour- or virus-specific immunity against T cell epitopes associated with impaired peptide processing.
  • Cytotoxic T lymphocytes are important for the immune control of viral infections and have also shown to exhibit the capacity to eradicate established tumours (Harty et al., 2000, Annu Rev Immunol 18:275; Melief et al., 2000, Adv. Immunol. 75:235; Offringa, 2005, Curr. Opin. Immunol. 17: 159; Dudley et al., 2002, Science 298:850).
  • the efficacy and safety of CTL-based immunotherapy are currently being evaluated in experimental clinical trials (Offringa et al., 2000, Curr. Opin. Immunol. 12:576; Rosenberg et al., 2004, Nat. Med.
  • TAP antigen processing
  • WO 98/25645 discloses practical applications of the concept that prevention of TAP-function leads to recognition of novel, endogenous MHC class I dependent antigens by host T-cells that are not recognized in the presence of a fully functional TAP-molecule.
  • WO 98/25645 discloses that immunization with TAP-deficient cells elicits T-cells directed against epitopes expressed preferentially by TAP-deficient cells and that induction of such T-cells can prevent growth of several tumour targets.
  • WO 98/25645 suggest the use of autologous cells, especially dendritic cells, that have been treated to express MHC class I dependent epitopes associated with impaired cellular peptide processing and to inject these cells into a patient in order to stimulate T cells to react on these epitopes as presented by tumour cells or virally infected cells.
  • autologous cells especially dendritic cells
  • WO 98/25645 suggest to use a variety of substances that include viral TAP-inhibitors such as e.g.
  • proteasome inhibitors such as the peptide aldehyde Z-Leu-Leu-Leu-H (Peptide Internationals Inc., Louisville, KY) or Lactacystin (Calbiochem, La Jolla, California), genes encoding inhibitors of components that take part in the peptide processing of the cell, and substances that inhibit the expression of cellular components that take part in the peptide processing, such as e.g. antisense oligonucleotides or ribozymes.
  • homologous when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species.
  • the term “homologous” means that one single- stranded nucleic acid sequence may hybridize to a complementary single- stranded nucleic acid sequence.
  • the degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as discussed above.
  • the region of identity is greater than about 5 bp, more preferably the region of identity is greater than 10 bp.
  • autologous is used herein to refer to proteins, nucleic acids, cells, tissues or organs that are obtained from one subject or patient and that are, preferably after some form of ex vivo treatment, returned to, administered to or reinplanted or reinfused into the same subject or patient.
  • promoter refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.
  • a “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions.
  • an “inducible” promoter is a promoter that is physiologically or developmentally regulated.
  • a “tissue specific” promoter is only active in specific types of tissues or cells.
  • operably linked refers to two or more nucleic acid or amino acid sequence elements that are physically linked in such a way that they are in a functional relationship with each other.
  • a promoter is operably linked to a coding sequence if the promoter is able to initiate or otherwise control/regulate the transcription and/or expression of a coding sequence, in which case the coding sequence should be understood as being “under the control of the promoter.
  • two nucleic acid sequences when operably linked, they will be in the same orientation and usually also in the same reading frame. They will usually also be essentially contiguous, although this may not be required.
  • signal sequence The terms “signal sequence”, “signal peptide” and “secretory leader” are used interchangeably and refer to a short (usually about 15-60 amino acids), continuous stretch of amino acids usually present at the amino-terminus of secreted and membrane-bound polypeptides and that directs their delivery to various locations outside the cytosol.
  • specific sorting or targeting signals which include signal sequences, may direct the delivery of polypeptides into the nucleus, ER, mitochondria, peroxisomes, etc.
  • Signal sequences usually contain a hydrophobic core of about 4-15 amino acids, which is often immediately preceded by a basic amino acid.
  • transgene is herein defined as a gene that has been newly introduced into a cell, i.e. a gene that does not normally occur in the cell.
  • the transgene may comprise sequences that are native to the cell, sequences that in naturally do not occur in the cell and it may comprise combinations of both.
  • a transgene may contain sequences coding for one or more proteins that may be operably linked to appropriate regulatory sequences for expression of the coding sequences in the cell.
  • the degree of identity i.e.
  • the match percentage, between two polypeptides, respectively two nucleic acid sequences is preferably determined using the optimal global alignment method CDA (Huang, 1994, A Context Dependent Method for Comparing Sequences, Proceedings of the 5th Symposium on Combinatorial Pattern Matching, Lecture Notes in Computer Science 807, Springer- Verlag, 54-63) with the parameters set as follows: (i) for (poly)peptide alignments: Mismatch:-2 Gap ⁇ pen: l l GapExtend: l ContextLength:10 MatchBonus: l, and (ii) for nucleotide sequence alignments Mismatch:-15 Gap ⁇ pen:5 GapExtend:2 ContextLength: 10 MatchBonus: l.
  • CDA Human, 1994, A Context Dependent Method for Comparing Sequences, Proceedings of the 5th Symposium on Combinatorial Pattern Matching, Lecture Notes in Computer Science 807, Springer- Verlag, 54-63
  • CDA Human, 1994, A Context Dependent Method for Comparing
  • degree of identity is used interchangeably to indicate the degree of identity between two polypeptides or nucleic acid sequences as calculated by the optimal global alignment method indicated above.
  • Examples of alternative programs used for alignments and determination of homology are Clustal method (Higgins, 1989, CABIOS 5 : 151-153) , the Wilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of the National Academy of Science USA 80: 726-730) using the LASERGENE.TM. MEGALIGN.TM.
  • amino acid positions, which are not identical differ by conservative amino acid substitutions.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic and/or hydrophobic side chains is glycine, alanine, valine, leucine, isoleucine and methionine; 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 glutamine; 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 sulphur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine- methionine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine- glutamine.
  • Herpesviruses are an ancient virus family whose members have long histories of co-evolution with their host species (1).
  • a hallmark of herpesvirus biology is the ability to colonize a naive host through the productive (lytic) infection of permissive cells, usually at a mucosal site of virus transmission, and thereafter to persist within that host as a non-productive (latent) infection of a different specialized cell type.
  • Persistence within the now-immune host is achieved through the down-regulation of all viral antigen expression in latently-infected cells. Subsequently, occasional reactivations from latency can serve to re-establish foci of virus replication at mucosal sites, providing a source of infectious virions for transmission to other individuals.
  • herpes simplex virus a member of the ⁇ - herpesvirus subfamily establishing latency in neurons
  • the chances of successful reactivation are increased if the virus initially establishes a high latent virus genome load (2, 3); this in turn is reliant upon the level of virus replication first achieved during primary infection of the naive host (3, 4).
  • the host CD8+ T cell response is the principal means of controlling this initial replication, any factor restricting the efficiency of that control would be to the advantage of the virus.
  • HSV was the first herpesvirus in which a CD8+ T cell evasion mechanism was recognized.
  • lymphocryptoviruses the most recently evolved ( ⁇ l) herpesvirus genus whose members are found only in Old World and some New World primate species and whose prototype is the Epstein-Barr virus (EBV) of man. These viruses are orally transmitted, replicate in oropharyngeal epithelial sites and establish latency in B lymphocytes (reviewed in (9)).
  • LCVs use their unique growth-transforming ability to directly drive the expansion of latently-infected B cells and only later down-regulate latent antigen expression to establish an immunologically silent infection of the memory B cell pool (10).
  • the invention relates to a polypeptide with TAP-inhibitor activity.
  • the polypeptide preferably comprises an amino acid sequence that has at least 45 50, 52, 53, 55, 60, 62, 63, 65, 70, 80, 90, 95, 99 or 100% amino acid identity with at least one of SEQ ID NO.'s 1, 2, 3, 4, 5 and 6.
  • the polypeptide with TAP-inhibitor activity of the invention (a "LCV TAP-inhibitor”) preferably has a length of 55 - 65 amino acids, more preferably 58 - 62 amino acids and most preferably 59 - 60 amino acids.
  • the amino acid sequence of the LCV TAP-inhibitor of the invention may thus comprise one or more amino acid substitution, insertion and/or deletions, whereby the amino acid substitutions are preferably conservative amino acid substitutions as hereinabove defined.
  • the amino acid sequence of the LCV TAP-inhibitor at least comprises the conserved amino acids indicated by the black regions in Figure 6 herein, except for the N-terminal methionine. More preferably, the amino acid sequence of the LCV TAP-inhibitor in addition the conserved amino acids at least comprises the conservative amino acids indicated by the shaded regions in Figure 6 herein.
  • a preferred LCV TAP-inhibitor of the invention comprises the amino acid sequence of, or is a BNLF2a homologues that is encoded by lymphocryptoviruses (LCV), more preferably an Old World ⁇ -1 primate herpesviruses.
  • LCV lymphocryptoviruses
  • Particularly preferred LCV TAP-inhibitor comprise the amino acid sequence of a BNLF2a homologues that is encoded by a genome of an LCV selected from the group consisting of Rhesus LCV, baboons LCV (papio herpesvirus), chimpanzee LCV (pan herpesvirus), orangutan LCV (orangutan herpesvirus) and gorilla LCV (gorilla herpesvirus).
  • a LCV TAP-inhibitor of the invention further preferably is a protein that reduces TAP-dependent peptide transport in a mammalian cell by at least 50, 60, 70, 80, 90, or 95%. More preferably the LCV TAP-inhibitor reduces TAP-dependent peptide transport in human and/or murine cells. Suitable assays for inhibition of TAP- dependent peptide transport are described in the Examples herein, in particular in Example 1.5 and for both human and mouse cells also in references (32) and (33).
  • a particularly preferred TAP-inhibitor of the invention reduces TAP-dependent transport of the fluorescein-conjugated synthetic peptide CVNKTERAY in cells of the human melanoma MEL-JUSO (MJS) cell line that stably express the LCV TAP-inhibitor by at least 50, 60, 70, 80, 90, or 95%, as compared to TAP-dependent transport of the peptide in untransformed human melanoma (MJS) cells, under the same conditions.
  • the human melanoma MEL-JUSO cell line is available from DSMZ, Braunschweig, Germany (www.dsmz.de) under accession no. ACC 74.
  • a polypeptide comprising a LCV TAP-inhibitor of the invention may be synthesised by standard methods of solid phase chemistry known to those of ordinary skill in the art.
  • synthetic LCV TAP-inhibitor peptides may be synthesised by solid phase chemistry techniques following the procedures described by Steward and Young (Steward, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company, Rockford, 111., (1984) using an Applied Biosystem synthesiser.
  • multiple peptide fragments may be synthesised then linked together to form larger fragments.
  • solid phase peptide synthesis a summary of the many techniques may be found in J. M. Stewart and J. D.
  • Synthetic LCV TAP-inhibitor peptides may e.g. be synthesised by Fmoc-based or Boc-based chemistry, on a normal solid support, e.g. on polystyrene based or polystyrene/polyethyleenglycol-copolymer based material containing a proper handle, e.g.
  • a safety catch handle in acceptable purity, e.g. preferably more than 80 % pure.
  • P carboxy-terminal proline
  • G amino -terminal glycine
  • the N-terminal G can than be modified with an protection/activation group like the N-2,3,4-trimethoxy-5-mercaptophenyl group which provides the glycine a reactivity towards thio esters that is comparable with that of a cystein and still yields a glycine in the endproduct (J. Am. Chem. Soc. 124, 4642 (2002)).
  • an protection/activation group like the N-2,3,4-trimethoxy-5-mercaptophenyl group which provides the glycine a reactivity towards thio esters that is comparable with that of a cystein and still yields a glycine in the endproduct (J. Am. Chem. Soc. 124, 4642 (2002)).
  • the invention thus also relates to a method for producing a LCV TAP-inhibitor of the invention, which method comprises chemical synthesis of at least one peptide bond or at least a part of the amino acid sequence of the LCV TAP- inhibitor.
  • An advantage of using chemical synthesis for the preparation a synthetic LCV TAP-inhibitor peptide is that non-naturally occurring LCV TAP-inhibitors may be produced, e.g. LCV TAP-inhibitors that comprise one or more of the following modifications.
  • (D)-amino acids can be substituted for (L)- amino acids, if desired.
  • the incorporation of one or more (D)-amino acids into a synthetic LCV TAP-inhibitor peptide analogue can confer, for example, additional stability of the peptide in vitro or, particularly, in vivo, since endogenous endoproteases generally are ineffective against peptides containing (D)-amino acids.
  • Naturally occurring peptides that have been chemically synthesized to contain (D)-amino acids maintain their activity (see e.g. Wade et al., 1990, Proc. Natl. Acad. Sci. USA 87: 4761-4765).
  • a synthetic LCV TAP-inhibitor peptide of the invention comprise at least two, three, four or five consecutive (L)-amino acids. More preferably a synthetic LCV TAP-inhibitor peptide of the invention comprise less than four alternating (D)- and (L)-amino acids, or less than four alternating (D)- and (L)- ⁇ - amino acids. If desired, the reactive side group of one or more amino acids in a synthetic LCV
  • TAP-inhibitor peptide of the invention can be modified or amino acid derivatives can be incorporated into the peptide (see, for example, Protein Engineering: A practical approach (IRL Press 1992); Bodanszky, Principles of Peptide Synthesis (Springer- Verlag 1984)).
  • Selective modification of a reactive group can impart desirable characteristics upon a cyclic compound of the invention. The choice of including such a modification is determined, in part, by the characteristics required of the peptide. Such modifications can result, for example, in synthetic LCV TAP-inhibitor peptide variants having greater LCV TAP-inhibitor peptide activity.
  • a peptide bond mimetic of the invention includes peptide backbone modifications well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the ⁇ -carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone cross-links. See, generally, Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. VII (Weinstein ed., 1983).
  • amino acids for incorporation into the synthetic LCV TAP- inhibitor peptide of the invention include amino acid mimetics.
  • An "amino acid mimetic" as used here is a moiety other than a naturally occurring amino acid that conformationally and functionally serves as a substitute for an amino acid in a cyclic compound of the present invention. Such a moiety serves as a substitute for an amino acid residue if it does not interfere with the antimicrobial activity of the compound. Preferably the substitute improves the antimicrobial activity of the compound.
  • Amino acid mimetics may include non-protein amino acids, such as ⁇ -, ⁇ -, ⁇ -amino acids, ⁇ -, ⁇ -, ⁇ imino acids (such as piperidine-4-carboxylic acid) ⁇ 2 amino acids, ⁇ 3 amino acids, ⁇ , ⁇ -di substituted amino acids, ⁇ -amino sulfonyl compounds, ⁇ -aminohydrazino acids, ⁇ -hydroxy acids, ⁇ -amino nitriles as well as many derivatives of L- ⁇ -amino acids.
  • Peptide mimetics suitable for peptides of the present invention are discussed by Morgan and Gainor, (1989) Ann. Repts. Med. Chem. 24:243-252.
  • a synthetic LCV TAP-inhibitor peptide of the invention comprises less than three consecutive homochiral ⁇ -amino acids, more preferably less than three homochiral ⁇ - amino acids.
  • the (synthetic) LCV TAP-inhibitor of the invention is a lipopeptide, i.e. the polypeptide comprising the amino acid sequence with TAP-inhibitor activity comprises one or more lipid moieties covalently attached to the polypeptide.
  • the lipid moieties covalently attached to the synthetic LCV TAP-inhibitor preferably effects translocation of exogenously added TAP-inhibitor into a cell.
  • Any lipid moiety that is capable of effecting translocation of the TAP-inhibitor into a cell may be attached to the inhibitor.
  • a lipid moiety that is effective in internalisation of a TAP-inhibitor covalently linked thereto is herein defined as a lipid moiety that increases the TAP inhibitor activity of the TAP inhibitor covalently linked to the moiety by at least a factor 2, 5, 10, 100 or 1000, as compared to the same TAP inhibitor without the moiety, when both are tested in an assays for inhibition of TAP-dependent peptide transport as described above, whereby the cells are preincubated with the inhibitors and then washed prior to that the labelled peptide is added.
  • Several different fatty acids are known for use in lipid moieties.
  • Exemplary fatty acids include, but are not limited to, palmitoyl, myristoyl, stearoyl and decanoyl groups or, more generally, any C2 to C30 saturated, monounsaturated, or polyunsaturated fatty acyl group is thought to be useful.
  • the at least one lipid moiety preferably is attached at or near the C-terminus of the amino acid sequence with TAP-inhibitor activity.
  • the at least one lipid moiety preferably is attached no more than 12, 6, 4, or 3 amino acid from the C-terminal amino acid of the amino acid sequence with TAP-inhibitor activity and/or of the polypeptide.
  • the invention in a third aspect relates to a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of a LCV TAP-inhibitor as defined hereinabove.
  • nucleotide sequences encoding amino acid sequences of LCV TAP-inhibitor of the invention are given in SEQ ID NO.'s 7, 8, 9, 10, and 11.
  • isocoding nucleotide sequences of SEQ ID NO.'s 7, 8, 9, 10, and 11 are particularly included in the invention, such as e.g. sequences the codon usage of which has been adapted to a given host cell as described hereinbelow.
  • the nucleic acid molecule comprising the nucleotide sequence encoding the LCV TAP-inhibitor may be a DNA molecule or it may be an RNA molecule.
  • the nucleic acid molecule comprising the nucleotide sequence encoding the LCV TAP-inhibitor is an expression construct.
  • the expression construct can be any nucleic acid construct comprising a nucleotide sequence encoding a LCV TAP- inhibitor of the invention that is suitable for introduction into the desired target cell and that is capable of expressing the LCV TAP-inhibitor upon introduction into the cell.
  • the nucleotide sequence encoding the LCV TAP-inhibitor is preferably operably linked to expression signals.
  • the nucleotide sequence encoding the LCV TAP-inhibitor preferably is at least operably linked to a promoter.
  • the promoter is a promoter that is preferably active or can be induced to be active in the mammalian target cell, preferably an antigen presenting cell, such as a dendritic cell.
  • the promoter may be a constitutive promoter, an inducible promoter or a tissue specific promoter, preferably specific for an antigen presenting cell, such as a dendritic cell.
  • Suitable promoters for expression of the nucleotide sequence encoding an LCV TAP-inhibitor include e.g.
  • CMV cytomegalovirus
  • LTRs viral long terminal repeat promoters
  • MMLV murine moloney leukaemia virus
  • HTLV-I viral long terminal repeat promoters
  • SV 40 simian virus 40
  • the expression construct may further comprise additional sequence elements for the expression of the nucleotide sequence encoding an LCV TAP-inhibitor, such as transcriptional enhancers and/or silencers, transcriptional terminators, and polyA- addition sites.
  • the expression construct may optionally comprise a second or one or more further nucleotide sequences coding for a second or further protein.
  • the second or further protein may be a (selectable) marker protein that allows for the identification, selection and/or screening for cells containing the expression construct. Suitable marker proteins for this purpose are e.g.
  • the fluorescent protein GFP and the selectable marker genes HSV thymidine kinase (for selection on HAT medium), bacterial hygromycin B phosphotransferase (for selection on hygromycin B), Tn5 aminoglycoside phosphotransferase (for selection on G418), and dihydrofolate reductase (DHFR) (for selection on methotrexate), CD20, the low affinity nerve growth factor gene.
  • HSV thymidine kinase for selection on HAT medium
  • bacterial hygromycin B phosphotransferase for selection on hygromycin B
  • Tn5 aminoglycoside phosphotransferase for selection on G418)
  • DHFR dihydrofolate reductase
  • the second or further nucleotide sequence may encode a protein that provides for fail-safe mechanism that allows to cure a subject from the LCV TAP- inhibitor transgenic cells of the invention, if deemed necessary.
  • a nucleotide sequence often referred to as a suicide gene, encodes a protein that is capable of converting a prodrug into a toxic substance that is capable of killing the transgenic cells in which the protein is expressed.
  • Suitable examples of such suicide genes include e.g.
  • the nucleotide sequence coding for the marker protein is preferably also operably linked to a promoter for expression in the mammalian target cell (e.g. an antigen presenting cell, such as a dendritic cell) as described above for the nucleotide sequence encoding an LCV TAP- inhibitor.
  • a promoter for expression in the mammalian target cell e.g. an antigen presenting cell, such as a dendritic cell
  • the expression construct may be in the form of any nucleic acid capable of being introduced into the mammalian target cell.
  • the expression construct may be DNA, RNA or a combination of both; it may be a naked nucleic acid molecule, such as a plasmid or a linear DNA or RNA fragment; and it may be a single or a double stranded nucleic acid molecule.
  • the expression construct may thus be a non-viral vector such as a plasmid or linear nucleic acid that may be packaged in e.g. a liposome for efficient delivery into the mammalian target cell.
  • the expression construct is a viral vector that may be used to transduce or infect the mammalian target cell.
  • the expression construct preferably is safe, efficient, and reliable and allows for expression, preferably controlled expression of the LCV TAP-inhibitor transgene, and for some therapeutic purposes long term expression of the transgene is preferred.
  • the construct may e.g. be a viral vector which are more efficient agents for gene transfer as compared to the non-viral agents.
  • Suitable viral expression constructs include e.g. vectors that are based on adenovirus, adeno-associated virus (AAV) or retroviruses as recently reviewed (Anderson, 1998, Nature 392: 25-30; Walther and Stein, 2000, Drugs 60: 249-71; Kay et al., 2001, Nat Med (2001) 7: 33-40).
  • Preferred retroviral expression constructs for use in the present invention are lentiviral based expression constructs.
  • Lentiviral vectors have the unique ability to infect non-dividing cells. Methods for the construction and use of lentiviral based expression constructs are described in U.S. Patent No.'s 6,165,782, 6,207,455, 6,218,181, 6,277,633 and 6,323,031.
  • the nucleic acid molecule comprising the nucleotide sequence encoding a LCV TAP-inhibitor of the present invention is a molecule that allows only transient expression of the LCV TAP-inhibitor.
  • the nucleic acid molecule is thus a molecule that does not stably transfect or transform the cell.
  • the nucleic acid molecule therefore preferably is an expression construct that does not integrate into the host cell's genome, e.g. the construct is a non-integrating construct, an episomal construct.
  • Such a construct integrates only with very low efficiency (preferably less than 10 "3 , 10 "4 , 10 "5 , or 10 "6 of all transduced cells).
  • nucleic acid molecule preferably also is a non-replicating construct, e.g. does not comprise an origin of replication that functions in the mammalian target host cell.
  • a particularly preferred nucleic acid molecule comprising the nucleotide sequence encoding a LCV TAP-inhibitor for transient expression thereof is a RNA molecule, which RNA molecule upon introduction into the cell is capable of being translated to produce LCV TAP-inhibitor protein in the mammalian target cell.
  • Suitable RNA molecules comprising the nucleotide sequence encoding the LCV TAP-inhibitor and that are capable of being translated upon introduction into the mammalian target cell may be obtained by in vitro transcription using e.g. a T7 polymerase in vitro transcription vector (e.g. pGEM4Z; Promega), comprising the LCV TAP-inhibitor coding sequence.
  • RNA may be transcribed in vitro using T7 RNA polymerase and a cap analogue, as described previously (45 ; Ambion mMessage mMachine kit).
  • a suitable a cap analogue is e.g. 5' 7-methyl guanosine nucleotide (m7G(5')ppp(5')G; Ribo m7G Cap Analog as obtainable from Promega).
  • RNA molecules encoding the LCV TAP-inhibitor at least 50, 60, 70, 80 or 90% of the RNA molecules comprise a cap or cap analog.
  • the in vitro transcribed RNA molecule comprising the nucleotide sequence encoding the LCV TAP-inhibitor may be electroporated into the mammalian target cell, preferably an antigen presenting cell, such as a dendritic cell as described (Boczkowski et al., 2000, Cancer Res. 60: 1028-1034; Coughlin et al., 2004, Blood 103: 2046-2054).
  • an antigen presenting cell such as a dendritic cell as described (Boczkowski et al., 2000, Cancer Res. 60: 1028-1034; Coughlin et al., 2004, Blood 103: 2046-2054).
  • the LCV TAP-inhibitor coding sequence is adapted for improved expression in the mammalian target cell.
  • the nucleotide sequence encoding the LCV TAP-inhibitor may be adapted to optimize its codon usage to that of the mammalian, preferably human, target host cell.
  • the adaptiveness of a nucleotide sequence encoding the LCV TAP-inhibitor to the codon usage of the host cell may be expressed as codon adaptation index (CAI).
  • CAI codon adaptation index
  • the host cell to which the codon usage is adapted preferably is a human cell, more preferably a hematopoetic cell.
  • the codon adaptation index is herein defined as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon usage of highly expressed genes.
  • the relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid.
  • the CAI index is defined as the geometric mean of these relative adaptiveness values. Non- synonymous codons and termination codons (dependent on genetic code) are excluded.
  • An adapted nucleotide sequence preferably has a CAI of at least 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9.
  • a preferred nucleotide sequence encoding a LCV TAP-inhibitor is a coding sequence wherein at least 50, 75, 90, 95, 98 or 99%, and preferably all of the non-common codons or less-common codons are replaced with a common codon encoding the same amino acid as outlined in Table 1.
  • a common codon is herein meant the most common codon encoding each particular amino acid residue in highly expressed human genes as shown in Table 1.
  • Common codons thus include: Ala (gcc); Arg (cgc); Asn (aac); Asp (gac); Cys (tgc); GIn (cag); GIy (ggc); His (cac); He (ate); Leu (ctg); Lys (aag); Pro (ccc); Phe (ttc); Ser (age); Thr (ace); Tyr (tac); GIu (gag); and VaI (gtg) (see Table 1).
  • "Less-common codons” are codons that occurs frequently in humans but are not the common codon: GIy (ggg); He (att); Leu (etc); Ser (tec); VaI (gtc); and Arg (agg).
  • the nucleotide sequence encoding the LCV TAP-inhibitor has a continuous stretch of at least 25, 50, 60, or 75 codons all of which are common codons.
  • the LCV TAP-inhibitor coding sequence may further be adapted for improved expression in the mammalian target cell by methods described in WO 2004/059556, and by modifying the CpG content of the coding sequence as described in WO 2006/015789.
  • the invention in a fourth aspect relates to a method for producing a cell that is capable of activating CD8 + T cells that selectively recognize cells presenting TEIPP, the method comprising treating the cell with a source of a LCV TAP-inhibitor of the invention as herein above defined.
  • the source of the LCV TAP-inhibitor preferably comprises at least one of: a) a polypeptide comprising an amino acid sequence of a LCV TAP-inhibitor as herein defined; b) a LCV TAP-inhibitor of the invention produced in a method comprising chemical synthesis of at least one peptide bond or at least a part of the amino acid sequence of the LCV TAP-inhibitor; and, c) a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of a LCV TAP-inhibitor as defined herein.
  • the source of the LCV TAP-inhibitor of the invention may be any composition that may be administered to the cells and that, when administered in an effective dose, is capable of effecting a functional level of LCV LCV TAP-inhibitor in the cell.
  • a functional level of LCV TAP-inhibitor in the cell is understood to mean a level that reduce TAP dependent peptide transport in the cell by at least 40, 50, 60, 70, or 80%.
  • the source of a LCV TAP-inhibitor may thus be a composition comprising the LCV TAP-inhibitor protein.
  • Such a LCV TAP-inhibitor protein composition may be any formulation that is suitable for introducing the protein into the cell, e.g.
  • LCV TAP-inhibitor of the invention is a nucleic acid molecule encoding the LCV TAP-inhibitor.
  • the treatment of the cell with the source of a LCV TAP-inhibitor of the invention is combined with treating the cell with at least one of a) a source of an ICP47 (derived from Herpes Simplex Virus) TAP-inhibitor; b) a source of a US6 (derived from Cytomegalo Virus) TAP- inhibitor; and, c) a source of varicellovirus UL49.5 TAP-inhibitor.
  • ICP47 derived from Herpes Simplex Virus
  • US6 derived from Cytomegalo Virus
  • An ICP47 TAP-inhibitor according to the present invention reduces TAP- dependent transport of the fluorescein-conjugated synthetic peptide CVNKTERAY in cells of the human melanoma MEL-JUSO (MJS) cell line that stably express the TAP- inhibitor by at least 50, 60, 70, 80, 90, or 95%, as compared to TAP-dependent transport of the peptide in untransformed human melanoma (MJS) cells, under the same conditions.
  • An ICP47 TAP-inhibitor according to the present invention may further be a protein that has at least 50, 60, 70, 80, 90, or 95% amino acid identity with at least one of SEQ ID NO.'s 12 and 13.
  • a US6 TAP-inhibitor according to the present invention reduces TAP-dependent transport of the fluorescein-conjugated synthetic peptide CVNKTERAY in cells of the human melanoma MEL-JUSO (MJS) cell line that stably express the TAP-inhibitor by at least 50, 60, 70, 80, 90, or 95%, as compared to TAP-dependent transport of the peptide in untransformed human melanoma (MJS) cells, under the same conditions.
  • a US6 TAP-inhibitor according to the present invention may further be a protein that has at least 50, 60, 70, 80, 90, or 95% amino acid identity with SEQ ID NO. 14.
  • a varicellovirus UL49.5 TAP-inhibitor according to the present invention reduces TAP-dependent transport of the fluorescein-conjugated synthetic peptide CVNKTERAY in cells of the human melanoma MEL-JUSO (MJS) cell line that stably express the TAP-inhibitor by at least 50, 60, 70, 80, 90, or 95%, as compared to TAP- dependent transport of the peptide in untransformed human melanoma (MJS) cells, under the same conditions.
  • MJS human melanoma MEL-JUSO
  • a varicellovirus UL49.5 TAP-inhibitor according to the present invention may further be a protein that has at least 50, 60, 70, 80, 90, or 95% amino acid identity with at least one of SEQ ID NO.'s 15 - 22.
  • the sources of ICP47, US6 and UL49.5 TAP-inhibitor may be any composition that may be administered to the cells and that, when administered in an effective dose, is capable of effecting a functional level of varicellovirus TAP-inhibitor in the cell.
  • a functional level of TAP-inhibitor in the cell is understood to mean a level that reduce TAP dependent peptide transport in the cell by at least 40, 50, 60, 70, or 80%.
  • the source of ICP47, US6 and/or UL49.5 TAP-inhibitor may thus be a composition comprising the ICP47, US6 and/or UL49.5 TAP-inhibitor protein as described above for source of the LCV TAP-inhibitor may thus be.
  • a preferred source of ICP47, US6 and/or UL49.5 TAP-inhibitor is however a nucleic acid molecule encoding the TAP- inhibitor(s) as described or defined above for the source of a LCV TAP-inhibitor.
  • the four herpes viral TAP -inhibiting proteins ICP47 (derived from Herpes Simplex Virus), US6 (derived from Cytomegalo Virus) UL49.5 (derived from Varicello Virus) and the LCV TAP-inhibitor BNLFa (Epstein-Barr Virus) of the present invention each act with a distinct mechanism: ICP47 blocks the pore, US6 prevents the energy supply, UL49.5 directs the breakdown of TAP and BNLF2a disables TAP function by blocking both the association of peptides with the peptide binding site and the interaction of ATP with the nucleotide binding domains of TAP. As a consequence, the combination of these inhibitors should result in highly efficient blockage of the peptide transport.
  • the combination of one or more of these inhibitors is synergistic. Therefore lower amounts of the individual TAP-inhibitors may be used when they are applied in combination. E.g. when applied in combination the dosage of each individual TAP-inhibitor in the combination is at least the amount that the reduces TAP dependent peptide transport in the cell by at least 30, 40, 50, 60, or 70% when the individual TAP-inhibitor is applied alone.
  • the method of the invention for producing a cell that is capable of activating CD8 + T cells that selectively recognize cells presenting TEIPP preferably is a method wherein the cell is treated in vitro or ex vivo with a source of a varicellovirus TAP- inhibitor, i.e. the method preferably is an in vitro method.
  • the cell that is treated with a source of a LCV TAP-inhibitor upon (re-)introduction activates CD8 + T cells that selectively recognize cells presenting TEIPP, in other words, the cell elicits, induces or arouses a TEIPP-specific CTL response in a system capable of exhibiting said response.
  • the TEIPP-specific CTL response preferably is a MHC class I dependent TEIPP-specific CTL response.
  • the system capable of exhibiting said response may be an in vitro system but preferably is a human or animal subject in need of a TEIPP- specific CTL response.
  • the human or animal in need of a TEIPP-specific CTL response may be a subject comprising tumour cells and/or virally infected cells that present T cell Epitopes associated with Impaired Peptide Processing (TEIPP).
  • the mammalian target cell that is treated with a source of a varicellovirus TAP- inhibitor preferably is a human or a murine cell.
  • the target cell preferably is a hematopoetic cell, such as e.g. lymphocytes, B cells, T cells, CD4+ cells, monocytes or dendritic cells (DC), MHC class II-positive or -negative cells, or combinations of these cells.
  • a hematopoetic cell such as e.g. lymphocytes, B cells, T cells, CD4+ cells, monocytes or dendritic cells (DC), MHC class II-positive or -negative cells, or combinations of these cells.
  • PBMC peripheral blood mononuclear cells
  • Specific subfractions of PBMCs may e.g. be enriched by red cell lysis, density centrifugation, by sorting on cell-sorter using fluorescent labelling of cell surface markers specific for a given subset of PBMCs, or by expanding specific subsets of PBMCs by incubation of the PBMCs under conditions that favour the proliferation and development of a given subset of
  • PBMCs e.g. using specific growth factors and/or interleukins (see e.g. Coligan et al.,
  • a preferred mammalian target cell that is treated with a source of a LCV TAP- inhibitor preferably is an antigen presenting cell, such as e.g. a dendritic cell.
  • Antigen presenting cells, such as dendritic cells can be enriched or isolated from peripheral blood by methods known in the art per se. They can e.g.
  • PBMC peripheral blood
  • IMDM Granulocyte- Macrophage colony stimulating factor
  • TGF- ⁇ Tumour Necrosis Factor ⁇
  • SCF stem cell factor
  • TGF- ⁇ Transforming Growth Factor - ⁇
  • Another preferred mammalian target cell that is treated with a source of a LCV TAP-inhibitor preferably is a B cell as may be enriched from PBMC as indicated (Coligan et al., 1994, supra).
  • the mammalian target cell preferably is an autologous cell.
  • the autologous cell is preferably obtained from human or animal subject in need of a TEIPP-specific CTL response.
  • the mammalian target cell preferably is a primary cell as opposed to a cell line. The cell therefore is a mortal cell (i.e. not immortalized) that is not tumorigenic and/or transformed.
  • the invention relates to a cell that has been treated with a source of a LCV TAP-inhibitor in a method as defined above, for use in the treatment of cancer or a virus infection.
  • the cancer is a tumour of cells with impaired peptide processing and/or the virus causes impaired peptide processing in cell infected by the virus, such as e.g. herpes viruses like EBV, CMV, VZV and HSV.
  • the cell is used for activating CD8 + T cells that selectively recognize cells presenting TEIPP.
  • the cells of the invention that have been treated to express TEIPP may be used for the manufacture of a pharmaceutical composition or a vaccine against cancer or virus infections and/or to activate CD8 + T cells that selectively recognize tumour- or virally infected cells presenting TEIPP, preferably MHC class I dependent TEIPP.
  • a composition of cells of the invention, that have been treated with a source of a LCV TAP-inhibitor in a method as defined above and that are capable of activating CD8 + T cells that selectively recognize cells presenting TEIPP may then be injected into a subject/patient in order to stimulate T cells (CTLs) to react on cells expressing these TEIPP.
  • CTLs T cells
  • Cells that have been treated with a source of a LCV TAP-inhibitor in a method as defined above, may be used for activation in vivo or in vitro of T cells (CD8 + ) against TEIPP.
  • the in vivo procedure is described above.
  • the in vitro procedure could be e.g. as follows: a) cells are treated with a source of a LCV TAP-inhibitor, as described above b) T cells are isolated (e.g. from PBMC) and stimulated in vitro with the cells obtained in step a; and c) activated T cells are given to the patient.
  • the activated T cells are autologous to the patient.
  • Stimulation of T-cells in vitro with dendritic cells that have been treated with a source of a LCV TAP-inhibitor may be done in accordance with to standard procedures, e.g. T-cells are sorted out from peripheral blood and cultured in the presence of dendritic cells in appropriate media and appropriate additives e.g. MEM media and IL-2 (Strobl et al., 1996 J. Immunology. 157(4): 1499-1507; Kiertscher and Roth, 1996, Journal of Leukocyte Biology 59(2):208-218).
  • MEM media and IL-2 Stimulation of T-cells in vitro with dendritic cells that have been treated with a source of a LCV TAP-inhibitor
  • the invention further relates to a pharmaceutical preparation comprising as active ingredient a cell or a source of a LCV TAP-inhibitor as defined above including combinations with sources of other viral TAP -inhibitors as defined above.
  • the composition preferably at least comprises a pharmaceutically acceptable carrier in addition to the active ingredient.
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • the pharmaceutical carrier can be any compatible, non-toxic substance suitable to deliver the polypeptides to the patient. Sterile water, alcohol, fats, waxes, and inert solids may be used as the carrier.
  • Pharmaceutically acceptable adjuvants, buffering agents, dispersing agents, and the like, may also be incorporated into the pharmaceutical compositions.
  • the cells obtained in any of the methods of the invention are administered parentally.
  • Preparations for parental administration must be sterile and physiologically tolerable. Sterilisation is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilisation and reconstitution.
  • the parental route for administration of the cells of the invention is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intramuscular, intraarterial or intralesional routes.
  • the cells may be administered continuously by infusion or by bolus injection.
  • Physiologically tolerable carriers are well known in the art.
  • liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, propylene glycol, polyethylene glycol and other solutes.
  • a typical composition for intravenous infusion could be made up to contain 10 to 50 ml of sterile 0.9% NaCl or 5% glucose optionally supplemented with a 20% albumin solution and 10 2 to 10 10 cells.
  • compositions are well known in the art and described in more detail in various sources, including, for example, Remington's Pharmaceutical Science (15th ed., Mack Publishing, Easton, PA, 1980) (incorporated by reference in its entirety for all purposes).
  • the invention in a further aspect relates to a nucleic acid molecule comprising a nucleotide sequence encoding a LCV TAP-inhibitor as herein defined above, including combinations with nucleotide sequences encoding other viral TAP-inhibitors as defined above, as well as to a composition comprising such a nucleic acid molecule.
  • the compositions may be used in any of the methods of the invention.
  • the invention pertains to a method for producing a cell that presents an empty MHC class I molecule at its cell surface, the method comprising treating the cell with a source of a LCV TAP-inhibitor.
  • the cell is a mammalian target cell, preferably is a hematopoetic cell, as herein defined above.
  • the cell is treated with a source of a LCV TAP-inhibitor as herein defined above, including combinations with other viral TAP-inhibitors as defined above.
  • the method preferably is an in vitro method. The method is thus used for induction of empty MHC class I molecules at the cell surface that can be loaded with peptides of a desired specificity, e.g.
  • synthetic peptides comprising a MHC class I epitope of a tumour- or microbial-antigen.
  • Cells presenting MHC class I molecules with exogenously added (synthetic) peptides may then be used to induce T cell immunity, e.g. a CTL response, against the tumour- or microbial-antigen in the treatment of cancer or an infectious disease.
  • the invention further relates to cells obtained in this method and compositions comprising those cells.
  • the invention pertains to a method for producing a cell having reduced surface expression of MHC class I molecules at its cell surface, the method comprising treating the cell with a source of a LCV TAP-inhibitor.
  • Reduced expression of MHC class I molecules is understood to mean a reduction of at least 20, 30, 40, 50, 60, 80 or 90% as compared to a cell that has not been treated with the TAP-inhibitor.
  • the cell is treated with a source of a LCV TAP-inhibitor as herein defined above, including combinations with other viral TAP-inhibitors as defined above.
  • the cell is a mammalian target cell, preferably is a human cell of a tissue to be transplanted.
  • the cell to be transplanted is treated with the source of TAP-inhibitors in order to reduce or the inhibit unwanted immune responses against transplanted tissues or organs, e.g. against transplanted (cells of) islets of Langerhans in type 1 diabetes, beta cells, allogeneic stem cells, or against self tissue/self antigens in the case of autoimmunity.
  • the source of viral TAP-inhibitors preferably is one or more nucleic acid expression constructs for long term expression, such as e.g. lentiviral based expression constructs.
  • the invention further relates to cells obtained in this method and compositions comprising those cells.
  • the invention relates to methods wherein the source of LCV
  • TAP-inhibitor is used in a method for immunisation (vaccination) of a subject against the LCV, whereby preferably the LCV is Epstein-Barr virus.
  • the subject may be immunised with a recombinant LCV, preferably Epstein-Barr virus, whereby sequences coding for (part) of the amino acid sequence of the LCV TAP-inhibitor are not present in the genome of the LCV.
  • FIG. 1 Diagrammatic alignment of the right hand ends of the sequenced ⁇ l- herpesviruses.
  • Open reading frames (ORFs) of EBV, the rhesus LCV and marmoset LCV are shown as boxes, filled symbols represent ORFs with homologues in other herpesviruses, open symbols represent genes found only in ⁇ l-hepresviruses and the shaded symbol represents a latent gene.
  • FIG. 1 Representative cytotoxicity assays testing recognition of target cells co- expressing EBV-unique genes and target proteins,
  • epithelial cells (upper panel) were infected with vaccinia viruses expressing EBNA3A (v Antigen) and combinations of the indicated viruses expressing genes unique to EBV or a control vaccinia virus lacking an inserted gene (vTk-), before being incubated with HLA-B*0801 restricted CD8+ T cell clones specific for EBNA3 A.
  • EBV transformed B cells (middle and lower panels) were co-infected with modified vaccinia Ankara expressing invariant chain- targeted EBNA3C (v Antigen) and the indicated combinations of vaccinia viruses.
  • the infected B cells were incubated with either HLA B*2705 restricted CD8+ T cell clones specific for EBNA3C (middle panel) or CD4+ T cell clones restricted by HLA DQ5 specific for EBNA3C (lower panel), (b) EBV transformed B cells were infected with vaccinia viruses expressing BNLF2a and the different EBV antigens indicated (v Antigen) encoding epitopes presented through a range of HLA types.
  • Infected cells were incubated with cognate CD8+ T cells specific for: an HLA- A*0201 epitope encoded by BMLFl, an HLA-A*2402 epitope encoded by BMRFl, an HLA-B*38 epitope encoded by EBNA2, an HLA-B*2705 epitope encoded by EBNA3B, an HLA-B*0801 epitope encoded by BZLFl or an HLA-C*0101 epitope encoded by BGLF4.
  • FIG. 3 Expression of BNLF2a prevents CD8+ T cell recognition by disrupting antigen presentation
  • MJS cells were retrovirally transduced to express BNLF2aHA (lower panels) or control GFP (upper panels). These cells were either infected with vaccinia viruses expressing BZLFl or a control TK- virus, or sensitized with RAKFKQLL peptide or DMSO as a control before being incubated with CD8+ T cells specific for the BZLFl -encoded RAKFKQLL epitope in 5 hr cytotoxicity assays, (b) Flow cytometry histograms of the same cell lines show surface staining for HLA class I (B9.12.1) and HLA class II (L243), or staining with an isotype control, (c) Lysates of the two cell lines were separated by SDS-PAGE and analyzed by immunoblotting with antibodies specific for the HA-tag (12CA5), TAPl (148.3), TAP2 (
  • EBV BNLF2a blocks peptide transport by TAP.
  • TAP-dependent peptide transport in BNLF2aHA-expressing and control MJS cells was assessed by permeabilizing the cells with streptolysin O and then incubating with a fluoresceinated peptide in the presence or absence of ATP.
  • Translocated peptides which had become glycosylated in the endoplasmic reticulum were recovered by adsorption to concanavalin A-sepharose beads. After elution, the recovered peptide was quantitated by fluorometry in arbitrary units.
  • EBV BNLF2a inhibits peptide and ATP binding to TAP.
  • Results are shown as the percentage of peptide binding relative to peptide binding in control MJS cells (set as 100%).
  • Digitonin- and NP40-lysates of MJS-BNLF2aHA and control MJS- GFP cells were incubated with ATP-agarose beads. ATP-agarose-bound (pellet) and unbound (supernatant) protein fractions were separated by SDS-PAGE and immunoblotted. Membranes were probed with antibodies specific for TAPl (148.3), TAP2 (435.4), tapasin (7F6), HLA class I heavy chain (HClO), and the HA-tag (12CA5).
  • FIG. 6 Old World primate ⁇ l -herpesvirus BNLF2a sequence and function
  • (a) The BNLF2 regions from the indicated herpesviruses were sequenced and the predicted amino acid sequence of the BNLF2a genes are shown. Black regions represent regions of homology between the different BNLF2a species, shaded regions represent conservative amino acid changes
  • (b) BNLF2a genes were subcloned into plasmid expression vectors which co-expressed GFP and these transiently transfected into MJS cells. At 48 hours surface levels of HLA I and HLA II were assessed on GFP positive cells by staining with the relevant antibody and analyzing the cells by flow cytometry.
  • Black histograms represent surface marker intensity of cells transfected with the relevant BNLF2a
  • open histograms represent the intensity of cells transfected with the empty vector plasmid.
  • Figure 7. Expression of lymphocryptovirus BNLF2a genes causes downregulation of surface MHC class I in cell lines of primate origin. LLC-MK2 cells (Rhesus macaque), orangutan B cells transformed with their endogenous lymphocryptovirus (orangutan herpesvirus) or Cos cells (African green monkey) were transfected with the plasmid vectors co-expressing BNLF2a and GFP used in Figure 6.
  • MHC class I molecules on GFP positive cells were measured at 48 hours after transfection by staining cells with the monoclonal antibody W6/32 and analysing by flow cytometry.
  • Black histograms represent surface marker intensity of cells transfected with the relevant BNLF2a
  • open histograms represent the intensity of cells transfected with the empty vector plasmid.
  • Geometric mean fluorescence values for MHC class I staining on the GFP positive cells are shown in the upper left of each histogram.
  • EBV-specific CD8+ T cell clones were generated from PBMCs of infectious mononucleosis patients by limiting dilution cloning as described previously (11).
  • CD8+ T cell clones used in this study were specific for the following epitopes derived from EBV gene products: RAKFKQLL from BZLFl, QAKWRLQTL from EBNA3A, both presented by HLA-B*0801 (50), (51), GLCTLVAML from BMLFl presented by HLA-A*0201 (11), CYDHAQTHL from BMRFl presented by HLA A*2402 (13), YHLIVDTDSL from EBNA2 presented by HLA-B*38 (52), HRCQAIRKK from EBNA3B presented by HLA-B*2705 (53), and an undefined epitope derived from BGLF4 presented by HLA-C*0101 (13).
  • CD4+ T cell clones specific for the HLA DQ5 presented epitope SDDELP YIDPNMEP from EBNA 3 C were produced by stimulation of PBMC from healthy donors with their autologous EBV-transformed B cell line and limiting dilution cloning these cells as described previously (54). All experiments were approved by the South Birmingham Health Authority Local Research Ethics Committee. Recombinant vaccinia viruses were used to express individual EBV B95.8 strain genes as described previously and a vaccinia virus lacking an insert (vTk-) used for control infections (13, 54).
  • Target cells infected with the vaccinia viruses for cytotoxicity assays were either SV40 transformed keratinocytes (SVK), HLA matched EBV transformed B lymphoblastoid cell lines, or the melanoma cell line MeIJuSo (MJS, HLA-A*01, B*08, Cw*07) (55).
  • Target cells were infected with vaccinia viruses at a multiplicity of infection of 10, incubated for 16 hours and then used as targets in standard 5 hr chromium release assays. Where indicated, target cells were sensitized with synthetic CTL epitope peptide (Alta Biosciences, Birmingham U.K.) at a concentration of 5 ⁇ M for 90 minutes before use in cytotoxicity assays.
  • Murine mAbs used to detect human cellular proteins were: W6/32 or B9.12.1, which recognizes ⁇ 2m-associated HLA class I complexes (Immunotech); HClO (provided by H. Ploegh, The Whitehead Institute, Cambridge, Massachusetts), specific for HLA class I heavy chains; anti -HLA-DR mAb L243 (ATCC); DA6-147, specific for HLA-DR ⁇ chains (provided by P. Cresswell, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, USA); 148.3 directed to TAPl and 435.4 to TAP2 (provided by R.
  • Anti-tapasin Abs used were a rabbit serum, R.gp46C, against the C-terminal region (provided by P. Cresswell) and a rat mAb, 7F6 (provided by R. Tampe).
  • HA haemagglutinin
  • the BNLF2a gene was cloned into the pLZRS retroviral vector, upstream from an internal ribosome entry site (IRES) and a marker gene, truncated nerve growth factor receptor, to allow co-expression of both proteins.
  • the BNLF2a sequence was modified at the carboxy terminus to encode four methionines and the influenza HA epitope to allow detection (denoted as BNLF2aHA).
  • Virus was produced using the
  • MJS cells Phoenix packaging cell line and used to transduce MJS cells, as previously described (55). As a control, MJS cells were transduced with an IRES-GFP expressing retrovirus.
  • HLA class I and II molecules were measured by flow cytometric analysis after staining with the antibodies W6/32 or B9.12.1 for class I molecules or L243 for class II molecules. In all cases, aliquots of cells were stained with appropriate isotype controls and bound antibodies detected using a goat -anti- mouse phycoerythrin antibody. Stained cells were analyzed on an Epics flow cytometer (Beckman Coulter) and data processed using Win MDI software (Scripps Research Institute). 1.5 Peptide transport assay
  • TAP-mediated peptide transport assays were conducted by permeablizing aliquots of 3x106 MJS cells with 2.5 IU/ml of streptolysin O (Murex diagnostics) and incubating these cells with 200 nmol of the fluoresceinated peptide CVNKTERAY (provided by W. Benckhuijsen and J.W. Drijfhout, Leiden University Medical Center, Leiden, The Netherlands) in the presence or absence of 10 mM ATP at 37°C for 10 minutes.
  • CVNKTERAY provided by W. Benckhuijsen and J.W. Drijfhout, Leiden University Medical Center, Leiden, The Netherlands
  • Translocation was terminated by lysing cells with ice-cold lysis buffer (1% Triton X-100, 500 mM NaCl, 2 mM MgC12, 50 mM Tris HCl, pH 8) and the nuclei were removed by centrifugation. Peptides which had been glycosylated in the endoplasmic reticulum were isolated from lysates by incubation with concanavalin A- sepharose beads (GE Healthcare).
  • MJS cells transfected with retroviruses expressing either BNLF2aHA or GFP were lysed using either 1% NP40 or 1% digitonin made in 50 mm Tris HCl pH 7.5, 5 mM MgC12, 150 mM NaCl, 1 mM leupeptin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride. Lysates of solubilized proteins, equivalent to 2 x 105 cells, were separated by SDS-PAGE and transferred to PVDF membranes (GE Healthcare).
  • Proteins of interest were detected by incubating the membranes with specific antibodies followed by HRP- conjugated secondary anti-species antibodies (Jackson Immunoresearch). Bound HRP labelled antibodies were visualized using ECL-plus (GE Healthcare). For immunoprecipitation experiments, cell lysates prepared with 1% digitonin solution were incubated with the different specific Abs for at least 2 hrs at 4 0 C. Protein- G sepharose beads (GE Healthcare) were used to isolate immune complexes, which were washed with 0.1% digitonin and subjected to western blot analysis. Antibodies which bound the blotted proteins were detected using ExactaCruz (Santa Cruz Biotechnology) as per the manufacturer's instructions. 1.7 Peptide binding assay
  • TAP-containing microsomal membranes were prepared from MJS cells transduced with BNLF2aHA or GFP by homogenization of the cells with an EMBL cell cracker and removal of the nuclei by centrifugation. The ability of peptides to bind to TAP complexes in the microsomes was assessed by incubation with an 125 I-labeled peptide 5PS2 (ERYDKSE-[BPA]-L (57)) containing a photoactivatable cross-linker, in the absence or presence of 2.5 ⁇ M unlabelled HSV-I ICP47 competitor peptide (16).
  • JS cells transduced with BNLF2aHA or GFP were lysed with 1% digitonin or 1% NP40 as described above. Nuclei were removed and lysates incubated with hydrated C- 8 ATP-agarose beads (final cone.13 ⁇ M; Sigma) at 4°C for 2 hrs. The beads were then centrifuged to give a supernatant fraction and a pellet fraction. Proteins bound to ATP- agarose pellet fraction were eluted with 500 mM EDTA and these as well as the supernatant fraction were analyzed by western blot.
  • BNLF2a homologues were cloned from the following LCVs: papio herpesvirus, rhesus herpesvirus, pan herpesvirus, orangutan herpesvirus and gorilla herpesvirus.
  • PCR primers were designed to hybridize to a sequence flanking the BNLF2 region which was conserved between EBV and the rhesus herpesvirus.
  • PCR products were amplified from either a genomic DNA fragment of rhesus herpesvirus (a kind gift from F. Wang, Harvard Medical School, Boston, Massachusetts, USA) or from DNA extracted from B cell lines spontaneously transformed with the endogenous LCV of each host species.
  • This amplified BNLF2 region of each virus was cloned into the plasmid pCR2. ITOPO (Invitrogen, Paisley, United Kingdom) and inserts sequenced using standard techniques. Sequences were deposited in GenBank with the following accession numbers: papio herpesvirus, EF207711; pan herpesvirus, EF207712; orangutan herpesvirus, EF207713; gorilla herpesvirus, EF207714.
  • BNLF2a genes were subcloned into a modified pCDNA3 vector (a kind gift from E. Reits) and expressed under the control of the CMV immediate early promoter.
  • IRES element Immediately downstream from BNLF2a was an IRES element followed by the GFP gene, allowing co-expression of this marker.
  • Plasmids were transfected into either MJS cells, the Rhesus macaque kidney cell line LLC-MK2 or the African green monkey kidney fibroblast like cell line Cos using Lipofectamine 2000 (Invitrogen), or transfected by electroporation into orangutan B cells transformed with their endogenous lymphocryptovirus. Transfected cells were cultured for 48 hours before staining for surface marker expression and flow cytometric analysis as described above. 2.
  • BARFl and BALFl have homologies to the cellular CSF-I receptor and bcl2 genes respectively, implying functions other than the interception of antigen processing pathways, while a third gene, LF3, is deleted in the B95.8 virus strain that displays HLA I downregulation in lytic cycle.
  • Figure 2A presents the results from a representative assay in the HLA-B*0801-positive epithelial cell line SVK using EBNA3 A as the target antigen and a CD8+ T cell clone specific for an HLA-B*0801-restricted EBNA3A epitope as the effector.
  • FIG. 2A presents data from an essentially similar assay conducted in an EBV transformed B lymphoblastoid cell line (LCL).
  • the cells were infected with a vaccinia virus encoding, as an indicator antigen, an invariant chain-tagged form of the EBNA3C protein that is processed by the HLA class I and class II pathways, thereby allowing both CD8+ and CD4+ T cell recognition to be assayed simultaneously on the same target cells.
  • Figure 2A (middle panel)
  • expression of BNLF2a (and of ICP47) reduced target cell killing by a HLA-B*2705-restricted EBNA3C- specific CD8+ T cell clone to the background level seen on unmanipulated LCL targets. This background level represents recognition of pre-existing epitopes derived from EBNA3C expressed from the resident EBV genome.
  • FIG. 2B shows the results of subsequent experiments conducted using EBV- transformed B cell lines with different HLA alleles to assess any allele-specific effects of BNLF2a on CD8+ T cell recognition.
  • EBV latent EBNA2, EBNA3B
  • lytic BMLFl, BMRFl, BZLFl, BGLF4
  • target antigens encoding epitopes recognized in the context of HLA-A (A*0201, A*2402), HLA-B (B*0801, B*2705, B*3801) and HLA-C (C*0101) molecules.
  • HLA-A A*0201, A*2402
  • HLA-B B*0801, B*2705, B*3801
  • HLA-C C*0101
  • BNLF2a containing an HA epitope tag (BNLF2aHA) was expressed in the melanoma cell line Mel JuSo (MJS) through retroviral transduction.
  • JS Mel JuSo
  • cytotoxicity assays on the transduced cells to confirm that retrovirus- mediated expression of BNLF2aHA in MJS cells recapitulates the BNLF2a-induced phenotype described above.
  • Figure 3A shows results of a cytotoxicity assay using as targets the HLA-B*0801-positive MJS cells expressing either BNLF2aHA or GFP.
  • HLA class I levels have been found to be markedly reduced during productive EBV infection (12), we next examined whether expression of BNLF2a affected the display of HLA molecules (Figure 3B).
  • levels of HLA class I were substantially reduced on BNLF2aHA-expressing cells compared to control cells. This was a specific effect, since expression of HLA class II ( Figure 3B) and transferrin receptor (data not shown) were unaffected.
  • Downregulation of surface HLA class I molecules can result from viral interference at specific stages of the class I antigen presentation pathway and may involve the degradation of HLA class I molecules and other components of the HLA class I peptide-loading complex (7, 8).
  • BNLF2a affects levels of the components of the HLA class I peptide loading complex
  • lysates of BNLF2aHA or control transduced MJS cells were subjected to western blot analysis for TAPl, TAP2, tapasin and HLA class I. No marked differences were observed in the steady-state levels of these proteins (Figure 3C).
  • FIG. 3C shows that despite reduced surface HLA class I levels, cells expressing BNLF2aHA contained similar levels of the major components of the HLA class I peptide-loading complex.
  • EBV BNLF2a blocks peptide transport by TAP
  • Surface HLA class I downregulation can also result from the reduced supply of peptides to nascent HLA class I molecules in the endoplasmic reticulum. This is turn relies on the efficient generation of peptides by the proteasome and other peptidases and the transport of these peptides in to the endoplasmic reticulum by the TAP complex.
  • surface HLA class I downregulation coincides with reduced peptide transport by TAP (12).
  • BNLF2a was associated with the peptide-loading complex by performing co-immunoprecipitation experiments.
  • BNLF2aHA-expressing or control cells were lysed using the mild detergent digitonin to preserve protein- protein interactions.
  • TAPl, TAP2, tapasin and HLA class I molecules were immunoprecipitated from the lysates and all precipitates were probed with an HA- specific mAb to detect BNLF2aHA.
  • Figure 4B shows that BNLF2a co-precipitates with TAPl, TAP2, tapasin and HLA class I molecules, but not with HLA class II and the transferrin receptor (used as controls; TfR data not shown).
  • EBV BNLF2a blocks TAP function by preventing the binding of peptides and ATP
  • TAP-mediated translocation of peptides into the endoplasmic reticulum is initiated by the interaction of peptides with the cytosolic peptide-binding domains of TAP, followed by the binding and hydrolysis of ATP, which facilitates the opening of the transmembrane pore and peptide translocation (14, 15).
  • BNLF2aHA a photoactivatable cross-linker with microsomes derived from BNLF2aHA- expressing or control MJS cells.
  • BNLF2a might also affect the binding of ATP to TAP.
  • Lysates of BNLF2aHA-transduced or control cells were made using either the detergents digitonin, which preserves protein interactions within the peptide-loading complex, or 1% NP40, which releases HLA class I, tapasin, and BNLF2a from TAP (see below).
  • Proteins capable of binding ATP were purified from the lysates by incubation with ATP-agarose beads and pelleting; these fractions, as well as the non- binding supernatant (free) fractions, were subjected to western blot analysis for the presence of TAPl, TAP2, tapasin, HLA I, or BNLF2aHA.
  • ATP binds to the TAP subunits solubilized either in digitonin or NP40 (Fig. 5C, lanes 1 and 3), while tapasin and HLA class I molecules were co-isolated only with ATP -bound TAP recovered from digitonin lysates (Fig. 5C, compare lanes 1 and 3).
  • the ATP -bound fraction from digitonin lysates of BNLF2aHA-expressing cells contained little of the four components of the peptide-loading complex (Fig. 5C, lane 2), indicating that the EBV protein inhibits the interaction of ATP with the TAP proteins.
  • BNLF2aHA was not detected in the ATP-agarose pellet fraction (lane 2), but was present in the supernatant fraction of BNLF2a transduced MJS cells (lane 6).
  • BNLF2a was released from TAP in NP40 lysates, which restored ATP- binding capacity of the TAP subunits, implying a low-affinity interaction of BNLF2a with TAP that prevents binding to ATP-agarose beads (lane 4).
  • 2.5 BNLF2a homologues are encoded by other Old World ⁇ -1 primate herpesviruses and downregulate HLA class I expression
  • proteins show between 53% and 63% overall identity, and share similar features; they are small 59-60 amino acid proteins, each has a hydrophobic C-terminus and a number of sites in the non-hydrophobic region where the sequence is conserved. Homology or alignment searches using programs such as the basic local alignment search tool (BLAST) reveals no significant matches with any other protein in the database (data not shown).
  • BLAST basic local alignment search tool
  • FIG. 6B shows the flow cytometric analysis of MJS cells that had been transfected with the BNLF2a genes derived from EBV, the rhesus LCV, pan herpesvirus (note, pan and orangutan BNLF2a genes had identical sequence) or gorilla herpesvirus.
  • BNLF2a encoding a CD8 immune evasion protein.
  • the BNLF2a protein When expressed from a vaccinia vector in target cells, the BNLF2a protein reproducibly inhibited recognition of various indicator antigens (EBV latent and lytic cycle proteins) by CD8+ T cell clones restricted through a number of HLA-A, -B and -C alleles.
  • class II antigen presentation in BNLF2a-expressing cells remained intact, only presentation by the HLA class I pathway was impeded.
  • the BNLF2a protein was found to function by blocking both peptide and ATP binding to the TAP complex, thereby impairing peptide loading of HLA class I molecules and their expression at the cell surface.
  • BNLF2a transcripts first appear in the early phase of the lytic cycle, peaking 8-12 hours after lytic cycle induction in a B cell line (18).
  • the majority of EBV lytic cycle transcripts are expressed in the late phase subsequent to BNLF2a expression, at a time when the protein's effects are likely to be well established. This would explain why the late proteins are poorly processed and presented by lytically- infected cells.
  • the two immediate early proteins, BZLFl and BRLFl expressed in the first wave of viral gene expression prior to BNLF2a are efficiently recognized by their cognate T cells (13).
  • the less efficient presentation of the four early antigens tested in our previous study likely reflects the fact they are expressed before BNLF2a's inhibitory effects are optimal.
  • These antigens are known to be BZLF 1/BRLFl -induced and therefore appear very soon after the immediate early to early transition, probably just prior to BNLF2a expression (19-23).
  • T cells were cloned from the joints of rheumatoid arthritis patients and tested against an EBV-cDNA expression library, only T cells specific for immediate early or a small number of early proteins were detected (24, 25). Thus, very few responses to late proteins have been described and, when they occur, these responses are weak (26). Yet, in principle, all lytic cycle proteins should be available as exogenous antigens for cross-presentation by dendritic cells during the priming of the CD8+ T cell response.
  • BNLF2a is one of a set of genes including two others with immunomodulatory function, BCRFl (viral IL-IO homologue) and BARFl (viral GCSF-receptor), that have been acquired by the ⁇ l viruses of Old World primates but are absent from the New World ⁇ l virus yet analyzed (27, 28). This implies acquisition, in the 50 million years or so since the Old World/New World fork in primate evolution. The Old World primate viruses have also acquired a more complex set of latent growth-transforming genes over this same period. Thus it may be the combination of these more sophisticated immune evasion and growth transforming strategies that underlies the apparently greater prevalence which the Old World viruses achieve in their host population compared to their New World counterpart (29).
  • BNLF2a is the first TAP-specific inhibitor identified in ⁇ 1 -herpesviruses and, like the other three herpesvirus TAP inhibitors identified so far, BNLF2a has its own unique structure and mechanism of action.
  • the simplexviruses HSVl and 2 encode a small cytosolic protein, ICP47, which acts as a high affinity competitor for peptide binding to TAP, yet does not affect ATP binding to the transporter complex (5, 6, 17, 30, 31).
  • BHVl varicellovirus bovine herpesvirus 1
  • HCMV prototypic virus of this subfamily
  • TAP This protein reduces association of ATP to TAPl and promotes binding to TAP2 but does not interfere with peptide binding, nor does it influence the stability of the transporter complex (34-38).
  • TAP is targeted secondarily for slow degradation by the virus-encoded ubiquitin ligase mK3, whose primary function is to rapidly target MHC class I heavy chains for proteasomal degradation (39-46).
  • BNLF2a disables TAP function by blocking both the association of peptides with the peptide binding site and the interaction of ATP with the nucleotide binding domains of the transporter.
  • BNLF2a may then interfere directly with peptide or ATP binding while simultaneously disrupting subsequent steps of the translocation cycle through an indirect mechanism, for example by inhibiting conformational transitions required for proper function of the TAP complex.
  • BGLF5 is at least one gene responsible for host protein synthesis shutoff (49).
  • Expression of BGLF5 is sufficient to inhibit synthesis of host proteins, including HLA class I molecules, causing a decrease in surface class I levels.
  • Co-expression of BNLF2a and BGLF5 by EBV could then represent an effective strategy for inhibiting the HLA class I antigen processing pathway (12).
  • the prototype ⁇ l herpesvirus, EBV evades CD8+ T cell detection in lytic cycle through a strategy that is unique in its molecular detail yet targets antigen presentation at the same point, TAP-mediated peptide transport, as is targeted by several other herpesviruses.
  • TAP inhibitors encoded by the different herpesvirus genera share no obvious homology presents a striking example of convergent evolution.
  • Their existence testifies to the strength of evolutionary pressure exerted on many herpesviruses by CD8+ T cell responses against virus replicative infections.
  • the present work indicates that, despite their ability to amplify viral loads in vivo through non-replicative latent infection, EBV and Old World ⁇ -1 herpesvirus relatives have indeed been subject to the same immune pressure.
  • MHC Major Histocompatibility Complex
  • TAP Antigen Processing
  • Epstein-Barr virus BMLFl promoter contains an enhancer element that is responsive to the BZLFl and BRLFl transactivators. J. Virol. 63:3878-3883.
  • Epstein-Barr virus (EBV) BMRFl promoter for early antigen (EA-D) is regulated by the EBV transactivators, BRLFl and BZLFl, in a cell-specific manner. J. Virol. 64:3753-3759.
  • Herpesvirus 68 with Major Histocompatibility Complex Class I Molecules with Impaired Peptide and ⁇ beta ⁇ 2-Microglobulin Assembly J. Virol. 76:2796-2803.

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Abstract

La présente invention concerne un nouveau type d'inhibiteur de TAP, le LCV. Les inhibiteurs de TAP de l'invention sont de petites protéines qui comprennent la protéine BNLF2a du virus d'Epstein-Barr et ses homologues codés dans des génomes d'herpèsvirus YI de primate d'Europe. Les inhibiteurs de TAP LCV de l'invention peuvent être utilisés facultativement en combinaison avec d'autres inhibiteurs de TAP dans des procédés pour la modulation de la réponse immunitaire, la dirigeant vers des antigènes spécifiques, loin des antigènes contre lesquels aucune réponse n'est souhaitée. Les inhibiteurs de TAP de l'invention sont utilisés pour : 1) l'induction d'une immunité spécifique de tumeur ou spécifique de virus dans des cas où une réponse immunitaire classique est absente en raison de défauts de traitement d'antigène ; 2) l'induction de molécules de MHC de classe I vide à la surface cellulaire qui peuvent être chargées par des peptides d'une spécificité désirée ; 3) l'inhibition de réponses immunitaires non souhaitée contre des tissus ou organes transplantés, par exemple, contre des îlots de Langerhans dans le diabète de type 1 ou des cellules souche allogéniques ou contre des antigènes du soi dans le cadre de l'auto-immunité.
PCT/NL2008/050440 2007-07-09 2008-07-01 Inhibiteurs de tap à partir d'herpèsvirus 1 de primate d'europe et leur utilisation Ceased WO2009008713A1 (fr)

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WO2013097226A1 (fr) 2011-12-31 2013-07-04 Beigene, Ltd. Pyridophthalazinones tétra ou pentacycliques fusionnés à utiliser en tant qu'inhibiteurs de parp
US10232003B2 (en) 2014-03-30 2019-03-19 Benevir Biopharm, Inc. Exogenous tap inhibitor armed oncolytic viruses and therapeutic uses thereof
WO2021154972A1 (fr) * 2020-01-29 2021-08-05 University Of Miami Vaccination contre des antigènes induits dans des cellules infectées par un pathogène

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012089338A1 (fr) * 2010-12-29 2012-07-05 Curevac Gmbh Combinaison de vaccination et d'inhibition de la présentation d'antigène restreinte à une classe de cmh
WO2012089225A1 (fr) * 2010-12-29 2012-07-05 Curevac Gmbh Combinaison de vaccination et d'inhibition de la présentation des antigènes restreinte par le cmh de classe i
US9737595B2 (en) 2010-12-29 2017-08-22 Curevac Ag Combination of vaccination and inhibition of MHC class I restricted antigen presentation
US11458193B2 (en) 2010-12-29 2022-10-04 Curevac Ag Combination of vaccination and inhibition of MHC class I restricted antigen presentation
WO2013097226A1 (fr) 2011-12-31 2013-07-04 Beigene, Ltd. Pyridophthalazinones tétra ou pentacycliques fusionnés à utiliser en tant qu'inhibiteurs de parp
US10232003B2 (en) 2014-03-30 2019-03-19 Benevir Biopharm, Inc. Exogenous tap inhibitor armed oncolytic viruses and therapeutic uses thereof
WO2021154972A1 (fr) * 2020-01-29 2021-08-05 University Of Miami Vaccination contre des antigènes induits dans des cellules infectées par un pathogène

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