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WO2017089786A1 - Peptides - Google Patents

Peptides Download PDF

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Publication number
WO2017089786A1
WO2017089786A1 PCT/GB2016/053668 GB2016053668W WO2017089786A1 WO 2017089786 A1 WO2017089786 A1 WO 2017089786A1 GB 2016053668 W GB2016053668 W GB 2016053668W WO 2017089786 A1 WO2017089786 A1 WO 2017089786A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq nos
mhc
polypeptide
cell
peptide
Prior art date
Application number
PCT/GB2016/053668
Other languages
English (en)
Inventor
Alex POWLESLAND
Original Assignee
Immunocore Limited
Adaptimmune Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB1520601.4A external-priority patent/GB201520601D0/en
Priority claimed from GBGB1520555.2A external-priority patent/GB201520555D0/en
Priority claimed from GBGB1520598.2A external-priority patent/GB201520598D0/en
Priority claimed from GBGB1520553.7A external-priority patent/GB201520553D0/en
Priority claimed from GBGB1520587.5A external-priority patent/GB201520587D0/en
Priority claimed from GBGB1520590.9A external-priority patent/GB201520590D0/en
Priority claimed from GBGB1520576.8A external-priority patent/GB201520576D0/en
Priority claimed from GBGB1520582.6A external-priority patent/GB201520582D0/en
Priority claimed from GBGB1520578.4A external-priority patent/GB201520578D0/en
Priority claimed from GBGB1520599.0A external-priority patent/GB201520599D0/en
Priority claimed from GBGB1520571.9A external-priority patent/GB201520571D0/en
Priority claimed from GBGB1520554.5A external-priority patent/GB201520554D0/en
Priority claimed from GBGB1520584.2A external-priority patent/GB201520584D0/en
Priority claimed from GBGB1520552.9A external-priority patent/GB201520552D0/en
Priority claimed from GBGB1520551.1A external-priority patent/GB201520551D0/en
Priority claimed from GBGB1520573.5A external-priority patent/GB201520573D0/en
Priority claimed from GBGB1520535.4A external-priority patent/GB201520535D0/en
Priority claimed from GBGB1520560.2A external-priority patent/GB201520560D0/en
Priority claimed from GBGB1520574.3A external-priority patent/GB201520574D0/en
Priority claimed from GBGB1520556.0A external-priority patent/GB201520556D0/en
Priority claimed from GBGB1520577.6A external-priority patent/GB201520577D0/en
Priority claimed from GBGB1520585.9A external-priority patent/GB201520585D0/en
Priority claimed from GBGB1520596.6A external-priority patent/GB201520596D0/en
Priority claimed from GBGB1520561.0A external-priority patent/GB201520561D0/en
Priority claimed from GBGB1520602.2A external-priority patent/GB201520602D0/en
Priority claimed from GBGB1520534.7A external-priority patent/GB201520534D0/en
Priority claimed from GBGB1520588.3A external-priority patent/GB201520588D0/en
Priority claimed from GBGB1520547.9A external-priority patent/GB201520547D0/en
Priority claimed from GBGB1520580.0A external-priority patent/GB201520580D0/en
Priority claimed from GBGB1520538.8A external-priority patent/GB201520538D0/en
Priority claimed from GBGB1520581.8A external-priority patent/GB201520581D0/en
Priority claimed from GBGB1520586.7A external-priority patent/GB201520586D0/en
Priority claimed from GBGB1520591.7A external-priority patent/GB201520591D0/en
Priority claimed from GBGB1520537.0A external-priority patent/GB201520537D0/en
Application filed by Immunocore Limited, Adaptimmune Limited filed Critical Immunocore Limited
Publication of WO2017089786A1 publication Critical patent/WO2017089786A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • 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
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/605—MHC molecules or ligands thereof

Definitions

  • the present invention relates to novel peptides derived from Centrosomal protein C10orf90 (C10orf90), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules.
  • binding moieties that bind to the peptides and/or complexes of the invention. Such moieties are useful for the development of immunotherapeutic reagents for the treatment of diseases such as cancer.
  • T cells are a key part of the cellular arm of the immune system. They specifically recognise peptide fragments that are derived from intracellular proteins and presented in complex with Major Histocompatibility Complex (MHC) molecules on the surface of antigen presenting cells (APCs).
  • MHC Major Histocompatibility Complex
  • MHC molecules are known as human leukocyte antigens (HLA), and both terms are used synonymously herein.
  • HLA human leukocyte antigens
  • MHC molecules have a binding groove in which the peptide fragments bind. Recognition of particular peptide-MHC antigens is mediated by a corresponding T cell receptor (TCR).
  • TCR T cell receptor
  • Tumour cells express various tumour associated antigens (TAA) and peptides derived from these antigens are frequently displayed on the tumour cell surface. Detection of a MHC class I- presented TAA-derived peptide by a CD8+ T cell bearing the corresponding T cell receptor, leads to targeted killing of the tumour cell.
  • TAA tumour associated antigens
  • tumour cells often escape detection.
  • TAA-derived peptides presented by MHC molecules on tumour cells enables the development of novel immunotherapeutic reagents designed to specifically target and destroy said tumour cells.
  • Such reagents may be moieties that bind to the TAA-derived peptide and/or peptide-MHC complexes of the invention, and typically involve the induction of a T cell response.
  • such reagents may be based, exclusively, or in part, on T cells, or T cell receptors (TCRs), or antibodies.
  • TAA-derived peptides that are presented on the cell surface in complex with a given MHC, only a small number of such peptides, covering a limited number of cancer indications, have been identified to date.
  • TAAs suitable for the development of immunotherapeutic reagents must have limited or no expression in normal cells, to prevent off target activity. It is therefore desirable to provide additional tumour-specific TAA-derived peptides and MHC complexes thereof that can be used for the development of new cancer therapies.
  • C10orf90 also known as centrosomal protein C10orf90 or fragile-site associated tumor suppressor homolog and having Uniprot accession number Q96M02
  • C10orf90 is a tumor suppressor that is required to sustain G2/M checkpoint after DNA damage.
  • Expression of C10orf90 has been linked to cancer (Zhang et al. Chin Med J (Engl). 201 1 Sep; 124(18):2894-8).
  • C10orf90 is an ideal target for immunotherapeutic applications.
  • the inventors have found novel peptides derived from C10orf90 that are presented on the cell surface in complex with MHC.
  • the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: A1 -A9, or
  • polypeptide forms a complex with a Major Histocompatibility Complex (MHC) molecule.
  • MHC Major Histocompatibility Complex
  • polypeptides of the invention are presented by MHC on the surface of tumour cells. Accordingly, the polypeptides of the invention, as well as moieties that bind the polypeptide-MHC complexes, can be used to develop therapeutic reagents.
  • the skilled person can determine whether or not a given polypeptide forms a complex with an MHC molecule by determining whether the MHC can be refolded in the presence of the polypeptide using the process set out in Example A2. If the polypeptide does not form a complex with MHC then MHC will not refold. Refolding is commonly confirmed using an antibody that recognises MHC in a folded state only. Further details can be found in Garboczi et al., Proc Natl Acad Sci
  • polypeptides of the invention are from about 8 to about 16 amino acids in length, and are most preferably 8, 9, or 10 or 11 amino acids in length.
  • polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQNOs: A1-A9.
  • amino acid residues comprising the polypeptides of the invention may be chemically modified.
  • chemical modifications include those corresponding to post translational modifications for example phosphorylation, acetylation and deamidation (Engelhard et al., Curr Opin Immunol. 2006 Feb;18(1 ):92-7).
  • Amino acid substitution means that an amino acid residue is substituted for a replacement amino acid residue at the same position.
  • Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are immediately adjacent one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent another inserted amino acid residue.
  • One, two or three amino acids may be deleted from the sequence of SEQNOs: A1-A9. Each deletion can take place at any position of SEQNOs: A1-A9.
  • the polypeptide of the invention may comprise one, two or three additional amino acids at the C-terminal end and/or at the N-terminal end of the sequence of SEQNOs: A1- A9.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQNOs: A1-A9 with the exception of one amino acid substitution and one amino acid insertion, one amino acid substitution and one amino acid deletion, or one amino acid insertion and one amino acid deletion.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQNOs: A1-A9, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.
  • Inserted amino acids and replacement amino acids may be naturally occurring amino acids or may be non-naturally occurring amino acids and, for example, may contain a non-natural side chain.
  • Such altered peptide ligands are discussed further in Douat-Casassus et al., J. Med. Chem, 2007 Apr 5;50(7): 1598-609 and Hoppes et ai, J. Immunol 2014 Nov 15;193(10):4803-13 and references therein). If more than one amino acid residue is substituted and/or inserted, the
  • replacement inserted amino acid residues may be the same as each other or different from one another. Each replacement amino acid may have a different side chain to the amino acid being replaced.
  • polypeptides of the invention bind to MHC in the peptide binding groove of the MHC molecule.
  • amino acid modifications described above will not impair the ability of the peptide to bind MHC.
  • the amino acid modifications improve the ability of the peptide to bind MHC.
  • mutations may be made at positions which anchor the peptide to MHC. Such anchor positions and the preferred residues at these locations are known in the art, particularly for peptides which bind HLA-A * 02 (see, e. g. Parkhurst et al., J. Immunol.
  • a polypeptide of the invention may be used to illicit an immune response. If this is the case, it is important that the immune response is specific to the intended target in order to avoid the risk of unwanted side effects that may be associated with an "off target" immune response. Therefore, it is preferred that the amino acid sequence of a polypeptide of the invention does not match the amino acid sequence of a peptide from any other protein(s), in particular, that of another human protein.
  • a person of skill in the art would understand how to search a database of known protein sequences to ascertain whether a polypeptide according to the invention is present in another protein.
  • Polypeptides of the invention can be synthesised easily by Merrifield synthesis, also known as solid phase synthesis, or any other peptide synthesis methodology.
  • GMP grade polypeptide is produced by solid-phase synthesis techniques by Multiple Peptide Systems, San Diego, CA.
  • the peptide may be recombinantly produced, if so desired, in accordance with methods known in the art.
  • Such methods typically involve the use of a vector comprising a nucleic acid sequence encoding the polypeptide to be expressed, to express the polypeptide in vivo; for example, in bacteria, yeast, insect or mammalian cells.
  • in vitro cell-free systems may be used. Such systems are known in the art and are commercially available for example from Life
  • polypeptides may be isolated and/or may be provided in substantially pure form. For example, they may be provided in a form which is substantially free of other polypeptides or proteins.
  • the invention provides a complex of the polypeptide of the first aspect and an MHC molecule.
  • the polypeptide is bound to the peptide binding groove of the MHC molecule.
  • the MHC molecule may be MHC class I.
  • the MHC class I molecule may be selected from HLA-A * 02, HLA-A * 01 , HLA-A * 03, HLA-A11 , HLA-A23, HLA-A24, HLA-ET07, HLA-ET08, HLA- B40, HLA-B44, HLA-B15, HLA-C * 04, HLA * C * 03 HLA-CW.
  • the complex of the invention may be isolated and/or in a substantially pure form.
  • the complex may be provided in a form which is substantially free of other polypeptides or proteins.
  • HC molecule includes recombinant MHC molecules, non-naturally occurring MHC molecules and functionally equivalent fragments of MHC, including derivatives or variants thereof, provided that peptide binding is retained.
  • MHC molecules may be fused to a therapeutic moiety, attached to a solid support, in soluble form, and/or in multimeric form.
  • MHC molecules with which polypeptides of the invention can form a complex
  • Suitable methods include, but are not limited to, expression and purification from E. co// cells or insect cells. A suitable method is provided in Example A2 herein.
  • MHC molecules may be produced synthetically, or using cell free systems.
  • Polypeptides and/or polypeptide-MHC complexes of the invention may be associated with a moiety capable of eliciting a therapeutic effect.
  • a moiety may be a carrier protein which is known to be immunogenic.
  • KLH keyhole limpet hemocyanin
  • the polypeptides and/or polypeptide-MHC complexes of the invention may be associated with a fusion partner. Fusion partners may be used for detection purposes, or for attaching said polypeptide or MHC to a solid support, or for MHC oligomerisation.
  • the MHC complexes may incorporate a biotinylation site to which biotin can be added, for example, using the BirA enzyme (O'Callaghan et al., 1999 Jan 1 ;266(1 ):9-15).
  • Other suitable fusion partners include, but are not limited to, fluorescent, or luminescent labels, radiolabels, nucleic acid probes and contrast reagents, antibodies, or enzymes that produce a detectable product. Detection methods may include flow cytometry, microscopy, electrophoresis or scintillation counting.
  • Polypeptide-MHC complexes of the invention may be provided in soluble form, or may be immobilised by attachment to a suitable solid support.
  • suitable solid supports include, but are not limited to, a bead, a membrane, sepharose, a magnetic bead, a plate, a tube, a column.
  • Polypeptide-MHC complexes may be attached to an ELISA plate, a magnetic bead, or a surface plasmon reasonance biosensor chip.
  • Methods of attaching peptide-MHC complexes to a solid support are known to the skilled person, and include, for example, using an affinity binding pair, e.g. biotin and streptavidin, or antibodies and antigens.
  • peptide-MHC complexes are labelled with biotin and attached to streptavidin-coated surfaces.
  • Polypeptide-MHC complexes of the invention may be in multimeric form, for example, dimeric, or tetrameric, or pentameric, or octomeric, or greater. Examples of suitable methods for the production of multimeric peptide MHC complexes are described in Greten et al., Clin. Diagn. Lab. Immunol. 2002 Mar;9(2):216-20 and references therein.
  • polypeptide-MHC multimers may be produced using peptide-MHC tagged with a biotin residue and complexed through fluorescent labelled streptavidin.
  • multimeric polypeptide-MHC complexes may be formed by using immunoglobulin as a molecular scaffold.
  • MHC molecules are fused with the constant region of an immunoglobulin heavy chain separated by a short amino acid linker.
  • Polypeptide-MHC multimers have also been produced using carrier molecules such as dextran (WO02072631 ). Multimeric peptide MHC complexes can be useful for improving the detection of binding moieties, such as T cell receptors, which bind said complex, because of avidity effects.
  • the polypeptides of the invention may be presented on the surface of a cell in complex with MHC.
  • the invention also provides a cell presenting on its surface a complex of the invention.
  • a cell may be a mammalian cell, preferably a cell of the immune system, and in particular a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • Other preferred cells include T2 cells (Hosken, et al., Science. 1990 Apr 20;248(4953):367-70).
  • Cells presenting the polypeptide or complex of the invention may be isolated, preferably in the form of a population, or provided in a substantially pure form.
  • Said cells may not naturally present the complex of the invention, or alternatively said cells may present the complex at a level higher than they would in nature.
  • Such cells may be obtained by pulsing said cells with the polypeptide of the invention. Pulsing involves incubating the cells with the polypeptide for several hours using polypeptide concentrations typically ranging from 10 "5 to 10 "12 M. Said cells may additionally be transduced with HLA-A * 02 molecules to further induce presentation of the peptide. Cells may be produced recombinantly. Cells presenting polypeptides of the invention may be used to isolate T cells and T cell receptors (TCRs) which are activated by, or bind to, said cells, as described in more detail below.
  • TCRs T cell receptors
  • the invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide of the first aspect of the invention.
  • the nucleic acid may be cDNA.
  • the nucleic acid molecule may consist essentially of a nucleic acid sequence encoding the polypeptide of the first aspect of the invention or may encode only the peptide of the invention, i.e. encode no other polypeptide.
  • Such a nucleic acid molecule can be synthesised in accordance with methods known in the art. Due to the degeneracy of the genetic code, one of ordinary skill in the art will appreciate that nucleic acid molecules of different nucleotide sequence can encode the same amino acid sequence.
  • the invention provides a vector comprising a nucleic acid sequence according to the third aspect of the invention.
  • the vector may include, in addition to a nucleic acid sequence encoding only a polypeptide of the invention, one or more additional nucleic acid sequences encoding one or more additional polypeptides. Such additional polypeptides may, once expressed, be fused to the N-terminus or the C-terminus of the polypeptide of the invention.
  • the vector includes a nucleic acid sequence encoding a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • Suitable vectors are known in the art as is vector construction, including the selection of promoters and other regulatory elements, such as enhancer elements.
  • the vector utilised in the context of the present invention desirably comprises sequences appropriate for introduction into cells.
  • the vector may be an expression vector, a vector in which the coding sequence of the polypeptide is under the control of its own cis-acting regulatory elements, a vector designed to facilitate gene integration or gene replacement in host cells, and the like.
  • the term "vector” encompasses a DNA molecule, such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • a DNA molecule such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • Appropriate phage and viral vectors include, but are not limited to, lambda (X) bacteriophage, EMBL bacteriophage, simian virus 40, bovine papilloma virus, Epstein-Barr virus, adenovirus, herpes virus, vaccinia virus, Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, lentivirus and Rous sarcoma virus.
  • the invention provides a cell comprising the vector of the fourth aspect of the invention.
  • the cell may be an antigen presenting cell and is preferably a cell of the immune system.
  • the cell may be a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • the cell may be a mammalian cell.
  • Polypeptides and complexes of the invention can be used to identify and/or isolate binding moieties that bind specifically to the polypeptide and/or the complex of the invention.
  • binding moieties may be used as immunotherapeutic reagents and may include antibodies and TCRs.
  • the invention provides a binding moiety that binds the polypeptide of the invention.
  • the binding moiety binds the polypeptide when said polypeptide is in complex with MHC.
  • the binding moiety may bind partially to the MHC, provided that it also binds to the polypeptide.
  • the binding moiety may bind only the polypeptide, and that binding may be specific.
  • the binding moiety may bind only the peptide MHC complex and that binding may be specific.
  • TCR T cell receptor
  • IMGT International Immunogenetics
  • the TCRs of the invention may be in any format known to those in the art.
  • the TCRs may be ⁇ heterodimers, or they may be in single chain format (such as those described in W09918129).
  • Single chain TCRs include ⁇ TCR polypeptides of the type: Va-L- ⁇ , ⁇ -L-Va, Va- Ca-L- ⁇ , ⁇ - ⁇ - ⁇ ⁇ -0 ⁇ or Va- Ca - ⁇ / ⁇ - ⁇ , optionally in the reverse orientation, wherein Va and ⁇ are TCR a and ⁇ variable regions respectively, Ca and C are TCR a and ⁇ constant regions respectively, and L is a linker sequence.
  • the TCR may be in a soluble form (i.e. having no transmembrane or cytoplasmic domains), or may contain full length alpha and beta chains.
  • the TCR may be provided on the surface of a cell, such as a T cell.
  • the alpha and/or beta chain constant domain may be truncated relative to the native/naturally occurring TRAC/TRBC sequences.
  • the TRAC/TRBC may contain modifications.
  • the alpha chain extracellular sequence may include a modification relative to the native/naturally occurring TRAC whereby amino acid T48 of TRAC, with reference to IMGT numbering, is replaced with C48.
  • the beta chain extracellular sequence may include a modification relative to the native/naturally occurring TRBC1 or TRBC2 whereby S57 of TRBC1 or TRBC2, with reference to IMGT numbering, is replaced with C57.
  • TCRs of the invention may be engineered to include mutations.
  • Methods for producing mutated high affinity TCR variants such as phage display and site directed mutagenesis and are known to those in the art (for example see WO 04/044004 and Li et al., Nat Biotechnol 2005 Mar;23(3):349- 54).
  • TCRs of the invention may also be may be labelled with an imaging compound, for example a label that is suitable for diagnostic purposes.
  • Such labelled high affinity TCRs are useful in a method for detecting a TCR ligand selected from CD1 -antigen complexes, bacterial superantigens, and MHC- peptide/superantigen complexes, which method comprises contacting the TCR ligand with a high affinity TCR (or a multimeric high affinity TCR complex) which is specific for the TCR ligand; and detecting binding to the TCR ligand.
  • a high affinity TCR or a multimeric high affinity TCR complex
  • multimeric high affinity TCR complexes such as those described in Zhu et ai, J. Immunol.
  • fluorescent streptavidin (commercially available) can be used to provide a detectable label.
  • a fluorescently-labelled multimer is suitable for use in FACS analysis, for example to detect antigen presenting cells carrying the peptide for which the high affinity TCR is specific.
  • a TCR of the present invention may alternatively or additionally be associated with (e.g. covalently or otherwise linked to) a therapeutic agent which may be, for example, a toxic moiety for use in cell killing, or an immunostimulating agent such as an interleukin or a cytokine.
  • a multivalent high affinity TCR complex of the present invention may have enhanced binding capability for a TCR ligand compared to a non-multimeric wild-type or high affinity T cell receptor heterodimer.
  • the multivalent high affinity TCR complexes according to the invention are particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo, and are also useful as intermediates for the production of further multivalent high affinity TCR complexes having such uses.
  • the high affinity TCR or multivalent high affinity TCR complex may therefore be provided in a pharmaceutically acceptable formulation for use in vivo.
  • High affinity TCRs of the invention may be used in the production of soluble bi-specific reagents.
  • a preferred embodiment is a reagent which comprises a soluble TCR, fused via a linker to an anti- CD3 specific antibody fragment. Further details including how to produce such reagents are described in W010/133828.
  • the invention provides nucleic acid encoding the TCR of the invention, a TCR expression vector comprising nucleic acid encoding a TCR of the invention, as well as a cell harbouring such a vector.
  • the TCR may be encoded either in a single open reading frame or two distinct open reading frames.
  • one vector may encode both an alpha and a beta chain of a TCR of the invention.
  • a further aspect of the invention provides a cell displaying on its surface a TCR of the invention.
  • the cell may be a T cell.
  • T cells expressing the TCRs of the invention are suitable for use in adoptive therapy-based treatment of diseases such as cancers.
  • adoptive therapy can be carried out (see for example Rosenberg et al., Nat Rev Cancer. 2008 Apr;8(4):299-308).
  • the TCRs of the invention intended for use in adoptive therapy are generally glycosylated when expressed by the transfected T cells.
  • the glycosylation pattern of transfected TCRs may be modified by mutations of the transfected gene (Kuball J et al., J Exp Med. 2009 Feb 16;206(2):463-75).
  • the binding moiety of the invention may be an antibody.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced.
  • antibody includes antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanised antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic and any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.
  • a humanised antibody may be a modified antibody having the variable regions of a non-human, e.g. murine, antibody and the constant region of a human antibody. Methods for making humanised antibodies are described in, for example, US Patent No. 5225539. Examples of antibodies are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. A monoclonal antibody may be referred to herein as "mab".
  • an antibody for example a monoclonal antibody
  • recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody.
  • Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementary determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin (see, for instance, EP-A-184187, GB 2188638A or EP-A-239400).
  • CDRs complementary determining regions
  • a hybridoma (or other cell that produces antibodies) may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. e£ al., Nature.
  • Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g.
  • antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).
  • bispecific antibodies these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Hollinger & Winter, Curr Opin
  • bispecific antibody fragments e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain "Janusins" described in Traunecker et al., EMBO J. 1991 Dec; 10(12):3655-9). Bispecific diabodies, as opposed to bispecific whole antibodies, may also be useful because they can be readily constructed and expressed in E. coli.
  • Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
  • An "antigen binding domain" is the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope.
  • An antigen binding domain may be provided by one or more antibody variable domains.
  • An antigen binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • the binding moiety may be an antibody-like molecule that has been designed to specifically bind a peptide-MHC complex of the invention.
  • TCR-mimic antibodies such as, for example those described in WO2007143104 and Sergeeva ef al., Blood. 201 1 Apr
  • binding moieties based on engineered protein scaffolds are derived from stable, soluble, natural protein structures which have been modified to provide a binding site for a target molecule of interest.
  • engineered protein scaffolds include, but are not limited to, affibodies, which are based on the Z-domain of staphylococcal protein A that provides a binding interface on two of its a-helices (Nygren, FEBS J. 2008 Jun;275(1 1 ):2668-76); anticalins, derived from lipocalins, that incorporate binding sites for small ligands at the open end of a beta-barrel fold (Skerra, FEBS J.
  • Engineered protein scaffolds are typically targeted to bind the same antigenic proteins as antibodies, and are potential therapeutic agents. They may act as inhibitors or antagonists, or as delivery vehicles to target molecules, such as toxins, to a specific tissue in vivo (Gebauer and Skerra, Curr Opin Chem Biol. 2009 Jun;13(3):245-55). Short peptides may also be used to bind a target protein.
  • Phylomers are natural structured peptides derived from bacterial genomes. Such peptides represent a diverse array of protein structural folds and can be used to inhibit/disrupt protein-protein interactions in vivo (Watt, Nat Biotechnol. 2006 Feb;24(2): 177-83)].
  • the invention further provides a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention for use in medicine.
  • the polypeptide, complex, nucleic acid, vector, cell or binding moiety may be used for in the treatment or prevention of cancer, in particular, uveal melanoma.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention together with a pharmaceutically acceptable carrier.
  • This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient). It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms. Suitable compositions and methods of administration are known to those skilled in the art, for example see, Johnson et a/., Blood.
  • Cells in accordance with the invention will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a
  • T cells transfected with TCRs of the invention may be provided in pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be adapted for administration by any appropriate route such as a parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation or intranasal routes.
  • a parenteral including subcutaneous, intramuscular, or intravenous
  • enteral including oral or rectal
  • inhalation or intranasal routes Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
  • Dosages of the substances of the present invention can vary between wide limits, depending upon the disease or disorder to be treated (such as cancer, viral infection or autoimmune disease), the age and condition of the individual to be treated, etc.
  • a suitable dose range for a reagent comprising a soluble TCR fused to an anti- CD3 domain may be between 25 ng/kg and 50 g/kg.
  • a physician will ultimately determine appropriate dosages to be used.
  • the polypeptide of the invention may be provided in the form of a vaccine composition.
  • the vaccine composition may be useful for the treatment or prevention of cancer. All such
  • compositions are encompassed in the present invention.
  • vaccines may take several forms (Schlom, J Natl Cancer Inst. 2012 Apr 18;104(8):599-613).
  • the peptide of the invention may be used directly to immunise patients (Salgaller, Cancer Res. 1996 Oct 15;56(20):4749-57 and Marchand, Int J Cancer. 1999 Jan 18;80(2):219-30).
  • the vaccine composition may include additional peptides such that the peptide of the invention is one of a mixture of peptides.
  • Adjuvants may be added to the vaccine composition to augment the immune response Alternatively the vaccine composition may take the form of an antigen presenting cell displaying the peptide of the invention in complex with MHC.
  • the antigen presenting cell is an immune cell, more preferably a dendritic cell.
  • the peptide may be pulsed onto the surface of the cell (Thurner, J Exp Med. 1999 Dec 6; 190(11 ): 1669-78), or nucleic acid encoding for the peptide of the invention may be introduced into dendritic cells (for example by electroporation. Van Tendeloo, Blood. 2001 Jul 1 ;98(1 ):49-56).
  • polypeptides, complexes, nucleic acid molecules, vectors, cells and binding moieties of the invention may be non-naturally occurring and/or purified and/or engineered and/or recombinant and/or isolated and/or synthetic.
  • the invention also provides a method of identifying a binding moiety that binds a complex of the invention, the method comprising contacting a candidate binding moiety with the complex and determining whether the candidate binding moiety binds the complex.
  • Methods to determine binding to polypeptide-MHC complexes are well known in the art. Preferred methods include, but are not limited to, surface plasmon resonance, or any other biosensor technique, ELISA, flow cytometry, chromatography, microscopy. Alternatively, or in addition, binding may be determined by functional assays in which a biological response is detected upon binding, for example, cytokine release or cell apoptosis.
  • the candidate binding moiety may be a binding moiety of the type already described, such as a TCR or an antibody.
  • T cells may be isolated from fresh blood obtained from volunteer donors. Such a method involves stimulating T cells using autologous DCs, followed by autologous B cells, pulsed with the polypeptide of the invention. Several round of stimulation may be carried out, for example three or four rounds. Activated T cells may then be tested for specificity by measuring cytokine release in the presence of T2 cells pulsed with the peptide of the invention (for example using an IFNy ELISpot assay). Activated cells may then be sorted by fluorescence-activated cell sorting (FACS) using labelled antibodies to detect intracellular cytokine production (e.g. IFNy), or expression of a cell surface marker (such as CD137).
  • FACS fluorescence-activated cell sorting
  • Sorted cells may be expanded and further validated, for example, by ELISpot assay and/or cytotoxicity against target cells and/or staining by peptide-MHC tetramer.
  • the TCR chains from validated clones may then be amplified by rapid amplification of cDNA ends (RACE) and sequenced.
  • RACE rapid amplification of cDNA ends
  • the polypeptide MHC complex of the invention may be used for screening a library of TCRs or antibodies.
  • the production of antibody libraries using phage display is well known in the art, for example see Aitken, Antibody phage display: Methods and Protocols (2009, Humana, New York). Further details on TCR libraries can be found in WO04044004.
  • the polypeptide-MHC complex of the invention is used to screen a library of diverse TCRs displayed on the surface of phage particles.
  • the TCRs displayed by said library may contain non- natural mutations.
  • Screening may involve panning the phage library with peptide-MHC complexes of the invention and subsequently isolating bound phage.
  • peptide-MHC complexes may be attached to a solid support, such as a magnet bead, or column matrix and phage bound peptide MHC complexes isolated, with a magnet, or by chromatography, respectively.
  • the panning steps may be repeated several times for example three or four times. Isolated phage may be further expanded in E. coli cells.
  • Isolated phage particles may be tested for specific binding peptide-MHC complexes of the invention. Binding can be detected using techniques including, but not limited to, ELISA, or SPR for example using a BiaCore instrument.
  • the DNA sequence of the T cell receptor displayed by peptide-MHC binding phage can be further identified by standard PCR methods.
  • Figures A1 to A9 show the respective fragmentation spectra for the peptides of SEQNOs: A1 to A9, eluted from cells.
  • a table highlighting the matching ions is shown below each spectrum.
  • Immortalised cell lines obtained from commercial sources were maintained and expanded under standard conditions.
  • Class I HLA complexes were purified by immunoaffinity using commercially available anti-HLA antibodies BB7.1 (anti-HLA-B * 07), BB7.2 (anti-HLA-A * 02) and W6/32 (anti-Class 1 ). Briefly, cells were lysed in buffer containing non-ionic detergent NP-40 (0.5% v/v) at 5 x 10 7 cells per ml and incubated at 4°C for 1 h with agitation/mixing. Cell debris was removed by centrifugation and supernatant pre-cleared using proteinA-Sepharose.
  • Peptides were separated by high pressure liquid chromatography (HPLC) on a Dionex Ultimate 3000 system using a C18 column (Phenomenex). Peptides were loaded in 98% buffer A (0.1 % aqueous trifluoroacetic acid (TFA)) and 2% buffer B (0.1% TFA in acetonitrile). Peptides were eluted using a stepped gradient of B (2-60%) over 20 min. Fractions were collected at one minute intervals and lyophilised.
  • HPLC high pressure liquid chromatography
  • Peptides were analysed by nanoLCMS/MS using a Dionex Ultimate 3000 nanoLC coupled to either AB Sciex Triple TOF 5600 or Thermo Orbitrap Fusion mass spectrometers. Both machines were equipped with nanoelectrospray ion sources. Peptides were loaded onto an Acclaim PepMap 100 trap column (Dionex) and separated using an Acclaim PepMap RSLC column (Dionex). Peptides were loaded in mobile phase A (0.5% formic acid: water) and eluted using a gradient of buffer B (acetonitrile:0.5% formic acid) directly into the nanospray ionisation source.
  • buffer B acetonitrile:0.5% formic acid
  • polypeptides set out in table A1 corresponding to SEQ NOs: A1 -A9, were detected by mass spec following extraction from cancer cell lines.
  • An example cell line from which the peptide was detected is indicated in the table along with the HLA antibody used for immunoaffinity purification.
  • Figures A1 -A9 show representative fragmentation patterns for the peptides of SEQNOs: A1-A9 respectively. A table highlighting the matching ions is shown below each spectrum.
  • Example A2 Preparation of recombinant peptide-HLA complexes
  • HLA-A * 02 molecules HLA-A * 02-heavy chain and HLA light-chain ( ⁇ 2 ⁇ ) were expressed separately in E. coli as inclusion bodies, using appropriate constructs.
  • HLA-A * 02-heavy chain additionally contained a C-terminal biotinylation tag which replaces the transmembrane and cytoplasmic domains (O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15).
  • E. coli cells were lysed and inclusion bodies processed to approximately 80% purity.
  • Inclusion bodies of ⁇ 2 ⁇ and heavy chain were denatured separately in 6 M guanidine-HCI, 50 mM Tris pH 8.1 , 100 mM NaCI, 10 mM DTT, 10 mM EDTA.
  • Refolding buffer was prepared containing 0.4 M L-Arginine, 100 mM Tris pH 8.1 , 3.7 mM cystamine dihydrochloride, 6.6 mM cysteamine hydrochloride and chilled to ⁇ 5°C.
  • Synthetic peptide dissolved in DMSO to a final concentration of 4mg/ml is added to the refold buffer at 4 mg/litre (final concentration). Then 30 mg/litre ⁇ 2 ⁇ followed by 30 mg/litre heavy chain (final concentrations) are added. Refolding was allowed to reach completion at 4 °C for at least 1 hour.
  • Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Two changes of buffer were necessary to reduce the ionic strength of the solution sufficiently.
  • the protein solution was then filtered through a 1.5 ⁇ cellulose acetate filter and loaded onto a POROS 50HQ anion exchange column (8 ml bed volume). Protein was eluted with a linear 0-500 mM NaCI gradient in 10 mM Tris pH 8.1 using an Akta purifier (GE Healthcare). HLA-A * 02-peptide complex eluted at approximately 250 mM NaCI, and peak fractions were collected, a cocktail of protease inhibitors (Calbiochem) was added and the fractions were chilled on ice.
  • Biotinylation tagged pHLA molecules were buffer exchanged into 10 mM Tris pH 8.1 , 5 mM NaCI using a GE Healthcare fast desalting column equilibrated in the same buffer. Immediately upon elution, the protein-containing fractions were chilled on ice and protease inhibitor cocktail (Calbiochem) was added. Biotinylation reagents were then added: 1 mM biotin, 5 mM ATP
  • Biotinylated pHLA-A * 02 molecules eluted as a single peak at approximately 15 ml. Fractions containing protein were pooled, chilled on ice, and protease inhibitor cocktail was added. Protein concentration was determined using a Coomassie-binding assay (PerBio) and aliquots of biotinylated pHLA-A * 02 molecules were stored frozen at -20 °C.
  • Such peptide-MHC complexes may be used in soluble form or may be immobilised through C terminal biotin moiety on to a solid support, to be used for the detection of T cells and T cell receptors which bind said complex.
  • TCR isolation methods are known to those skilled in the art; for example, DNA sequences corresponding to TCR alpha and beta chains may be amplified from a T cell clone (obtained from volunteer blood donors) that is activated by cells presenting the peptide-HLA complex of interest.
  • T cell clone obtained from volunteer blood donors
  • Other suitable methods include isolation from TCR libraries (as described in WO2005/1 16646 and WO2005/116074 for example).
  • TCR alpha and beta chain sequences are expressed in E. coli as soluble TCRs, (WO2003020763; Boulter, et al., Protein Eng, 2003. 16: 707-711 ). Binding of the soluble TCRs to the complexes is analysed by surface plasmon resonance using a BiaCore 3000 instrument. Biotinylated peptide-HLA monomers are prepared as previously described (Example A2) and immobilized on to a streptavidin-coupled CM-5 sensor chip.
  • The[LSi] present invention also relates to novel peptides derived from Cyclin-dependent kinase 5 activator 2 (CDK5R2), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules.
  • CDK5R2 Cyclin-dependent kinase 5 activator 2
  • binding moieties that bind to the peptides and/or complexes of the invention. Such moieties are useful for the development of immunotherapeutic reagents for the treatment of diseases such as cancer. T cells are a key part of the cellular arm of the immune system.
  • MHC molecules are known as human leukocyte antigens (HLA), and both terms are used synonymously herein. MHC molecules have a binding groove in which the peptide fragments bind. Recognition of particular peptide-MHC antigens is mediated by a corresponding T cell receptor (TCR). Tumour cells express various tumour associated antigens (TAA) and peptides derived from these antigens are frequently displayed on the tumour cell surface.
  • TAA tumour associated antigens
  • TAA-derived peptides presented by MHC molecules on tumour cells enables the development of novel immunotherapeutic reagents designed to specifically target and destroy said tumour cells.
  • Such reagents may be moieties that bind to the TAA-derived peptide and/or peptide-MHC complexes of the invention, and typically involve the induction of a T cell response.
  • such reagents may be based, exclusively, or in part, on T cells, or T cell receptors (TCRs), or antibodies.
  • TAA-derived peptides that are presented on the cell surface in complex with a given MHC, only a small number of such peptides, covering a limited number of cancer indications, have been identified to date.
  • TAAs suitable for the development of immunotherapeutic reagents must have limited or no expression in normal cells, to prevent off target activity. It is therefore desirable to provide additional tumour-specific TAA-derived peptides and MHC complexes thereof that can be used for the development of new cancer therapies.
  • CDK5R2 (also known as cyclin-dependent kinase 5 activator 2 or p39 or p39l and having Uniprot accession number Q13319) is a cyclin-dependent protein kinase activator. Expression of CDK5R2 has been linked to cancer (Arif, Biochem Pharmacol. 2012 Oct 15;84(8):985-93). CDK5R2 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from CDK5R2 that are presented on the cell surface in complex with MHC. These peptides are particularly useful for the development of reagents that can target cells expressing CDK5R2 and for the treatment of cancers, including small cell lung cancer.
  • the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: B1 -B3, or
  • polypeptides of the invention are presented by MHC on the surface of tumour cells. Accordingly, the polypeptides of the invention, as well as moieties that bind the polypeptide-MHC complexes, can be used to develop therapeutic reagents.
  • MHC Major Histocompatibility Complex
  • the skilled person can determine whether or not a given polypeptide forms a complex with an MHC molecule by determining whether the MHC can be refolded in the presence of the polypeptide using the process set out in Example B2. If the polypeptide does not form a complex with MHC then MHC will not refold. Refolding is commonly confirmed using an antibody that recognises MHC in a folded state only. Further details can be found in Garboczi et al., Proc Natl Acad Sci
  • polypeptides of the invention are from about 8 to about 16 amino acids in length, and are most preferably 8, 9, or 10 or 11 amino acids in length.
  • the polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQNOs: B1-B3.
  • amino acid residues comprising the polypeptides of the invention may be chemically modified.
  • chemical modifications include those corresponding to post translational modifications for example phosphorylation, acetylation and deamidation (Engelhard et al., Curr Opin Immunol. 2006 Feb;18(1 ):92-7).
  • Amino acid substitution means that an amino acid residue is substituted for a replacement amino acid residue at the same position.
  • Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are immediately adjacent one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent another inserted amino acid residue.
  • One, two or three amino acids may be deleted from the sequence of SEQNOs: B1-B3. Each deletion can take place at any position of SEQNOs: B1-B3.
  • the polypeptide of the invention may comprise one, two or three additional amino acids at the C-terminal end and/or at the N-terminal end of the sequence of SEQNOs: BIBS.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQNOs: B1-B3 with the exception of one amino acid substitution and one amino acid insertion, one amino acid substitution and one amino acid deletion, or one amino acid insertion and one amino acid deletion.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQNOs: B1-B3, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.
  • Inserted amino acids and replacement amino acids may be naturally occurring amino acids or may be non-naturally occurring amino acids and, for example, may contain a non-natural side chain.
  • Such altered peptide ligands are discussed further in Douat-Casassus et al., J. Med. Chem, 2007 Apr 5;50(7): 1598-609 and Hoppes et al., J. Immunol 2014 Nov 15;193(10):4803-13 and references therein). If more than one amino acid residue is substituted and/or inserted, the
  • replacement/inserted amino acid residues may be the same as each other or different from one another.
  • Each replacement amino acid may have a different side chain to the amino acid being replaced.
  • polypeptides of the invention bind to MHC in the peptide binding groove of the MHC molecule.
  • amino acid modifications described above will not impair the ability of the peptide to bind MHC.
  • the amino acid modifications improve the ability of the peptide to bind MHC.
  • mutations may be made at positions which anchor the peptide to MHC. Such anchor positions and the preferred residues at these locations are known in the art, particularly for peptides which bind HLA-A * 02 (see, e. g. Parkhurst et al., J. Immunol. 1996 Sep 15;157(6):2539-48 and Parker et al. J Immunol.
  • a polypeptide of the invention may be used to illicit an immune response. If this is the case, it is important that the immune response is specific to the intended target in order to avoid the risk of unwanted side effects that may be associated with an "off target" immune response. Therefore, it is preferred that the amino acid sequence of a polypeptide of the invention does not match the amino acid sequence of a peptide from any other protein(s), in particular, that of another human protein.
  • a person of skill in the art would understand how to search a database of known protein sequences to ascertain whether a polypeptide according to the invention is present in another protein.
  • Polypeptides of the invention can be synthesised easily by Merrifield synthesis, also known as solid phase synthesis, or any other peptide synthesis methodology.
  • GMP grade polypeptide is produced by solid-phase synthesis techniques by Multiple Peptide Systems, San Diego, CA.
  • the peptide may be recombinantly produced, if so desired, in accordance with methods known in the art.
  • Such methods typically involve the use of a vector comprising a nucleic acid sequence encoding the polypeptide to be expressed, to express the polypeptide in vivo; for example, in bacteria, yeast, insect or mammalian cells.
  • in vitro cell-free systems may be used. Such systems are known in the art and are commercially available for example from Life
  • polypeptides may be isolated and/or may be provided in substantially pure form. For example, they may be provided in a form which is substantially free of other polypeptides or proteins.
  • the invention provides a complex of the polypeptide of the first aspect and an MHC molecule.
  • the polypeptide is bound to the peptide binding groove of the MHC molecule.
  • the MHC molecule may be MHC class I.
  • the MHC class I molecule may be selected from HLA-A * 02, HLA-A * 01 , HLA-A * 03, HLA-A11 , HLA-A23, HLA-A24, HLA-B * 07, HLA-B*08, HLA- B40, HLA-B44, HLA-B15, HLA-C * 04, HLA * C * 03 HLA-CW.
  • HLA alleles As is known to those skilled in the art there are allelic variants of the above HLA types, all of which are encompassed by the present invention. A full list of HLA alleles can be found on the EMBL Immune Polymorphism Database (http://www.ebi.ac.uk/ipd/imqt/hla/allele.html; Robinson et al. Nucleic Acids Research (2015) 43:D423-431 ).
  • the complex of the invention may be isolated and/or in a substantially pure form.
  • the complex may be provided in a form which is substantially free of other polypeptides or proteins.
  • MHC molecule includes recombinant MHC molecules, non-naturally occurring MHC molecules and functionally equivalent fragments of MHC, including derivatives or variants thereof, provided that peptide binding is retained.
  • MHC molecules may be fused to a therapeutic moiety, attached to a solid support, in soluble form, and/or in multimeric form.
  • Methods to produce soluble recombinant MHC molecules with which polypeptides of the invention can form a complex are known in the art. Suitable methods include, but are not limited to, expression and purification from E. coli cells or insect cells. A suitable method is provided in Example B2 herein.
  • MHC molecules may be produced synthetically, or using cell free systems.
  • Polypeptides and/or polypeptide-MHC complexes of the invention may be associated with a moiety capable of eliciting a therapeutic effect.
  • a moiety may be a carrier protein which is known to be immunogenic.
  • KLH keyhole limpet hemocyanin
  • the polypeptides and/or polypeptide-MHC complexes of the invention may be associated with a fusion partner. Fusion partners may be used for detection purposes, or for attaching said polypeptide or MHC to a solid support, or for MHC oligomerisation.
  • the MHC complexes may incorporate a biotinylation site to which biotin can be added, for example, using the BirA enzyme (O'Callaghan et al., 1999 Jan 1 ;266(1 ):9-15).
  • Other suitable fusion partners include, but are not limited to, fluorescent, or luminescent labels, radiolabels, nucleic acid probes and contrast reagents, antibodies, or enzymes that produce a detectable product. Detection methods may include flow cytometry, microscopy, electrophoresis or scintillation counting.
  • Polypeptide-MHC complexes of the invention may be provided in soluble form, or may be immobilised by attachment to a suitable solid support.
  • suitable solid supports include, but are not limited to, a bead, a membrane, sepharose, a magnetic bead, a plate, a tube, a column.
  • Polypeptide-MHC complexes may be attached to an ELISA plate, a magnetic bead, or a surface plasmon reasonance biosensor chip.
  • Methods of attaching peptide-MHC complexes to a solid support are known to the skilled person, and include, for example, using an affinity binding pair, e.g. biotin and streptavidin, or antibodies and antigens.
  • peptide-MHC complexes are labelled with biotin and attached to streptavidin-coated surfaces.
  • Polypeptide-MHC complexes of the invention may be in multimeric form, for example, dimeric, or tetrameric, or pentameric, or octomeric, or greater. Examples of suitable methods for the production of multimeric peptide MHC complexes are described in Greten et al., Clin. Diagn. Lab. Immunol. 2002 Mar;9(2):216-20 and references therein.
  • polypeptide-MHC multimers may be produced using peptide-MHC tagged with a biotin residue and complexed through fluorescent labelled streptavidin.
  • multimeric polypeptide-MHC complexes may be formed by using immunoglobulin as a molecular scaffold.
  • the extracellular domains of MHC molecules are fused with the constant region of an immunoglobulin heavy chain separated by a short amino acid linker.
  • Polypeptide-MHC multimers have also been produced using carrier molecules such as dextran (WO02072631 ). Multimeric peptide MHC complexes can be useful for improving the detection of binding moieties, such as T cell receptors, which bind said complex, because of avidity effects.
  • the polypeptides of the invention may be presented on the surface of a cell in complex with MHC.
  • the invention also provides a cell presenting on its surface a complex of the invention.
  • Such a cell may be a mammalian cell, preferably a cell of the immune system, and in particular a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • Other preferred cells include T2 cells (Hosken, et al., Science. 1990 Apr 20;248(4953):367-70).
  • Cells presenting the polypeptide or complex of the invention may be isolated, preferably in the form of a population, or provided in a substantially pure form. Said cells may not naturally present the complex of the invention, or alternatively said cells may present the complex at a level higher than they would in nature. Such cells may be obtained by pulsing said cells with the polypeptide of the invention.
  • Pulsing involves incubating the cells with the polypeptide for several hours using polypeptide concentrations typically ranging from 10 "5 to 10 "12 M. Said cells may additionally be transduced with HLA-A * 02 molecules to further induce presentation of the peptide. Cells may be produced recombinantly. Cells presenting polypeptides of the invention may be used to isolate T cells and T cell receptors (TCRs) which are activated by, or bind to, said cells, as described in more detail below.
  • TCRs T cell receptors
  • the invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide of the first aspect of the invention.
  • the nucleic acid may be cDNA.
  • the nucleic acid molecule may consist essentially of a nucleic acid sequence encoding the polypeptide of the first aspect of the invention or may encode only the peptide of the invention, i.e. encode no other polypeptide.
  • nucleic acid molecule can be synthesised in accordance with methods known in the art. Due to the degeneracy of the genetic code, one of ordinary skill in the art will appreciate that nucleic acid molecules of different nucleotide sequence can encode the same amino acid sequence.
  • the invention provides a vector comprising a nucleic acid sequence according to the third aspect of the invention.
  • the vector may include, in addition to a nucleic acid sequence encoding only a polypeptide of the invention, one or more additional nucleic acid sequences encoding one or more additional polypeptides. Such additional polypeptides may, once expressed, be fused to the N-terminus or the C-terminus of the polypeptide of the invention.
  • the vector includes a nucleic acid sequence encoding a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • Suitable vectors are known in the art as is vector construction, including the selection of promoters and other regulatory elements, such as enhancer elements.
  • the vector utilised in the context of the present invention desirably comprises sequences appropriate for introduction into cells.
  • the vector may be an expression vector, a vector in which the coding sequence of the polypeptide is under the control of its own cis-acting regulatory elements, a vector designed to facilitate gene integration or gene replacement in host cells, and the like.
  • the term "vector” encompasses a DNA molecule, such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • a DNA molecule such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • Appropriate phage and viral vectors include, but are not limited to, lambda (X) bacteriophage, EMBL bacteriophage, simian virus 40, bovine papilloma virus, Epstein-Barr virus, adenovirus, herpes virus, vaccinia virus, Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, lentivirus and Rous sarcoma virus.
  • the invention provides a cell comprising the vector of the fourth aspect of the invention.
  • the cell may be an antigen presenting cell and is preferably a cell of the immune system.
  • the cell may be a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • the cell may be a mammalian cell.
  • Polypeptides and complexes of the invention can be used to identify and/or isolate binding moieties that bind specifically to the polypeptide and/or the complex of the invention.
  • binding moieties may be used as immunotherapeutic reagents and may include antibodies and TCRs.
  • the invention provides a binding moiety that binds the polypeptide of the invention.
  • the binding moiety binds the polypeptide when said polypeptide is in complex with MHC.
  • the binding moiety may bind partially to the MHC, provided that it also binds to the polypeptide.
  • the binding moiety may bind only the polypeptide, and that binding may be specific.
  • the binding moiety may bind only the peptide MHC complex and that binding may be specific.
  • TCR T cell receptor
  • IMGT International Immunogenetics
  • the TCRs of the invention may be in any format known to those in the art.
  • the TCRs may be ⁇ heterodimers, or they may be in single chain format (such as those described in
  • Single chain TCRs include ⁇ TCR polypeptides of the type: Va-L- ⁇ , ⁇ -L-Va, Va- Ca-L- ⁇ , Va-L- ⁇ - ⁇ or Va- Ca - ⁇ ⁇ - ⁇ , optionally in the reverse orientation, wherein Va and ⁇ are TCR a and ⁇ variable regions respectively, Ca and C are TCR a and ⁇ constant regions respectively, and L is a linker sequence.
  • the TCR may be in a soluble form (i.e. having no transmembrane or cytoplasmic domains), or may contain full length alpha and beta chains.
  • the TCR may be provided on the surface of a cell, such as a T cell.
  • the alpha and/or beta chain constant domain may be truncated relative to the native/naturally occurring TRAC/TRBC sequences.
  • the TRAC/TRBC may contain modifications.
  • the alpha chain extracellular sequence may include a modification relative to the native/naturally occurring TRAC whereby amino acid T48 of TRAC, with reference to IMGT numbering, is replaced with C48.
  • the beta chain extracellular sequence may include a modification relative to the native/naturally occurring TRBC1 or TRBC2 whereby S57 of TRBC1 or TRBC2, with reference to IMGT numbering, is replaced with C57.
  • TCRs of the invention may be engineered to include mutations.
  • Methods for producing mutated high affinity TCR variants such as phage display and site directed mutagenesis and are known to those in the art (for example see WO 04/044004 and Li et al., Nat Biotechnol 2005 Mar;23(3):349- 54).
  • TCRs of the invention may also be may be labelled with an imaging compound, for example a label that is suitable for diagnostic purposes.
  • Such labelled high affinity TCRs are useful in a method for detecting a TCR ligand selected from CD1 -antigen complexes, bacterial superantigens, and MHC- peptide/superantigen complexes, which method comprises contacting the TCR ligand with a high affinity TCR (or a multimeric high affinity TCR complex) which is specific for the TCR ligand; and detecting binding to the TCR ligand.
  • a high affinity TCR or a multimeric high affinity TCR complex
  • multimeric high affinity TCR complexes such as those described in Zhu et al., J. Immunol.
  • a fluorescent streptavidin (commercially available) can be used to provide a detectable label.
  • a fluorescently-labelled multimer is suitable for use in FACS analysis, for example to detect antigen presenting cells carrying the peptide for which the high affinity TCR is specific.
  • a TCR of the present invention (or multivalent complex thereof) may alternatively or additionally be associated with (e.g. covalently or otherwise linked to) a therapeutic agent which may be, for example, a toxic moiety for use in cell killing, or an immunostimulating agent such as an interleukin or a cytokine.
  • a multivalent high affinity TCR complex of the present invention may have enhanced binding capability for a TCR ligand compared to a non-multimeric wild-type or high affinity T cell receptor heterodimer.
  • the multivalent high affinity TCR complexes according to the invention are particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo, and are also useful as intermediates for the production of further multivalent high affinity TCR complexes having such uses.
  • the high affinity TCR or multivalent high affinity TCR complex may therefore be provided in a pharmaceutically acceptable formulation for use in vivo.
  • High affinity TCRs of the invention may be used in the production of soluble bi-specific reagents.
  • a preferred embodiment is a reagent which comprises a soluble TCR, fused via a linker to an anti- CD3 specific antibody fragment. Further details including how to produce such reagents are described in W010/133828.
  • the invention provides nucleic acid encoding the TCR of the invention, a TCR expression vector comprising nucleic acid encoding a TCR of the invention, as well as a cell harbouring such a vector.
  • the TCR may be encoded either in a single open reading frame or two distinct open reading frames.
  • one vector may encode both an alpha and a beta chain of a TCR of the invention.
  • a further aspect of the invention provides a cell displaying on its surface a TCR of the invention.
  • the cell may be a T cell.
  • T cells expressing the TCRs of the invention are suitable for use in adoptive therapy-based treatment of diseases such as cancers.
  • adoptive therapy see for example Rosenberg et al., Nat Rev Cancer. 2008 Apr;8(4):299-308.
  • the TCRs of the invention intended for use in adoptive therapy are generally glycosylated when expressed by the transfected T cells.
  • the glycosylation pattern of transfected TCRs may be modified by mutations of the transfected gene (Kuball J et al., J Exp Med. 2009 Feb 16;206(2):463-75).
  • the binding moiety of the invention may be an antibody.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced.
  • antibody includes antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanised antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic and any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.
  • a humanised antibody may be a modified antibody having the variable regions of a non-human, e.g. murine, antibody and the constant region of a human antibody. Methods for making humanised antibodies are described in, for example, US Patent No. 5225539. Examples of antibodies are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. A monoclonal antibody may be referred to herein as "mab".
  • an antibody for example a monoclonal antibody
  • recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody.
  • Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementary determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin (see, for instance, EP-A-184187, GB 2188638A or EP-A-239400).
  • CDRs complementary determining regions
  • a hybridoma (or other cell that produces antibodies) may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. et al., Nature.
  • Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g.
  • antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).
  • bispecific antibodies these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Hollinger & Winter, Curr Opin
  • bispecific antibody fragments e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain "Janusins" described in Traunecker et al., EMBO J. 1991 Dec; 10(12):3655-9). Bispecific diabodies, as opposed to bispecific whole antibodies, may also be useful because they can be readily constructed and expressed in E. coli.
  • Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
  • An "antigen binding domain" is the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope.
  • An antigen binding domain may be provided by one or more antibody variable domains.
  • An antigen binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • the binding moiety may be an antibody-like molecule that has been designed to specifically bind a peptide-MHC complex of the invention.
  • TCR-mimic antibodies such as, for example those described in WO2007143104 and Sergeeva et al., Blood. 201 1 Apr
  • binding moieties based on engineered protein scaffolds are derived from stable, soluble, natural protein structures which have been modified to provide a binding site for a target molecule of interest.
  • engineered protein scaffolds include, but are not limited to, affibodies, which are based on the Z-domain of staphylococcal protein A that provides a binding interface on two of its a-helices (Nygren, FEBS J. 2008 Jun;275(1 1 ):2668-76); anticalins, derived from lipocalins, that incorporate binding sites for small ligands at the open end of a beta-barrel fold (Skerra, FEBS J.
  • Engineered protein scaffolds are typically targeted to bind the same antigenic proteins as antibodies, and are potential therapeutic agents. They may act as inhibitors or antagonists, or as delivery vehicles to target molecules, such as toxins, to a specific tissue in vivo (Gebauer and Skerra, Curr Opin Chem Biol. 2009 Jun;13(3):245-55). Short peptides may also be used to bind a target protein.
  • Phylomers are natural structured peptides derived from bacterial genomes. Such peptides represent a diverse array of protein structural folds and can be used to inhibit/disrupt protein-protein interactions in vivo (Watt, Nat Biotechnol. 2006 Feb;24(2): 177-83)].
  • the invention further provides a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention for use in medicine.
  • the polypeptide, complex, nucleic acid, vector, cell or binding moiety may be used for in the treatment or prevention of cancer, in particular, small cell lung cancer.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention together with a pharmaceutically acceptable carrier.
  • This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient). It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms. Suitable compositions and methods of administration are known to those skilled in the art, for example see, Johnson et al., Blood.
  • Cells in accordance with the invention will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a
  • T cells transfected with TCRs of the invention may be provided in pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be adapted for administration by any appropriate route such as a parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation or intranasal routes.
  • a parenteral including subcutaneous, intramuscular, or intravenous
  • enteral including oral or rectal
  • inhalation or intranasal routes Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
  • Dosages of the substances of the present invention can vary between wide limits, depending upon the disease or disorder to be treated (such as cancer, viral infection or autoimmune disease), the age and condition of the individual to be treated, etc.
  • a suitable dose range for a reagent comprising a soluble TCR fused to an anti- CD3 domain may be between 25 ng/kg and 50 pg/kg.
  • a physician will ultimately determine appropriate dosages to be used.
  • the polypeptide of the invention may be provided in the form of a vaccine composition.
  • the vaccine composition may be useful for the treatment or prevention of cancer. All such
  • compositions are encompassed in the present invention.
  • vaccines may take several forms (Schlom, J Natl Cancer Inst. 2012 Apr 18;104(8):599-613).
  • the peptide of the invention may be used directly to immunise patients (Salgaller, Cancer Res. 1996 Oct 15;56(20):4749-57 and Marchand, Int J Cancer. 1999 Jan 18;80(2):219-30).
  • the vaccine composition may include additional peptides such that the peptide of the invention is one of a mixture of peptides.
  • Adjuvants may be added to the vaccine composition to augment the immune response
  • the vaccine composition may take the form of an antigen presenting cell displaying the peptide of the invention in complex with MHC.
  • the antigen presenting cell is an immune cell, more preferably a dendritic cell.
  • the peptide may be pulsed onto the surface of the cell (Thurner, J Exp Med. 1999 Dec 6; 190(11 ): 1669-78), or nucleic acid encoding for the peptide of the invention may be introduced into dendritic cells (for example by electroporation. Van Tendeloo, Blood. 2001 Jul 1 ;98(1 ):49-56).
  • polypeptides, complexes, nucleic acid molecules, vectors, cells and binding moieties of the invention may be non-naturally occurring and/or purified and/or engineered and/or recombinant and/or isolated and/or synthetic.
  • the invention also provides a method of identifying a binding moiety that binds a complex of the invention, the method comprising contacting a candidate binding moiety with the complex and determining whether the candidate binding moiety binds the complex.
  • Methods to determine binding to polypeptide-MHC complexes are well known in the art. Preferred methods include, but are not limited to, surface plasmon resonance, or any other biosensor technique, ELISA, flow cytometry, chromatography, microscopy. Alternatively, or in addition, binding may be determined by functional assays in which a biological response is detected upon binding, for example, cytokine release or cell apoptosis.
  • the candidate binding moiety may be a binding moiety of the type already described, such as a TCR or an antibody.
  • T cells may be isolated from fresh blood obtained from volunteer donors. Such a method involves stimulating T cells using autologous DCs, followed by autologous B cells, pulsed with the polypeptide of the invention. Several round of stimulation may be carried out, for example three or four rounds. Activated T cells may then be tested for specificity by measuring cytokine release in the presence of T2 cells pulsed with the peptide of the invention (for example using an IFNy ELISpot assay). Activated cells may then be sorted by fluorescence-activated cell sorting (FACS) using labelled antibodies to detect intracellular cytokine production (e.g. IFNy), or expression of a cell surface marker (such as CD137).
  • FACS fluorescence-activated cell sorting
  • Sorted cells may be expanded and further validated, for example, by ELISpot assay and/or cytotoxicity against target cells and/or staining by peptide-MHC tetramer.
  • the TCR chains from validated clones may then be amplified by rapid amplification of cDNA ends (RACE) and sequenced.
  • the polypeptide MHC complex of the invention may be used for screening a library of TCRs or antibodies.
  • the production of antibody libraries using phage display is well known in the art, for example see Aitken, Antibody phage display: Methods and Protocols (2009, Humana, New York). Further details on TCR libraries can be found in WO04044004.
  • the polypeptide-MHC complex of the invention is used to screen a library of diverse TCRs displayed on the surface of phage particles.
  • the TCRs displayed by said library may contain non- natural mutations. Screening may involve panning the phage library with peptide-MHC complexes of the invention and subsequently isolating bound phage.
  • peptide-MHC complexes may be attached to a solid support, such as a magnet bead, or column matrix and phage bound peptide MHC complexes isolated, with a magnet, or by chromatography, respectively.
  • the panning steps may be repeated several times for example three or four times.
  • Isolated phage may be further expanded in E. coli cells.
  • Isolated phage particles may be tested for specific binding peptide-MHC complexes of the invention. Binding can be detected using techniques including, but not limited to, ELISA, or SPR for example using a BiaCore instrument.
  • the DNA sequence of the T cell receptor displayed by peptide-MHC binding phage can be further identified by standard PCR methods.
  • Figures B1 to B3 show the respective fragmentation spectra for the peptides of SEQNOs: B1 to B3, eluted from cells. A table highlighting the matching ions is shown below each spectrum.
  • Example B1 Identification of target-derived peptides by Mass spectrometry
  • Immortalised cell lines obtained from commercial sources were maintained and expanded under standard conditions.
  • Class I HLA complexes were purified by immunoaffinity using commercially available anti-HLA antibodies BB7.1 (anti-HLA-B * 07), BB7.2 (anti-HLA-A * 02) and W6/32 (anti-Class 1 ). Briefly, cells were lysed in buffer containing non-ionic detergent NP-40 (0.5% v/v) at 5 x 10 7 cells per ml and incubated at 4°C for 1 h with agitation/mixing. Cell debris was removed by centrifugation and supernatant pre-cleared using proteinA-Sepharose.
  • Peptides were separated by high pressure liquid chromatography (HPLC) on a Dionex Ultimate 3000 system using a C18 column (Phenomenex). Peptides were loaded in 98% buffer A (0.1 % aqueous trifluoroacetic acid (TFA)) and 2% buffer B (0.1% TFA in acetonitrile). Peptides were eluted using a stepped gradient of B (2-60%) over 20 min. Fractions were collected at one minute intervals and lyophilised.
  • HPLC high pressure liquid chromatography
  • Peptides were analysed by nanoLCMS/MS using a Dionex Ultimate 3000 nanoLC coupled to either AB Sciex Triple TOF 5600 or Thermo Orbitrap Fusion mass spectrometers. Both machines were equipped with nanoelectrospray ion sources. Peptides were loaded onto an Acclaim PepMap 100 trap column (Dionex) and separated using an Acclaim PepMap RSLC column (Dionex). Peptides were loaded in mobile phase A (0.5% formic acid: water) and eluted using a gradient of buffer B (acetonitrile:0.5% formic acid) directly into the nanospray ionisation source.
  • buffer B acetonitrile:0.5% formic acid
  • IDA information dependent acquisition
  • Information acquired in these experiments was used to search the Uniprot database of human proteins for peptides consistent with the fragmentation patterns seen, using Protein pilot software (Ab Sciex) and PEAKS software (Bioinformatics solutions). Peptides identified are assigned a score by the software, based on the match between the observed and expected fragmentation patterns.
  • polypeptides set out in table B1 corresponding to SEQNOs: B1-B3, were detected by mass spec following extraction from cancer cell lines.
  • An example cell line from which the peptide was detected is indicated in the table along with the HLA antibody used for immunoaffinity purification.
  • Figures B1 -B3 show representative fragmentation patterns for the peptides of SEQNOs: B1-B3 respectively. A table highlighting the matching ions is shown below each spectrum.
  • Example B2 Preparation of recombinant peptide-HLA complexes
  • HLA-A*02 molecules HLA-A*02-heavy chain and HLA light-chain ( ⁇ 2 ⁇ ) were expressed separately in E. coli as inclusion bodies, using appropriate constructs.
  • HLA-A * 02-heavy chain additionally contained a C-terminal biotinylation tag which replaces the transmembrane and cytoplasmic domains (O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15).
  • E. coli cells were lysed and inclusion bodies processed to approximately 80% purity.
  • Inclusion bodies of ⁇ 2 ⁇ and heavy chain were denatured separately in 6 M guanidine-HCI, 50 mM Tris pH 8.1 , 100 mM NaCI, 10 mM DTT, 10 mM EDTA.
  • Refolding buffer was prepared containing 0.4 M L-Arginine, 100 mM Tris pH 8.1 , 3.7 mM cystamine dihydrochloride, 6.6 mM cysteamine hydrochloride and chilled to ⁇ 5°C.
  • Synthetic peptide dissolved in DMSO to a final concentration of 4mg/ml is added to the refold buffer at 4 mg/litre (final concentration). Then 30 mg/litre ⁇ 2 ⁇ followed by 30 mg/litre heavy chain (final concentrations) are added. Refolding was allowed to reach completion at 4 °C for at least 1 hour.
  • Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Two changes of buffer were necessary to reduce the ionic strength of the solution sufficiently.
  • the protein solution was then filtered through a 1.5 pm cellulose acetate filter and loaded onto a POROS 50HQ anion exchange column (8 ml bed volume). Protein was eluted with a linear 0-500 mM NaCI gradient in 10 mM Tris pH 8.1 using an Akta purifier (GE Healthcare). HLA-A * 02-peptide complex eluted at approximately 250 mM NaCI, and peak fractions were collected, a cocktail of protease inhibitors (Calbiochem) was added and the fractions were chilled on ice.
  • Biotinylation tagged pHLA molecules were buffer exchanged into 10 mM Tris pH 8.1 , 5 mM NaCI using a GE Healthcare fast desalting column equilibrated in the same buffer. Immediately upon elution, the protein-containing fractions were chilled on ice and protease inhibitor cocktail (Calbiochem) was added. Biotinylation reagents were then added: 1 mM biotin, 5 mM ATP (buffered to pH 8), 7.5 mM MgCI2, and 5 pg/ml BirA enzyme (purified according to O'Callaghan et al., (1999) Anal. Biochem. 266: 9-15). The mixture was then allowed to incubate at room temperature overnight.
  • the biotinylated pHLA-A * 02 molecules were purified using gel filtration chromatography.
  • a GE Healthcare Superdex 75 HR 10/30 column was pre-equilibrated with filtered PBS and 1 ml of the biotinylation reaction mixture was loaded and the column was developed with PBS at 0.5 ml/min using an Akta purifier (GE Healthcare).
  • Biotinylated pHLA-A * 02 molecules eluted as a single peak at approximately 15 ml. Fractions containing protein were pooled, chilled on ice, and protease inhibitor cocktail was added. Protein concentration was determined using a Coomassie-binding assay (PerBio) and aliquots of biotinylated pHLA-A * 02 molecules were stored frozen at -20 °C.
  • Such peptide-MHC complexes may be used in soluble form or may be immobilised through C terminal biotin moiety on to a solid support, to be used for the detection of T cells and T cell receptors which bind said complex.
  • TCR isolation methods are known to those skilled in the art; for example, DNA sequences corresponding to TCR alpha and beta chains may be amplified from a T cell clone (obtained from volunteer blood donors) that is activated by cells presenting the peptide-HLA complex of interest.
  • T cell clone obtained from volunteer blood donors
  • Other suitable methods include isolation from TCR libraries (as described in WO2005/1 16646 and WO2005/116074 for example).
  • TCR alpha and beta chain sequences are expressed in E. coli as soluble TCRs, (WO2003020763; Boulter, et al., Protein Eng, 2003. 16: 707-711 ). Binding of the soluble TCRs to the complexes is analysed by surface plasmon resonance using a BiaCore 3000 instrument. Biotinylated peptide-HLA monomers are prepared as previously described (Example B2) and immobilized on to a streptavidin-coupled CM-5 sensor chip.
  • The[LS2] present invention also relates to novel peptides derived from Cyclic nucleotide-gated cation channel beta-1 (CNGB1 ), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules.
  • binding moieties that bind to the peptides and/or complexes of the invention. Such moieties are useful for the development of immunotherapeutic reagents for the treatment of diseases such as cancer.
  • T cells are a key part of the cellular arm of the immune system. They specifically recognise peptide fragments that are derived from intracellular proteins and presented in complex with Major Histocompatibility Complex (MHC) molecules on the surface of antigen presenting cells (APCs).
  • MHC molecules are known as human leukocyte antigens (HLA), and both terms are used synonymously herein.
  • HLA human leukocyte antigens
  • MHC molecules have a binding groove in which the peptide fragments bind. Recognition of particular peptide-MHC antigens is mediated by a corresponding T cell receptor (TCR).
  • TCR T cell receptor
  • Tumour cells express various tumour associated antigens (TAA) and peptides derived from these antigens are frequently displayed on the tumour cell surface.
  • TAA tumour associated antigens
  • TAA-derived peptides presented by MHC molecules on tumour cells enables the development of novel immunotherapeutic reagents designed to specifically target and destroy said tumour cells.
  • Such reagents may be moieties that bind to the TAA-derived peptide and/or peptide-MHC complexes of the invention, and typically involve the induction of a T cell response.
  • such reagents may be based, exclusively, or in part, on T cells, or T cell receptors (TCRs), or antibodies.
  • TAA-derived peptides that are presented on the cell surface in complex with a given MHC, only a small number of such peptides, covering a limited number of cancer indications, have been identified to date.
  • TAAs suitable for the development of immunotherapeutic reagents must have limited or no expression in normal cells, to prevent off target activity. It is therefore desirable to provide additional tumour-specific TAA-derived peptides and MHC complexes thereof that can be used for the development of new cancer therapies.
  • SYFPETHEI Randommensee, et al., Immunogenetics. 1999 Nov; 50(3- 4):213-9 (access via www.syfpeithi.de) and BIMAS (Parker, et al., J. Immunol. 1994 Jan
  • CNGB1 also known as cyclic nucleotide-gated cation channel beta-1 or CNG channel 4 or GARP and having Uniprot accession number Q14028
  • CNG cyclic nucleotide-gated channels
  • CNGB1 is an ideal target for immunotherapeutic applications.
  • the inventors have found novel peptides derived from CNGB1 that are presented on the cell surface in complex with MHC. These peptides are particularly useful for the development of reagents that can target cells expressing CNGB1 and for the treatment of cancers, including head and neck cancer and oesophageal cancer.
  • the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: C1 -C9, or
  • polypeptides of the invention are presented by MHC on the surface of tumour cells. Accordingly, the polypeptides of the invention, as well as moieties that bind the polypeptide-MHC complexes, can be used to develop therapeutic reagents.
  • the skilled person can determine whether or not a given polypeptide forms a complex with an MHC molecule by determining whether the MHC can be refolded in the presence of the polypeptide using the process set out in Example C2. If the polypeptide does not form a complex with MHC then MHC will not refold. Refolding is commonly confirmed using an antibody that recognises MHC in a folded state only. Further details can be found in Garboczi et al., Proc Natl Acad Sci
  • polypeptides of the invention are from about 8 to about 16 amino acids in length, and are most preferably 8, 9, or 10 or 11 amino acids in length.
  • polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: C1-C9.
  • amino acid residues comprising the polypeptides of the invention may be chemically modified.
  • chemical modifications include those corresponding to post translational modifications for example phosphorylation, acetylation and deamidation (Engelhard et al., Curr Opin Immunol. 2006 Feb;18(1 ):92-7).
  • Amino acid substitution means that an amino acid residue is substituted for a replacement amino acid residue at the same position.
  • Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are immediately adjacent one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent another inserted amino acid residue.
  • One, two or three amino acids may be deleted from the sequence of SEQ NOs: C1-C9. Each deletion can take place at any position of SEQ NOs: C1 -C9.
  • the polypeptide of the invention may comprise one, two or three additional amino acids at the C-terminal end and/or at the N-terminal end of the sequence of SEQ NOs: C1- C9.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: C1-C9 with the exception of one amino acid substitution and one amino acid insertion, one amino acid substitution and one amino acid deletion, or one amino acid insertion and one amino acid deletion.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: C1-C9, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.
  • Inserted amino acids and replacement amino acids may be naturally occurring amino acids or may be non-naturally occurring amino acids and, for example, may contain a non-natural side chain.
  • Such altered peptide ligands are discussed further in Douat-Casassus et al., J. Med. Chem, 2007 Apr 5;50(7): 1598-609 and Hoppes et al., J. Immunol 2014 Nov 15;193(10):4803-13 and references therein). If more than one amino acid residue is substituted and/or inserted, the
  • replacement/inserted amino acid residues may be the same as each other or different from one another.
  • Each replacement amino acid may have a different side chain to the amino acid being replaced.
  • polypeptides of the invention bind to MHC in the peptide binding groove of the MHC molecule.
  • amino acid modifications described above will not impair the ability of the peptide to bind MHC.
  • the amino acid modifications improve the ability of the peptide to bind MHC.
  • mutations may be made at positions which anchor the peptide to MHC. Such anchor positions and the preferred residues at these locations are known in the art, particularly for peptides which bind HLA-A * 02 (see, e. g. Parkhurst et al., J. Immunol. 1996 Sep 15;157(6):2539-48 and Parker et al. J Immunol.
  • a polypeptide of the invention may be used to illicit an immune response. If this is the case, it is important that the immune response is specific to the intended target in order to avoid the risk of unwanted side effects that may be associated with an "off target" immune response. Therefore, it is preferred that the amino acid sequence of a polypeptide of the invention does not match the amino acid sequence of a peptide from any other protein(s), in particular, that of another human protein.
  • a person of skill in the art would understand how to search a database of known protein sequences to ascertain whether a polypeptide according to the invention is present in another protein.
  • Polypeptides of the invention can be synthesised easily by Merrifield synthesis, also known as solid phase synthesis, or any other peptide synthesis methodology.
  • GMP grade polypeptide is produced by solid-phase synthesis techniques by Multiple Peptide Systems, San Diego, CA.
  • the peptide may be recombinantly produced, if so desired, in accordance with methods known in the art.
  • Such methods typically involve the use of a vector comprising a nucleic acid sequence encoding the polypeptide to be expressed, to express the polypeptide in vivo; for example, in bacteria, yeast, insect or mammalian cells.
  • in vitro cell-free systems may be used. Such systems are known in the art and are commercially available for example from Life
  • polypeptides may be isolated and/or may be provided in substantially pure form. For example, they may be provided in a form which is substantially free of other polypeptides or proteins.
  • the invention provides a complex of the polypeptide of the first aspect and an MHC molecule.
  • the polypeptide is bound to the peptide binding groove of the MHC molecule.
  • the MHC molecule may be MHC class I.
  • the MHC class I molecule may be selected from HLA-A * 02, HLA-A * 01 , HLA-A * 03, HLA-A11 , HLA-A23, HLA-A24, HLA-B * 07, HLA-B*08, HLA- B40, HLA-B44, HLA-B15, HLA-C * 04, HLA * C * 03 HLA-CW.
  • HLA alleles As is known to those skilled in the art there are allelic variants of the above HLA types, all of which are encompassed by the present invention. A full list of HLA alleles can be found on the EMBL Immune Polymorphism Database (http://www.ebi.ac.uk/ipcl/imqt/hla/allele.html; Robinson et al. Nucleic Acids Research (2015) 43:D423-431 ).
  • the complex of the invention may be isolated and/or in a substantially pure form.
  • the complex may be provided in a form which is substantially free of other polypeptides or proteins.
  • MHC molecule includes recombinant MHC molecules, non-naturally occurring MHC molecules and functionally equivalent fragments of MHC, including derivatives or variants thereof, provided that peptide binding is retained.
  • MHC molecules may be fused to a therapeutic moiety, attached to a solid support, in soluble form, and/or in multimeric form.
  • KLH keyhole limpet hemocyanin
  • the polypeptides and/or polypeptide-MHC complexes of the invention may be associated with a fusion partner. Fusion partners may be used for detection purposes, or for attaching said polypeptide or MHC to a solid support, or for MHC oligomerisation.
  • the MHC complexes may incorporate a biotinylation site to which biotin can be added, for example, using the BirA enzyme (O'Callaghan et al., 1999 Jan 1 ;266(1 ):9-15).
  • Other suitable fusion partners include, but are not limited to, fluorescent, or luminescent labels, radiolabels, nucleic acid probes and contrast reagents, antibodies, or enzymes that produce a detectable product. Detection methods may include flow cytometry, microscopy, electrophoresis or scintillation counting.
  • Polypeptide-MHC complexes of the invention may be provided in soluble form, or may be immobilised by attachment to a suitable solid support.
  • suitable solid supports include, but are not limited to, a bead, a membrane, sepharose, a magnetic bead, a plate, a tube, a column.
  • Polypeptide-MHC complexes may be attached to an ELISA plate, a magnetic bead, or a surface plasmon reasonance biosensor chip.
  • Methods of attaching peptide-MHC complexes to a solid support are known to the skilled person, and include, for example, using an affinity binding pair, e.g. biotin and streptavidin, or antibodies and antigens.
  • peptide-MHC complexes are labelled with biotin and attached to streptavidin-coated surfaces.
  • Polypeptide-MHC complexes of the invention may be in multimeric form, for example, dimeric, or tetrameric, or pentameric, or octomeric, or greater. Examples of suitable methods for the production of multimeric peptide MHC complexes are described in Greten et al., Clin. Diagn. Lab. Immunol. 2002 Mar;9(2):216-20 and references therein.
  • polypeptide-MHC multimers may be produced using peptide-MHC tagged with a biotin residue and complexed through fluorescent labelled streptavidin.
  • multimeric polypeptide-MHC complexes may be formed by using immunoglobulin as a molecular scaffold.
  • MHC molecules are fused with the constant region of an immunoglobulin heavy chain separated by a short amino acid linker.
  • Polypeptide-MHC multimers have also been produced using carrier molecules such as dextran (WO02072631 ). Multimeric peptide MHC complexes can be useful for improving the detection of binding moieties, such as T cell receptors, which bind said complex, because of avidity effects.
  • the polypeptides of the invention may be presented on the surface of a cell in complex with MHC.
  • the invention also provides a cell presenting on its surface a complex of the invention.
  • a cell may be a mammalian cell, preferably a cell of the immune system, and in particular a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • Other preferred cells include T2 cells (Hosken, et al., Science. 1990 Apr 20;248(4953):367-70).
  • Cells presenting the polypeptide or complex of the invention may be isolated, preferably in the form of a population, or provided in a substantially pure form.
  • Said cells may not naturally present the complex of the invention, or alternatively said cells may present the complex at a level higher than they would in nature.
  • Such cells may be obtained by pulsing said cells with the polypeptide of the invention. Pulsing involves incubating the cells with the polypeptide for several hours using polypeptide concentrations typically ranging from 10 "5 to 10 "12 M. Said cells may additionally be transduced with HLA-A * 02 molecules to further induce presentation of the peptide. Cells may be produced recombinantly. Cells presenting polypeptides of the invention may be used to isolate T cells and T cell receptors (TCRs) which are activated by, or bind to, said cells, as described in more detail below.
  • TCRs T cell receptors
  • the invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide of the first aspect of the invention.
  • the nucleic acid may be cDNA.
  • the nucleic acid molecule may consist essentially of a nucleic acid sequence encoding the polypeptide of the first aspect of the invention or may encode only the peptide of the invention, i.e. encode no other polypeptide.
  • Such a nucleic acid molecule can be synthesised in accordance with methods known in the art. Due to the degeneracy of the genetic code, one of ordinary skill in the art will appreciate that nucleic acid molecules of different nucleotide sequence can encode the same amino acid sequence.
  • the invention provides a vector comprising a nucleic acid sequence according to the third aspect of the invention.
  • the vector may include, in addition to a nucleic acid sequence encoding only a polypeptide of the invention, one or more additional nucleic acid sequences encoding one or more additional polypeptides. Such additional polypeptides may, once expressed, be fused to the N-terminus or the C-terminus of the polypeptide of the invention.
  • the vector includes a nucleic acid sequence encoding a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • Suitable vectors are known in the art as is vector construction, including the selection of promoters and other regulatory elements, such as enhancer elements.
  • the vector utilised in the context of the present invention desirably comprises sequences appropriate for introduction into cells.
  • the vector may be an expression vector, a vector in which the coding sequence of the polypeptide is under the control of its own cis-acting regulatory elements, a vector designed to facilitate gene integration or gene replacement in host cells, and the like.
  • the term "vector” encompasses a DNA molecule, such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • a DNA molecule such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • Appropriate phage and viral vectors include, but are not limited to, lambda (X) bacteriophage, EMBL bacteriophage, simian virus 40, bovine papilloma virus, Epstein-Barr virus, adenovirus, herpes virus, vaccinia virus, Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, lentivirus and Rous sarcoma virus.
  • the invention provides a cell comprising the vector of the fourth aspect of the invention.
  • the cell may be an antigen presenting cell and is preferably a cell of the immune system.
  • the cell may be a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • the cell may be a mammalian cell.
  • Polypeptides and complexes of the invention can be used to identify and/or isolate binding moieties that bind specifically to the polypeptide and/or the complex of the invention.
  • binding moieties may be used as immunotherapeutic reagents and may include antibodies and TCRs.
  • the invention provides a binding moiety that binds the polypeptide of the invention.
  • the binding moiety binds the polypeptide when said polypeptide is in complex with MHC.
  • the binding moiety may bind partially to the MHC, provided that it also binds to the polypeptide.
  • the binding moiety may bind only the polypeptide, and that binding may be specific.
  • the binding moiety may bind only the peptide MHC complex and that binding may be specific.
  • binding moieties that bind the complex of the invention When used with reference to binding moieties that bind the complex of the invention, "specific" is generally used herein to refer to the situation in which the binding moiety does not show any significant binding to one or more alternative polypeptide-MHC complexes other than the polypeptide-MHC complex of the invention.
  • the binding moiety may be a T cell receptor (TCR).
  • TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature, and links to the IMGT public database of TCR sequences. The unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the TCR field. For example, they can be found in the "T cell Receptor Factsbook", (2001 ) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8;
  • the TCRs of the invention may be in any format known to those in the art.
  • the TCRs may be ⁇ heterodimers, or they may be in single chain format (such as those described in W09918129).
  • Single chain TCRs include ⁇ TCR polypeptides of the type: Va-L- ⁇ , ⁇ -L-Va, Va- Ca-L- ⁇ , ⁇ - ⁇ - ⁇ ⁇ -0 ⁇ or Va- Ca - ⁇ / ⁇ - ⁇ , optionally in the reverse orientation, wherein Va and ⁇ are TCR a and ⁇ variable regions respectively, Ca and C are TCR a and ⁇ constant regions respectively, and L is a linker sequence.
  • the TCR may be in a soluble form (i.e.
  • the TCR may be provided on the surface of a cell, such as a T cell.
  • the alpha and/or beta chain constant domain may be truncated relative to the native/naturally occurring TRAC/TRBC sequences.
  • the TRAC/TRBC may contain modifications.
  • the alpha chain extracellular sequence may include a modification relative to the native/naturally occurring TRAC whereby amino acid T48 of TRAC, with reference to IMGT numbering, is replaced with C48.
  • the beta chain extracellular sequence may include a modification relative to the native/naturally occurring TRBC1 or TRBC2 whereby S57 of TRBC1 or TRBC2, with reference to IMGT numbering, is replaced with C57.
  • These cysteine substitutions relative to the native alpha and beta chain extracellular sequences enable the formation of a non- native interchain disulphide bond which stabilises the refolded soluble TCR, i.e. the TCR formed by refolding extracellular alpha and beta chains (WO 03/020763). This non-native disulphide bond facilitates the display of correctly folded TCRs on phage.
  • TCRs of the invention may be engineered to include mutations. Methods for producing mutated high affinity TCR variants such as phage display and site directed mutagenesis and are known to those in the art (for example see WO 04/044004 and Li et al., Nat Biotechnol 2005 Mar;23(3):349- 54). TCRs of the invention may also be may be labelled with an imaging compound, for example a label that is suitable for diagnostic purposes.
  • Such labelled high affinity TCRs are useful in a method for detecting a TCR ligand selected from CD1 -antigen complexes, bacterial superantigens, and MHC- peptide/superantigen complexes, which method comprises contacting the TCR ligand with a high affinity TCR (or a multimeric high affinity TCR complex) which is specific for the TCR ligand; and detecting binding to the TCR ligand.
  • a high affinity TCR or a multimeric high affinity TCR complex
  • fluorescent streptavidin (commercially available) can be used to provide a detectable label.
  • a fluorescently-labelled multimer is suitable for use in FACS analysis, for example to detect antigen presenting cells carrying the peptide for which the high affinity TCR is specific.
  • a TCR of the present invention may alternatively or additionally be associated with (e.g. covalently or otherwise linked to) a therapeutic agent which may be, for example, a toxic moiety for use in cell killing, or an immunostimulating agent such as an interleukin or a cytokine.
  • a multivalent high affinity TCR complex of the present invention may have enhanced binding capability for a TCR ligand compared to a non-multimeric wild-type or high affinity T cell receptor heterodimer.
  • the multivalent high affinity TCR complexes according to the invention are particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo, and are also useful as intermediates for the production of further multivalent high affinity TCR complexes having such uses.
  • the high affinity TCR or multivalent high affinity TCR complex may therefore be provided in a pharmaceutically acceptable formulation for use in vivo.
  • High affinity TCRs of the invention may be used in the production of soluble bi-specific reagents.
  • a preferred embodiment is a reagent which comprises a soluble TCR, fused via a linker to an anti- CD3 specific antibody fragment. Further details including how to produce such reagents are described in W010/133828.
  • the invention provides nucleic acid encoding the TCR of the invention, a TCR expression vector comprising nucleic acid encoding a TCR of the invention, as well as a cell harbouring such a vector.
  • the TCR may be encoded either in a single open reading frame or two distinct open reading frames.
  • one vector may encode both an alpha and a beta chain of a TCR of the invention.
  • a further aspect of the invention provides a cell displaying on its surface a TCR of the invention.
  • the cell may be a T cell.
  • T cells expressing the TCRs of the invention are suitable for use in adoptive therapy-based treatment of diseases such as cancers.
  • adoptive therapy see for example Rosenberg et al., Nat Rev Cancer. 2008 Apr;8(4):299-308.
  • the TCRs of the invention intended for use in adoptive therapy are generally glycosylated when expressed by the transfected T cells.
  • the glycosylation pattern of transfected TCRs may be modified by mutations of the transfected gene (Kuball J et ai, J Exp Med. 2009 Feb 16;206(2):463-75).
  • the binding moiety of the invention may be an antibody.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced.
  • antibody includes antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanised antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic and any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.
  • a humanised antibody may be a modified antibody having the variable regions of a non-human, e.g. murine, antibody and the constant region of a human antibody. Methods for making humanised antibodies are described in, for example, US Patent No. 5225539. Examples of antibodies are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. A monoclonal antibody may be referred to herein as "mab".
  • an antibody for example a monoclonal antibody
  • recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody.
  • Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementary determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin (see, for instance, EP-A-184187, GB 2188638A or EP-A-239400).
  • CDRs complementary determining regions
  • a hybridoma (or other cell that produces antibodies) may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. et ai, Nature.
  • Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g.
  • antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).
  • bispecific antibodies these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Hollinger & Winter, Curr Opin
  • bispecific antibody fragments e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain "Janusins" described in Traunecker et al., EMBO J. 1991 Dec; 10(12):3655-9). Bispecific diabodies, as opposed to bispecific whole antibodies, may also be useful because they can be readily constructed and expressed in E. coli.
  • Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
  • An "antigen binding domain" is the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope.
  • An antigen binding domain may be provided by one or more antibody variable domains.
  • An antigen binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • the binding moiety may be an antibody-like molecule that has been designed to specifically bind a peptide-MHC complex of the invention.
  • TCR-mimic antibodies such as, for example those described in WO2007143104 and Sergeeva ef al., Blood. 201 1 Apr
  • binding moieties based on engineered protein scaffolds are derived from stable, soluble, natural protein structures which have been modified to provide a binding site for a target molecule of interest.
  • engineered protein scaffolds include, but are not limited to, affibodies, which are based on the Z-domain of staphylococcal protein A that provides a binding interface on two of its a-helices (Nygren, FEBS J.
  • Engineered protein scaffolds are typically targeted to bind the same antigenic proteins as antibodies, and are potential therapeutic agents. They may act as inhibitors or antagonists, or as delivery vehicles to target molecules, such as toxins, to a specific tissue in vivo (Gebauer and Skerra, Curr Opin Chem Biol. 2009 Jun;13(3):245-55). Short peptides may also be used to bind a target protein.
  • Phylomers are natural structured peptides derived from bacterial genomes. Such peptides represent a diverse array of protein structural folds and can be used to inhibit/disrupt protein-protein interactions in vivo (Watt, Nat Biotechnol. 2006 Feb;24(2): 177-83)].
  • the invention further provides a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention for use in medicine.
  • the polypeptide, complex, nucleic acid, vector, cell or binding moiety may be used for in the treatment or prevention of cancer, in particular, head and neck cancer and oesophageal cancer.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention together with a pharmaceutically acceptable carrier.
  • This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient). It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms. Suitable compositions and methods of administration are known to those skilled in the art, for example see, Johnson et a/., Blood.
  • Cells in accordance with the invention will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a
  • T cells transfected with TCRs of the invention may be provided in pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be adapted for administration by any appropriate route such as a parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation or intranasal routes.
  • a parenteral including subcutaneous, intramuscular, or intravenous
  • enteral including oral or rectal
  • inhalation or intranasal routes Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
  • Dosages of the substances of the present invention can vary between wide limits, depending upon the disease or disorder to be treated (such as cancer, viral infection or autoimmune disease), the age and condition of the individual to be treated, etc.
  • a suitable dose range for a reagent comprising a soluble TCR fused to an anti- CD3 domain may be between 25 ng/kg and 50 pg/kg. A physician will ultimately determine appropriate dosages to be used.
  • the polypeptide of the invention may be provided in the form of a vaccine composition.
  • the vaccine composition may be useful for the treatment or prevention of cancer. All such
  • compositions are encompassed in the present invention.
  • vaccines may take several forms (Schlom, J Natl Cancer Inst. 2012 Apr 18;104(8):599-613).
  • the peptide of the invention may be used directly to immunise patients (Salgaller, Cancer Res. 1996 Oct 15;56(20):4749-57 and Marchand, Int J Cancer. 1999 Jan 18;80(2):219-30).
  • the vaccine composition may include additional peptides such that the peptide of the invention is one of a mixture of peptides.
  • Adjuvants may be added to the vaccine composition to augment the immune response
  • the vaccine composition may take the form of an antigen presenting cell displaying the peptide of the invention in complex with MHC.
  • the antigen presenting cell is an immune cell, more preferably a dendritic cell.
  • the peptide may be pulsed onto the surface of the cell (Thurner, J Exp Med. 1999 Dec 6; 190(11 ): 1669-78), or nucleic acid encoding for the peptide of the invention may be introduced into dendritic cells (for example by electroporation. Van Tendeloo, Blood. 2001 Jul 1 ;98(1 ):49-56).
  • the polypeptides, complexes, nucleic acid molecules, vectors, cells and binding moieties of the invention may be non-naturally occurring and/or purified and/or engineered and/or recombinant and/or isolated and/or synthetic.
  • the invention also provides a method of identifying a binding moiety that binds a complex of the invention, the method comprising contacting a candidate binding moiety with the complex and determining whether the candidate binding moiety binds the complex.
  • Methods to determine binding to polypeptide-MHC complexes are well known in the art. Preferred methods include, but are not limited to, surface plasmon resonance, or any other biosensor technique, ELISA, flow cytometry, chromatography, microscopy. Alternatively, or in addition, binding may be determined by functional assays in which a biological response is detected upon binding, for example, cytokine release or cell apoptosis.
  • the candidate binding moiety may be a binding moiety of the type already described, such as a TCR or an antibody.
  • T cells may be isolated from fresh blood obtained from volunteer donors. Such a method involves stimulating T cells using autologous DCs, followed by autologous B cells, pulsed with the polypeptide of the invention. Several round of stimulation may be carried out, for example three or four rounds. Activated T cells may then be tested for specificity by measuring cytokine release in the presence of T2 cells pulsed with the peptide of the invention (for example using an IFNY ELISpot assay). Activated cells may then be sorted by fluorescence-activated cell sorting (FACS) using labelled antibodies to detect intracellular cytokine production (e.g. IFNy), or expression of a cell surface marker (such as CD137).
  • FACS fluorescence-activated cell sorting
  • Sorted cells may be expanded and further validated, for example, by ELISpot assay and/or cytotoxicity against target cells and/or staining by peptide-MHC tetramer.
  • the TCR chains from validated clones may then be amplified by rapid amplification of cDNA ends (RACE) and sequenced.
  • the polypeptide MHC complex of the invention may be used for screening a library of TCRs or antibodies.
  • the production of antibody libraries using phage display is well known in the art, for example see Aitken, Antibody phage display: Methods and Protocols (2009, Humana, New York). Further details on TCR libraries can be found in WO04044004.
  • the polypeptide-MHC complex of the invention is used to screen a library of diverse TCRs displayed on the surface of phage particles.
  • the TCRs displayed by said library may contain non- natural mutations. Screening may involve panning the phage library with peptide-MHC complexes of the invention and subsequently isolating bound phage.
  • peptide-MHC complexes may be attached to a solid support, such as a magnet bead, or column matrix and phage bound peptide MHC complexes isolated, with a magnet, or by chromatography, respectively.
  • the panning steps may be repeated several times for example three or four times.
  • Isolated phage may be further expanded in E. coli cells.
  • Isolated phage particles may be tested for specific binding peptide-MHC complexes of the invention. Binding can be detected using techniques including, but not limited to, ELISA, or SPR for example using a BiaCore instrument.
  • the DNA sequence of the T cell receptor displayed by peptide-MHC binding phage can be further identified by standard PCR methods.
  • Figures C1 to C9 show the respective fragmentation spectra for the peptides of SEQNOs: C1 to C9, eluted from cells. A table highlighting the matching ions is shown below each spectrum.
  • Example C1 Identification of target-derived peptides by Mass spectrometry Presentation of HLA-restricted peptides derived from CNGB1 on the surface of tumour cell lines was investigated using mass spectrometry.
  • Immortalised cell lines obtained from commercial sources were maintained and expanded under standard conditions.
  • Class I HLA complexes were purified by immunoaffinity using commercially available anti-HLA antibodies BB7.1 (anti-HLA-B * 07), BB7.2 (anti-HLA-A * 02) and W6/32 (anti-Class 1 ). Briefly, cells were lysed in buffer containing non-ionic detergent NP-40 (0.5% v/v) at 5 x 10 7 cells per ml and incubated at 4°C for 1 h with agitation/mixing. Cell debris was removed by centrifugation and supernatant pre-cleared using proteinA-Sepharose. Supernatant was passed over 5 ml of resin containing 8 mg of anti-HLA antibody immobilised on a proteinA-Sepharose scaffold.
  • Peptides were separated by high pressure liquid chromatography (HPLC) on a Dionex Ultimate 3000 system using a C18 column (Phenomenex). Peptides were loaded in 98% buffer A (0.1 % aqueous trifluoroacetic acid (TFA)) and 2% buffer B (0.1% TFA in acetonitrile). Peptides were eluted using a stepped gradient of B (2-60%) over 20 min. Fractions were collected at one minute intervals and lyophilised. Peptides were analysed by nanoLCMS/MS using a Dionex Ultimate 3000 nanoLC coupled to either AB Sciex Triple TOF 5600 or Thermo Orbitrap Fusion mass spectrometers.
  • HPLC high pressure liquid chromatography
  • IDA information dependent acquisition
  • Information acquired in these experiments was used to search the Uniprot database of human proteins for peptides consistent with the fragmentation patterns seen, using Protein pilot software (Ab Sciex) and PEAKS software (Bioinformatics solutions). Peptides identified are assigned a score by the software, based on the match between the observed and expected fragmentation patterns.
  • Figures C1 -C9 show representative fragmentation patterns for the peptides of SEQ NOs: C1-C9 respectively. A table highlighting the matching ions is shown below each spectrum.
  • HLA-A * 02 molecules (HLA-A * 02-heavy chain and HLA light-chain ( ⁇ 2 ⁇ )) were expressed separately in E. coli as inclusion bodies, using appropriate constructs.
  • HLA-A * 02-heavy chain additionally contained a C-terminal biotinylation tag which replaces the transmembrane and cytoplasmic domains (O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15).
  • E. coli cells were lysed and inclusion bodies processed to approximately 80% purity.
  • Inclusion bodies of ⁇ 2 ⁇ and heavy chain were denatured separately in 6 M guanidine-HCI, 50 mM Tris pH 8.1 , 100 mM NaCI, 10 mM DTT, 10 mM EDTA.
  • Refolding buffer was prepared containing 0.4 M L-Arginine, 100 mM Tris pH 8.1 , 3.7 mM cystamine dihydrochloride, 6.6 mM cysteamine hydrochloride and chilled to ⁇ 5°C.
  • Synthetic peptide dissolved in DMSO to a final concentration of 4mg/ml is added to the refold buffer at 4 mg/litre (final concentration). Then 30 mg/litre ⁇ 2 ⁇ followed by 30 mg/litre heavy chain (final concentrations) are added. Refolding was allowed to reach completion at 4 °C for at least 1 hour.
  • Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Two changes of buffer were necessary to reduce the ionic strength of the solution sufficiently.
  • the protein solution was then filtered through a 1.5 ⁇ cellulose acetate filter and loaded onto a POROS 50HQ anion exchange column (8 ml bed volume). Protein was eluted with a linear 0-500 mM NaCI gradient in 10 mM Tris pH 8.1 using an Akta purifier (GE Healthcare). HLA-A * 02-peptide complex eluted at approximately 250 mM NaCI, and peak fractions were collected, a cocktail of protease inhibitors (Calbiochem) was added and the fractions were chilled on ice.
  • Biotinylation tagged pHLA molecules were buffer exchanged into 10 mM Tris pH 8.1 , 5 mM NaCI using a GE Healthcare fast desalting column equilibrated in the same buffer. Immediately upon elution, the protein-containing fractions were chilled on ice and protease inhibitor cocktail (Calbiochem) was added. Biotinylation reagents were then added: 1 mM biotin, 5 mM ATP
  • Biotinylated pHLA-A * 02 molecules eluted as a single peak at approximately 15 ml. Fractions containing protein were pooled, chilled on ice, and protease inhibitor cocktail was added. Protein concentration was determined using a Coomassie-binding assay (PerBio) and aliquots of biotinylated pHLA-A * 02 molecules were stored frozen at -20 °C.
  • Such peptide-MHC complexes may be used in soluble form or may be immobilised through C terminal biotin moiety on to a solid support, to be used for the detection of T cells and T cell receptors which bind said complex.
  • TCR isolation methods are known to those skilled in the art; for example, DNA sequences corresponding to TCR alpha and beta chains may be amplified from a T cell clone (obtained from volunteer blood donors) that is activated by cells presenting the peptide-HLA complex of interest.
  • T cell clone obtained from volunteer blood donors
  • Other suitable methods include isolation from TCR libraries (as described in WO2005/1 16646 and WO2005/116074 for example).
  • TCR alpha and beta chain sequences are expressed in E. coli as soluble TCRs, (WO2003020763; Boulter, et al., Protein Eng, 2003. 16: 707-711 ). Binding of the soluble TCRs to the complexes is analysed by surface plasmon resonance using a BiaCore 3000 instrument. Biotinylated peptide-HLA monomers are prepared as previously described (Example C2) and immobilized on to a streptavidin-coupled CM-5 sensor chip.
  • The[LS3] present invention also relates to novel peptides derived from Contactin-associated proteinlike 2 (CNTNAP2), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules.
  • CNTNAP2 Contactin-associated proteinlike 2
  • binding moieties that bind to the peptides and/or complexes of the invention. Such moieties are useful for the development of immunotherapeutic reagents for the treatment of diseases such as cancer.
  • T cells are a key part of the cellular arm of the immune system. They specifically recognise peptide fragments that are derived from intracellular proteins and presented in complex with Major Histocompatibility Complex (MHC) molecules on the surface of antigen presenting cells (APCs).
  • MHC Major Histocompatibility Complex
  • MHC molecules are known as human leukocyte antigens (HLA), and both terms are used synonymously herein.
  • HLA human leukocyte antigens
  • MHC molecules have a binding groove in which the peptide fragments bind. Recognition of particular peptide-MHC antigens is mediated by a corresponding T cell receptor (TCR).
  • TCR T cell receptor
  • Tumour cells express various tumour associated antigens (TAA) and peptides derived from these antigens are frequently displayed on the tumour cell surface. Detection of a MHC class I- presented TAA-derived peptide by a CD8+ T cell bearing the corresponding T cell receptor, leads to targeted killing of the tumour cell.
  • TAA tumour associated antigens
  • tumour cells often escape detection.
  • TAA-derived peptides presented by MHC molecules on tumour cells enables the development of novel immunotherapeutic reagents designed to specifically target and destroy said tumour cells.
  • Such reagents may be moieties that bind to the TAA-derived peptide and/or peptide-MHC complexes of the invention, and typically involve the induction of a T cell response.
  • such reagents may be based, exclusively, or in part, on T cells, or T cell receptors (TCRs), or antibodies.
  • TAA-derived peptides that are presented on the cell surface in complex with a given MHC, only a small number of such peptides, covering a limited number of cancer indications, have been identified to date.
  • TAAs suitable for the development of immunotherapeutic reagents must have limited or no expression in normal cells, to prevent off target activity. It is therefore desirable to provide additional tumour-specific TAA-derived peptides and MHC complexes thereof that can be used for the development of new cancer therapies.
  • CNTNAP2 also known as contactin-associated protein-like 2 or cell recognition molecule Caspr2 and having Uniprot accession number Q9UHC6
  • CNTNAP2 is reported to play a role in cancer (Parris et al. BMC Cancer. 2014 May 7;14:324; Bralten et al. Oncogene. 2010 Nov 18;29(46):6138-48)
  • CNTNAP2 is an ideal target for immunotherapeutic applications).
  • the inventors have found novel peptides derived from CNTNAP2 that are presented on the cell surface in complex with MHC. These peptides are particularly useful for the development of reagents that can targets cells expressing CNTNAP2 and for the treatment of cancers, including breast cancer, non small cell lung cancer (squamous) and prostate cancer.
  • the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NO: D1 , or
  • polypeptides of the invention are presented by MHC on the surface of tumour cells. Accordingly, the polypeptides of the invention, as well as moieties that bind the polypeptide-MHC complexes, can be used to develop therapeutic reagents.
  • the skilled person can determine whether or not a given polypeptide forms a complex with an MHC molecule by determining whether the MHC can be refolded in the presence of the polypeptide using the process set out in Example D2. If the polypeptide does not form a complex with MHC then MHC will not refold. Refolding is commonly confirmed using an antibody that recognises MHC in a folded state only. Further details can be found in Garboczi et al., Proc Natl Acad Sci
  • polypeptides of the invention are from about 8 to about 16 amino acids in length, and are most preferably 8, 9, or 10 or 11 amino acids in length.
  • the polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NO: D1.
  • the amino acid residues comprising the polypeptides of the invention may be chemically modified. Examples of chemical modifications include those corresponding to post translational modifications for example phosphorylation, acetylation and deamidation (Engelhard et al., Curr Opin Immunol. 2006 Feb;18(1 ):92-7).
  • Amino acid substitution means that an amino acid residue is substituted for a replacement amino acid residue at the same position.
  • Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are immediately adjacent one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent another inserted amino acid residue.
  • One, two or three amino acids may be deleted from the sequence of SEQ NO: D1. Each deletion can take place at any position of SEQ NO: D1.
  • the polypeptide of the invention may comprise one, two or three additional amino acids at the C-terminal end and/or at the N-terminal end of the sequence of SEQ NO: D1.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQ NO: D1 with the exception of one amino acid substitution and one amino acid insertion, one amino acid substitution and one amino acid deletion, or one amino acid insertion and one amino acid deletion.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQ NO: D1 , with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.
  • Inserted amino acids and replacement amino acids may be naturally occurring amino acids or may be non-naturally occurring amino acids and, for example, may contain a non-natural side chain.
  • Such altered peptide ligands are discussed further in Douat-Casassus et al., J. Med. Chem, 2007 Apr 5;50(7): 1598-609 and Hoppes et al., J. Immunol 2014 Nov 15;193(10):4803-13 and references therein). If more than one amino acid residue is substituted and/or inserted, the
  • replacement inserted amino acid residues may be the same as each other or different from one another. Each replacement amino acid may have a different side chain to the amino acid being replaced.
  • polypeptides of the invention bind to MHC in the peptide binding groove of the MHC molecule.
  • the amino acid modifications described above will not impair the ability of the peptide to bind MHC.
  • the amino acid modifications improve the ability of the peptide to bind MHC.
  • mutations may be made at positions which anchor the peptide to MHC. Such anchor positions and the preferred residues at these locations are known in the art, particularly for peptides which bind HLA-A * 02 (see, e. g. Parkhurst et al., J. Immunol. 1996 Sep 15;157(6):2539-48 and Parker et al. J Immunol. 1992 Dec 1 ; 149(1 1 ):3580-7).
  • a polypeptide of the invention may be used to illicit an immune response. If this is the case, it is important that the immune response is specific to the intended target in order to avoid the risk of unwanted side effects that may be associated with an "off target" immune response. Therefore, it is preferred that the amino acid sequence of a polypeptide of the invention does not match the amino acid sequence of a peptide from any other protein(s), in particular, that of another human protein.
  • a person of skill in the art would understand how to search a database of known protein sequences to ascertain whether a polypeptide according to the invention is present in another protein.
  • Polypeptides of the invention can be synthesised easily by Merrifield synthesis, also known as solid phase synthesis, or any other peptide synthesis methodology.
  • GMP grade polypeptide is produced by solid-phase synthesis techniques by Multiple Peptide Systems, San Diego, CA.
  • the peptide may be recombinantly produced, if so desired, in accordance with methods known in the art.
  • Such methods typically involve the use of a vector comprising a nucleic acid sequence encoding the polypeptide to be expressed, to express the polypeptide in vivo; for example, in bacteria, yeast, insect or mammalian cells.
  • in vitro cell-free systems may be used. Such systems are known in the art and are commercially available for example from Life
  • polypeptides may be isolated and/or may be provided in substantially pure form. For example, they may be provided in a form which is substantially free of other polypeptides or proteins.
  • the invention provides a complex of the polypeptide of the first aspect and an MHC molecule.
  • the polypeptide is bound to the peptide binding groove of the MHC molecule.
  • the MHC molecule may be MHC class I.
  • the MHC class I molecule may be selected from HLA-A * 02, HLA-A * 01 , HLA-A * 03, HLA-A11 , HLA-A23, HLA-A24, HLA-B * 07, HLA-B*08, HLA- B40, HLA-B44, HLA-B15, HLA-C * 04, HLA * C * 03 HLA-CW.
  • HLA-A * 02, HLA-A * 01 , HLA-A * 03, HLA-A11 , HLA-A23, HLA-A24, HLA-B * 07, HLA-B*08, HLA- B40, HLA-B44, HLA-B15, HLA-C * 04, HLA * C * 03 HLA-CW As is known to those skilled in the art there are allelic variants of the above HLA types, all of which are encompassed by the present invention.
  • the complex of the invention may be isolated and/or in a substantially pure form.
  • the complex may be provided in a form which is substantially free of other polypeptides or proteins.
  • the term "MHC molecule" includes recombinant MHC molecules, non-naturally occurring MHC molecules and functionally equivalent fragments of MHC, including derivatives or variants thereof, provided that peptide binding is retained.
  • MHC molecules may be fused to a therapeutic moiety, attached to a solid support, in soluble form, and/or in multimeric form.
  • KLH keyhole limpet hemocyanin
  • the polypeptides and/or polypeptide-MHC complexes of the invention may be associated with a fusion partner. Fusion partners may be used for detection purposes, or for attaching said polypeptide or MHC to a solid support, or for MHC oligomerisation.
  • the MHC complexes may incorporate a biotinylation site to which biotin can be added, for example, using the BirA enzyme (O'Callaghan et al., 1999 Jan 1 ;266(1 ):9-15).
  • Other suitable fusion partners include, but are not limited to, fluorescent, or luminescent labels, radiolabels, nucleic acid probes and contrast reagents, antibodies, or enzymes that produce a detectable product. Detection methods may include flow cytometry, microscopy, electrophoresis or scintillation counting.
  • Polypeptide-MHC complexes of the invention may be provided in soluble form, or may be immobilised by attachment to a suitable solid support.
  • suitable solid supports include, but are not limited to, a bead, a membrane, sepharose, a magnetic bead, a plate, a tube, a column.
  • Polypeptide-MHC complexes may be attached to an ELISA plate, a magnetic bead, or a surface plasmon reasonance biosensor chip.
  • Methods of attaching peptide-MHC complexes to a solid support are known to the skilled person, and include, for example, using an affinity binding pair, e.g. biotin and streptavidin, or antibodies and antigens.
  • peptide-MHC complexes are labelled with biotin and attached to streptavidin-coated surfaces.
  • Polypeptide-MHC complexes of the invention may be in multimeric form, for example, dimeric, or tetrameric, or pentameric, or octomeric, or greater. Examples of suitable methods for the production of multimeric peptide MHC complexes are described in Greten et al., Clin. Diagn. Lab. Immunol. 2002 Mar;9(2):216-20 and references therein.
  • polypeptide-MHC multimers may be produced using peptide-MHC tagged with a biotin residue and complexed through fluorescent labelled streptavidin.
  • multimeric polypeptide-MHC complexes may be formed by using immunoglobulin as a molecular scaffold.
  • MHC molecules are fused with the constant region of an immunoglobulin heavy chain separated by a short amino acid linker.
  • Polypeptide-MHC multimers have also been produced using carrier molecules such as dextran (WO02072631 ). Multimeric peptide MHC complexes can be useful for improving the detection of binding moieties, such as T cell receptors, which bind said complex, because of avidity effects.
  • the polypeptides of the invention may be presented on the surface of a cell in complex with MHC.
  • the invention also provides a cell presenting on its surface a complex of the invention.
  • a cell may be a mammalian cell, preferably a cell of the immune system, and in particular a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • Other preferred cells include T2 cells (Hosken, et al., Science. 1990 Apr 20;248(4953):367-70).
  • Cells presenting the polypeptide or complex of the invention may be isolated, preferably in the form of a population, or provided in a substantially pure form.
  • Said cells may not naturally present the complex of the invention, or alternatively said cells may present the complex at a level higher than they would in nature.
  • Such cells may be obtained by pulsing said cells with the polypeptide of the invention. Pulsing involves incubating the cells with the polypeptide for several hours using polypeptide concentrations typically ranging from 10 "5 to 10 "12 M. Said cells may additionally be transduced with HLA-A * 02 molecules to further induce presentation of the peptide. Cells may be produced recombinantly. Cells presenting polypeptides of the invention may be used to isolate T cells and T cell receptors (TCRs) which are activated by, or bind to, said cells, as described in more detail below.
  • TCRs T cell receptors
  • the invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide of the first aspect of the invention.
  • the nucleic acid may be cDNA.
  • the nucleic acid molecule may consist essentially of a nucleic acid sequence encoding the polypeptide of the first aspect of the invention or may encode only the peptide of the invention, i.e. encode no other polypeptide.
  • Such a nucleic acid molecule can be synthesised in accordance with methods known in the art. Due to the degeneracy of the genetic code, one of ordinary skill in the art will appreciate that nucleic acid molecules of different nucleotide sequence can encode the same amino acid sequence.
  • the invention provides a vector comprising a nucleic acid sequence according to the third aspect of the invention.
  • the vector may include, in addition to a nucleic acid sequence encoding only a polypeptide of the invention, one or more additional nucleic acid sequences encoding one or more additional polypeptides. Such additional polypeptides may, once expressed, be fused to the N-terminus or the C-terminus of the polypeptide of the invention.
  • the vector includes a nucleic acid sequence encoding a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • Suitable vectors are known in the art as is vector construction, including the selection of promoters and other regulatory elements, such as enhancer elements.
  • the vector utilised in the context of the present invention desirably comprises sequences appropriate for introduction into cells.
  • the vector may be an expression vector, a vector in which the coding sequence of the polypeptide is under the control of its own cis-acting regulatory elements, a vector designed to facilitate gene integration or gene replacement in host cells, and the like.
  • the term "vector” encompasses a DNA molecule, such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • a DNA molecule such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • Appropriate phage and viral vectors include, but are not limited to, lambda (X) bacteriophage, EMBL bacteriophage, simian virus 40, bovine papilloma virus, Epstein-Barr virus, adenovirus, herpes virus, vaccinia virus, Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, lentivirus and Rous sarcoma virus.
  • the invention provides a cell comprising the vector of the fourth aspect of the invention.
  • the cell may be an antigen presenting cell and is preferably a cell of the immune system.
  • the cell may be a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • the cell may be a mammalian cell.
  • Polypeptides and complexes of the invention can be used to identify and/or isolate binding moieties that bind specifically to the polypeptide and/or the complex of the invention.
  • binding moieties may be used as immunotherapeutic reagents and may include antibodies and TCRs.
  • the invention provides a binding moiety that binds the polypeptide of the invention.
  • the binding moiety binds the polypeptide when said polypeptide is in complex with MHC.
  • the binding moiety may bind partially to the MHC, provided that it also binds to the polypeptide.
  • the binding moiety may bind only the polypeptide, and that binding may be specific.
  • the binding moiety may bind only the peptide MHC complex and that binding may be specific.
  • binding moieties that bind the complex of the invention When used with reference to binding moieties that bind the complex of the invention, "specific" is generally used herein to refer to the situation in which the binding moiety does not show any significant binding to one or more alternative polypeptide-MHC complexes other than the polypeptide-MHC complex of the invention.
  • the binding moiety may be a T cell receptor (TCR).
  • TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature, and links to the IMGT public database of TCR sequences. The unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the TCR field. For example, they can be found in the "T cell Receptor Factsbook", (2001 ) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8;
  • the TCRs of the invention may be in any format known to those in the art.
  • the TCRs may be ⁇ heterodimers, or they may be in single chain format (such as those described in W09918129).
  • Single chain TCRs include ⁇ TCR polypeptides of the type: Va-L- ⁇ , ⁇ -L-Va, Va- Ca-L- ⁇ , ⁇ - ⁇ - ⁇ ⁇ -0 ⁇ or Va- Ca - ⁇ / ⁇ - ⁇ , optionally in the reverse orientation, wherein Va and ⁇ are TCR a and ⁇ variable regions respectively, Ca and C are TCR a and ⁇ constant regions respectively, and L is a linker sequence.
  • the TCR may be in a soluble form (i.e.
  • the TCR may be provided on the surface of a cell, such as a T cell.
  • the alpha and/or beta chain constant domain may be truncated relative to the native/naturally occurring TRAC/TRBC sequences.
  • the TRAC/TRBC may contain modifications.
  • the alpha chain extracellular sequence may include a modification relative to the native/naturally occurring TRAC whereby amino acid T48 of TRAC, with reference to IMGT numbering, is replaced with C48.
  • the beta chain extracellular sequence may include a modification relative to the native/naturally occurring TRBC1 or TRBC2 whereby S57 of TRBC1 or TRBC2, with reference to IMGT numbering, is replaced with C57.
  • These cysteine substitutions relative to the native alpha and beta chain extracellular sequences enable the formation of a non- native interchain disulphide bond which stabilises the refolded soluble TCR, i.e. the TCR formed by refolding extracellular alpha and beta chains (WO 03/020763). This non-native disulphide bond facilitates the display of correctly folded TCRs on phage.
  • TCRs of the invention may be engineered to include mutations. Methods for producing mutated high affinity TCR variants such as phage display and site directed mutagenesis and are known to those in the art (for example see WO 04/044004 and Li et al., Nat Biotechnol 2005 Mar;23(3):349- 54). TCRs of the invention may also be may be labelled with an imaging compound, for example a label that is suitable for diagnostic purposes.
  • Such labelled high affinity TCRs are useful in a method for detecting a TCR ligand selected from CD1 -antigen complexes, bacterial superantigens, and MHC- peptide/superantigen complexes, which method comprises contacting the TCR ligand with a high affinity TCR (or a multimeric high affinity TCR complex) which is specific for the TCR ligand; and detecting binding to the TCR ligand.
  • a high affinity TCR or a multimeric high affinity TCR complex
  • fluorescent streptavidin (commercially available) can be used to provide a detectable label.
  • a fluorescently-labelled multimer is suitable for use in FACS analysis, for example to detect antigen presenting cells carrying the peptide for which the high affinity TCR is specific.
  • a TCR of the present invention may alternatively or additionally be associated with (e.g. covalently or otherwise linked to) a therapeutic agent which may be, for example, a toxic moiety for use in cell killing, or an immunostimulating agent such as an interleukin or a cytokine.
  • a multivalent high affinity TCR complex of the present invention may have enhanced binding capability for a TCR ligand compared to a non-multimeric wild-type or high affinity T cell receptor heterodimer.
  • the multivalent high affinity TCR complexes according to the invention are particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo, and are also useful as intermediates for the production of further multivalent high affinity TCR complexes having such uses.
  • the high affinity TCR or multivalent high affinity TCR complex may therefore be provided in a pharmaceutically acceptable formulation for use in vivo.
  • High affinity TCRs of the invention may be used in the production of soluble bi-specific reagents.
  • a preferred embodiment is a reagent which comprises a soluble TCR, fused via a linker to an anti- CD3 specific antibody fragment. Further details including how to produce such reagents are described in W010/133828.
  • the invention provides nucleic acid encoding the TCR of the invention, a TCR expression vector comprising nucleic acid encoding a TCR of the invention, as well as a cell harbouring such a vector.
  • the TCR may be encoded either in a single open reading frame or two distinct open reading frames.
  • one vector may encode both an alpha and a beta chain of a TCR of the invention.
  • a further aspect of the invention provides a cell displaying on its surface a TCR of the invention.
  • the cell may be a T cell.
  • T cells expressing the TCRs of the invention are suitable for use in adoptive therapy-based treatment of diseases such as cancers.
  • adoptive therapy see for example Rosenberg et al., Nat Rev Cancer. 2008 Apr;8(4):299-308.
  • the TCRs of the invention intended for use in adoptive therapy are generally glycosylated when expressed by the transfected T cells.
  • the glycosylation pattern of transfected TCRs may be modified by mutations of the transfected gene (Kuball J et ai, J Exp Med. 2009 Feb 16;206(2):463-75).
  • the binding moiety of the invention may be an antibody.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced.
  • antibody includes antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanised antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic and any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.
  • a humanised antibody may be a modified antibody having the variable regions of a non-human, e.g. murine, antibody and the constant region of a human antibody. Methods for making humanised antibodies are described in, for example, US Patent No. 5225539. Examples of antibodies are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. A monoclonal antibody may be referred to herein as "mab".
  • an antibody for example a monoclonal antibody
  • recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody.
  • Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementary determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin (see, for instance, EP-A-184187, GB 2188638A or EP-A-239400).
  • CDRs complementary determining regions
  • a hybridoma (or other cell that produces antibodies) may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. et ai, Nature.
  • Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g.
  • antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).
  • bispecific antibodies these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Hollinger & Winter, Curr Opin
  • bispecific antibody fragments e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain "Janusins" described in Traunecker et al., EMBO J. 1991 Dec; 10(12):3655-9). Bispecific diabodies, as opposed to bispecific whole antibodies, may also be useful because they can be readily constructed and expressed in E. coli.
  • Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
  • An "antigen binding domain" is the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope.
  • An antigen binding domain may be provided by one or more antibody variable domains.
  • An antigen binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • the binding moiety may be an antibody-like molecule that has been designed to specifically bind a peptide-MHC complex of the invention.
  • TCR-mimic antibodies such as, for example those described in WO2007143104 and Sergeeva ef al., Blood. 201 1 Apr
  • binding moieties based on engineered protein scaffolds are derived from stable, soluble, natural protein structures which have been modified to provide a binding site for a target molecule of interest.
  • engineered protein scaffolds include, but are not limited to, affibodies, which are based on the Z-domain of staphylococcal protein A that provides a binding interface on two of its a-helices (Nygren, FEBS J.
  • Engineered protein scaffolds are typically targeted to bind the same antigenic proteins as antibodies, and are potential therapeutic agents. They may act as inhibitors or antagonists, or as delivery vehicles to target molecules, such as toxins, to a specific tissue in vivo (Gebauer and Skerra, Curr Opin Chem Biol. 2009 Jun;13(3):245-55). Short peptides may also be used to bind a target protein.
  • Phylomers are natural structured peptides derived from bacterial genomes. Such peptides represent a diverse array of protein structural folds and can be used to inhibit/disrupt protein-protein interactions in vivo (Watt, Nat Biotechnol. 2006 Feb;24(2): 177-83)].
  • the invention further provides a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention for use in medicine.
  • the polypeptide, complex, nucleic acid, vector, cell or binding moiety may be used for in the treatment or prevention of cancer, in particular, breast cancer, non-small cell lung cancer (squamous) and head and neck cancer.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention together with a pharmaceutically acceptable carrier.
  • This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient). It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms. Suitable compositions and methods of administration are known to those skilled in the art, for example see, Johnson et a/., Blood.
  • Cells in accordance with the invention will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a
  • T cells transfected with TCRs of the invention may be provided in pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be adapted for administration by any appropriate route such as a parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation or intranasal routes.
  • a parenteral including subcutaneous, intramuscular, or intravenous
  • enteral including oral or rectal
  • inhalation or intranasal routes Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
  • Dosages of the substances of the present invention can vary between wide limits, depending upon the disease or disorder to be treated (such as cancer, viral infection or autoimmune disease), the age and condition of the individual to be treated, etc.
  • a suitable dose range for a reagent comprising a soluble TCR fused to an anti- CD3 domain may be between 25 ng/kg and 50 pg/kg. A physician will ultimately determine appropriate dosages to be used.
  • the polypeptide of the invention may be provided in the form of a vaccine composition.
  • the vaccine composition may be useful for the treatment or prevention of cancer. All such
  • compositions are encompassed in the present invention.
  • vaccines may take several forms (Schlom, J Natl Cancer Inst. 2012 Apr 18;104(8):599-613).
  • the peptide of the invention may be used directly to immunise patients (Salgaller, Cancer Res. 1996 Oct 15;56(20):4749-57 and Marchand, Int J Cancer. 1999 Jan 18;80(2):219-30).
  • the vaccine composition may include additional peptides such that the peptide of the invention is one of a mixture of peptides.
  • Adjuvants may be added to the vaccine composition to augment the immune response
  • the vaccine composition may take the form of an antigen presenting cell displaying the peptide of the invention in complex with MHC.
  • the antigen presenting cell is an immune cell, more preferably a dendritic cell.
  • the peptide may be pulsed onto the surface of the cell (Thurner, J Exp Med. 1999 Dec 6; 190(11 ): 1669-78), or nucleic acid encoding for the peptide of the invention may be introduced into dendritic cells (for example by electroporation. Van Tendeloo, Blood. 2001 Jul 1 ;98(1 ):49-56).
  • the polypeptides, complexes, nucleic acid molecules, vectors, cells and binding moieties of the invention may be non-naturally occurring and/or purified and/or engineered and/or recombinant and/or isolated and/or synthetic.
  • the invention also provides a method of identifying a binding moiety that binds a complex of the invention, the method comprising contacting a candidate binding moiety with the complex and determining whether the candidate binding moiety binds the complex.
  • Methods to determine binding to polypeptide-MHC complexes are well known in the art. Preferred methods include, but are not limited to, surface plasmon resonance, or any other biosensor technique, ELISA, flow cytometry, chromatography, microscopy. Alternatively, or in addition, binding may be determined by functional assays in which a biological response is detected upon binding, for example, cytokine release or cell apoptosis.
  • the candidate binding moiety may be a binding moiety of the type already described, such as a TCR or an antibody.
  • T cells may be isolated from fresh blood obtained from volunteer donors. Such a method involves stimulating T cells using autologous DCs, followed by autologous B cells, pulsed with the polypeptide of the invention. Several round of stimulation may be carried out, for example three or four rounds. Activated T cells may then be tested for specificity by measuring cytokine release in the presence of T2 cells pulsed with the peptide of the invention (for example using an IFNY ELISpot assay). Activated cells may then be sorted by fluorescence-activated cell sorting (FACS) using labelled antibodies to detect intracellular cytokine production (e.g. IFNy), or expression of a cell surface marker (such as CD137).
  • FACS fluorescence-activated cell sorting
  • Sorted cells may be expanded and further validated, for example, by ELISpot assay and/or cytotoxicity against target cells and/or staining by peptide-MHC tetramer.
  • the TCR chains from validated clones may then be amplified by rapid amplification of cDNA ends (RACE) and sequenced.
  • the polypeptide MHC complex of the invention may be used for screening a library of TCRs or antibodies.
  • the production of antibody libraries using phage display is well known in the art, for example see Aitken, Antibody phage display: Methods and Protocols (2009, Humana, New York). Further details on TCR libraries can be found in WO04044004.
  • the polypeptide-MHC complex of the invention is used to screen a library of diverse TCRs displayed on the surface of phage particles.
  • the TCRs displayed by said library may contain non- natural mutations. Screening may involve panning the phage library with peptide-MHC complexes of the invention and subsequently isolating bound phage.
  • peptide-MHC complexes may be attached to a solid support, such as a magnet bead, or column matrix and phage bound peptide MHC complexes isolated, with a magnet, or by chromatography, respectively.
  • the panning steps may be repeated several times for example three or four times.
  • Isolated phage may be further expanded in E. coli cells.
  • Isolated phage particles may be tested for specific binding peptide-MHC complexes of the invention. Binding can be detected using techniques including, but not limited to, ELISA, or SPR for example using a BiaCore instrument.
  • the DNA sequence of the T cell receptor displayed by peptide-MHC binding phage can be further identified by standard PCR methods.
  • Figure D1 shows the fragmentation spectra for the peptide of SEQ NO: D1 , eluted from cells. A table highlighting the matching ions is shown below the spectrum.
  • Example D1 Identification of target-derived peptides by Mass spectrometry Presentation of HLA-restricted peptides derived from CNTNAP2 on the surface of tumour cell lines was investigated using mass spectrometry.
  • Immortalised cell lines obtained from commercial sources were maintained and expanded under standard conditions.
  • Class I HLA complexes were purified by immunoaffinity using commercially available anti-HLA antibodies BB7.1 (anti-HLA-B * 07), BB7.2 (anti-HLA-A * 02) and W6/32 (anti-Class 1 ). Briefly, cells were lysed in buffer containing non-ionic detergent NP-40 (0.5% v/v) at 5 x 10 7 cells per ml and incubated at 4°C for 1 h with agitation/mixing. Cell debris was removed by centrifugation and supernatant pre-cleared using proteinA-Sepharose. Supernatant was passed over 5 ml of resin containing 8 mg of anti-HLA antibody immobilised on a proteinA-Sepharose scaffold.
  • Peptides were separated by high pressure liquid chromatography (HPLC) on a Dionex Ultimate 3000 system using a C18 column (Phenomenex). Peptides were loaded in 98% buffer A (0.1 % aqueous trifluoroacetic acid (TFA)) and 2% buffer B (0.1% TFA in acetonitrile). Peptides were eluted using a stepped gradient of B (2-60%) over 20 min. Fractions were collected at one minute intervals and lyophilised. Peptides were analysed by nanoLCMS/MS using a Dionex Ultimate 3000 nanoLC coupled to either AB Sciex Triple TOF 5600 or Thermo Orbitrap Fusion mass spectrometers.
  • HPLC high pressure liquid chromatography
  • IDA information dependent acquisition
  • Information acquired in these experiments was used to search the Uniprot database of human proteins for peptides consistent with the fragmentation patterns seen, using Protein pilot software (Ab Sciex) and PEAKS software (Bioinformatics solutions). Peptides identified are assigned a score by the software, based on the match between the observed and expected fragmentation patterns.
  • results The polypeptide set out in table D1 , corresponding to SEQ NO: D1 , was detected by mass spec following extraction from cancer cell lines.
  • An example cell line from which the peptide was detected is indicated in the table along with the HLA antibody used for immunoaffinity purification.
  • Figure D1 shows representative fragmentation patterns for the peptide of SEQ NO: D1.
  • a table highlighting the matching ions is shown below the spectrum.
  • HLA-A*02 molecules HLA-A*02-heavy chain and HLA light-chain ( ⁇ 2 ⁇ ) were expressed separately in E. coli as inclusion bodies, using appropriate constructs.
  • HLA-A * 02-heavy chain additionally contained a C-terminal biotinylation tag which replaces the transmembrane and cytoplasmic domains (O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15).
  • E. coli cells were lysed and inclusion bodies processed to approximately 80% purity.
  • Inclusion bodies of ⁇ 2 ⁇ and heavy chain were denatured separately in 6 M guanidine-HCI, 50 mM Tris pH 8.1 , 100 mM NaCI, 10 mM DTT, 10 mM EDTA.
  • Refolding buffer was prepared containing 0.4 M L-Arginine, 100 mM Tris pH 8.1 , 3.7 mM cystamine dihydrochloride, 6.6 mM cysteamine hydrochloride and chilled to ⁇ 5°C.
  • Synthetic peptide dissolved in DMSO to a final concentration of 4mg/ml is added to the refold buffer at 4 mg/litre (final concentration). Then 30 mg/litre ⁇ 2 ⁇ followed by 30 mg/litre heavy chain (final concentrations) are added. Refolding was allowed to reach completion at 4 °C for at least 1 hour.
  • Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Two changes of buffer were necessary to reduce the ionic strength of the solution sufficiently.
  • the protein solution was then filtered through a 1.5 ⁇ cellulose acetate filter and loaded onto a POROS 50HQ anion exchange column (8 ml bed volume). Protein was eluted with a linear 0-500 mM NaCI gradient in 10 mM Tris pH 8.1 using an Akta purifier (GE Healthcare). HLA-A * 02-peptide complex eluted at approximately 250 mM NaCI, and peak fractions were collected, a cocktail of protease inhibitors (Calbiochem) was added and the fractions were chilled on ice.
  • Biotinylation tagged pHLA molecules were buffer exchanged into 10 mM Tris pH 8.1 , 5 mM NaCI using a GE Healthcare fast desalting column equilibrated in the same buffer. Immediately upon elution, the protein-containing fractions were chilled on ice and protease inhibitor cocktail
  • Biotinylation reagents were then added: 1 mM biotin, 5 mM ATP (buffered to pH 8), 7.5 mM MgCI2, and 5 pg/ml BirA enzyme (purified according to O'Callaghan et al., (1999) Anal. Biochem. 266: 9-15). The mixture was then allowed to incubate at room temperature overnight.
  • the biotinylated pHLA-A * 02 molecules were purified using gel filtration chromatography.
  • a GE Healthcare Superdex 75 HR 10/30 column was pre-equilibrated with filtered PBS and 1 ml of the biotinylation reaction mixture was loaded and the column was developed with PBS at 0.5 ml/min using an Akta purifier (GE Healthcare).
  • Biotinylated pHLA-A * 02 molecules eluted as a single peak at approximately 15 ml. Fractions containing protein were pooled, chilled on ice, and protease inhibitor cocktail was added.
  • Protein concentration was determined using a Coomassie-binding assay (PerBio) and aliquots of biotinylated pHLA-A * 02 molecules were stored frozen at -20 °C.
  • Such peptide-MHC complexes may be used in soluble form or may be immobilised through C terminal biotin moiety on to a solid support, to be used for the detection of T cells and T cell receptors which bind said complex.
  • Example D3 identification of TCRs that bind to a peptide-MHC complex of the invention
  • TCR isolation methods are known to those skilled in the art; for example, DNA sequences corresponding to TCR alpha and beta chains may be amplified from a T cell clone (obtained from volunteer blood donors) that is activated by cells presenting the peptide-HLA complex of interest.
  • T cell clone obtained from volunteer blood donors
  • Other suitable methods include isolation from TCR libraries (as described in WO2005/1 16646 and WO2005/116074 for example).
  • TCR alpha and beta chain sequences are expressed in E. coli as soluble TCRs, (WO2003020763; Boulter, et al., Protein Eng, 2003. 16: 707-711 ). Binding of the soluble TCRs to the complexes is analysed by surface plasmon resonance using a BiaCore 3000 instrument. Biotinylated peptide-HLA monomers are prepared as previously described (Example D2) and immobilized on to a streptavidin-coupled CM-5 sensor chip.
  • The[LS4] present invention also relates to novel peptides derived from Cancer/testis antigen family 45 member A3 (CT45A3), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules.
  • C45A3 Cancer/testis antigen family 45 member A3
  • binding moieties that bind to the peptides and/or complexes of the invention. Such moieties are useful for the development of immunotherapeutic reagents for the treatment of diseases such as cancer.
  • T cells are a key part of the cellular arm of the immune system. They specifically recognise peptide fragments that are derived from intracellular proteins and presented in complex with Major Histocompatibility Complex (MHC) molecules on the surface of antigen presenting cells (APCs).
  • MHC molecules are known as human leukocyte antigens (HLA), and both terms are used synonymously herein.
  • HLA human leukocyte antigens
  • MHC molecules have a binding groove in which the peptide fragments bind.
  • TCR T cell receptor
  • TAA-derived peptides presented by MHC molecules on tumour cells enables the development of novel immunotherapeutic reagents designed to specifically target and destroy said tumour cells.
  • Such reagents may be moieties that bind to the TAA-derived peptide and/or peptide-MHC complexes of the invention, and typically involve the induction of a T cell response.
  • such reagents may be based, exclusively, or in part, on T cells, or T cell receptors (TCRs), or antibodies.
  • TAA-derived peptides that are presented on the cell surface in complex with a given MHC, only a small number of such peptides, covering a limited number of cancer indications, have been identified to date.
  • TAAs suitable for the development of immunotherapeutic reagents must have limited or no expression in normal cells, to prevent off target activity. It is therefore desirable to provide additional tumour-specific TAA-derived peptides and MHC complexes thereof that can be used for the development of new cancer therapies.
  • CT45A3 (also known as cancer/testis antigen family 45 member A3 and having Uniprot accession number Q8NHU0) belongs to the cancer/testis family of germline encoded tumour antigens.
  • CT45A3 is therefore a particularly attractive target for therapeutic intervention.
  • the inventors have found novel peptides derived from CT45A3 that are presented on the cell surface in complex with MHC. These peptides are particularly useful for the development of reagents that can targets cells expressing CT45A3 and for the treatment of cancers, including non small cell lung cancer (squamous) and oesophageal cancer.
  • the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: E1 -E2, or
  • polypeptide forms a complex with a Major Histocompatibility Complex (MHC) molecule.
  • MHC Major Histocompatibility Complex
  • polypeptides of the invention are presented by MHC on the surface of tumour cells. Accordingly, the polypeptides of the invention, as well as moieties that bind the polypeptide-MHC complexes, can be used to develop therapeutic reagents.
  • the skilled person can determine whether or not a given polypeptide forms a complex with an MHC molecule by determining whether the MHC can be refolded in the presence of the polypeptide using the process set out in Example E2. If the polypeptide does not form a complex with MHC then MHC will not refold. Refolding is commonly confirmed using an antibody that recognises MHC in a folded state only. Further details can be found in Garboczi et al., Proc Natl Acad Sci
  • polypeptides of the invention are from about 8 to about 16 amino acids in length, and are most preferably 8, 9, or 10 or 11 amino acids in length.
  • the polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: E1-E2.
  • the amino acid residues comprising the polypeptides of the invention may be chemically modified. Examples of chemical modifications include those corresponding to post translational modifications for example phosphorylation, acetylation and deamidation (Engelhard et al., Curr Opin Immunol. 2006 Feb;18(1 ):92-7). Amino acid substitution means that an amino acid residue is substituted for a replacement amino acid residue at the same position.
  • Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are immediately adjacent one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent another inserted amino acid residue.
  • One, two or three amino acids may be deleted from the sequence of SEQ NOs: E1-E2. Each deletion can take place at any position of SEQ NOs: E1-E2.
  • the polypeptide of the invention may comprise one, two or three additional amino acids at the C-terminal end and/or at the N-terminal end of the sequence of SEQ NOs: E1- E2.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: E1-E2 with the exception of one amino acid substitution and one amino acid insertion, one amino acid substitution and one amino acid deletion, or one amino acid insertion and one amino acid deletion.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: E1-E2, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.
  • Inserted amino acids and replacement amino acids may be naturally occurring amino acids or may be non-naturally occurring amino acids and, for example, may contain a non-natural side chain.
  • Such altered peptide ligands are discussed further in Douat-Casassus et al., J. Med. Chem, 2007 Apr 5;50(7): 1598-609 and Hoppes et al., J. Immunol 2014 Nov 15;193(10):4803-13 and references therein). If more than one amino acid residue is substituted and/or inserted, the
  • replacement/inserted amino acid residues may be the same as each other or different from one another. Each replacement amino acid may have a different side chain to the amino acid being replaced.
  • polypeptides of the invention bind to MHC in the peptide binding groove of the MHC molecule.
  • the amino acid modifications described above will not impair the ability of the peptide to bind MHC.
  • the amino acid modifications improve the ability of the peptide to bind MHC.
  • mutations may be made at positions which anchor the peptide to MHC. Such anchor positions and the preferred residues at these locations are known in the art, particularly for peptides which bind HLA-A * 02 (see, e. g. Parkhurst et al., J. Immunol. 1996 Sep 15;157(6):2539-48 and Parker et al. J Immunol. 1992 Dec 1 ; 149(1 1 ):3580-7).
  • a polypeptide of the invention may be used to illicit an immune response. If this is the case, it is important that the immune response is specific to the intended target in order to avoid the risk of unwanted side effects that may be associated with an "off target" immune response. Therefore, it is preferred that the amino acid sequence of a polypeptide of the invention does not match the amino acid sequence of a peptide from any other protein(s), in particular, that of another human protein.
  • a person of skill in the art would understand how to search a database of known protein sequences to ascertain whether a polypeptide according to the invention is present in another protein.
  • Polypeptides of the invention can be synthesised easily by Merrifield synthesis, also known as solid phase synthesis, or any other peptide synthesis methodology.
  • GMP grade polypeptide is produced by solid-phase synthesis techniques by Multiple Peptide Systems, San Diego, CA.
  • the peptide may be recombinantly produced, if so desired, in accordance with methods known in the art.
  • Such methods typically involve the use of a vector comprising a nucleic acid sequence encoding the polypeptide to be expressed, to express the polypeptide in vivo; for example, in bacteria, yeast, insect or mammalian cells.
  • in vitro cell-free systems may be used. Such systems are known in the art and are commercially available for example from Life
  • polypeptides may be isolated and/or may be provided in substantially pure form. For example, they may be provided in a form which is substantially free of other polypeptides or proteins.
  • the invention provides a complex of the polypeptide of the first aspect and an MHC molecule.
  • the polypeptide is bound to the peptide binding groove of the MHC molecule.
  • the MHC molecule may be MHC class I.
  • the MHC class I molecule may be selected from HLA-A * 02, HLA-A * 01 , HLA-A * 03, HLA-A11 , HLA-A23, HLA-A24, HLA-B * 07, HLA-B*08, HLA- B40, HLA-B44, HLA-B15, HLA-C * 04, HLA * C * 03 HLA-CW.
  • HLA-A * 02, HLA-A * 01 , HLA-A * 03, HLA-A11 , HLA-A23, HLA-A24, HLA-B * 07, HLA-B*08, HLA- B40, HLA-B44, HLA-B15, HLA-C * 04, HLA * C * 03 HLA-CW As is known to those skilled in the art there are allelic variants of the above HLA types, all of which are encompassed by the present invention.
  • the complex of the invention may be isolated and/or in a substantially pure form.
  • the complex may be provided in a form which is substantially free of other polypeptides or proteins.
  • the term "MHC molecule" includes recombinant MHC molecules, non-naturally occurring MHC molecules and functionally equivalent fragments of MHC, including derivatives or variants thereof, provided that peptide binding is retained.
  • MHC molecules may be fused to a therapeutic moiety, attached to a solid support, in soluble form, and/or in multimeric form.
  • KLH keyhole limpet hemocyanin
  • the polypeptides and/or polypeptide-MHC complexes of the invention may be associated with a fusion partner. Fusion partners may be used for detection purposes, or for attaching said polypeptide or MHC to a solid support, or for MHC oligomerisation.
  • the MHC complexes may incorporate a biotinylation site to which biotin can be added, for example, using the BirA enzyme (O'Callaghan et al., 1999 Jan 1 ;266(1 ):9-15).
  • Other suitable fusion partners include, but are not limited to, fluorescent, or luminescent labels, radiolabels, nucleic acid probes and contrast reagents, antibodies, or enzymes that produce a detectable product. Detection methods may include flow cytometry, microscopy, electrophoresis or scintillation counting.
  • Polypeptide-MHC complexes of the invention may be provided in soluble form, or may be immobilised by attachment to a suitable solid support.
  • suitable solid supports include, but are not limited to, a bead, a membrane, sepharose, a magnetic bead, a plate, a tube, a column.
  • Polypeptide-MHC complexes may be attached to an ELISA plate, a magnetic bead, or a surface plasmon reasonance biosensor chip.
  • Methods of attaching peptide-MHC complexes to a solid support are known to the skilled person, and include, for example, using an affinity binding pair, e.g. biotin and streptavidin, or antibodies and antigens.
  • peptide-MHC complexes are labelled with biotin and attached to streptavidin-coated surfaces.
  • Polypeptide-MHC complexes of the invention may be in multimeric form, for example, dimeric, or tetrameric, or pentameric, or octomeric, or greater. Examples of suitable methods for the production of multimeric peptide MHC complexes are described in Greten et al., Clin. Diagn. Lab. Immunol. 2002 Mar;9(2):216-20 and references therein.
  • polypeptide-MHC multimers may be produced using peptide-MHC tagged with a biotin residue and complexed through fluorescent labelled streptavidin.
  • multimeric polypeptide-MHC complexes may be formed by using immunoglobulin as a molecular scaffold.
  • MHC molecules are fused with the constant region of an immunoglobulin heavy chain separated by a short amino acid linker.
  • Polypeptide-MHC multimers have also been produced using carrier molecules such as dextran (WO02072631 ). Multimeric peptide MHC complexes can be useful for improving the detection of binding moieties, such as T cell receptors, which bind said complex, because of avidity effects.
  • the polypeptides of the invention may be presented on the surface of a cell in complex with MHC.
  • the invention also provides a cell presenting on its surface a complex of the invention.
  • a cell may be a mammalian cell, preferably a cell of the immune system, and in particular a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • Other preferred cells include T2 cells (Hosken, et al., Science. 1990 Apr 20;248(4953):367-70).
  • Cells presenting the polypeptide or complex of the invention may be isolated, preferably in the form of a population, or provided in a substantially pure form.
  • Said cells may not naturally present the complex of the invention, or alternatively said cells may present the complex at a level higher than they would in nature.
  • Such cells may be obtained by pulsing said cells with the polypeptide of the invention. Pulsing involves incubating the cells with the polypeptide for several hours using polypeptide concentrations typically ranging from 10 "5 to 10 "12 M. Said cells may additionally be transduced with HLA-A * 02 molecules to further induce presentation of the peptide. Cells may be produced recombinantly. Cells presenting polypeptides of the invention may be used to isolate T cells and T cell receptors (TCRs) which are activated by, or bind to, said cells, as described in more detail below.
  • TCRs T cell receptors
  • the invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide of the first aspect of the invention.
  • the nucleic acid may be cDNA.
  • the nucleic acid molecule may consist essentially of a nucleic acid sequence encoding the polypeptide of the first aspect of the invention or may encode only the peptide of the invention, i.e. encode no other polypeptide.
  • Such a nucleic acid molecule can be synthesised in accordance with methods known in the art. Due to the degeneracy of the genetic code, one of ordinary skill in the art will appreciate that nucleic acid molecules of different nucleotide sequence can encode the same amino acid sequence.
  • the invention provides a vector comprising a nucleic acid sequence according to the third aspect of the invention.
  • the vector may include, in addition to a nucleic acid sequence encoding only a polypeptide of the invention, one or more additional nucleic acid sequences encoding one or more additional polypeptides. Such additional polypeptides may, once expressed, be fused to the N-terminus or the C-terminus of the polypeptide of the invention.
  • the vector includes a nucleic acid sequence encoding a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • Suitable vectors are known in the art as is vector construction, including the selection of promoters and other regulatory elements, such as enhancer elements.
  • the vector utilised in the context of the present invention desirably comprises sequences appropriate for introduction into cells.
  • the vector may be an expression vector, a vector in which the coding sequence of the polypeptide is under the control of its own cis-acting regulatory elements, a vector designed to facilitate gene integration or gene replacement in host cells, and the like.
  • the term "vector” encompasses a DNA molecule, such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • a DNA molecule such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • Appropriate phage and viral vectors include, but are not limited to, lambda (X) bacteriophage, EMBL bacteriophage, simian virus 40, bovine papilloma virus, Epstein-Barr virus, adenovirus, herpes virus, vaccinia virus, Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, lentivirus and Rous sarcoma virus.
  • the invention provides a cell comprising the vector of the fourth aspect of the invention.
  • the cell may be an antigen presenting cell and is preferably a cell of the immune system.
  • the cell may be a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • the cell may be a mammalian cell.
  • Polypeptides and complexes of the invention can be used to identify and/or isolate binding moieties that bind specifically to the polypeptide and/or the complex of the invention.
  • binding moieties may be used as immunotherapeutic reagents and may include antibodies and TCRs.
  • the invention provides a binding moiety that binds the polypeptide of the invention.
  • the binding moiety binds the polypeptide when said polypeptide is in complex with MHC.
  • the binding moiety may bind partially to the MHC, provided that it also binds to the polypeptide.
  • the binding moiety may bind only the polypeptide, and that binding may be specific.
  • the binding moiety may bind only the peptide MHC complex and that binding may be specific.
  • binding moieties that bind the complex of the invention When used with reference to binding moieties that bind the complex of the invention, "specific" is generally used herein to refer to the situation in which the binding moiety does not show any significant binding to one or more alternative polypeptide-MHC complexes other than the polypeptide-MHC complex of the invention.
  • the binding moiety may be a T cell receptor (TCR).
  • TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature, and links to the IMGT public database of TCR sequences. The unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the TCR field.
  • IMGT International Immunogenetics
  • TCRs of the invention may be in any format known to those in the art.
  • the TCRs may be ⁇ heterodimers, or they may be in single chain format (such as those described in W09918129).
  • Single chain TCRs include ⁇ TCR polypeptides of the type: Va-L- ⁇ , ⁇ -L-Va, Va- Ca-L- ⁇ , ⁇ - ⁇ - ⁇ ⁇ -0 ⁇ or Va- Ca - ⁇ / ⁇ - ⁇ , optionally in the reverse orientation, wherein Va and ⁇ are TCR a and ⁇ variable regions respectively, Ca and C are TCR a and ⁇ constant regions respectively, and L is a linker sequence.
  • the TCR may be in a soluble form (i.e. having no transmembrane or cytoplasmic domains), or may contain full length alpha and beta chains.
  • the TCR may be provided on the surface of a cell, such as a T cell.
  • the alpha and/or beta chain constant domain may be truncated relative to the native/naturally occurring TRAC/TRBC sequences.
  • the TRAC/TRBC may contain modifications.
  • the alpha chain extracellular sequence may include a modification relative to the native/naturally occurring TRAC whereby amino acid T48 of TRAC, with reference to IMGT numbering, is replaced with C48.
  • the beta chain extracellular sequence may include a modification relative to the native/naturally occurring TRBC1 or TRBC2 whereby S57 of TRBC1 or TRBC2, with reference to IMGT numbering, is replaced with C57.
  • TCRs of the invention may be engineered to include mutations.
  • Methods for producing mutated high affinity TCR variants such as phage display and site directed mutagenesis and are known to those in the art (for example see WO 04/044004 and Li et al., Nat Biotechnol 2005 Mar;23(3):349- 54).
  • TCRs of the invention may also be may be labelled with an imaging compound, for example a label that is suitable for diagnostic purposes.
  • Such labelled high affinity TCRs are useful in a method for detecting a TCR ligand selected from CD1 -antigen complexes, bacterial superantigens, and MHC- peptide/superantigen complexes, which method comprises contacting the TCR ligand with a high affinity TCR (or a multimeric high affinity TCR complex) which is specific for the TCR ligand; and detecting binding to the TCR ligand.
  • a high affinity TCR or a multimeric high affinity TCR complex
  • multimeric high affinity TCR complexes such as those described in Zhu et al., J. Immunol.
  • fluorescent streptavidin (commercially available) can be used to provide a detectable label.
  • a fluorescently-labelled multimer is suitable for use in FACS analysis, for example to detect antigen presenting cells carrying the peptide for which the high affinity TCR is specific.
  • a TCR of the present invention may alternatively or additionally be associated with (e.g. covalently or otherwise linked to) a therapeutic agent which may be, for example, a toxic moiety for use in cell killing, or an immunostimulating agent such as an interleukin or a cytokine.
  • a multivalent high affinity TCR complex of the present invention may have enhanced binding capability for a TCR ligand compared to a non-multimeric wild-type or high affinity T cell receptor heterodimer.
  • the multivalent high affinity TCR complexes according to the invention are particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo, and are also useful as intermediates for the production of further multivalent high affinity TCR complexes having such uses.
  • the high affinity TCR or multivalent high affinity TCR complex may therefore be provided in a pharmaceutically acceptable formulation for use in vivo.
  • High affinity TCRs of the invention may be used in the production of soluble bi-specific reagents.
  • a preferred embodiment is a reagent which comprises a soluble TCR, fused via a linker to an anti- CD3 specific antibody fragment. Further details including how to produce such reagents are described in W010/133828.
  • the invention provides nucleic acid encoding the TCR of the invention, a TCR expression vector comprising nucleic acid encoding a TCR of the invention, as well as a cell harbouring such a vector.
  • the TCR may be encoded either in a single open reading frame or two distinct open reading frames.
  • one vector may encode both an alpha and a beta chain of a TCR of the invention.
  • a further aspect of the invention provides a cell displaying on its surface a TCR of the invention.
  • the cell may be a T cell.
  • T cells expressing the TCRs of the invention are suitable for use in adoptive therapy-based treatment of diseases such as cancers.
  • adoptive therapy see for example Rosenberg et al., Nat Rev Cancer. 2008 Apr;8(4):299-308.
  • the TCRs of the invention intended for use in adoptive therapy are generally glycosylated when expressed by the transfected T cells.
  • the glycosylation pattern of transfected TCRs may be modified by mutations of the transfected gene (Kuball J et al., J Exp Med. 2009 Feb 16;206(2):463-75).
  • the binding moiety of the invention may be an antibody.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced.
  • antibody includes antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanised antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic and any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.
  • a humanised antibody may be a modified antibody having the variable regions of a non-human, e.g. murine, antibody and the constant region of a human antibody. Methods for making humanised antibodies are described in, for example, US Patent No. 5225539. Examples of antibodies are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. A monoclonal antibody may be referred to herein as "mab".
  • an antibody for example a monoclonal antibody
  • recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody.
  • Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementary determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin (see, for instance, EP-A-184187, GB 2188638A or EP-A-239400).
  • CDRs complementary determining regions
  • a hybridoma (or other cell that produces antibodies) may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. et al., Nature.
  • Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g.
  • antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).
  • bispecific antibodies these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Hollinger & Winter, Curr Opin
  • bispecific antibody fragments e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain "Janusins" described in Traunecker et al., EMBO J. 1991 Dec; 10(12):3655-9). Bispecific diabodies, as opposed to bispecific whole antibodies, may also be useful because they can be readily constructed and expressed in E. coli.
  • Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
  • An "antigen binding domain" is the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope.
  • An antigen binding domain may be provided by one or more antibody variable domains.
  • An antigen binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • the binding moiety may be an antibody-like molecule that has been designed to specifically bind a peptide-MHC complex of the invention.
  • TCR-mimic antibodies such as, for example those described in WO2007143104 and Sergeeva et al., Blood. 201 1 Apr
  • binding moieties based on engineered protein scaffolds are derived from stable, soluble, natural protein structures which have been modified to provide a binding site for a target molecule of interest.
  • engineered protein scaffolds include, but are not limited to, affibodies, which are based on the Z-domain of staphylococcal protein A that provides a binding interface on two of its a-helices (Nygren, FEBS J. 2008 Jun;275(1 1 ):2668-76); anticalins, derived from lipocalins, that incorporate binding sites for small ligands at the open end of a beta-barrel fold (Skerra, FEBS J.
  • Engineered protein scaffolds are typically targeted to bind the same antigenic proteins as antibodies, and are potential therapeutic agents. They may act as inhibitors or antagonists, or as delivery vehicles to target molecules, such as toxins, to a specific tissue in vivo (Gebauer and Skerra, Curr Opin Chem Biol. 2009 Jun;13(3):245-55). Short peptides may also be used to bind a target protein.
  • Phylomers are natural structured peptides derived from bacterial genomes. Such peptides represent a diverse array of protein structural folds and can be used to inhibit/disrupt protein-protein interactions in vivo (Watt, Nat Biotechnol. 2006 Feb;24(2): 177-83)].
  • the invention further provides a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention for use in medicine.
  • the polypeptide, complex, nucleic acid, vector, cell or binding moiety may be used for in the treatment or prevention of cancer, in particular, non small cell lung cancer (squamous) and oesophageal cancer.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention together with a pharmaceutically acceptable carrier.
  • This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient). It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms. Suitable compositions and methods of administration are known to those skilled in the art, for example see, Johnson et al., Blood.
  • Cells in accordance with the invention will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a
  • T cells transfected with TCRs of the invention may be provided in pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be adapted for administration by any appropriate route such as a parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation or intranasal routes.
  • a parenteral including subcutaneous, intramuscular, or intravenous
  • enteral including oral or rectal
  • inhalation or intranasal routes Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
  • Dosages of the substances of the present invention can vary between wide limits, depending upon the disease or disorder to be treated (such as cancer, viral infection or autoimmune disease), the age and condition of the individual to be treated, etc.
  • a suitable dose range for a reagent comprising a soluble TCR fused to an anti- CD3 domain may be between 25 ng/kg and 50 pg/kg.
  • a physician will ultimately determine appropriate dosages to be used.
  • the polypeptide of the invention may be provided in the form of a vaccine composition.
  • the vaccine composition may be useful for the treatment or prevention of cancer. All such
  • compositions are encompassed in the present invention.
  • vaccines may take several forms (Schlom, J Natl Cancer Inst. 2012 Apr 18;104(8):599-613).
  • the peptide of the invention may be used directly to immunise patients (Salgaller, Cancer Res. 1996 Oct 15;56(20):4749-57 and Marchand, Int J Cancer. 1999 Jan 18;80(2):219-30).
  • the vaccine composition may include additional peptides such that the peptide of the invention is one of a mixture of peptides.
  • Adjuvants may be added to the vaccine composition to augment the immune response
  • the vaccine composition may take the form of an antigen presenting cell displaying the peptide of the invention in complex with MHC.
  • the antigen presenting cell is an immune cell, more preferably a dendritic cell.
  • the peptide may be pulsed onto the surface of the cell (Thurner, J Exp Med. 1999 Dec 6; 190(11 ): 1669-78), or nucleic acid encoding for the peptide of the invention may be introduced into dendritic cells (for example by electroporation. Van Tendeloo, Blood. 2001 Jul 1 ;98(1 ):49-56).
  • polypeptides, complexes, nucleic acid molecules, vectors, cells and binding moieties of the invention may be non-naturally occurring and/or purified and/or engineered and/or recombinant and/or isolated and/or synthetic.
  • the invention also provides a method of identifying a binding moiety that binds a complex of the invention, the method comprising contacting a candidate binding moiety with the complex and determining whether the candidate binding moiety binds the complex.
  • Methods to determine binding to polypeptide-MHC complexes are well known in the art. Preferred methods include, but are not limited to, surface plasmon resonance, or any other biosensor technique, ELISA, flow cytometry, chromatography, microscopy. Alternatively, or in addition, binding may be determined by functional assays in which a biological response is detected upon binding, for example, cytokine release or cell apoptosis.
  • the candidate binding moiety may be a binding moiety of the type already described, such as a TCR or an antibody.
  • T cells may be isolated from fresh blood obtained from volunteer donors. Such a method involves stimulating T cells using autologous DCs, followed by autologous B cells, pulsed with the polypeptide of the invention. Several round of stimulation may be carried out, for example three or four rounds. Activated T cells may then be tested for specificity by measuring cytokine release in the presence of T2 cells pulsed with the peptide of the invention (for example using an IFNy ELISpot assay). Activated cells may then be sorted by fluorescence-activated cell sorting (FACS) using labelled antibodies to detect intracellular cytokine production (e.g. IFNy), or expression of a cell surface marker (such as CD137).
  • FACS fluorescence-activated cell sorting
  • Sorted cells may be expanded and further validated, for example, by ELISpot assay and/or cytotoxicity against target cells and/or staining by peptide-MHC tetramer.
  • the TCR chains from validated clones may then be amplified by rapid amplification of cDNA ends (RACE) and sequenced.
  • the polypeptide MHC complex of the invention may be used for screening a library of TCRs or antibodies.
  • the production of antibody libraries using phage display is well known in the art, for example see Aitken, Antibody phage display: Methods and Protocols (2009, Humana, New York). Further details on TCR libraries can be found in WO04044004.
  • the polypeptide-MHC complex of the invention is used to screen a library of diverse TCRs displayed on the surface of phage particles.
  • the TCRs displayed by said library may contain non- natural mutations. Screening may involve panning the phage library with peptide-MHC complexes of the invention and subsequently isolating bound phage.
  • peptide-MHC complexes may be attached to a solid support, such as a magnet bead, or column matrix and phage bound peptide MHC complexes isolated, with a magnet, or by chromatography, respectively.
  • the panning steps may be repeated several times for example three or four times.
  • Isolated phage may be further expanded in E. coli cells.
  • Isolated phage particles may be tested for specific binding peptide-MHC complexes of the invention. Binding can be detected using techniques including, but not limited to, ELISA, or SPR for example using a BiaCore instrument.
  • the DNA sequence of the T cell receptor displayed by peptide-MHC binding phage can be further identified by standard PCR methods.
  • Figures E1 to E2 show the respective fragmentation spectra for the peptides of SEQ NOs: E1 to E2, eluted from cells. A table highlighting the matching ions is shown below each spectrum.
  • Immortalised cell lines obtained from commercial sources were maintained and expanded under standard conditions.
  • Class I HLA complexes were purified by immunoaffinity using commercially available anti-HLA antibodies BB7.1 (anti-HLA-B * 07), BB7.2 (anti-HLA-A * 02) and W6/32 (anti-Class 1 ). Briefly, cells were lysed in buffer containing non-ionic detergent NP-40 (0.5% v/v) at 5 x 10 7 cells per ml and incubated at 4°C for 1 h with agitation/mixing. Cell debris was removed by centrifugation and supernatant pre-cleared using proteinA-Sepharose.
  • Peptides were separated by high pressure liquid chromatography (HPLC) on a Dionex Ultimate 3000 system using a C18 column (Phenomenex). Peptides were loaded in 98% buffer A (0.1 % aqueous trifluoroacetic acid (TFA)) and 2% buffer B (0.1% TFA in acetonitrile). Peptides were eluted using a stepped gradient of B (2-60%) over 20 min. Fractions were collected at one minute intervals and lyophilised.
  • HPLC high pressure liquid chromatography
  • Peptides were analysed by nanoLCMS/MS using a Dionex Ultimate 3000 nanoLC coupled to either AB Sciex Triple TOF 5600 or Thermo Orbitrap Fusion mass spectrometers. Both machines were equipped with nanoelectrospray ion sources. Peptides were loaded onto an Acclaim PepMap 100 trap column (Dionex) and separated using an Acclaim PepMap RSLC column (Dionex). Peptides were loaded in mobile phase A (0.5% formic acid: water) and eluted using a gradient of buffer B (acetonitrile:0.5% formic acid) directly into the nanospray ionisation source.
  • buffer B acetonitrile:0.5% formic acid
  • IDA information dependent acquisition
  • Information acquired in these experiments was used to search the Uniprot database of human proteins for peptides consistent with the fragmentation patterns seen, using Protein pilot software (Ab Sciex) and PEAKS software (Bioinformatics solutions). Peptides identified are assigned a score by the software, based on the match between the observed and expected fragmentation patterns.
  • polypeptides set out in table E1 corresponding to SEQ NOs: E1-E2, were detected by mass spec following extraction from cancer cell lines.
  • An example cell line from which the peptide was detected is indicated in the table along with the HLA antibody used for immunoaffinity purification.
  • Figures E1 -E2 show representative fragmentation patterns for the peptides of SEQ NOs: E1-E2 respectively. A table highlighting the matching ions is shown below each spectrum.
  • HLA-A*02 molecules HLA-A*02-heavy chain and HLA light-chain ( ⁇ 2 ⁇ ) were expressed separately in E. coli as inclusion bodies, using appropriate constructs.
  • HLA-A * 02-heavy chain additionally contained a C-terminal biotinylation tag which replaces the transmembrane and cytoplasmic domains (O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15).
  • E. coli cells were lysed and inclusion bodies processed to approximately 80% purity.
  • Inclusion bodies of ⁇ 2 ⁇ and heavy chain were denatured separately in 6 M guanidine-HCI, 50 mM Tris pH 8.1 , 100 mM NaCI, 10 mM DTT, 10 mM EDTA.
  • Refolding buffer was prepared containing 0.4 M L-Arginine, 100 mM Tris pH 8.1 , 3.7 mM cystamine dihydrochloride, 6.6 mM cysteamine hydrochloride and chilled to ⁇ 5°C.
  • Synthetic peptide dissolved in DMSO to a final concentration of 4mg/ml is added to the refold buffer at 4 mg/litre (final concentration). Then 30 mg/litre ⁇ 2 ⁇ followed by 30 mg/litre heavy chain (final concentrations) are added. Refolding was allowed to reach completion at 4 °C for at least 1 hour.
  • Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Two changes of buffer were necessary to reduce the ionic strength of the solution sufficiently.
  • the protein solution was then filtered through a 1.5 Mm cellulose acetate filter and loaded onto a POROS 50HQ anion exchange column (8 ml bed volume). Protein was eluted with a linear 0-500 mM NaCI gradient in 10 mM Tris pH 8.1 using an Akta purifier (GE Healthcare). HLA-A * 02-peptide complex eluted at approximately 250 mM NaCI, and peak fractions were collected, a cocktail of protease inhibitors (Calbiochem) was added and the fractions were chilled on ice.
  • Biotinylation tagged pHLA molecules were buffer exchanged into 10 mM Tris pH 8.1 , 5 mM NaCI using a GE Healthcare fast desalting column equilibrated in the same buffer. Immediately upon elution, the protein-containing fractions were chilled on ice and protease inhibitor cocktail (Calbiochem) was added. Biotinylation reagents were then added: 1 mM biotin, 5 mM ATP
  • the biotinylated pHLA-A * 02 molecules were purified using gel filtration chromatography.
  • a GE Healthcare Superdex 75 HR 10/30 column was pre-equilibrated with filtered PBS and 1 ml of the biotinylation reaction mixture was loaded and the column was developed with PBS at 0.5 ml/min using an Akta purifier (GE Healthcare).
  • Biotinylated pHLA-A * 02 molecules eluted as a single peak at approximately 15 ml. Fractions containing protein were pooled, chilled on ice, and protease inhibitor cocktail was added. Protein concentration was determined using a Coomassie-binding assay (PerBio) and aliquots of biotinylated pHLA-A * 02 molecules were stored frozen at -20 °C.
  • Such peptide-MHC complexes may be used in soluble form or may be immobilised through C terminal biotin moiety on to a solid support, to be used for the detection of T cells and T cell receptors which bind said complex.
  • TCR isolation methods are known to those skilled in the art; for example, DNA sequences corresponding to TCR alpha and beta chains may be amplified from a T cell clone (obtained from volunteer blood donors) that is activated by cells presenting the peptide-HLA complex of interest.
  • T cell clone obtained from volunteer blood donors
  • Other suitable methods include isolation from TCR libraries (as described in WO2005/1 16646 and WO2005/116074 for example).
  • TCR alpha and beta chain sequences are expressed in E. coli as soluble TCRs, (WO2003020763; Boulter, et al., Protein Eng, 2003. 16: 707-711 ). Binding of the soluble TCRs to the complexes is analysed by surface plasmon resonance using a BiaCore 3000 instrument. Biotinylated peptide-HLA monomers are prepared as previously described (Example E2) and immobilized on to a streptavidin-coupled CM-5 sensor chip.
  • The[LS5] present invention also relates to novel peptides derived from Cancer/testis antigen family 45 member A5 (CT45A5) complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules.
  • C45A5 Cancer/testis antigen family 45 member A5
  • binding moieties that bind to the peptides and/or complexes of the invention. Such moieties are useful for the development of immunotherapeutic reagents for the treatment of diseases such as cancer.
  • T cells are a key part of the cellular arm of the immune system. They specifically recognise peptide fragments that are derived from intracellular proteins and presented in complex with Major Histocompatibility Complex (MHC) molecules on the surface of antigen presenting cells (APCs).
  • MHC molecules are known as human leukocyte antigens (HLA), and both terms are used synonymously herein.
  • HLA human leukocyte antigens
  • MHC molecules have a binding groove in which the peptide fragments bind.
  • TCR T cell receptor
  • TAA-derived peptides presented by MHC molecules on tumour cells enables the development of novel immunotherapeutic reagents designed to specifically target and destroy said tumour cells.
  • Such reagents may be moieties that bind to the TAA-derived peptide and/or peptide-MHC complexes of the invention, and typically involve the induction of a T cell response.
  • such reagents may be based, exclusively, or in part, on T cells, or T cell receptors (TCRs), or antibodies.
  • TAA-derived peptides that are presented on the cell surface in complex with a given MHC, only a small number of such peptides, covering a limited number of cancer indications, have been identified to date.
  • TAAs suitable for the development of immunotherapeutic reagents must have limited or no expression in normal cells, to prevent off target activity. It is therefore desirable to provide additional tumour-specific TAA-derived peptides and MHC complexes thereof that can be used for the development of new cancer therapies.
  • CT45A5 (also known as cancer/testis antigen family 45 member A5 and having Uniprot accession number P0DMU8 (formally Q6NSH3)) belongs to the cancer/testis family of germline encoded tumour antigens. Expression of CT45A5 has been reported in cancer cells, while expression in normal tissue is restricted to testis (Chen et al. Proc Natl Acad Sci U S A. 2005 May
  • CT45A5 is therefore a particularly attractive target for therapeutic intervention.
  • the inventors have found novel peptides derived from CT45A5 that are presented on the cell surface in complex with MHC. These peptides are particularly useful for the development of reagents that can targets cells expressing CT45A5 and for the treatment of cancers, including non- small cell lung cancer (squamous) and oesophageal cancer.
  • the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: F1 -F2, or
  • polypeptide forms a complex with a Major Histocompatibility Complex (MHC) molecule.
  • MHC Major Histocompatibility Complex
  • polypeptides of the invention are presented by MHC on the surface of tumour cells. Accordingly, the polypeptides of the invention, as well as moieties that bind the polypeptide-MHC complexes, can be used to develop therapeutic reagents.
  • the skilled person can determine whether or not a given polypeptide forms a complex with an MHC molecule by determining whether the MHC can be refolded in the presence of the polypeptide using the process set out in Example F2. If the polypeptide does not form a complex with MHC then MHC will not refold. Refolding is commonly confirmed using an antibody that recognises MHC in a folded state only. Further details can be found in Garboczi et al., Proc Natl Acad Sci
  • polypeptides of the invention are from about 8 to about 16 amino acids in length, and are most preferably 8, 9, or 10 or 11 amino acids in length.
  • the polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: F1-F2.
  • the amino acid residues comprising the polypeptides of the invention may be chemically modified. Examples of chemical modifications include those corresponding to post translational modifications for example phosphorylation, acetylation and deamidation (Engelhard et al., Curr Opin Immunol. 2006 Feb;18(1 ):92-7). Amino acid substitution means that an amino acid residue is substituted for a replacement amino acid residue at the same position.
  • Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are immediately adjacent one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent another inserted amino acid residue.
  • One, two or three amino acids may be deleted from the sequence of SEQ NOs: F1-F2. Each deletion can take place at any position of SEQ NOs: F1-F2.
  • the polypeptide of the invention may comprise one, two or three additional amino acids at the C-terminal end and/or at the N-terminal end of the sequence of SEQ NOs: F1- F2.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: F1-F2 with the exception of one amino acid substitution and one amino acid insertion, one amino acid substitution and one amino acid deletion, or one amino acid insertion and one amino acid deletion.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: F1-F2, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.
  • Inserted amino acids and replacement amino acids may be naturally occurring amino acids or may be non-naturally occurring amino acids and, for example, may contain a non-natural side chain.
  • Such altered peptide ligands are discussed further in Douat-Casassus et al., J. Med. Chem, 2007 Apr 5;50(7): 1598-609 and Hoppes et al., J. Immunol 2014 Nov 15;193(10):4803-13 and references therein). If more than one amino acid residue is substituted and/or inserted, the
  • replacement inserted amino acid residues may be the same as each other or different from one another. Each replacement amino acid may have a different side chain to the amino acid being replaced.
  • polypeptides of the invention bind to MHC in the peptide binding groove of the MHC molecule.
  • the amino acid modifications described above will not impair the ability of the peptide to bind MHC.
  • the amino acid modifications improve the ability of the peptide to bind MHC.
  • mutations may be made at positions which anchor the peptide to MHC. Such anchor positions and the preferred residues at these locations are known in the art, particularly for peptides which bind HLA-A * 02 (see, e. g. Parkhurst et al., J. Immunol. 1996 Sep 15;157(6):2539-48 and Parker et al. J Immunol. 1992 Dec 1 ; 149(1 1 ):3580-7).
  • a polypeptide of the invention may be used to illicit an immune response. If this is the case, it is important that the immune response is specific to the intended target in order to avoid the risk of unwanted side effects that may be associated with an "off target" immune response. Therefore, it is preferred that the amino acid sequence of a polypeptide of the invention does not match the amino acid sequence of a peptide from any other protein(s), in particular, that of another human protein.
  • a person of skill in the art would understand how to search a database of known protein sequences to ascertain whether a polypeptide according to the invention is present in another protein.
  • Polypeptides of the invention can be synthesised easily by Merrifield synthesis, also known as solid phase synthesis, or any other peptide synthesis methodology.
  • GMP grade polypeptide is produced by solid-phase synthesis techniques by Multiple Peptide Systems, San Diego, CA.
  • the peptide may be recombinantly produced, if so desired, in accordance with methods known in the art.
  • Such methods typically involve the use of a vector comprising a nucleic acid sequence encoding the polypeptide to be expressed, to express the polypeptide in vivo; for example, in bacteria, yeast, insect or mammalian cells.
  • in vitro cell-free systems may be used. Such systems are known in the art and are commercially available for example from Life
  • polypeptides may be isolated and/or may be provided in substantially pure form. For example, they may be provided in a form which is substantially free of other polypeptides or proteins.
  • the invention provides a complex of the polypeptide of the first aspect and an MHC molecule.
  • the polypeptide is bound to the peptide binding groove of the MHC molecule.
  • the MHC molecule may be MHC class I.
  • the MHC class I molecule may be selected from HLA-A * 02, HLA-A * 01 , HLA-A * 03, HLA-A11 , HLA-A23, HLA-A24, HLA-B * 07, HLA-B*08, HLA- B40, HLA-B44, HLA-B15, HLA-C * 04, HLA * C * 03 HLA-CW.
  • HLA-A * 02, HLA-A * 01 , HLA-A * 03, HLA-A11 , HLA-A23, HLA-A24, HLA-B * 07, HLA-B*08, HLA- B40, HLA-B44, HLA-B15, HLA-C * 04, HLA * C * 03 HLA-CW As is known to those skilled in the art there are allelic variants of the above HLA types, all of which are encompassed by the present invention.
  • the complex of the invention may be isolated and/or in a substantially pure form.
  • the complex may be provided in a form which is substantially free of other polypeptides or proteins.
  • the term "MHC molecule" includes recombinant MHC molecules, non-naturally occurring MHC molecules and functionally equivalent fragments of MHC, including derivatives or variants thereof, provided that peptide binding is retained.
  • MHC molecules may be fused to a therapeutic moiety, attached to a solid support, in soluble form, and/or in multimeric form.
  • KLH keyhole limpet hemocyanin
  • the polypeptides and/or polypeptide-MHC complexes of the invention may be associated with a fusion partner. Fusion partners may be used for detection purposes, or for attaching said polypeptide or MHC to a solid support, or for MHC oligomerisation.
  • the MHC complexes may incorporate a biotinylation site to which biotin can be added, for example, using the BirA enzyme (O'Callaghan et al., 1999 Jan 1 ;266(1 ):9-15).
  • Other suitable fusion partners include, but are not limited to, fluorescent, or luminescent labels, radiolabels, nucleic acid probes and contrast reagents, antibodies, or enzymes that produce a detectable product. Detection methods may include flow cytometry, microscopy, electrophoresis or scintillation counting.
  • Polypeptide-MHC complexes of the invention may be provided in soluble form, or may be immobilised by attachment to a suitable solid support.
  • suitable solid supports include, but are not limited to, a bead, a membrane, sepharose, a magnetic bead, a plate, a tube, a column.
  • Polypeptide-MHC complexes may be attached to an ELISA plate, a magnetic bead, or a surface plasmon reasonance biosensor chip.
  • Methods of attaching peptide-MHC complexes to a solid support are known to the skilled person, and include, for example, using an affinity binding pair, e.g. biotin and streptavidin, or antibodies and antigens.
  • peptide-MHC complexes are labelled with biotin and attached to streptavidin-coated surfaces.
  • Polypeptide-MHC complexes of the invention may be in multimeric form, for example, dimeric, or tetrameric, or pentameric, or octomeric, or greater. Examples of suitable methods for the production of multimeric peptide MHC complexes are described in Greten et al., Clin. Diagn. Lab. Immunol. 2002 Mar;9(2):216-20 and references therein.
  • polypeptide-MHC multimers may be produced using peptide-MHC tagged with a biotin residue and complexed through fluorescent labelled streptavidin.
  • multimeric polypeptide-MHC complexes may be formed by using immunoglobulin as a molecular scaffold.
  • MHC molecules are fused with the constant region of an immunoglobulin heavy chain separated by a short amino acid linker.
  • Polypeptide-MHC multimers have also been produced using carrier molecules such as dextran (WO02072631 ). Multimeric peptide MHC complexes can be useful for improving the detection of binding moieties, such as T cell receptors, which bind said complex, because of avidity effects.
  • the polypeptides of the invention may be presented on the surface of a cell in complex with MHC.
  • the invention also provides a cell presenting on its surface a complex of the invention.
  • a cell may be a mammalian cell, preferably a cell of the immune system, and in particular a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • Other preferred cells include T2 cells (Hosken, et al., Science. 1990 Apr 20;248(4953):367-70).
  • Cells presenting the polypeptide or complex of the invention may be isolated, preferably in the form of a population, or provided in a substantially pure form.
  • Said cells may not naturally present the complex of the invention, or alternatively said cells may present the complex at a level higher than they would in nature.
  • Such cells may be obtained by pulsing said cells with the polypeptide of the invention. Pulsing involves incubating the cells with the polypeptide for several hours using polypeptide concentrations typically ranging from 10 "5 to 10 "12 M. Said cells may additionally be transduced with HLA-A * 02 molecules to further induce presentation of the peptide. Cells may be produced recombinantly. Cells presenting polypeptides of the invention may be used to isolate T cells and T cell receptors (TCRs) which are activated by, or bind to, said cells, as described in more detail below.
  • TCRs T cell receptors
  • the invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide of the first aspect of the invention.
  • the nucleic acid may be cDNA.
  • the nucleic acid molecule may consist essentially of a nucleic acid sequence encoding the polypeptide of the first aspect of the invention or may encode only the peptide of the invention, i.e. encode no other polypeptide.
  • Such a nucleic acid molecule can be synthesised in accordance with methods known in the art. Due to the degeneracy of the genetic code, one of ordinary skill in the art will appreciate that nucleic acid molecules of different nucleotide sequence can encode the same amino acid sequence.
  • the invention provides a vector comprising a nucleic acid sequence according to the third aspect of the invention.
  • the vector may include, in addition to a nucleic acid sequence encoding only a polypeptide of the invention, one or more additional nucleic acid sequences encoding one or more additional polypeptides. Such additional polypeptides may, once expressed, be fused to the N-terminus or the C-terminus of the polypeptide of the invention.
  • the vector includes a nucleic acid sequence encoding a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • Suitable vectors are known in the art as is vector construction, including the selection of promoters and other regulatory elements, such as enhancer elements.
  • the vector utilised in the context of the present invention desirably comprises sequences appropriate for introduction into cells.
  • the vector may be an expression vector, a vector in which the coding sequence of the polypeptide is under the control of its own cis-acting regulatory elements, a vector designed to facilitate gene integration or gene replacement in host cells, and the like.
  • the term "vector” encompasses a DNA molecule, such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • a DNA molecule such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • Appropriate phage and viral vectors include, but are not limited to, lambda (X) bacteriophage, EMBL bacteriophage, simian virus 40, bovine papilloma virus, Epstein-Barr virus, adenovirus, herpes virus, vaccinia virus, Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, lentivirus and Rous sarcoma virus.
  • the invention provides a cell comprising the vector of the fourth aspect of the invention.
  • the cell may be an antigen presenting cell and is preferably a cell of the immune system.
  • the cell may be a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • the cell may be a mammalian cell.
  • Polypeptides and complexes of the invention can be used to identify and/or isolate binding moieties that bind specifically to the polypeptide and/or the complex of the invention.
  • binding moieties may be used as immunotherapeutic reagents and may include antibodies and TCRs.
  • the invention provides a binding moiety that binds the polypeptide of the invention.
  • the binding moiety binds the polypeptide when said polypeptide is in complex with MHC.
  • the binding moiety may bind partially to the MHC, provided that it also binds to the polypeptide.
  • the binding moiety may bind only the polypeptide, and that binding may be specific.
  • the binding moiety may bind only the peptide MHC complex and that binding may be specific.
  • binding moieties that bind the complex of the invention When used with reference to binding moieties that bind the complex of the invention, "specific" is generally used herein to refer to the situation in which the binding moiety does not show any significant binding to one or more alternative polypeptide-MHC complexes other than the polypeptide-MHC complex of the invention.
  • the binding moiety may be a T cell receptor (TCR).
  • TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature, and links to the IMGT public database of TCR sequences. The unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the TCR field.
  • IMGT International Immunogenetics
  • TCRs of the invention may be in any format known to those in the art.
  • the TCRs may be ⁇ heterodimers, or they may be in single chain format (such as those described in W09918129).
  • Single chain TCRs include ⁇ TCR polypeptides of the type: Va-L- ⁇ , ⁇ -L-Va, Va- Ca-L- ⁇ , ⁇ - ⁇ - ⁇ ⁇ -0 ⁇ or Va- Ca - ⁇ / ⁇ - ⁇ , optionally in the reverse orientation, wherein Va and ⁇ are TCR a and ⁇ variable regions respectively, Ca and C are TCR a and ⁇ constant regions respectively, and L is a linker sequence.
  • the TCR may be in a soluble form (i.e. having no transmembrane or cytoplasmic domains), or may contain full length alpha and beta chains.
  • the TCR may be provided on the surface of a cell, such as a T cell.
  • the alpha and/or beta chain constant domain may be truncated relative to the native/naturally occurring TRAC/TRBC sequences.
  • the TRAC/TRBC may contain modifications.
  • the alpha chain extracellular sequence may include a modification relative to the native/naturally occurring TRAC whereby amino acid T48 of TRAC, with reference to IMGT numbering, is replaced with C48.
  • the beta chain extracellular sequence may include a modification relative to the native/naturally occurring TRBC1 or TRBC2 whereby S57 of TRBC1 or TRBC2, with reference to IMGT numbering, is replaced with C57.
  • TCRs of the invention may be engineered to include mutations.
  • Methods for producing mutated high affinity TCR variants such as phage display and site directed mutagenesis and are known to those in the art (for example see WO 04/044004 and Li et al., Nat Biotechnol 2005 Mar;23(3):349- 54).
  • TCRs of the invention may also be may be labelled with an imaging compound, for example a label that is suitable for diagnostic purposes.
  • Such labelled high affinity TCRs are useful in a method for detecting a TCR ligand selected from CD1 -antigen complexes, bacterial superantigens, and MHC- peptide/superantigen complexes, which method comprises contacting the TCR ligand with a high affinity TCR (or a multimeric high affinity TCR complex) which is specific for the TCR ligand; and detecting binding to the TCR ligand.
  • a high affinity TCR or a multimeric high affinity TCR complex
  • multimeric high affinity TCR complexes such as those described in Zhu et al., J. Immunol.
  • fluorescent streptavidin (commercially available) can be used to provide a detectable label.
  • a fluorescently-labelled multimer is suitable for use in FACS analysis, for example to detect antigen presenting cells carrying the peptide for which the high affinity TCR is specific.
  • a TCR of the present invention may alternatively or additionally be associated with (e.g. covalently or otherwise linked to) a therapeutic agent which may be, for example, a toxic moiety for use in cell killing, or an immunostimulating agent such as an interleukin or a cytokine.
  • a multivalent high affinity TCR complex of the present invention may have enhanced binding capability for a TCR ligand compared to a non-multimeric wild-type or high affinity T cell receptor heterodimer.
  • the multivalent high affinity TCR complexes according to the invention are particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo, and are also useful as intermediates for the production of further multivalent high affinity TCR complexes having such uses.
  • the high affinity TCR or multivalent high affinity TCR complex may therefore be provided in a pharmaceutically acceptable formulation for use in vivo.
  • High affinity TCRs of the invention may be used in the production of soluble bi-specific reagents.
  • a preferred embodiment is a reagent which comprises a soluble TCR, fused via a linker to an anti- CD3 specific antibody fragment. Further details including how to produce such reagents are described in W010/133828.
  • the invention provides nucleic acid encoding the TCR of the invention, a TCR expression vector comprising nucleic acid encoding a TCR of the invention, as well as a cell harbouring such a vector.
  • the TCR may be encoded either in a single open reading frame or two distinct open reading frames.
  • one vector may encode both an alpha and a beta chain of a TCR of the invention.
  • a further aspect of the invention provides a cell displaying on its surface a TCR of the invention.
  • the cell may be a T cell.
  • T cells expressing the TCRs of the invention are suitable for use in adoptive therapy-based treatment of diseases such as cancers.
  • adoptive therapy see for example Rosenberg et al., Nat Rev Cancer. 2008 Apr;8(4):299-308.
  • the TCRs of the invention intended for use in adoptive therapy are generally glycosylated when expressed by the transfected T cells.
  • the glycosylation pattern of transfected TCRs may be modified by mutations of the transfected gene (Kuball J et al., J Exp Med. 2009 Feb 16;206(2):463-75).
  • the binding moiety of the invention may be an antibody.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced.
  • antibody includes antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanised antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic and any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.
  • a humanised antibody may be a modified antibody having the variable regions of a non-human, e.g. murine, antibody and the constant region of a human antibody. Methods for making humanised antibodies are described in, for example, US Patent No. 5225539. Examples of antibodies are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. A monoclonal antibody may be referred to herein as "mab".
  • an antibody for example a monoclonal antibody
  • recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody.
  • Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementary determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin (see, for instance, EP-A-184187, GB 2188638A or EP-A-239400).
  • CDRs complementary determining regions
  • a hybridoma (or other cell that produces antibodies) may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. et al., Nature.
  • Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g.
  • antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).
  • bispecific antibodies these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Hollinger & Winter, Curr Opin
  • bispecific antibody fragments e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain "Janusins" described in Traunecker et al., EMBO J. 1991 Dec; 10(12):3655-9). Bispecific diabodies, as opposed to bispecific whole antibodies, may also be useful because they can be readily constructed and expressed in E. coli.
  • Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
  • An "antigen binding domain" is the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope.
  • An antigen binding domain may be provided by one or more antibody variable domains.
  • An antigen binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • the binding moiety may be an antibody-like molecule that has been designed to specifically bind a peptide-MHC complex of the invention.
  • TCR-mimic antibodies such as, for example those described in WO2007143104 and Sergeeva et al., Blood. 201 1 Apr
  • binding moieties based on engineered protein scaffolds are derived from stable, soluble, natural protein structures which have been modified to provide a binding site for a target molecule of interest.
  • engineered protein scaffolds include, but are not limited to, affibodies, which are based on the Z-domain of staphylococcal protein A that provides a binding interface on two of its a-helices (Nygren, FEBS J. 2008 Jun;275(1 1 ):2668-76); anticalins, derived from lipocalins, that incorporate binding sites for small ligands at the open end of a beta-barrel fold (Skerra, FEBS J.
  • Engineered protein scaffolds are typically targeted to bind the same antigenic proteins as antibodies, and are potential therapeutic agents. They may act as inhibitors or antagonists, or as delivery vehicles to target molecules, such as toxins, to a specific tissue in vivo (Gebauer and Skerra, Curr Opin Chem Biol. 2009 Jun;13(3):245-55). Short peptides may also be used to bind a target protein.
  • Phylomers are natural structured peptides derived from bacterial genomes. Such peptides represent a diverse array of protein structural folds and can be used to inhibit/disrupt protein-protein interactions in vivo (Watt, Nat Biotechnol. 2006 Feb;24(2): 177-83)].
  • the invention further provides a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention for use in medicine.
  • the polypeptide, complex, nucleic acid, vector, cell or binding moiety may be used for in the treatment or prevention of cancer, in particular, Non-small cell lung cancer (squamous) and oesophageal cancer.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention together with a pharmaceutically acceptable carrier.
  • This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient). It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms. Suitable compositions and methods of administration are known to those skilled in the art, for example see, Johnson et al., Blood.
  • Cells in accordance with the invention will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a
  • T cells transfected with TCRs of the invention may be provided in pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be adapted for administration by any appropriate route such as a parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation or intranasal routes.
  • a parenteral including subcutaneous, intramuscular, or intravenous
  • enteral including oral or rectal
  • inhalation or intranasal routes Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
  • Dosages of the substances of the present invention can vary between wide limits, depending upon the disease or disorder to be treated (such as cancer, viral infection or autoimmune disease), the age and condition of the individual to be treated, etc.
  • a suitable dose range for a reagent comprising a soluble TCR fused to an anti- CD3 domain may be between 25 ng/kg and 50 pg/kg.
  • a physician will ultimately determine appropriate dosages to be used.
  • the polypeptide of the invention may be provided in the form of a vaccine composition.
  • the vaccine composition may be useful for the treatment or prevention of cancer. All such
  • compositions are encompassed in the present invention.
  • vaccines may take several forms (Schlom, J Natl Cancer Inst. 2012 Apr 18;104(8):599-613).
  • the peptide of the invention may be used directly to immunise patients (Salgaller, Cancer Res. 1996 Oct 15;56(20):4749-57 and Marchand, Int J Cancer. 1999 Jan 18;80(2):219-30).
  • the vaccine composition may include additional peptides such that the peptide of the invention is one of a mixture of peptides.
  • Adjuvants may be added to the vaccine composition to augment the immune response
  • the vaccine composition may take the form of an antigen presenting cell displaying the peptide of the invention in complex with MHC.
  • the antigen presenting cell is an immune cell, more preferably a dendritic cell.
  • the peptide may be pulsed onto the surface of the cell (Thurner, J Exp Med. 1999 Dec 6; 190(11 ): 1669-78), or nucleic acid encoding for the peptide of the invention may be introduced into dendritic cells (for example by electroporation. Van Tendeloo, Blood. 2001 Jul 1 ;98(1 ):49-56).
  • polypeptides, complexes, nucleic acid molecules, vectors, cells and binding moieties of the invention may be non-naturally occurring and/or purified and/or engineered and/or recombinant and/or isolated and/or synthetic.
  • the invention also provides a method of identifying a binding moiety that binds a complex of the invention, the method comprising contacting a candidate binding moiety with the complex and determining whether the candidate binding moiety binds the complex.
  • Methods to determine binding to polypeptide-MHC complexes are well known in the art. Preferred methods include, but are not limited to, surface plasmon resonance, or any other biosensor technique, ELISA, flow cytometry, chromatography, microscopy. Alternatively, or in addition, binding may be determined by functional assays in which a biological response is detected upon binding, for example, cytokine release or cell apoptosis.
  • the candidate binding moiety may be a binding moiety of the type already described, such as a TCR or an antibody.
  • T cells may be isolated from fresh blood obtained from volunteer donors. Such a method involves stimulating T cells using autologous DCs, followed by autologous B cells, pulsed with the polypeptide of the invention. Several round of stimulation may be carried out, for example three or four rounds. Activated T cells may then be tested for specificity by measuring cytokine release in the presence of T2 cells pulsed with the peptide of the invention (for example using an IFNy ELISpot assay). Activated cells may then be sorted by fluorescence-activated cell sorting (FACS) using labelled antibodies to detect intracellular cytokine production (e.g. IFNy), or expression of a cell surface marker (such as CD137).
  • FACS fluorescence-activated cell sorting
  • Sorted cells may be expanded and further validated, for example, by ELISpot assay and/or cytotoxicity against target cells and/or staining by peptide-MHC tetramer.
  • the TCR chains from validated clones may then be amplified by rapid amplification of cDNA ends (RACE) and sequenced.
  • the polypeptide MHC complex of the invention may be used for screening a library of TCRs or antibodies.
  • the production of antibody libraries using phage display is well known in the art, for example see Aitken, Antibody phage display: Methods and Protocols (2009, Humana, New York). Further details on TCR libraries can be found in WO04044004.
  • the polypeptide-MHC complex of the invention is used to screen a library of diverse TCRs displayed on the surface of phage particles.
  • the TCRs displayed by said library may contain non- natural mutations. Screening may involve panning the phage library with peptide-MHC complexes of the invention and subsequently isolating bound phage.
  • peptide-MHC complexes may be attached to a solid support, such as a magnet bead, or column matrix and phage bound peptide MHC complexes isolated, with a magnet, or by chromatography, respectively.
  • the panning steps may be repeated several times for example three or four times.
  • Isolated phage may be further expanded in E. coli cells.
  • Isolated phage particles may be tested for specific binding peptide-MHC complexes of the invention. Binding can be detected using techniques including, but not limited to, ELISA, or SPR for example using a BiaCore instrument.
  • the DNA sequence of the T cell receptor displayed by peptide-MHC binding phage can be further identified by standard PCR methods.
  • Figures F1 and F2 show the respective fragmentation spectra for the peptides of SEQ NOs: F1 and F2, eluted from cells. A table highlighting the matching ions is shown below each spectrum.
  • Example F1 Identification of target-derived peptides by Mass spectrometry
  • Immortalised cell lines obtained from commercial sources were maintained and expanded under standard conditions.
  • Class I HLA complexes were purified by immunoaffinity using commercially available anti-HLA antibodies BB7.1 (anti-HLA-B * 07), BB7.2 (anti-HLA-A * 02) and W6/32 (anti-Class 1 ). Briefly, cells were lysed in buffer containing non-ionic detergent NP-40 (0.5% v/v) at 5 x 10 7 cells per ml and incubated at 4°C for 1 h with agitation/mixing. Cell debris was removed by centrifugation and supernatant pre-cleared using proteinA-Sepharose.
  • Peptides were separated by high pressure liquid chromatography (HPLC) on a Dionex Ultimate 3000 system using a C18 column (Phenomenex). Peptides were loaded in 98% buffer A (0.1 % aqueous trifluoroacetic acid (TFA)) and 2% buffer B (0.1% TFA in acetonitrile). Peptides were eluted using a stepped gradient of B (2-60%) over 20 min. Fractions were collected at one minute intervals and lyophilised.
  • HPLC high pressure liquid chromatography
  • Peptides were analysed by nanoLCMS/MS using a Dionex Ultimate 3000 nanoLC coupled to either AB Sciex Triple TOF 5600 or Thermo Orbitrap Fusion mass spectrometers. Both machines were equipped with nanoelectrospray ion sources. Peptides were loaded onto an Acclaim PepMap 100 trap column (Dionex) and separated using an Acclaim PepMap RSLC column (Dionex). Peptides were loaded in mobile phase A (0.5% formic acid: water) and eluted using a gradient of buffer B (acetonitrile:0.5% formic acid) directly into the nanospray ionisation source.
  • buffer B acetonitrile:0.5% formic acid
  • IDA information dependent acquisition
  • Information acquired in these experiments was used to search the Uniprot database of human proteins for peptides consistent with the fragmentation patterns seen, using Protein pilot software (Ab Sciex) and PEAKS software (Bioinformatics solutions). Peptides identified are assigned a score by the software, based on the match between the observed and expected fragmentation patterns.
  • polypeptides set out in table F1 were detected by mass spec following extraction from cancer cell lines.
  • An example cell line from which the peptide was detected is indicated in the table along with the HLA antibody used for immunoaffinity purification.
  • Figures F1 -F2 show representative fragmentation patterns for the peptides of SEQ NOs: F1-F2 respectively. A table highlighting the matching ions is shown below each spectrum.
  • HLA-A*02 molecules HLA-A*02-heavy chain and HLA light-chain ( ⁇ 2 ⁇ ) were expressed separately in E. coli as inclusion bodies, using appropriate constructs.
  • HLA-A * 02-heavy chain additionally contained a C-terminal biotinylation tag which replaces the transmembrane and cytoplasmic domains (O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15).
  • E. coli cells were lysed and inclusion bodies processed to approximately 80% purity.
  • Inclusion bodies of ⁇ 2 ⁇ and heavy chain were denatured separately in 6 M guanidine-HCI, 50 mM Tris pH 8.1 , 100 mM NaCI, 10 mM DTT, 10 mM EDTA.
  • Refolding buffer was prepared containing 0.4 M L-Arginine, 100 mM Tris pH 8.1 , 3.7 mM cystamine dihydrochloride, 6.6 mM cysteamine hydrochloride and chilled to ⁇ 5°C.
  • Synthetic peptide dissolved in DMSO to a final concentration of 4mg/ml is added to the refold buffer at 4 mg/litre (final concentration). Then 30 mg/litre ⁇ 2 ⁇ followed by 30 mg/litre heavy chain (final concentrations) are added. Refolding was allowed to reach completion at 4 °C for at least 1 hour.
  • Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Two changes of buffer were necessary to reduce the ionic strength of the solution sufficiently.
  • the protein solution was then filtered through a 1.5 pm cellulose acetate filter and loaded onto a POROS 50HQ anion exchange column (8 ml bed volume). Protein was eluted with a linear 0-500 mM NaCI gradient in 10 mM Tris pH 8.1 using an Akta purifier (GE Healthcare). HLA-A * 02-peptide complex eluted at approximately 250 mM NaCI, and peak fractions were collected, a cocktail of protease inhibitors (Calbiochem) was added and the fractions were chilled on ice.
  • Biotinylation tagged pHLA molecules were buffer exchanged into 10 mM Tris pH 8.1 , 5 mM NaCI using a GE Healthcare fast desalting column equilibrated in the same buffer. Immediately upon elution, the protein-containing fractions were chilled on ice and protease inhibitor cocktail (Calbiochem) was added. Biotinylation reagents were then added: 1 mM biotin, 5 mM ATP (buffered to pH 8), 7.5 mM MgCI2, and 5 pg/ml BirA enzyme (purified according to O'Callaghan et al., (1999) Anal. Biochem. 266: 9-15). The mixture was then allowed to incubate at room temperature overnight.
  • the biotinylated pHLA-A * 02 molecules were purified using gel filtration chromatography.
  • a GE Healthcare Superdex 75 HR 10/30 column was pre-equilibrated with filtered PBS and 1 ml of the biotinylation reaction mixture was loaded and the column was developed with PBS at 0.5 ml/min using an Akta purifier (GE Healthcare).
  • Biotinylated pHLA-A * 02 molecules eluted as a single peak at approximately 15 ml. Fractions containing protein were pooled, chilled on ice, and protease inhibitor cocktail was added. Protein concentration was determined using a Coomassie-binding assay (PerBio) and aliquots of biotinylated pHLA-A * 02 molecules were stored frozen at -20 °C.
  • Such peptide-MHC complexes may be used in soluble form or may be immobilised through C terminal biotin moiety on to a solid support, to be used for the detection of T cells and T cell receptors which bind said complex.
  • Example F3 identification of TCRs that bind to a peptide-MHC complex of the invention
  • TCR isolation methods are known to those skilled in the art; for example, DNA sequences corresponding to TCR alpha and beta chains may be amplified from a T cell clone (obtained from volunteer blood donors) that is activated by cells presenting the peptide-HLA complex of interest.
  • T cell clone obtained from volunteer blood donors
  • Other suitable methods include isolation from TCR libraries (as described in WO2005/1 16646 and WO2005/116074 for example).
  • TCR alpha and beta chain sequences are expressed in E. coli as soluble TCRs, (WO2003020763; Boulter, et al., Protein Eng, 2003. 16: 707-711 ). Binding of the soluble TCRs to the complexes is analysed by surface plasmon resonance using a BiaCore 3000 instrument. Biotinylated peptide-HLA monomers are prepared as previously described (Example F2) and immobilized on to a streptavidin-coupled CM-5 sensor chip.
  • The[LS6] present invention also relates to novel peptides derived from Doublesex- and mab-3- related transcription factor A2 (DMRTA2), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules.
  • DRTA2 Doublesex- and mab-3- related transcription factor A2
  • binding moieties that bind to the peptides and/or complexes of the invention. Such moieties are useful for the development of immunotherapeutic reagents for the treatment of diseases such as cancer.
  • T cells are a key part of the cellular arm of the immune system. They specifically recognise peptide fragments that are derived from intracellular proteins and presented in complex with Major Histocompatibility Complex (MHC) molecules on the surface of antigen presenting cells (APCs).
  • MHC molecules are known as human leukocyte antigens (HLA), and both terms are used synonymously herein.
  • HLA human leukocyte antigens
  • MHC molecules have a binding groove in which the peptide fragments bind.
  • TCR T cell receptor
  • TAA-derived peptides presented by MHC molecules on tumour cells enables the development of novel immunotherapeutic reagents designed to specifically target and destroy said tumour cells.
  • Such reagents may be moieties that bind to the TAA-derived peptide and/or peptide-MHC complexes of the invention, and typically involve the induction of a T cell response.
  • such reagents may be based, exclusively, or in part, on T cells, or T cell receptors (TCRs), or antibodies.
  • TAA-derived peptides that are presented on the cell surface in complex with a given MHC, only a small number of such peptides, covering a limited number of cancer indications, have been identified to date.
  • TAAs suitable for the development of immunotherapeutic reagents must have limited or no expression in normal cells, to prevent off target activity. It is therefore desirable to provide additional tumour-specific TAA-derived peptides and MHC complexes thereof that can be used for the development of new cancer therapies.
  • DMRTA2 (also known as Doublesex- and mab-3-related transcription factor A2 or Doublesex- and mab-3-related transcription factor 5 and having Uniprot accession number Q96SC8) is a transcription factor with a possible role in sexual development (Ottolenghi et al. Genomics. 2002 Mar;79(3):333-43). DMRTA2 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from DMRTA2 that are presented on the cell surface in complex with HC. These peptides are particularly useful for the development of reagents that can target cells expressing DMRTA2 and for the treatment of cancers, including non small cell lung cancer small cell lung cancer and head and neck cancer.
  • the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: G1 -G8, or
  • polypeptide forms a complex with a Major Histocompatibility Complex (MHC) molecule.
  • MHC Major Histocompatibility Complex
  • polypeptides of the invention are presented by MHC on the surface of tumour cells. Accordingly, the polypeptides of the invention, as well as moieties that bind the polypeptide-MHC complexes, can be used to develop therapeutic reagents.
  • the skilled person can determine whether or not a given polypeptide forms a complex with an MHC molecule by determining whether the MHC can be refolded in the presence of the polypeptide using the process set out in Example G2. If the polypeptide does not form a complex with MHC then MHC will not refold. Refolding is commonly confirmed using an antibody that recognises MHC in a folded state only. Further details can be found in Garboczi et al., Proc Natl Acad Sci
  • polypeptides of the invention are from about 8 to about 16 amino acids in length, and are most preferably 8, 9, or 10 or 11 amino acids in length.
  • the polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: G1-G8.
  • the amino acid residues comprising the polypeptides of the invention may be chemically modified. Examples of chemical modifications include those corresponding to post translational modifications for example phosphorylation, acetylation and deamidation (Engelhard et al., Curr Opin Immunol. 2006 Feb;18(1 ):92-7).
  • Amino acid substitution means that an amino acid residue is substituted for a replacement amino acid residue at the same position.
  • Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are immediately adjacent one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent another inserted amino acid residue.
  • One, two or three amino acids may be deleted from the sequence of SEQ NOs: G1-G8. Each deletion can take place at any position of SEQ NOs: G1-G8.
  • the polypeptide of the invention may comprise one, two or three additional amino acids at the C-terminal end and/or at the N-terminal end of the sequence of SEQ NOs: G1- G8.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: G1-G8 with the exception of one amino acid substitution and one amino acid insertion, one amino acid substitution and one amino acid deletion, or one amino acid insertion and one amino acid deletion.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: G1-G8, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.
  • Inserted amino acids and replacement amino acids may be naturally occurring amino acids or may be non-naturally occurring amino acids and, for example, may contain a non-natural side chain.
  • Such altered peptide ligands are discussed further in Douat-Casassus et al., J. Med. Chem, 2007 Apr 5;50(7): 1598-609 and Hoppes et ai, J. Immunol 2014 Nov 15;193(10):4803-13 and references therein). If more than one amino acid residue is substituted and/or inserted, the
  • replacement/inserted amino acid residues may be the same as each other or different from one another. Each replacement amino acid may have a different side chain to the amino acid being replaced.
  • polypeptides of the invention bind to MHC in the peptide binding groove of the MHC molecule.
  • the amino acid modifications described above will not impair the ability of the peptide to bind MHC.
  • the amino acid modifications improve the ability of the peptide to bind MHC.
  • mutations may be made at positions which anchor the peptide to MHC. Such anchor positions and the preferred residues at these locations are known in the art, particularly for peptides which bind HLA-A * 02 (see, e. g. Parkhurst et al., J. Immunol. 1996 Sep 15;157(6):2539-48 and Parker et al. J Immunol. 1992 Dec 1 ; 149(1 1 ):3580-7).
  • a polypeptide of the invention may be used to illicit an immune response. If this is the case, it is important that the immune response is specific to the intended target in order to avoid the risk of unwanted side effects that may be associated with an "off target" immune response. Therefore, it is preferred that the amino acid sequence of a polypeptide of the invention does not match the amino acid sequence of a peptide from any other protein(s), in particular, that of another human protein.
  • a person of skill in the art would understand how to search a database of known protein sequences to ascertain whether a polypeptide according to the invention is present in another protein.
  • Polypeptides of the invention can be synthesised easily by Merrifield synthesis, also known as solid phase synthesis, or any other peptide synthesis methodology.
  • GMP grade polypeptide is produced by solid-phase synthesis techniques by Multiple Peptide Systems, San Diego, CA.
  • the peptide may be recombinantly produced, if so desired, in accordance with methods known in the art.
  • Such methods typically involve the use of a vector comprising a nucleic acid sequence encoding the polypeptide to be expressed, to express the polypeptide in vivo; for example, in bacteria, yeast, insect or mammalian cells.
  • in vitro cell-free systems may be used. Such systems are known in the art and are commercially available for example from Life
  • polypeptides may be isolated and/or may be provided in substantially pure form. For example, they may be provided in a form which is substantially free of other polypeptides or proteins.
  • the invention provides a complex of the polypeptide of the first aspect and an MHC molecule.
  • the polypeptide is bound to the peptide binding groove of the MHC molecule.
  • the MHC molecule may be MHC class I.
  • the MHC class I molecule may be selected from HLA-A * 02, HLA-A * 01 , HLA-A * 03, HLA-A11 , HLA-A23, HLA-A24, HLA-B * 07, HLA-B * 08, HLA- B40, HLA-B44, HLA-B15, HLA-C * 04, HLA * C * 03 HLA-CW.
  • HLA-A * 02 HLA-A * 01
  • HLA-A * 03 HLA-A11
  • HLA-A23, HLA-A24 HLA-B * 07, HLA-B * 08, HLA- B40, HLA-B44, HLA-B15, HLA-C * 04, HLA * C * 03 HLA-CW.
  • the complex of the invention may be isolated and/or in a substantially pure form.
  • the complex may be provided in a form which is substantially free of other polypeptides or proteins.
  • HC molecule includes recombinant MHC molecules, non-naturally occurring MHC molecules and functionally equivalent fragments of MHC, including derivatives or variants thereof, provided that peptide binding is retained.
  • MHC molecules may be fused to a therapeutic moiety, attached to a solid support, in soluble form, and/or in multimeric form.
  • MHC molecules with which polypeptides of the invention can form a complex are known in the art. Suitable methods include, but are not limited to, expression and purification from E. co// cells or insect cells. A suitable method is provided in Example G2 herein. Alternatively, MHC molecules may be produced synthetically, or using cell free systems. Polypeptides and/or polypeptide-MHC complexes of the invention may be associated with a moiety capable of eliciting a therapeutic effect. Such a moiety may be a carrier protein which is known to be immunogenic. KLH (keyhole limpet hemocyanin) is an example of a suitable carrier protein used in vaccine compositions.
  • KLH keyhole limpet hemocyanin
  • the polypeptides and/or polypeptide-MHC complexes of the invention may be associated with a fusion partner. Fusion partners may be used for detection purposes, or for attaching said polypeptide or MHC to a solid support, or for MHC oligomerisation.
  • the MHC complexes may incorporate a biotinylation site to which biotin can be added, for example, using the BirA enzyme (O'Callaghan et al., 1999 Jan 1 ;266(1 ):9-15).
  • Other suitable fusion partners include, but are not limited to, fluorescent, or luminescent labels, radiolabels, nucleic acid probes and contrast reagents, antibodies, or enzymes that produce a detectable product. Detection methods may include flow cytometry, microscopy, electrophoresis or scintillation counting.
  • Polypeptide-MHC complexes of the invention may be provided in soluble form, or may be immobilised by attachment to a suitable solid support.
  • suitable solid supports include, but are not limited to, a bead, a membrane, sepharose, a magnetic bead, a plate, a tube, a column.
  • Polypeptide-MHC complexes may be attached to an ELISA plate, a magnetic bead, or a surface plasmon reasonance biosensor chip.
  • Methods of attaching peptide-MHC complexes to a solid support are known to the skilled person, and include, for example, using an affinity binding pair, e.g. biotin and streptavidin, or antibodies and antigens.
  • peptide-MHC complexes are labelled with biotin and attached to streptavidin-coated surfaces.
  • Polypeptide-MHC complexes of the invention may be in multimeric form, for example, dimeric, or tetrameric, or pentameric, or octomeric, or greater. Examples of suitable methods for the production of multimeric peptide MHC complexes are described in Greten et al., Clin. Diagn. Lab. Immunol. 2002 Mar;9(2):216-20 and references therein.
  • polypeptide-MHC multimers may be produced using peptide-MHC tagged with a biotin residue and complexed through fluorescent labelled streptavidin.
  • multimeric polypeptide-MHC complexes may be formed by using immunoglobulin as a molecular scaffold.
  • MHC molecules are fused with the constant region of an immunoglobulin heavy chain separated by a short amino acid linker.
  • Polypeptide-MHC multimers have also been produced using carrier molecules such as dextran (WO02072631 ). Multimeric peptide MHC complexes can be useful for improving the detection of binding moieties, such as T cell receptors, which bind said complex, because of avidity effects.
  • the polypeptides of the invention may be presented on the surface of a cell in complex with MHC.
  • the invention also provides a cell presenting on its surface a complex of the invention.
  • a cell may be a mammalian cell, preferably a cell of the immune system, and in particular a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • Other preferred cells include T2 cells (Hosken, et al., Science. 1990 Apr 20;248(4953):367-70).
  • Cells presenting the polypeptide or complex of the invention may be isolated, preferably in the form of a population, or provided in a substantially pure form.
  • Said cells may not naturally present the complex of the invention, or alternatively said cells may present the complex at a level higher than they would in nature.
  • Such cells may be obtained by pulsing said cells with the polypeptide of the invention. Pulsing involves incubating the cells with the polypeptide for several hours using polypeptide concentrations typically ranging from 10 "5 to 10 "12 M. Said cells may additionally be transduced with HLA-A * 02 molecules to further induce presentation of the peptide. Cells may be produced recombinantly. Cells presenting polypeptides of the invention may be used to isolate T cells and T cell receptors (TCRs) which are activated by, or bind to, said cells, as described in more detail below.
  • TCRs T cell receptors
  • the invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide of the first aspect of the invention.
  • the nucleic acid may be cDNA.
  • the nucleic acid molecule may consist essentially of a nucleic acid sequence encoding the polypeptide of the first aspect of the invention or may encode only the peptide of the invention, i.e. encode no other polypeptide.
  • Such a nucleic acid molecule can be synthesised in accordance with methods known in the art. Due to the degeneracy of the genetic code, one of ordinary skill in the art will appreciate that nucleic acid molecules of different nucleotide sequence can encode the same amino acid sequence.
  • the invention provides a vector comprising a nucleic acid sequence according to the third aspect of the invention.
  • the vector may include, in addition to a nucleic acid sequence encoding only a polypeptide of the invention, one or more additional nucleic acid sequences encoding one or more additional polypeptides. Such additional polypeptides may, once expressed, be fused to the N-terminus or the C-terminus of the polypeptide of the invention.
  • the vector includes a nucleic acid sequence encoding a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • Suitable vectors are known in the art as is vector construction, including the selection of promoters and other regulatory elements, such as enhancer elements.
  • the vector utilised in the context of the present invention desirably comprises sequences appropriate for introduction into cells.
  • the vector may be an expression vector, a vector in which the coding sequence of the polypeptide is under the control of its own cis-acting regulatory elements, a vector designed to facilitate gene integration or gene replacement in host cells, and the like.
  • the term "vector” encompasses a DNA molecule, such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • a DNA molecule such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • Appropriate phage and viral vectors include, but are not limited to, lambda (X) bacteriophage, EMBL bacteriophage, simian virus 40, bovine papilloma virus, Epstein-Barr virus, adenovirus, herpes virus, vaccinia virus, Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, lentivirus and Rous sarcoma virus.
  • the invention provides a cell comprising the vector of the fourth aspect of the invention.
  • the cell may be an antigen presenting cell and is preferably a cell of the immune system.
  • the cell may be a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • the cell may be a mammalian cell.
  • Polypeptides and complexes of the invention can be used to identify and/or isolate binding moieties that bind specifically to the polypeptide and/or the complex of the invention.
  • binding moieties may be used as immunotherapeutic reagents and may include antibodies and TCRs.
  • the invention provides a binding moiety that binds the polypeptide of the invention.
  • the binding moiety binds the polypeptide when said polypeptide is in complex with MHC.
  • the binding moiety may bind partially to the MHC, provided that it also binds to the polypeptide.
  • the binding moiety may bind only the polypeptide, and that binding may be specific.
  • the binding moiety may bind only the peptide MHC complex and that binding may be specific.
  • TCR T cell receptor
  • IMGT International Immunogenetics
  • the TCRs of the invention may be in any format known to those in the art.
  • the TCRs may be ⁇ heterodimers, or they may be in single chain format (such as those described in W09918129).
  • Single chain TCRs include ⁇ TCR polypeptides of the type: Va-L- ⁇ , ⁇ -L-Va, Va- Ca-L- ⁇ , ⁇ - ⁇ - ⁇ ⁇ -0 ⁇ or Va- Ca - ⁇ / ⁇ - ⁇ , optionally in the reverse orientation, wherein Va and ⁇ are TCR a and ⁇ variable regions respectively, Ca and C are TCR a and ⁇ constant regions respectively, and L is a linker sequence.
  • the TCR may be in a soluble form (i.e. having no transmembrane or cytoplasmic domains), or may contain full length alpha and beta chains.
  • the TCR may be provided on the surface of a cell, such as a T cell.
  • the alpha and/or beta chain constant domain may be truncated relative to the native/naturally occurring TRAC/TRBC sequences.
  • the TRAC/TRBC may contain modifications.
  • the alpha chain extracellular sequence may include a modification relative to the native/naturally occurring TRAC whereby amino acid T48 of TRAC, with reference to IMGT numbering, is replaced with C48.
  • the beta chain extracellular sequence may include a modification relative to the native/naturally occurring TRBC1 or TRBC2 whereby S57 of TRBC1 or TRBC2, with reference to IMGT numbering, is replaced with C57.
  • TCRs of the invention may be engineered to include mutations.
  • Methods for producing mutated high affinity TCR variants such as phage display and site directed mutagenesis and are known to those in the art (for example see WO 04/044004 and Li et al., Nat Biotechnol 2005 Mar;23(3):349- 54).
  • TCRs of the invention may also be may be labelled with an imaging compound, for example a label that is suitable for diagnostic purposes.
  • Such labelled high affinity TCRs are useful in a method for detecting a TCR ligand selected from CD1 -antigen complexes, bacterial superantigens, and MHC- peptide/superantigen complexes, which method comprises contacting the TCR ligand with a high affinity TCR (or a multimeric high affinity TCR complex) which is specific for the TCR ligand; and detecting binding to the TCR ligand.
  • a high affinity TCR or a multimeric high affinity TCR complex
  • multimeric high affinity TCR complexes such as those described in Zhu et ai, J. Immunol.
  • fluorescent streptavidin (commercially available) can be used to provide a detectable label.
  • a fluorescently-labelled multimer is suitable for use in FACS analysis, for example to detect antigen presenting cells carrying the peptide for which the high affinity TCR is specific.
  • a TCR of the present invention may alternatively or additionally be associated with (e.g. covalently or otherwise linked to) a therapeutic agent which may be, for example, a toxic moiety for use in cell killing, or an immunostimulating agent such as an interleukin or a cytokine.
  • a multivalent high affinity TCR complex of the present invention may have enhanced binding capability for a TCR ligand compared to a non-multimeric wild-type or high affinity T cell receptor heterodimer.
  • the multivalent high affinity TCR complexes according to the invention are particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo, and are also useful as intermediates for the production of further multivalent high affinity TCR complexes having such uses.
  • the high affinity TCR or multivalent high affinity TCR complex may therefore be provided in a pharmaceutically acceptable formulation for use in vivo.
  • High affinity TCRs of the invention may be used in the production of soluble bi-specific reagents.
  • a preferred embodiment is a reagent which comprises a soluble TCR, fused via a linker to an anti- CD3 specific antibody fragment. Further details including how to produce such reagents are described in W010/133828.
  • the invention provides nucleic acid encoding the TCR of the invention, a TCR expression vector comprising nucleic acid encoding a TCR of the invention, as well as a cell harbouring such a vector.
  • the TCR may be encoded either in a single open reading frame or two distinct open reading frames.
  • one vector may encode both an alpha and a beta chain of a TCR of the invention.
  • a further aspect of the invention provides a cell displaying on its surface a TCR of the invention.
  • the cell may be a T cell.
  • T cells expressing the TCRs of the invention are suitable for use in adoptive therapy-based treatment of diseases such as cancers.
  • adoptive therapy can be carried out (see for example Rosenberg et al., Nat Rev Cancer. 2008 Apr;8(4):299-308).
  • the TCRs of the invention intended for use in adoptive therapy are generally glycosylated when expressed by the transfected T cells.
  • the glycosylation pattern of transfected TCRs may be modified by mutations of the transfected gene (Kuball J et al., J Exp Med. 2009 Feb 16;206(2):463-75).
  • the binding moiety of the invention may be an antibody.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced.
  • antibody includes antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanised antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic and any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.
  • a humanised antibody may be a modified antibody having the variable regions of a non-human, e.g. murine, antibody and the constant region of a human antibody. Methods for making humanised antibodies are described in, for example, US Patent No. 5225539. Examples of antibodies are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. A monoclonal antibody may be referred to herein as "mab".
  • an antibody for example a monoclonal antibody
  • recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody.
  • Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementary determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin (see, for instance, EP-A-184187, GB 2188638A or EP-A-239400).
  • CDRs complementary determining regions
  • a hybridoma (or other cell that produces antibodies) may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. e£ al., Nature.
  • Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g.
  • antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).
  • bispecific antibodies these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Hollinger & Winter, Curr Opin
  • bispecific antibody fragments e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain "Janusins" described in Traunecker et al., EMBO J. 1991 Dec; 10(12):3655-9). Bispecific diabodies, as opposed to bispecific whole antibodies, may also be useful because they can be readily constructed and expressed in E. coli.
  • Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
  • An "antigen binding domain" is the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope.
  • An antigen binding domain may be provided by one or more antibody variable domains.
  • An antigen binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • the binding moiety may be an antibody-like molecule that has been designed to specifically bind a peptide-MHC complex of the invention.
  • TCR-mimic antibodies such as, for example those described in WO2007143104 and Sergeeva ef al., Blood. 201 1 Apr
  • binding moieties based on engineered protein scaffolds are derived from stable, soluble, natural protein structures which have been modified to provide a binding site for a target molecule of interest.
  • engineered protein scaffolds include, but are not limited to, affibodies, which are based on the Z-domain of staphylococcal protein A that provides a binding interface on two of its a-helices (Nygren, FEBS J. 2008 Jun;275(1 1 ):2668-76); anticalins, derived from lipocalins, that incorporate binding sites for small ligands at the open end of a beta-barrel fold (Skerra, FEBS J.
  • Engineered protein scaffolds are typically targeted to bind the same antigenic proteins as antibodies, and are potential therapeutic agents. They may act as inhibitors or antagonists, or as delivery vehicles to target molecules, such as toxins, to a specific tissue in vivo (Gebauer and Skerra, Curr Opin Chem Biol. 2009 Jun;13(3):245-55). Short peptides may also be used to bind a target protein.
  • Phylomers are natural structured peptides derived from bacterial genomes. Such peptides represent a diverse array of protein structural folds and can be used to inhibit/disrupt protein-protein interactions in vivo (Watt, Nat Biotechnol. 2006 Feb;24(2): 177-83)].
  • the invention further provides a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention for use in medicine.
  • the polypeptide, complex, nucleic acid, vector, cell or binding moiety may be used for in the treatment or prevention of cancer, in particular, non small cell lung cancer small cell lung cancer and head and neck cancer.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention together with a pharmaceutically acceptable carrier.
  • This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient). It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms. Suitable compositions and methods of administration are known to those skilled in the art, for example see, Johnson et a/., Blood.
  • Cells in accordance with the invention will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a
  • T cells transfected with TCRs of the invention may be provided in pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be adapted for administration by any appropriate route such as a parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation or intranasal routes.
  • Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
  • Dosages of the substances of the present invention can vary between wide limits, depending upon the disease or disorder to be treated (such as cancer, viral infection or autoimmune disease), the age and condition of the individual to be treated, etc.
  • a suitable dose range for a reagent comprising a soluble TCR fused to an anti- CD3 domain may be between 25 ng/kg and 50 Mg/kg. A physician will ultimately determine appropriate dosages to be used.
  • the polypeptide of the invention may be provided in the form of a vaccine composition.
  • the vaccine composition may be useful for the treatment or prevention of cancer. All such
  • compositions are encompassed in the present invention.
  • vaccines may take several forms (Schlom, J Natl Cancer Inst. 2012 Apr 18;104(8):599-613).
  • the peptide of the invention may be used directly to immunise patients (Salgaller, Cancer Res. 1996 Oct 15;56(20):4749-57 and Marchand, Int J Cancer. 1999 Jan 18;80(2):219-30).
  • the vaccine composition may include additional peptides such that the peptide of the invention is one of a mixture of peptides.
  • Adjuvants may be added to the vaccine composition to augment the immune response
  • the vaccine composition may take the form of an antigen presenting cell displaying the peptide of the invention in complex with MHC.
  • the antigen presenting cell is an immune cell, more preferably a dendritic cell.
  • the peptide may be pulsed onto the surface of the cell (Thurner, J Exp Med. 1999 Dec 6; 190(11 ): 1669-78), or nucleic acid encoding for the peptide of the invention may be introduced into dendritic cells (for example by electroporation. Van Tendeloo, Blood. 2001 Jul 1 ;98(1 ):49-56).
  • polypeptides, complexes, nucleic acid molecules, vectors, cells and binding moieties of the invention may be non-naturally occurring and/or purified and/or engineered and/or recombinant and/or isolated and/or synthetic.
  • the invention also provides a method of identifying a binding moiety that binds a complex of the invention, the method comprising contacting a candidate binding moiety with the complex and determining whether the candidate binding moiety binds the complex.
  • Methods to determine binding to polypeptide-MHC complexes are well known in the art. Preferred methods include, but are not limited to, surface plasmon resonance, or any other biosensor technique, ELISA, flow cytometry, chromatography, microscopy. Alternatively, or in addition, binding may be determined by functional assays in which a biological response is detected upon binding, for example, cytokine release or cell apoptosis.
  • the candidate binding moiety may be a binding moiety of the type already described, such as a TCR or an antibody.
  • T cells may be isolated from fresh blood obtained from volunteer donors. Such a method involves stimulating T cells using autologous DCs, followed by autologous B cells, pulsed with the polypeptide of the invention. Several round of stimulation may be carried out, for example three or four rounds. Activated T cells may then be tested for specificity by measuring cytokine release in the presence of T2 cells pulsed with the peptide of the invention (for example using an IFNy ELISpot assay). Activated cells may then be sorted by fluorescence-activated cell sorting (FACS) using labelled antibodies to detect intracellular cytokine production (e.g.
  • FACS fluorescence-activated cell sorting
  • Sorted cells may be expanded and further validated, for example, by ELISpot assay and/or cytotoxicity against target cells and/or staining by peptide-MHC tetramer.
  • the TCR chains from validated clones may then be amplified by rapid amplification of cDNA ends (RACE) and sequenced.
  • the polypeptide MHC complex of the invention may be used for screening a library of TCRs or antibodies.
  • the production of antibody libraries using phage display is well known in the art, for example see Aitken, Antibody phage display: Methods and Protocols (2009, Humana, New York). Further details on TCR libraries can be found in WO04044004.
  • the polypeptide-MHC complex of the invention is used to screen a library of diverse TCRs displayed on the surface of phage particles.
  • the TCRs displayed by said library may contain non- natural mutations. Screening may involve panning the phage library with peptide-MHC complexes of the invention and subsequently isolating bound phage.
  • peptide-MHC complexes may be attached to a solid support, such as a magnet bead, or column matrix and phage bound peptide MHC complexes isolated, with a magnet, or by chromatography, respectively.
  • the panning steps may be repeated several times for example three or four times.
  • Isolated phage may be further expanded in E. coli cells.
  • Isolated phage particles may be tested for specific binding peptide-MHC complexes of the invention. Binding can be detected using techniques including, but not limited to, ELISA, or SPR for example using a BiaCore instrument.
  • the DNA sequence of the T cell receptor displayed by peptide-MHC binding phage can be further identified by standard PCR methods. Preferred or optional features of each aspect of the invention are as for each of the other aspects mutatis mutandis.
  • Figures G1 to G8 show the respective fragmentation spectra for the peptides of SEQ NOs: G1 to G8, eluted from cells. A table highlighting the matching ions is shown below each spectrum. Examples
  • Example G1 Identification of target-derived peptides by Mass spectrometry Presentation of HLA-restricted peptides derived from DMRTA2 on the surface of tumour cell lines was investigated using mass spectrometry.
  • Immortalised cell lines obtained from commercial sources were maintained and expanded under standard conditions.
  • Class I HLA complexes were purified by immunoaffinity using commercially available anti-HLA antibodies BB7.1 (anti-HLA-B * 07), BB7.2 (anti-HLA-A * 02) and W6/32 (anti-Class 1 ). Briefly, cells were lysed in buffer containing non-ionic detergent NP-40 (0.5% v/v) at 5 x 10 7 cells per ml and incubated at 4°C for 1 h with agitation/mixing. Cell debris was removed by centrifugation and supernatant pre-cleared using proteinA-Sepharose. Supernatant was passed over 5 ml of resin containing 8 mg of anti-HLA antibody immobilised on a proteinA-Sepharose scaffold.
  • Peptides were separated by high pressure liquid chromatography (HPLC) on a Dionex Ultimate 3000 system using a C18 column (Phenomenex). Peptides were loaded in 98% buffer A (0.1% aqueous trifluoroacetic acid (TFA)) and 2% buffer B (0.1% TFA in acetonitrile). Peptides were eluted using a stepped gradient of B (2-60%) over 20 min. Fractions were collected at one minute intervals and lyophilised.
  • HPLC high pressure liquid chromatography
  • Peptides were analysed by nanoLCMS/MS using a Dionex Ultimate 3000 nanoLC coupled to either AB Sciex Triple TOF 5600 or Thermo Orbitrap Fusion mass spectrometers. Both machines were equipped with nanoelectrospray ion sources. Peptides were loaded onto an Acclaim PepMap 100 trap column (Dionex) and separated using an Acclaim PepMap RSLC column (Dionex). Peptides were loaded in mobile phase A (0.5% formic acid: water) and eluted using a gradient of buffer B (acetonitrile:0.5% formic acid) directly into the nanospray ionisation source.
  • buffer B acetonitrile:0.5% formic acid
  • IDA information dependent acquisition
  • Information acquired in these experiments was used to search the Uniprot database of human proteins for peptides consistent with the fragmentation patterns seen, using Protein pilot software (Ab Sciex) and PEAKS software (Bioinformatics solutions). Peptides identified are assigned a score by the software, based on the match between the observed and expected fragmentation patterns.
  • polypeptides set out in table G1 corresponding to SEQ NOs: G1 -G8, were detected by mass spec following extraction from cancer cell lines.
  • An example cell line from which the peptide was detected is indicated in the table along with the HLA antibody used for immunoaffinity purification.
  • Figures G1 -G8 show representative fragmentation patterns for the peptides of SEQ NOs: G1-G8 respectively. A table highlighting the matching ions is shown below each spectrum.
  • Example G2 Preparation of recombinant peptide-HLA complexes The following describes a suitable method for the preparation of soluble recombinant HLA loaded with TAA peptide.
  • HLA-A*02 molecules HLA-A*02-heavy chain and HLA light-chain ( ⁇ 2 ⁇ ) were expressed separately in E. coli as inclusion bodies, using appropriate constructs.
  • HLA-A * 02-heavy chain additionally contained a C-terminal biotinylation tag which replaces the transmembrane and cytoplasmic domains (O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15).
  • E. coli cells were lysed and inclusion bodies processed to approximately 80% purity.
  • Inclusion bodies of ⁇ 2 ⁇ and heavy chain were denatured separately in 6 M guanidine-HCI, 50 mM Tris pH 8.1 , 100 mM NaCI, 10 mM DTT, 10 mM EDTA.
  • Refolding buffer was prepared containing 0.4 M L-Arginine, 100 mM Tris pH 8.1 , 3.7 mM cystamine dihydrochloride, 6.6 mM cysteamine hydrochloride and chilled to ⁇ 5°C.
  • Synthetic peptide dissolved in DMSO to a final concentration of 4mg/ml is added to the refold buffer at 4 mg/litre (final concentration). Then 30 mg/litre ⁇ 2 ⁇ followed by 30 mg/litre heavy chain (final concentrations) are added.
  • Refolding was allowed to reach completion at 4 °C for at least 1 hour.
  • Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Two changes of buffer were necessary to reduce the ionic strength of the solution sufficiently.
  • the protein solution was then filtered through a 1.5 ⁇ cellulose acetate filter and loaded onto a POROS 50HQ anion exchange column (8 ml bed volume). Protein was eluted with a linear 0-500 mM NaCI gradient in 10 mM Tris pH 8.1 using an Akta purifier (GE Healthcare). HLA-A * 02-peptide complex eluted at approximately 250 mM NaCI, and peak fractions were collected, a cocktail of protease inhibitors (Calbiochem) was added and the fractions were chilled on ice.
  • Biotinylation tagged pHLA molecules were buffer exchanged into 10 mM Tris pH 8.1 , 5 mM NaCI using a GE Healthcare fast desalting column equilibrated in the same buffer. Immediately upon elution, the protein-containing fractions were chilled on ice and protease inhibitor cocktail (Calbiochem) was added. Biotinylation reagents were then added: 1 mM biotin, 5 mM ATP (buffered to pH 8), 7.5 mM MgCl2, and 5 pg/ml BirA enzyme (purified according to O'Callaghan et al., (1999) Anal. Biochem. 266: 9-15). The mixture was then allowed to incubate at room temperature overnight.
  • the biotinylated pHLA-A * 02 molecules were purified using gel filtration chromatography.
  • a GE Healthcare Superdex 75 HR 10/30 column was pre-equilibrated with filtered PBS and 1 ml of the biotinylation reaction mixture was loaded and the column was developed with PBS at 0.5 ml/min using an Akta purifier (GE Healthcare).
  • Biotinylated pHLA-A * 02 molecules eluted as a single peak at approximately 15 ml. Fractions containing protein were pooled, chilled on ice, and protease inhibitor cocktail was added.
  • Protein concentration was determined using a Coomassie-binding assay (PerBio) and aliquots of biotinylated pHLA-A * 02 molecules were stored frozen at -20 °C.
  • Such peptide-MHC complexes may be used in soluble form or may be immobilised through C terminal biotin moiety on to a solid support, to be used for the detection of T cells and T cell receptors which bind said complex.
  • TCR isolation methods are known to those skilled in the art; for example, DNA sequences corresponding to TCR alpha and beta chains may be amplified from a T cell clone (obtained from volunteer blood donors) that is activated by cells presenting the peptide-HLA complex of interest.
  • T cell clone obtained from volunteer blood donors
  • Other suitable methods include isolation from TCR libraries (as described in WO2005/1 16646 and WO2005/116074 for example).
  • TCR alpha and beta chain sequences are expressed in E. coli as soluble TCRs, (WO2003020763; Boulter, et al., Protein Eng, 2003. 16: 707-711 ). Binding of the soluble TCRs to the complexes is analysed by surface plasmon resonance using a BiaCore 3000 instrument. Biotinylated peptide-HLA monomers are prepared as previously described (Example G2) and immobilized on to a streptavidin-coupled CM-5 sensor chip.
  • The[LS7] present invention also relates to novel peptides derived from DNA
  • nucleotidylexotransferase DNTT
  • binding moieties that bind to the peptides and/or complexes of the invention. Such moieties are useful for the development of immunotherapeutic reagents for the treatment of diseases such as cancer.
  • T cells are a key part of the cellular arm of the immune system. They specifically recognise peptide fragments that are derived from intracellular proteins and presented in complex with Major Histocompatibility Complex (MHC) molecules on the surface of antigen presenting cells (APCs).
  • MHC Major Histocompatibility Complex
  • MHC molecules are known as human leukocyte antigens (HLA), and both terms are used synonymously herein.
  • HLA human leukocyte antigens
  • MHC molecules have a binding groove in which the peptide fragments bind. Recognition of particular peptide-MHC antigens is mediated by a corresponding T cell receptor (TCR).
  • TCR T cell receptor
  • Tumour cells express various tumour associated antigens (TAA) and peptides derived from these antigens are frequently displayed on the tumour cell surface. Detection of a MHC class I- presented TAA-derived peptide by a CD8+ T cell bearing the corresponding T cell receptor, leads to targeted killing of the tumour cell.
  • TAA tumour associated antigens
  • tumour cells often escape detection.
  • TAA-derived peptides presented by MHC molecules on tumour cells enables the development of novel immunotherapeutic reagents designed to specifically target and destroy said tumour cells.
  • Such reagents may be moieties that bind to the TAA-derived peptide and/or peptide-MHC complexes of the invention, and typically involve the induction of a T cell response.
  • such reagents may be based, exclusively, or in part, on T cells, or T cell receptors (TCRs), or antibodies.
  • TAA-derived peptides that are presented on the cell surface in complex with a given MHC, only a small number of such peptides, covering a limited number of cancer indications, have been identified to date.
  • TAAs suitable for the development of immunotherapeutic reagents must have limited or no expression in normal cells, to prevent off target activity. It is therefore desirable to provide additional tumour-specific TAA-derived peptides and MHC complexes thereof that can be used for the development of new cancer therapies.
  • DNTT also known as DNA nucleotidylexotransferase or terminal deoxynucleotidyltransferase or terminal addition enzyme and having Uniprot accession number P04053
  • DNTT is a template- independent DNA polymerase. Expression of DNTT is associated with cancer (Kolhe et al. Int J Clin Exp Pathol. 2013;6(2): 142-7). DNTT is an ideal target for immunotherapeutic applications.
  • the inventors have found novel peptides derived from DNTT that are presented on the cell surface in complex with MHC. These peptides are particularly useful for the development of reagents that can targets cells expressing DNTT and for the treatment of cancers, including acute myeloid leukemia.
  • the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: H1 -H18, or
  • polypeptide forms a complex with a Major Histocompatibility Complex (MHC) molecule.
  • MHC Major Histocompatibility Complex
  • polypeptides of the invention are presented by MHC on the surface of tumour cells. Accordingly, the polypeptides of the invention, as well as moieties that bind the polypeptide-MHC complexes, can be used to develop therapeutic reagents.
  • polypeptides of the invention are from about 8 to about 16 amino acids in length, and are most preferably 8, 9, or 10 or 11 amino acids in length.
  • polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: H1-H18.
  • amino acid residues comprising the polypeptides of the invention may be chemically modified.
  • chemical modifications include those corresponding to post translational modifications for example phosphorylation, acetylation and deamidation (Engelhard et al., Curr Opin Immunol. 2006 Feb;18(1 ):92-7).
  • Amino acid substitution means that an amino acid residue is substituted for a replacement amino acid residue at the same position.
  • Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are immediately adjacent one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent another inserted amino acid residue.
  • One, two or three amino acids may be deleted from the sequence of SEQ NOs: H1-H18. Each deletion can take place at any position of SEQ NOs: H1 -H18.
  • the polypeptide of the invention may comprise one, two or three additional amino acids at the C-terminal end and/or at the N-terminal end of the sequence of SEQ NOs: H1- H18.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: H1-H18 with the exception of one amino acid substitution and one amino acid insertion, one amino acid substitution and one amino acid deletion, or one amino acid insertion and one amino acid deletion.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: H1-H18, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.
  • Inserted amino acids and replacement amino acids may be naturally occurring amino acids or may be non-naturally occurring amino acids and, for example, may contain a non-natural side chain.
  • Such altered peptide ligands are discussed further in Douat-Casassus et a/., J. Med. Chem, 2007 Apr 5;50(7): 1598-609 and Hoppes et al., J. Immunol 2014 Nov 15;193(10):4803-13 and references therein). If more than one amino acid residue is substituted and/or inserted, the
  • replacement/inserted amino acid residues may be the same as each other or different from one another.
  • Each replacement amino acid may have a different side chain to the amino acid being replaced.
  • polypeptides of the invention bind to MHC in the peptide binding groove of the MHC molecule.
  • amino acid modifications described above will not impair the ability of the peptide to bind MHC.
  • the amino acid modifications improve the ability of the peptide to bind MHC.
  • mutations may be made at positions which anchor the peptide to MHC. Such anchor positions and the preferred residues at these locations are known in the art, particularly for peptides which bind HLA-A * 02 (see, e. g. Parkhurst et al., J. Immunol. 1996 Sep 15;157(6):2539-48 and Parker et al. J Immunol. 1992 Dec 1 ; 149(1 1 ):3580-7).
  • a polypeptide of the invention may be used to illicit an immune response. If this is the case, it is important that the immune response is specific to the intended target in order to avoid the risk of unwanted side effects that may be associated with an "off target" immune response. Therefore, it is preferred that the amino acid sequence of a polypeptide of the invention does not match the amino acid sequence of a peptide from any other protein(s), in particular, that of another human protein.
  • a person of skill in the art would understand how to search a database of known protein sequences to ascertain whether a polypeptide according to the invention is present in another protein.
  • Polypeptides of the invention can be synthesised easily by Merrifield synthesis, also known as solid phase synthesis, or any other peptide synthesis methodology.
  • GMP grade polypeptide is produced by solid-phase synthesis techniques by Multiple Peptide Systems, San Diego, CA.
  • the peptide may be recombinantly produced, if so desired, in accordance with methods known in the art.
  • Such methods typically involve the use of a vector comprising a nucleic acid sequence encoding the polypeptide to be expressed, to express the polypeptide in vivo; for example, in bacteria, yeast, insect or mammalian cells.
  • in vitro cell-free systems may be used. Such systems are known in the art and are commercially available for example from Life
  • polypeptides may be isolated and/or may be provided in substantially pure form. For example, they may be provided in a form which is substantially free of other polypeptides or proteins.
  • the invention provides a complex of the polypeptide of the first aspect and an MHC molecule.
  • the polypeptide is bound to the peptide binding groove of the MHC molecule.
  • the MHC molecule may be MHC class I.
  • the MHC class I molecule may be selected from HLA-A * 02, HLA-A * 01 , HLA-A * 03, HLA-A11 , HLA-A23, HLA-A24, HLA-B * 07, HLA-B * 08, HLA- B40, HLA-B44, HLA-B15, HLA-C * 04, HLA * C * 03 HLA-CW.
  • HLA-A * 02 HLA-A * 01
  • HLA-A * 03 HLA-A11
  • HLA-A23, HLA-A24 HLA-B * 07, HLA-B * 08, HLA- B40, HLA-B44, HLA-B15, HLA-C * 04, HLA * C * 03 HLA-CW.
  • HLA alleles can be found on the EMBL Immune Polymorphism Database (http://www.ebi.ac.uk/ipd/imqt/hla/allele.html; Robinson et al. Nucleic Acids Research (2015) 43:D423-431 ).
  • the complex of the invention may be isolated and/or in a substantially pure form.
  • the complex may be provided in a form which is substantially free of other polypeptides or proteins.
  • MHC molecule includes recombinant MHC molecules, non-naturally occurring MHC molecules and functionally equivalent fragments of MHC, including derivatives or variants thereof, provided that peptide binding is retained.
  • MHC molecules may be fused to a therapeutic moiety, attached to a solid support, in soluble form, and/or in multimeric form.
  • Methods to produce soluble recombinant MHC molecules with which polypeptides of the invention can form a complex are known in the art. Suitable methods include, but are not limited to, expression and purification from E. coli cells or insect cells. A suitable method is provided in Example H2 herein.
  • MHC molecules may be produced synthetically, or using cell free systems.
  • Polypeptides and/or polypeptide-MHC complexes of the invention may be associated with a moiety capable of eliciting a therapeutic effect.
  • a moiety may be a carrier protein which is known to be immunogenic.
  • KLH keyhole limpet hemocyanin
  • the polypeptides and/or polypeptide-MHC complexes of the invention may be associated with a fusion partner. Fusion partners may be used for detection purposes, or for attaching said polypeptide or MHC to a solid support, or for MHC oligomerisation.
  • the MHC complexes may incorporate a biotinylation site to which biotin can be added, for example, using the BirA enzyme (O'Callaghan et al., 1999 Jan 1 ;266(1 ):9-15).
  • Other suitable fusion partners include, but are not limited to, fluorescent, or luminescent labels, radiolabels, nucleic acid probes and contrast reagents, antibodies, or enzymes that produce a detectable product. Detection methods may include flow cytometry, microscopy, electrophoresis or scintillation counting.
  • Polypeptide-MHC complexes of the invention may be provided in soluble form, or may be immobilised by attachment to a suitable solid support.
  • solid supports include, but are not limited to, a bead, a membrane, sepharose, a magnetic bead, a plate, a tube, a column.
  • Polypeptide-MHC complexes may be attached to an ELISA plate, a magnetic bead, or a surface plasmon reasonance biosensor chip.
  • Methods of attaching peptide-MHC complexes to a solid support are known to the skilled person, and include, for example, using an affinity binding pair, e.g. biotin and streptavidin, or antibodies and antigens.
  • peptide-MHC complexes are labelled with biotin and attached to streptavidin-coated surfaces.
  • Polypeptide-MHC complexes of the invention may be in multimeric form, for example, dimeric, or tetrameric, or pentameric, or octomeric, or greater. Examples of suitable methods for the production of multimeric peptide MHC complexes are described in Greten et al., Clin. Diagn. Lab. Immunol. 2002 Mar;9(2):216-20 and references therein.
  • polypeptide-MHC multimers may be produced using peptide-MHC tagged with a biotin residue and complexed through fluorescent labelled streptavidin.
  • multimeric polypeptide-MHC complexes may be formed by using immunoglobulin as a molecular scaffold.
  • the extracellular domains of MHC molecules are fused with the constant region of an immunoglobulin heavy chain separated by a short amino acid linker.
  • Polypeptide-MHC multimers have also been produced using carrier molecules such as dextran (WO02072631 ). Multimeric peptide MHC complexes can be useful for improving the detection of binding moieties, such as T cell receptors, which bind said complex, because of avidity effects.
  • the polypeptides of the invention may be presented on the surface of a cell in complex with MHC.
  • the invention also provides a cell presenting on its surface a complex of the invention.
  • Such a cell may be a mammalian cell, preferably a cell of the immune system, and in particular a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • Other preferred cells include T2 cells (Hosken, et al., Science. 1990 Apr 20;248(4953):367-70).
  • Cells presenting the polypeptide or complex of the invention may be isolated, preferably in the form of a population, or provided in a substantially pure form. Said cells may not naturally present the complex of the invention, or alternatively said cells may present the complex at a level higher than they would in nature. Such cells may be obtained by pulsing said cells with the polypeptide of the invention.
  • Pulsing involves incubating the cells with the polypeptide for several hours using polypeptide concentrations typically ranging from 10 "5 to 10 "12 M. Said cells may additionally be transduced with HLA-A * 02 molecules to further induce presentation of the peptide. Cells may be produced recombinantly. Cells presenting polypeptides of the invention may be used to isolate T cells and T cell receptors (TCRs) which are activated by, or bind to, said cells, as described in more detail below.
  • TCRs T cell receptors
  • the invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide of the first aspect of the invention.
  • the nucleic acid may be cDNA.
  • the nucleic acid molecule may consist essentially of a nucleic acid sequence encoding the polypeptide of the first aspect of the invention or may encode only the peptide of the invention, i.e. encode no other polypeptide. Such a nucleic acid molecule can be synthesised in accordance with methods known in the art. Due to the degeneracy of the genetic code, one of ordinary skill in the art will appreciate that nucleic acid molecules of different nucleotide sequence can encode the same amino acid sequence.
  • the invention provides a vector comprising a nucleic acid sequence according to the third aspect of the invention.
  • the vector may include, in addition to a nucleic acid sequence encoding only a polypeptide of the invention, one or more additional nucleic acid sequences encoding one or more additional polypeptides. Such additional polypeptides may, once expressed, be fused to the N-terminus or the C-terminus of the polypeptide of the invention.
  • the vector includes a nucleic acid sequence encoding a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • Suitable vectors are known in the art as is vector construction, including the selection of promoters and other regulatory elements, such as enhancer elements.
  • the vector utilised in the context of the present invention desirably comprises sequences appropriate for introduction into cells.
  • the vector may be an expression vector, a vector in which the coding sequence of the polypeptide is under the control of its own cis-acting regulatory elements, a vector designed
  • the term "vector” encompasses a DNA molecule, such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • a DNA molecule such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • Appropriate phage and viral vectors include, but are not limited to, lambda (X) bacteriophage, EMBL bacteriophage, simian virus 40, bovine papilloma virus, Epstein-Barr virus, adenovirus, herpes virus, vaccinia virus, Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, lentivirus and Rous sarcoma virus.
  • the invention provides a cell comprising the vector of the fourth aspect of the invention.
  • the cell may be an antigen presenting cell and is preferably a cell of the immune system.
  • the cell may be a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • the cell may be a mammalian cell.
  • Polypeptides and complexes of the invention can be used to identify and/or isolate binding moieties that bind specifically to the polypeptide and/or the complex of the invention.
  • binding moieties may be used as immunotherapeutic reagents and may include antibodies and TCRs.
  • the invention provides a binding moiety that binds the polypeptide of the invention.
  • the binding moiety binds the polypeptide when said polypeptide is in complex with MHC.
  • the binding moiety may bind partially to the MHC, provided that it also binds to the polypeptide.
  • the binding moiety may bind only the polypeptide, and that binding may be specific.
  • the binding moiety may bind only the peptide MHC complex and that binding may be specific.
  • binding moieties that bind the complex of the invention When used with reference to binding moieties that bind the complex of the invention, "specific" is generally used herein to refer to the situation in which the binding moiety does not show any significant binding to one or more alternative polypeptide-MHC complexes other than the polypeptide-MHC complex of the invention.
  • the binding moiety may be a T cell receptor (TCR).
  • TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature, and links to the IMGT public database of TCR sequences. The unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the TCR field. For example, they can be found in the "T cell Receptor Factsbook", (2001 ) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8;
  • the TCRs of the invention may be in any format known to those in the art.
  • the TCRs may be ⁇ heterodimers, or they may be in single chain format (such as those described in W09918129).
  • Single chain TCRs include ⁇ TCR polypeptides of the type: Va-L- ⁇ , ⁇ -L-Va, Va- Ca-L- ⁇ , ⁇ - ⁇ - ⁇ ⁇ -0 ⁇ or Va- Ca - ⁇ / ⁇ - ⁇ , optionally in the reverse orientation, wherein Va and ⁇ are TCR a and ⁇ variable regions respectively, Ca and C are TCR a and ⁇ constant regions respectively, and L is a linker sequence.
  • the TCR may be in a soluble form (i.e.
  • the TCR may be provided on the surface of a cell, such as a T cell.
  • the alpha and/or beta chain constant domain may be truncated relative to the native/naturally occurring TRAC/TRBC sequences.
  • the TRAC/TRBC may contain modifications.
  • the alpha chain extracellular sequence may include a modification relative to the native/naturally occurring TRAC whereby amino acid T48 of TRAC, with reference to IMGT numbering, is replaced with C48.
  • the beta chain extracellular sequence may include a modification relative to the native/naturally occurring TRBC1 or TRBC2 whereby S57 of TRBC1 or TRBC2, with reference to IMGT numbering, is replaced with C57.
  • These cysteine substitutions relative to the native alpha and beta chain extracellular sequences enable the formation of a non- native interchain disulphide bond which stabilises the refolded soluble TCR, i.e. the TCR formed by refolding extracellular alpha and beta chains (WO 03/020763). This non-native disulphide bond facilitates the display of correctly folded TCRs on phage. (Li et a/., Nat Biotechnol 2005 Mar;23(3):349-54).
  • TCRs of the invention may be engineered to include mutations. Methods for producing mutated high affinity TCR variants such as phage display and site directed mutagenesis and are known to those in the art (for example see WO 04/044004 and Li et al., Nat Biotechnol 2005 Mar;23(3):349- 54). TCRs of the invention may also be may be labelled with an imaging compound, for example a label that is suitable for diagnostic purposes.
  • Such labelled high affinity TCRs are useful in a method for detecting a TCR ligand selected from CD1 -antigen complexes, bacterial superantigens, and MHC- peptide/superantigen complexes, which method comprises contacting the TCR ligand with a high affinity TCR (or a multimeric high affinity TCR complex) which is specific for the TCR ligand; and detecting binding to the TCR ligand.
  • a high affinity TCR or a multimeric high affinity TCR complex
  • fluorescent streptavidin (commercially available) can be used to provide a detectable label.
  • a fluorescently-labelled multimer is suitable for use in FACS analysis, for example to detect antigen presenting cells carrying the peptide for which the high affinity TCR is specific.
  • a TCR of the present invention may alternatively or additionally be associated with (e.g. covalently or otherwise linked to) a therapeutic agent which may be, for example, a toxic moiety for use in cell killing, or an immunostimulating agent such as an interleukin or a cytokine.
  • a multivalent high affinity TCR complex of the present invention may have enhanced binding capability for a TCR ligand compared to a non-multimeric wild-type or high affinity T cell receptor heterodimer.
  • the multivalent high affinity TCR complexes according to the invention are particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo, and are also useful as intermediates for the production of further multivalent high affinity TCR complexes having such uses.
  • the high affinity TCR or multivalent high affinity TCR complex may therefore be provided in a pharmaceutically acceptable formulation for use in vivo.
  • High affinity TCRs of the invention may be used in the production of soluble bi-specific reagents.
  • a preferred embodiment is a reagent which comprises a soluble TCR, fused via a linker to an anti- CD3 specific antibody fragment. Further details including how to produce such reagents are described in W010/133828.
  • the invention provides nucleic acid encoding the TCR of the invention, a TCR expression vector comprising nucleic acid encoding a TCR of the invention, as well as a cell harbouring such a vector.
  • the TCR may be encoded either in a single open reading frame or two distinct open reading frames.
  • one vector may encode both an alpha and a beta chain of a TCR of the invention.
  • a further aspect of the invention provides a cell displaying on its surface a TCR of the invention.
  • the cell may be a T cell.
  • T cells expressing the TCRs of the invention are suitable for use in adoptive therapy-based treatment of diseases such as cancers.
  • adoptive therapy see for example Rosenberg et al., Nat Rev Cancer. 2008 Apr;8(4):299-308.
  • the TCRs of the invention intended for use in adoptive therapy are generally glycosylated when expressed by the transfected T cells.
  • the glycosylation pattern of transfected TCRs may be modified by mutations of the transfected gene (Kuball J et al., J Exp Med. 2009 Feb 16;206(2):463-75).
  • the binding moiety of the invention may be an antibody.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced.
  • antibody includes antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanised antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic and any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023.
  • a humanised antibody may be a modified antibody having the variable regions of a non-human, e.g. murine, antibody and the constant region of a human antibody. Methods for making humanised antibodies are described in, for example, US Patent No.
  • antibodies are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies.
  • Antibodies may be polyclonal or monoclonal. A monoclonal antibody may be referred to herein as "mab".
  • an antibody for example a monoclonal antibody
  • recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody.
  • Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementary determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin (see, for instance, EP-A-184187, GB 2188638A or EP-A-239400).
  • CDRs complementary determining regions
  • a hybridoma (or other cell that produces antibodies) may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. e£ al., Nature.
  • Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g.
  • antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).
  • bispecific antibodies these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Hollinger & Winter, Curr Opin
  • bispecific antibody fragments e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain "Janusins" described in Traunecker et al., EMBO J. 1991 Dec; 10(12):3655-9). Bispecific diabodies, as opposed to bispecific whole antibodies, may also be useful because they can be readily constructed and expressed in E. coli.
  • Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
  • An "antigen binding domain" is the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope.
  • An antigen binding domain may be provided by one or more antibody variable domains.
  • An antigen binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • the binding moiety may be an antibody-like molecule that has been designed to specifically bind a peptide-MHC complex of the invention.
  • TCR-mimic antibodies such as, for example those described in WO2007143104 and Sergeeva ef a/., Blood. 201 1 Apr 21 ;1 17(16):4262-72 and/or Dahan and Reiter. Expert Rev Mol Med. 2012 Feb 24;14:e6.
  • binding moieties based on engineered protein scaffolds are derived from stable, soluble, natural protein structures which have been modified to provide a binding site for a target molecule of interest.
  • engineered protein scaffolds include, but are not limited to, affibodies, which are based on the Z-domain of staphylococcal protein A that provides a binding interface on two of its a-helices (Nygren, FEBS J. 2008 Jun;275(1 1 ):2668-76); anticalins, derived from lipocalins, that incorporate binding sites for small ligands at the open end of a beta-barrel fold (Skerra, FEBS J.
  • Engineered protein scaffolds are typically targeted to bind the same antigenic proteins as antibodies, and are potential therapeutic agents. They may act as inhibitors or antagonists, or as delivery vehicles to target molecules, such as toxins, to a specific tissue in vivo (Gebauer and Skerra, Curr Opin Chem Biol. 2009 Jun;13(3):245-55). Short peptides may also be used to bind a target protein.
  • Phylomers are natural structured peptides derived from bacterial genomes. Such peptides represent a diverse array of protein structural folds and can be used to inhibit/disrupt protein-protein interactions in vivo (Watt, Nat Biotechnol. 2006 Feb;24(2): 177-83)].
  • the invention further provides a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention for use in medicine.
  • the polypeptide, complex, nucleic acid, vector, cell or binding moiety may be used for in the treatment or prevention of cancer, in particular, acute myeloid leukaemia.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention together with a pharmaceutically acceptable carrier.
  • This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient). It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms. Suitable compositions and methods of administration are known to those skilled in the art, for example see, Johnson et al., Blood.
  • Cells in accordance with the invention will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a
  • T cells transfected with TCRs of the invention may be provided in pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be adapted for administration by any appropriate route such as a parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation or intranasal routes.
  • Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
  • Dosages of the substances of the present invention can vary between wide limits, depending upon the disease or disorder to be treated (such as cancer, viral infection or autoimmune disease), the age and condition of the individual to be treated, etc.
  • a suitable dose range for a reagent comprising a soluble TCR fused to an anti- CD3 domain may be between 25 ng/kg and 50 ⁇ g/kg. A physician will ultimately determine appropriate dosages to be used.
  • the polypeptide of the invention may be provided in the form of a vaccine composition.
  • the vaccine composition may be useful for the treatment or prevention of cancer. All such
  • compositions are encompassed in the present invention.
  • vaccines may take several forms (Schlom, J Natl Cancer Inst. 2012 Apr 18;104(8):599-613).
  • the peptide of the invention may be used directly to immunise patients (Salgaller, Cancer Res. 1996 Oct 15;56(20):4749-57 and Marchand, Int J Cancer. 1999 Jan 18;80(2):219-30).
  • the vaccine composition may include additional peptides such that the peptide of the invention is one of a mixture of peptides.
  • Adjuvants may be added to the vaccine composition to augment the immune response
  • the vaccine composition may take the form of an antigen presenting cell displaying the peptide of the invention in complex with MHC.
  • the antigen presenting cell is an immune cell, more preferably a dendritic cell.
  • the peptide may be pulsed onto the surface of the cell (Thurner, J Exp Med. 1999 Dec 6; 190(11 ): 1669-78), or nucleic acid encoding for the peptide of the invention may be introduced into dendritic cells (for example by electroporation. Van Tendeloo, Blood. 2001 Jul 1 ;98(1 ):49-56).
  • polypeptides, complexes, nucleic acid molecules, vectors, cells and binding moieties of the invention may be non-naturally occurring and/or purified and/or engineered and/or recombinant and/or isolated and/or synthetic.
  • the invention also provides a method of identifying a binding moiety that binds a complex of the invention, the method comprising contacting a candidate binding moiety with the complex and determining whether the candidate binding moiety binds the complex.
  • Methods to determine binding to polypeptide-MHC complexes are well known in the art. Preferred methods include, but are not limited to, surface plasmon resonance, or any other biosensor technique, ELISA, flow cytometry, chromatography, microscopy. Alternatively, or in addition, binding may be determined by functional assays in which a biological response is detected upon binding, for example, cytokine release or cell apoptosis.
  • the candidate binding moiety may be a binding moiety of the type already described, such as a TCR or an antibody.
  • T cells may be isolated from fresh blood obtained from volunteer donors. Such a method involves stimulating T cells using autologous DCs, followed by autologous B cells, pulsed with the polypeptide of the invention. Several round of stimulation may be carried out, for example three or four rounds. Activated T cells may then be tested for specificity by measuring cytokine release in the presence of T2 cells pulsed with the peptide of the invention (for example using an IFNy ELISpot assay). Activated cells may then be sorted by fluorescence-activated cell sorting (FACS) using labelled antibodies to detect intracellular cytokine production (e.g. IFNy), or expression of a cell surface marker (such as CD137).
  • FACS fluorescence-activated cell sorting
  • Sorted cells may be expanded and further validated, for example, by ELISpot assay and/or cytotoxicity against target cells and/or staining by peptide-MHC tetramer.
  • the TCR chains from validated clones may then be amplified by rapid amplification of cDNA ends (RACE) and sequenced.
  • the polypeptide MHC complex of the invention may be used for screening a library of TCRs or antibodies.
  • the production of antibody libraries using phage display is well known in the art, for example see Aitken, Antibody phage display: Methods and Protocols (2009, Humana, New York). Further details on TCR libraries can be found in WO04044004.
  • the polypeptide-MHC complex of the invention is used to screen a library of diverse TCRs displayed on the surface of phage particles.
  • the TCRs displayed by said library may contain non- natural mutations. Screening may involve panning the phage library with peptide-MHC complexes of the invention and subsequently isolating bound phage.
  • peptide-MHC complexes may be attached to a solid support, such as a magnet bead, or column matrix and phage bound peptide MHC complexes isolated, with a magnet, or by chromatography, respectively.
  • the panning steps may be repeated several times for example three or four times.
  • Isolated phage may be further expanded in E. coli cells.
  • Isolated phage particles may be tested for specific binding peptide-MHC complexes of the invention. Binding can be detected using techniques including, but not limited to, ELISA, or SPR for example using a BiaCore instrument.
  • the DNA sequence of the T cell receptor displayed by peptide-MHC binding phage can be further identified by standard PCR methods.
  • Figures H1 to H18 show the respective fragmentation spectra for the peptides of SEQ NOs: H1 to H18, eluted from cells. A table highlighting the matching ions is shown below each spectrum.
  • Immortalised cell lines obtained from commercial sources were maintained and expanded under standard conditions.
  • Class I HLA complexes were purified by immunoaffinity using commercially available anti-HLA antibodies BB7.1 (anti-HLA-B * 07), BB7.2 (anti-HLA-A * 02) and W6/32 (anti-Class 1 ). Briefly, cells were lysed in buffer containing non-ionic detergent NP-40 (0.5% v/v) at 5 x 10 7 cells per ml and incubated at 4°C for 1 h with agitation/mixing. Cell debris was removed by centrifugation and supernatant pre-cleared using proteinA-Sepharose.
  • Peptides were separated by high pressure liquid chromatography (HPLC) on a Dionex Ultimate 3000 system using a C18 column (Phenomenex). Peptides were loaded in 98% buffer A (0.1 % aqueous trifluoroacetic acid (TFA)) and 2% buffer B (0.1% TFA in acetonitrile). Peptides were eluted using a stepped gradient of B (2-60%) over 20 min. Fractions were collected at one minute intervals and lyophilised.
  • HPLC high pressure liquid chromatography
  • Peptides were analysed by nanoLCMS/MS using a Dionex Ultimate 3000 nanoLC coupled to either AB Sciex Triple TOF 5600 or Thermo Orbitrap Fusion mass spectrometers. Both machines were equipped with nanoelectrospray ion sources. Peptides were loaded onto an Acclaim PepMap 100 trap column (Dionex) and separated using an Acclaim PepMap RSLC column (Dionex). Peptides were loaded in mobile phase A (0.5% formic acid: water) and eluted using a gradient of buffer B (acetonitrile:0.5% formic acid) directly into the nanospray ionisation source.
  • buffer B acetonitrile:0.5% formic acid
  • IDA information dependent acquisition
  • Information acquired in these experiments was used to search the Uniprot database of human proteins for peptides consistent with the fragmentation patterns seen, using Protein pilot software (Ab Sciex) and PEAKS software (Bioinformatics solutions). Peptides identified are assigned a score by the software, based on the match between the observed and expected fragmentation patterns.
  • polypeptides set out in table H1 corresponding to SEQ NOs: H1 -H18, were detected by mass spec following extraction from cancer cell lines.
  • An example cell line from which the peptide was detected is indicated in the table along with the HLA antibody used for immunoaffinity purification.
  • Figures H1 -H18 show representative fragmentation patterns for the peptides of SEQ NOs: H1-H18 respectively. A table highlighting the matching ions is shown below each spectrum.
  • HLA-A*02 molecules HLA-A*02-heavy chain and HLA light-chain ( ⁇ 2 ⁇ ) were expressed separately in E. coli as inclusion bodies, using appropriate constructs.
  • HLA-A * 02-heavy chain additionally contained a C-terminal biotinylation tag which replaces the transmembrane and cytoplasmic domains (O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15).
  • E. coli cells were lysed and inclusion bodies processed to approximately 80% purity.
  • Inclusion bodies of ⁇ 2 ⁇ and heavy chain were denatured separately in 6 M guanidine-HCI, 50 mM Tris pH 8.1 , 100 mM NaCI, 10 mM DTT, 10 mM EDTA.
  • Refolding buffer was prepared containing 0.4 M L-Arginine, 100 mM Tris pH 8.1 , 3.7 mM cystamine dihydrochloride, 6.6 mM cysteamine hydrochloride and chilled to ⁇ 5°C.
  • Synthetic peptide dissolved in DMSO to a final concentration of 4mg/ml is added to the refold buffer at 4 mg/litre (final concentration). Then 30 mg/litre ⁇ 2 ⁇ followed by 30 mg/litre heavy chain (final concentrations) are added. Refolding was allowed to reach completion at 4 °C for at least 1 hour.
  • Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Two changes of buffer were necessary to reduce the ionic strength of the solution sufficiently.
  • the protein solution was then filtered through a 1.5 pm cellulose acetate filter and loaded onto a POROS 50HQ anion exchange column (8 ml bed volume). Protein was eluted with a linear 0-500 mM NaCI gradient in 10 mM Tris pH 8.1 using an Akta purifier (GE Healthcare). HLA-A * 02-peptide complex eluted at approximately 250 mM NaCI, and peak fractions were collected, a cocktail of protease inhibitors (Calbiochem) was added and the fractions were chilled on ice.
  • Biotinylation tagged pHLA molecules were buffer exchanged into 10 mM Tris pH 8.1 , 5 mM NaCI using a GE Healthcare fast desalting column equilibrated in the same buffer. Immediately upon elution, the protein-containing fractions were chilled on ice and protease inhibitor cocktail (Calbiochem) was added. Biotinylation reagents were then added: 1 mM biotin, 5 mM ATP (buffered to pH 8), 7.5 mM MgCI2, and 5 pg/ml BirA enzyme (purified according to O'Callaghan et al., (1999) Anal. Biochem. 266: 9-15). The mixture was then allowed to incubate at room temperature overnight.
  • the biotinylated pHLA-A * 02 molecules were purified using gel filtration chromatography.
  • a GE Healthcare Superdex 75 HR 10/30 column was pre-equilibrated with filtered PBS and 1 ml of the biotinylation reaction mixture was loaded and the column was developed with PBS at 0.5 ml/min using an Akta purifier (GE Healthcare).
  • Biotinylated pHLA-A * 02 molecules eluted as a single peak at approximately 15 ml. Fractions containing protein were pooled, chilled on ice, and protease inhibitor cocktail was added. Protein concentration was determined using a Coomassie-binding assay (PerBio) and aliquots of biotinylated pHLA-A * 02 molecules were stored frozen at -20 °C.
  • Such peptide-MHC complexes may be used in soluble form or may be immobilised through C terminal biotin moiety on to a solid support, to be used for the detection of T cells and T cell receptors which bind said complex.
  • TCR isolation methods are known to those skilled in the art; for example, DNA sequences corresponding to TCR alpha and beta chains may be amplified from a T cell clone (obtained from volunteer blood donors) that is activated by cells presenting the peptide-HLA complex of interest.
  • T cell clone obtained from volunteer blood donors
  • Other suitable methods include isolation from TCR libraries (as described in WO2005/1 16646 and WO2005/116074 for example).
  • TCR alpha and beta chain sequences are expressed in E. coli as soluble TCRs, (WO2003020763; Boulter, et al., Protein Eng, 2003. 16: 707-711 ). Binding of the soluble TCRs to the complexes is analysed by surface plasmon resonance using a BiaCore 3000 instrument. Biotinylated peptide-HLA monomers are prepared as previously described (Example H2) and immobilized on to a streptavidin-coupled CM-5 sensor chip.
  • The[LS8] present invention also relates to novel peptides derived from ETS translocation variant 4 (ETV4), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules.
  • ETV4 ETS translocation variant 4
  • binding moieties that bind to the peptides and/or complexes of the invention. Such moieties are useful for the development of immunotherapeutic reagents for the treatment of diseases such as cancer.
  • T cells are a key part of the cellular arm of the immune system. They specifically recognise peptide fragments that are derived from intracellular proteins and presented in complex with Major Histocompatibility Complex (MHC) molecules on the surface of antigen presenting cells (APCs).
  • MHC Major Histocompatibility Complex
  • MHC molecules are known as human leukocyte antigens (HLA), and both terms are used synonymously herein.
  • HLA human leukocyte antigens
  • MHC molecules have a binding groove in which the peptide fragments bind. Recognition of particular peptide-MHC antigens is mediated by a corresponding T cell receptor (TCR).
  • TCR T cell receptor
  • Tumour cells express various tumour associated antigens (TAA) and peptides derived from these antigens are frequently displayed on the tumour cell surface. Detection of a MHC class I- presented TAA-derived peptide by a CD8+ T cell bearing the corresponding T cell receptor, leads to targeted killing of the tumour cell.
  • TAA tumour associated antigens
  • tumour cells often escape detection.
  • TAA-derived peptides presented by MHC molecules on tumour cells enables the development of novel immunotherapeutic reagents designed to specifically target and destroy said tumour cells.
  • Such reagents may be moieties that bind to the TAA-derived peptide and/or peptide-MHC complexes of the invention, and typically involve the induction of a T cell response.
  • such reagents may be based, exclusively, or in part, on T cells, or T cell receptors (TCRs), or antibodies.
  • TAA-derived peptides that are presented on the cell surface in complex with a given MHC, only a small number of such peptides, covering a limited number of cancer indications, have been identified to date.
  • TAAs suitable for the development of immunotherapeutic reagents must have limited or no expression in normal cells, to prevent off target activity. It is therefore desirable to provide additional tumour-specific TAA-derived peptides and MHC complexes thereof that can be used for the development of new cancer therapies.
  • ETV4 (also known as ETS translocation variant 4 or Adenovirus E1 A enhancer-binding protein or E1 A-F or PEA3 and having Uniprot accession number P43268) is a member of the family of ETS transcription factors. ETV4 is a known oncogene Oh et al. Biochim Biophys Acta. 2012
  • ETV4 is an ideal target for immunotherapeutic applications.
  • the inventors have found novel peptides derived from ETV4 that are presented on the cell surface in complex with MHC. These peptides are particularly useful for the development of reagents that can target cells expressing ETV4 and for the treatment of cancers, including ovarian cancer.
  • the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: 11 -18, or
  • polypeptide forms a complex with a Major Histocompatibility Complex (MHC) molecule.
  • MHC Major Histocompatibility Complex
  • polypeptides of the invention are presented by MHC on the surface of tumour cells. Accordingly, the polypeptides of the invention, as well as moieties that bind the polypeptide-MHC complexes, can be used to develop therapeutic reagents.
  • the skilled person can determine whether or not a given polypeptide forms a complex with an MHC molecule by determining whether the MHC can be refolded in the presence of the polypeptide using the process set out in Example I2. If the polypeptide does not form a complex with MHC then MHC will not refold. Refolding is commonly confirmed using an antibody that recognises MHC in a folded state only. Further details can be found in Garboczi et al., Proc Natl Acad Sci
  • polypeptides of the invention are from about 8 to about 16 amino acids in length, and are most preferably 8, 9, or 10 or 11 amino acids in length.
  • the polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: 11-18.
  • amino acid residues comprising the polypeptides of the invention may be chemically modified.
  • chemical modifications include those corresponding to post translational modifications for example phosphorylation, acetylation and deamidation (Engelhard et al., Curr Opin Immunol. 2006 Feb;18(1 ):92-7).
  • Amino acid substitution means that an amino acid residue is substituted for a replacement amino acid residue at the same position.
  • Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are immediately adjacent one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent another inserted amino acid residue.
  • One, two or three amino acids may be deleted from the sequence of SEQ NOs: 11-18. Each deletion can take place at any position of SEQ NOs: 11 -18.
  • the polypeptide of the invention may comprise one, two or three additional amino acids at the C-terminal end and/or at the N-terminal end of the sequence of SEQ NOs: 11-18.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: 11-18 with the exception of one amino acid substitution and one amino acid insertion, one amino acid substitution and one amino acid deletion, or one amino acid insertion and one amino acid deletion.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: 11-18, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.
  • Inserted amino acids and replacement amino acids may be naturally occurring amino acids or may be non-naturally occurring amino acids and, for example, may contain a non-natural side chain.
  • Such altered peptide ligands are discussed further in Douat-Casassus et al., J. Med. Chem, 2007 Apr 5;50(7): 1598-609 and Hoppes et al., J. Immunol 2014 Nov 15;193(10):4803-13 and references therein). If more than one amino acid residue is substituted and/or inserted, the
  • replacement/inserted amino acid residues may be the same as each other or different from one another.
  • Each replacement amino acid may have a different side chain to the amino acid being replaced.
  • polypeptides of the invention bind to MHC in the peptide binding groove of the MHC molecule.
  • amino acid modifications described above will not impair the ability of the peptide to bind MHC.
  • the amino acid modifications improve the ability of the peptide to bind MHC.
  • mutations may be made at positions which anchor the peptide to MHC. Such anchor positions and the preferred residues at these locations are known in the art, particularly for peptides which bind HLA-A * 02 (see, e. g. Parkhurst et al., J. Immunol. 1996 Sep 15;157(6):2539-48 and Parker et al. J Immunol. 1992 Dec 1 ; 149(1 1 ):3580-7).
  • a polypeptide of the invention may be used to illicit an immune response. If this is the case, it is important that the immune response is specific to the intended target in order to avoid the risk of unwanted side effects that may be associated with an "off target" immune response. Therefore, it is preferred that the amino acid sequence of a polypeptide of the invention does not match the amino acid sequence of a peptide from any other protein(s), in particular, that of another human protein.
  • a person of skill in the art would understand how to search a database of known protein sequences to ascertain whether a polypeptide according to the invention is present in another protein.
  • Polypeptides of the invention can be synthesised easily by Merrifield synthesis, also known as solid phase synthesis, or any other peptide synthesis methodology.
  • GMP grade polypeptide is produced by solid-phase synthesis techniques by Multiple Peptide Systems, San Diego, CA.
  • the peptide may be recombinantly produced, if so desired, in accordance with methods known in the art.
  • Such methods typically involve the use of a vector comprising a nucleic acid sequence encoding the polypeptide to be expressed, to express the polypeptide in vivo; for example, in bacteria, yeast, insect or mammalian cells.
  • in vitro cell-free systems may be used. Such systems are known in the art and are commercially available for example from Life
  • polypeptides may be isolated and/or may be provided in substantially pure form. For example, they may be provided in a form which is substantially free of other polypeptides or proteins.
  • the invention provides a complex of the polypeptide of the first aspect and an MHC molecule.
  • the polypeptide is bound to the peptide binding groove of the MHC molecule.
  • the MHC molecule may be MHC class I.
  • the MHC class I molecule may be selected from HLA-A * 02, HLA-A * 01 , HLA-A * 03, HLA-A11 , HLA-A23, HLA-A24, HLA-B * 07, HLA-B*08, HLA- B40, HLA-B44, HLA-B15, HLA-C * 04, HLA * C * 03 HLA-CW.
  • HLA alleles As is known to those skilled in the art there are allelic variants of the above HLA types, all of which are encompassed by the present invention. A full list of HLA alleles can be found on the EMBL Immune Polymorphism Database (http://www.ebi.ac.uk/ipcl/imqt/hla/allele.html; Robinson et al. Nucleic Acids Research (2015) 43:D423-431 ).
  • the complex of the invention may be isolated and/or in a substantially pure form.
  • the complex may be provided in a form which is substantially free of other polypeptides or proteins.
  • MHC molecule includes recombinant MHC molecules, non-naturally occurring MHC molecules and functionally equivalent fragments of MHC, including derivatives or variants thereof, provided that peptide binding is retained.
  • MHC molecules may be fused to a therapeutic moiety, attached to a solid support, in soluble form, and/or in multimeric form.
  • Methods to produce soluble recombinant MHC molecules with which polypeptides of the invention can form a complex are known in the art. Suitable methods include, but are not limited to, expression and purification from E. coli cells or insect cells. A suitable method is provided in Example 12 herein.
  • MHC molecules may be produced synthetically, or using cell free systems.
  • Polypeptides and/or polypeptide-MHC complexes of the invention may be associated with a moiety capable of eliciting a therapeutic effect.
  • a moiety may be a carrier protein which is known to be immunogenic.
  • KLH keyhole limpet hemocyanin
  • the polypeptides and/or polypeptide-MHC complexes of the invention may be associated with a fusion partner. Fusion partners may be used for detection purposes, or for attaching said polypeptide or MHC to a solid support, or for MHC oligomerisation.
  • the MHC complexes may incorporate a biotinylation site to which biotin can be added, for example, using the BirA enzyme (O'Callaghan et al., 1999 Jan 1 ;266(1 ):9-15).
  • Other suitable fusion partners include, but are not limited to, fluorescent, or luminescent labels, radiolabels, nucleic acid probes and contrast reagents, antibodies, or enzymes that produce a detectable product. Detection methods may include flow cytometry, microscopy, electrophoresis or scintillation counting.
  • Polypeptide-MHC complexes of the invention may be provided in soluble form, or may be immobilised by attachment to a suitable solid support.
  • suitable solid supports include, but are not limited to, a bead, a membrane, sepharose, a magnetic bead, a plate, a tube, a column.
  • Polypeptide-MHC complexes may be attached to an ELISA plate, a magnetic bead, or a surface plasmon reasonance biosensor chip.
  • Methods of attaching peptide-MHC complexes to a solid support are known to the skilled person, and include, for example, using an affinity binding pair, e.g. biotin and streptavidin, or antibodies and antigens.
  • peptide-MHC complexes are labelled with biotin and attached to streptavidin-coated surfaces.
  • Polypeptide-MHC complexes of the invention may be in multimeric form, for example, dimeric, or tetrameric, or pentameric, or octomeric, or greater. Examples of suitable methods for the production of multimeric peptide MHC complexes are described in Greten et al., Clin. Diagn. Lab. Immunol. 2002 Mar;9(2):216-20 and references therein.
  • polypeptide-MHC multimers may be produced using peptide-MHC tagged with a biotin residue and complexed through fluorescent labelled streptavidin.
  • multimeric polypeptide-MHC complexes may be formed by using immunoglobulin as a molecular scaffold.
  • the extracellular domains of MHC molecules are fused with the constant region of an immunoglobulin heavy chain separated by a short amino acid linker.
  • Polypeptide-MHC multimers have also been produced using carrier molecules such as dextran (WO02072631 ). Multimeric peptide MHC complexes can be useful for improving the detection of binding moieties, such as T cell receptors, which bind said complex, because of avidity effects.
  • the polypeptides of the invention may be presented on the surface of a cell in complex with MHC.
  • the invention also provides a cell presenting on its surface a complex of the invention.
  • Such a cell may be a mammalian cell, preferably a cell of the immune system, and in particular a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • Other preferred cells include T2 cells (Hosken, et al., Science. 1990 Apr 20;248(4953):367-70).
  • Cells presenting the polypeptide or complex of the invention may be isolated, preferably in the form of a population, or provided in a substantially pure form. Said cells may not naturally present the complex of the invention, or alternatively said cells may present the complex at a level higher than they would in nature. Such cells may be obtained by pulsing said cells with the polypeptide of the invention.
  • Pulsing involves incubating the cells with the polypeptide for several hours using polypeptide concentrations typically ranging from 10 "5 to 10 "12 M. Said cells may additionally be transduced with HLA-A * 02 molecules to further induce presentation of the peptide. Cells may be produced recombinantly. Cells presenting polypeptides of the invention may be used to isolate T cells and T cell receptors (TCRs) which are activated by, or bind to, said cells, as described in more detail below.
  • the invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide of the first aspect of the invention.
  • the nucleic acid may be cDNA.
  • the nucleic acid molecule may consist essentially of a nucleic acid sequence encoding the polypeptide of the first aspect of the invention or may encode only the peptide of the invention, i.e. encode no other polypeptide.
  • nucleic acid molecule can be synthesised in accordance with methods known in the art. Due to the degeneracy of the genetic code, one of ordinary skill in the art will appreciate that nucleic acid molecules of different nucleotide sequence can encode the same amino acid sequence.
  • the invention provides a vector comprising a nucleic acid sequence according to the third aspect of the invention.
  • the vector may include, in addition to a nucleic acid sequence encoding only a polypeptide of the invention, one or more additional nucleic acid sequences encoding one or more additional polypeptides. Such additional polypeptides may, once expressed, be fused to the N-terminus or the C-terminus of the polypeptide of the invention.
  • the vector includes a nucleic acid sequence encoding a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly-histidine tag.
  • Suitable vectors are known in the art as is vector construction, including the selection of promoters and other regulatory elements, such as enhancer elements.
  • the vector utilised in the context of the present invention desirably comprises sequences appropriate for introduction into cells.
  • the vector may be an expression vector, a vector in which the coding sequence of the polypeptide is under the control of its own cis-acting regulatory elements, a vector designed
  • the term "vector” encompasses a DNA molecule, such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • a DNA molecule such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant nucleotide sequences (e.g., an above-described nucleic acid molecule of the invention, under the control of a functional promoter and, possibly, also an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.
  • Appropriate phage and viral vectors include, but are not limited to, lambda (X) bacteriophage, EMBL bacteriophage, simian virus 40, bovine papilloma virus, Epstein-Barr virus, adenovirus, herpes virus, vaccinia virus, Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, lentivirus and Rous sarcoma virus.
  • the invention provides a cell comprising the vector of the fourth aspect of the invention.
  • the cell may be an antigen presenting cell and is preferably a cell of the immune system.
  • the cell may be a specialised antigen presenting cell such as a dendritic cell or a B cell.
  • the cell may be a mammalian cell.
  • Polypeptides and complexes of the invention can be used to identify and/or isolate binding moieties that bind specifically to the polypeptide and/or the complex of the invention.
  • binding moieties may be used as immunotherapeutic reagents and may include antibodies and TCRs.
  • the invention provides a binding moiety that binds the polypeptide of the invention.
  • the binding moiety binds the polypeptide when said polypeptide is in complex with MHC.
  • the binding moiety may bind partially to the MHC, provided that it also binds to the polypeptide.
  • the binding moiety may bind only the polypeptide, and that binding may be specific.
  • the binding moiety may bind only the peptide MHC complex and that binding may be specific.
  • binding moieties that bind the complex of the invention When used with reference to binding moieties that bind the complex of the invention, "specific" is generally used herein to refer to the situation in which the binding moiety does not show any significant binding to one or more alternative polypeptide-MHC complexes other than the polypeptide-MHC complex of the invention.
  • the binding moiety may be a T cell receptor (TCR).
  • TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature, and links to the IMGT public database of TCR sequences. The unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the TCR field. For example, they can be found in the "T cell Receptor Factsbook", (2001 ) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8;
  • the TCRs of the invention may be in any format known to those in the art.
  • the TCRs may be ⁇ heterodimers, or they may be in single chain format (such as those described in W09918129).
  • Single chain TCRs include ⁇ TCR polypeptides of the type: Va-L- ⁇ , ⁇ -L-Va, Va- Ca-L- ⁇ , ⁇ - ⁇ - ⁇ ⁇ -0 ⁇ or Va- Ca - ⁇ / ⁇ - ⁇ , optionally in the reverse orientation, wherein Va and ⁇ are TCR a and ⁇ variable regions respectively, Ca and C are TCR a and ⁇ constant regions respectively, and L is a linker sequence.
  • the TCR may be in a soluble form (i.e. having no transmembrane or cytoplasmic domains), or may contain full length alpha and beta chains.
  • the TCR may be provided on the surface of a cell, such as a T cell.
  • the alpha and/or beta chain constant domain may be truncated relative to the native/naturally occurring TRAC/TRBC sequences.
  • the TRAC/TRBC may contain modifications.
  • the alpha chain extracellular sequence may include a modification relative to the native/naturally occurring TRAC whereby amino acid T48 of TRAC, with reference to IMGT numbering, is replaced with C48.
  • the beta chain extracellular sequence may include a modification relative to the native/naturally occurring TRBC1 or TRBC2 whereby S57 of TRBC1 or TRBC2, with reference to IMGT numbering, is replaced with C57.
  • TCRs of the invention may be engineered to include mutations.
  • Methods for producing mutated high affinity TCR variants such as phage display and site directed mutagenesis and are known to those in the art (for example see WO 04/044004 and Li et al., Nat Biotechnol 2005 Mar;23(3):349- 54).
  • TCRs of the invention may also be may be labelled with an imaging compound, for example a label that is suitable for diagnostic purposes.
  • Such labelled high affinity TCRs are useful in a method for detecting a TCR ligand selected from CD1 -antigen complexes, bacterial superantigens, and MHC- peptide/superantigen complexes, which method comprises contacting the TCR ligand with a high affinity TCR (or a multimeric high affinity TCR complex) which is specific for the TCR ligand; and detecting binding to the TCR ligand.
  • a high affinity TCR or a multimeric high affinity TCR complex
  • multimeric high affinity TCR complexes such as those described in Zhu et ai, J. Immunol.
  • fluorescent streptavidin (commercially available) can be used to provide a detectable label.
  • a fluorescently-labelled multimer is suitable for use in FACS analysis, for example to detect antigen presenting cells carrying the peptide for which the high affinity TCR is specific.
  • a TCR of the present invention may alternatively or additionally be associated with (e.g. covalently or otherwise linked to) a therapeutic agent which may be, for example, a toxic moiety for use in cell killing, or an immunostimulating agent such as an interleukin or a cytokine.
  • a multivalent high affinity TCR complex of the present invention may have enhanced binding capability for a TCR ligand compared to a non-multimeric wild-type or high affinity T cell receptor heterodimer.
  • the multivalent high affinity TCR complexes according to the invention are particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo, and are also useful as intermediates for the production of further multivalent high affinity TCR complexes having such uses.
  • the high affinity TCR or multivalent high affinity TCR complex may therefore be provided in a pharmaceutically acceptable formulation for use in vivo.
  • High affinity TCRs of the invention may be used in the production of soluble bi-specific reagents.
  • a preferred embodiment is a reagent which comprises a soluble TCR, fused via a linker to an anti- CD3 specific antibody fragment. Further details including how to produce such reagents are described in W010/133828.
  • the invention provides nucleic acid encoding the TCR of the invention, a TCR expression vector comprising nucleic acid encoding a TCR of the invention, as well as a cell harbouring such a vector.
  • the TCR may be encoded either in a single open reading frame or two distinct open reading frames.
  • one vector may encode both an alpha and a beta chain of a TCR of the invention.
  • a further aspect of the invention provides a cell displaying on its surface a TCR of the invention.
  • the cell may be a T cell.
  • T cells expressing the TCRs of the invention are suitable for use in adoptive therapy-based treatment of diseases such as cancers.
  • adoptive therapy can be carried out (see for example Rosenberg et al., Nat Rev Cancer. 2008 Apr;8(4):299-308).
  • the TCRs of the invention intended for use in adoptive therapy are generally glycosylated when expressed by the transfected T cells.
  • the glycosylation pattern of transfected TCRs may be modified by mutations of the transfected gene (Kuball J et ai, J Exp Med. 2009 Feb 16;206(2):463-75).
  • the binding moiety of the invention may be an antibody.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced.
  • antibody includes antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanised antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic and any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.
  • a humanised antibody may be a modified antibody having the variable regions of a non-human, e.g. murine, antibody and the constant region of a human antibody. Methods for making humanised antibodies are described in, for example, US Patent No. 5225539. Examples of antibodies are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. A monoclonal antibody may be referred to herein as "mab".
  • an antibody for example a monoclonal antibody
  • recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody.
  • Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementary determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin (see, for instance, EP-A-184187, GB 2188638A or EP-A-239400).
  • CDRs complementary determining regions
  • a hybridoma (or other cell that produces antibodies) may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. et ai, Nature.
  • Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g.
  • antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).
  • bispecific antibodies these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Hollinger & Winter, Curr Opin
  • bispecific antibody fragments e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain "Janusins" described in Traunecker et al., EMBO J. 1991 Dec; 10(12):3655-9). Bispecific diabodies, as opposed to bispecific whole antibodies, may also be useful because they can be readily constructed and expressed in E. coli.
  • Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
  • An "antigen binding domain" is the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope.
  • An antigen binding domain may be provided by one or more antibody variable domains.
  • An antigen binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • the binding moiety may be an antibody-like molecule that has been designed to specifically bind a peptide-MHC complex of the invention.
  • TCR-mimic antibodies such as, for example those described in WO2007143104 and Sergeeva ef al., Blood. 201 1 Apr
  • binding moieties based on engineered protein scaffolds are derived from stable, soluble, natural protein structures which have been modified to provide a binding site for a target molecule of interest.
  • engineered protein scaffolds include, but are not limited to, affibodies, which are based on the Z-domain of staphylococcal protein A that provides a binding interface on two of its a-helices (Nygren, FEBS J.
  • Engineered protein scaffolds are typically targeted to bind the same antigenic proteins as antibodies, and are potential therapeutic agents. They may act as inhibitors or antagonists, or as delivery vehicles to target molecules, such as toxins, to a specific tissue in vivo (Gebauer and Skerra, Curr Opin Chem Biol. 2009 Jun;13(3):245-55). Short peptides may also be used to bind a target protein.
  • Phylomers are natural structured peptides derived from bacterial genomes. Such peptides represent a diverse array of protein structural folds and can be used to inhibit/disrupt protein-protein interactions in vivo (Watt, Nat Biotechnol. 2006 Feb;24(2): 177-83)].
  • the invention further provides a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention for use in medicine.
  • the polypeptide, complex, nucleic acid, vector, cell or binding moiety may be used for in the treatment or prevention of cancer, in particular, ovarian cancer.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a binding moiety of the invention together with a pharmaceutically acceptable carrier.
  • This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient). It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally
  • compositions and methods of administration are known to those skilled in the art, for example see, Johnson et al., Blood. 2009 Jul 16; 1 14(3):535-46, with reference to clinical trial numbers NCI-07-C-0175 and NCI-07-C-0174.
  • Cells in accordance with the invention will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a
  • T cells transfected with TCRs of the invention may be provided in pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be adapted for administration by any appropriate route such as a parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation or intranasal routes.
  • Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
  • Dosages of the substances of the present invention can vary between wide limits, depending upon the disease or disorder to be treated (such as cancer, viral infection or autoimmune disease), the age and condition of the individual to be treated, etc.
  • a suitable dose range for a reagent comprising a soluble TCR fused to an anti- CD3 domain may be between 25 ng/kg and 50 pg/kg.
  • a physician will ultimately determine appropriate dosages to be used.
  • the polypeptide of the invention may be provided in the form of a vaccine composition.
  • the vaccine composition may be useful for the treatment or prevention of cancer. All such
  • compositions are encompassed in the present invention.
  • vaccines may take several forms (Schlom, J Natl Cancer Inst. 2012 Apr 18;104(8):599-613).
  • the peptide of the invention may be used directly to immunise patients (Salgaller, Cancer Res. 1996 Oct 15;56(20):4749-57 and Marchand, Int J Cancer. 1999 Jan 18;80(2):219-30).
  • the vaccine composition may include additional peptides such that the peptide of the invention is one of a mixture of peptides.
  • Adjuvants may be added to the vaccine composition to augment the immune response
  • the vaccine composition may take the form of an antigen presenting cell displaying the peptide of the invention in complex with MHC.
  • the antigen presenting cell is an immune cell, more preferably a dendritic cell.
  • the peptide may be pulsed onto the surface of the cell (Thurner, J Exp Med. 1999 Dec 6; 190(11 ): 1669-78), or nucleic acid encoding for the peptide of the invention may be introduced into dendritic cells (for example by electroporation. Van Tendeloo, Blood. 2001 Jul 1 ;98(1 ):49-56).
  • polypeptides, complexes, nucleic acid molecules, vectors, cells and binding moieties of the invention may be non-naturally occurring and/or purified and/or engineered and/or recombinant and/or isolated and/or synthetic.
  • the invention also provides a method of identifying a binding moiety that binds a complex of the invention, the method comprising contacting a candidate binding moiety with the complex and determining whether the candidate binding moiety binds the complex.
  • Methods to determine binding to polypeptide-MHC complexes are well known in the art. Preferred methods include, but are not limited to, surface plasmon resonance, or any other biosensor technique, ELISA, flow cytometry, chromatography, microscopy. Alternatively, or in addition, binding may be determined by functional assays in which a biological response is detected upon binding, for example, cytokine release or cell apoptosis.
  • the candidate binding moiety may be a binding moiety of the type already described, such as a TCR or an antibody.
  • T cells may be isolated from fresh blood obtained from volunteer donors. Such a method involves stimulating T cells using autologous DCs, followed by autologous B cells, pulsed with the polypeptide of the invention. Several round of stimulation may be carried out, for example three or four rounds. Activated T cells may then be tested for specificity by measuring cytokine release in the presence of T2 cells pulsed with the peptide of the invention (for example using an IFNy ELISpot assay). Activated cells may then be sorted by fluorescence-activated cell sorting (FACS) using labelled antibodies to detect intracellular cytokine production (e.g.
  • FACS fluorescence-activated cell sorting
  • Sorted cells may be expanded and further validated, for example, by ELISpot assay and/or cytotoxicity against target cells and/or staining by peptide-MHC tetramer.
  • the TCR chains from validated clones may then be amplified by rapid amplification of cDNA ends (RACE) and sequenced.
  • the polypeptide MHC complex of the invention may be used for screening a library of TCRs or antibodies.
  • the production of antibody libraries using phage display is well known in the art, for example see Aitken, Antibody phage display: Methods and Protocols (2009, Humana, New York). Further details on TCR libraries can be found in WO04044004.
  • the polypeptide-MHC complex of the invention is used to screen a library of diverse TCRs displayed on the surface of phage particles.
  • the TCRs displayed by said library may contain non- natural mutations. Screening may involve panning the phage library with peptide-MHC complexes of the invention and subsequently isolating bound phage.
  • peptide-MHC complexes may be attached to a solid support, such as a magnet bead, or column matrix and phage bound peptide MHC complexes isolated, with a magnet, or by chromatography, respectively.
  • the panning steps may be repeated several times for example three or four times.
  • Isolated phage may be further expanded in E. coli cells.
  • Isolated phage particles may be tested for specific binding peptide-MHC complexes of the invention. Binding can be detected using techniques including, but not limited to, ELISA, or SPR for example using a BiaCore instrument.
  • the DNA sequence of the T cell receptor displayed by peptide-MHC binding phage can be further identified by standard PCR methods.
  • Figures 11 to I8 show the respective fragmentation spectra for the peptides of SEQ NOs: 11 to I8, eluted from cells. A table highlighting the matching ions is shown below each spectrum.
  • Immortalised cell lines obtained from commercial sources were maintained and expanded under standard conditions.
  • Class I HLA complexes were purified by immunoaffinity using commercially available anti-HLA antibodies BB7.1 (anti-HLA-B * 07), BB7.2 (anti-HLA-A * 02) and W6/32 (anti-Class 1 ). Briefly, cells were lysed in buffer containing non-ionic detergent NP-40 (0.5% v/v) at 5 x 10 7 cells per ml and incubated at 4°C for 1 h with agitation/mixing. Cell debris was removed by centrifugation and supernatant pre-cleared using proteinA-Sepharose.
  • Peptides were separated by high pressure liquid chromatography (HPLC) on a Dionex Ultimate 3000 system using a C18 column (Phenomenex). Peptides were loaded in 98% buffer A (0.1 % aqueous trifluoroacetic acid (TFA)) and 2% buffer B (0.1% TFA in acetonitrile). Peptides were eluted using a stepped gradient of B (2-60%) over 20 min. Fractions were collected at one minute intervals and lyophilised.
  • HPLC high pressure liquid chromatography
  • Peptides were analysed by nanoLCMS/MS using a Dionex Ultimate 3000 nanoLC coupled to either AB Sciex Triple TOF 5600 or Thermo Orbitrap Fusion mass spectrometers. Both machines were equipped with nanoelectrospray ion sources. Peptides were loaded onto an Acclaim PepMap 100 trap column (Dionex) and separated using an Acclaim PepMap RSLC column (Dionex). Peptides were loaded in mobile phase A (0.5% formic acid: water) and eluted using a gradient of buffer B (acetonitrile:0.5% formic acid) directly into the nanospray ionisation source.
  • buffer B acetonitrile:0.5% formic acid
  • polypeptides set out in table 11 were detected by mass spec following extraction from cancer cell lines.
  • An example cell line from which the peptide was detected is indicated in the table along with the HLA antibody used for immunoaffinity purification.
  • Figures 11 -18 show representative fragmentation patterns for the peptides of SEQ NOs: 11-18 respectively. A table highlighting the matching ions is shown below each spectrum.
  • Example I2 Preparation of recombinant peptide-HLA complexes
  • HLA-A*02 molecules HLA-A*02-heavy chain and HLA light-chain ( ⁇ 2 ⁇ ) were expressed separately in E. coli as inclusion bodies, using appropriate constructs.
  • HLA-A * 02-heavy chain additionally contained a C-terminal biotinylation tag which replaces the transmembrane and cytoplasmic domains (O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15).
  • E. coli cells were lysed and inclusion bodies processed to approximately 80% purity.
  • Inclusion bodies of ⁇ 2 ⁇ and heavy chain were denatured separately in 6 M guanidine-HCI, 50 mM Tris pH 8.1 , 100 mM NaCI, 10 mM DTT, 10 mM EDTA.
  • Refolding buffer was prepared containing 0.4 M L-Arginine, 100 mM Tris pH 8.1 , 3.7 mM cystamine dihydrochloride, 6.6 mM cysteamine hydrochloride and chilled to ⁇ 5°C.
  • Synthetic peptide dissolved in DMSO to a final concentration of 4mg/ml is added to the refold buffer at 4 mg/litre (final concentration). Then 30 mg/litre ⁇ 2 ⁇ followed by 30 mg/litre heavy chain (final concentrations) are added.
  • Refolding was allowed to reach completion at 4 °C for at least 1 hour.
  • Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Two changes of buffer were necessary to reduce the ionic strength of the solution sufficiently.
  • the protein solution was then filtered through a 1.5 ⁇ cellulose acetate filter and loaded onto a POROS 50HQ anion exchange column (8 ml bed volume). Protein was eluted with a linear 0-500 mM NaCI gradient in 10 mM Tris pH 8.1 using an Akta purifier (GE Healthcare). HLA-A * 02-peptide complex eluted at approximately 250 mM NaCI, and peak fractions were collected, a cocktail of protease inhibitors (Calbiochem) was added and the fractions were chilled on ice.
  • Biotinylation tagged pHLA molecules were buffer exchanged into 10 mM Tris pH 8.1 , 5 mM NaCI using a GE Healthcare fast desalting column equilibrated in the same buffer. Immediately upon elution, the protein-containing fractions were chilled on ice and protease inhibitor cocktail (Calbiochem) was added. Biotinylation reagents were then added: 1 mM biotin, 5 mM ATP (buffered to pH 8), 7.5 mM MgCl2, and 5 pg/ml BirA enzyme (purified according to O'Callaghan et al., (1999) Anal. Biochem. 266: 9-15). The mixture was then allowed to incubate at room temperature overnight.
  • the biotinylated pHLA-A * 02 molecules were purified using gel filtration chromatography.
  • a GE Healthcare Superdex 75 HR 10/30 column was pre-equilibrated with filtered PBS and 1 ml of the biotinylation reaction mixture was loaded and the column was developed with PBS at 0.5 ml/min using an Akta purifier (GE Healthcare).
  • Biotinylated pHLA-A * 02 molecules eluted as a single peak at approximately 15 ml. Fractions containing protein were pooled, chilled on ice, and protease inhibitor cocktail was added.
  • Protein concentration was determined using a Coomassie-binding assay (PerBio) and aliquots of biotinylated pHLA-A * 02 molecules were stored frozen at -20 °C.
  • Such peptide-MHC complexes may be used in soluble form or may be immobilised through C terminal biotin moiety on to a solid support, to be used for the detection of T cells and T cell receptors which bind said complex.
  • TCR isolation methods are known to those skilled in the art; for example, DNA sequences corresponding to TCR alpha and beta chains may be amplified from a T cell clone (obtained from volunteer blood donors) that is activated by cells presenting the peptide-HLA complex of interest.
  • T cell clone obtained from volunteer blood donors
  • Other suitable methods include isolation from TCR libraries (as described in WO2005/1 16646 and WO2005/116074 for example).
  • TCR alpha and beta chain sequences are expressed in E. coli as soluble TCRs, (WO2003020763; Boulter, et al., Protein Eng, 2003. 16: 707-711 ). Binding of the soluble TCRs to the complexes is analysed by surface plasmon resonance using a BiaCore 3000 instrument. Biotinylated peptide-HLA monomers are prepared as previously described (Example I2) and immobilized on to a streptavidin-coupled CM-5 sensor chip.
  • The[LS9] present invention also relates to novel peptides derived from Fc receptor-like A (FCRLA), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules.
  • FCRLA Fc receptor-like A
  • binding moieties that bind to the peptides and/or complexes of the invention. Such moieties are useful for the development of immunotherapeutic reagents for the treatment of diseases such as cancer.
  • T cells are a key part of the cellular arm of the immune system. They specifically recognise peptide fragments that are derived from intracellular proteins and presented in complex with Major
  • MHC Histocompatibility Complex
  • APCs antigen presenting cells
  • HLA human leukocyte antigens
  • MHC molecules have a binding groove in which the peptide fragments bind. Recognition of particular peptide-MHC antigens is mediated by a corresponding T cell receptor (TCR). Tumour cells express various tumour associated antigens (TAA) and peptides derived from these antigens are frequently displayed on the tumour cell surface. Detection of a MHC class I- presented TAA-derived peptide by a CD8+ T cell bearing the corresponding T cell receptor, leads to targeted killing of the tumour cell.
  • TAA tumour associated antigens
  • tumour cells often escape detection.
  • TAA-derived peptides presented by MHC molecules on tumour cells enables the development of novel immunotherapeutic reagents designed to specifically target and destroy said tumour cells.
  • Such reagents may be moieties that bind to the TAA-derived peptide and/or peptide-MHC complexes of the invention, and typically involve the induction of a T cell response.
  • such reagents may be based, exclusively, or in part, on T cells, or T cell receptors (TCRs), or antibodies.
  • TAA-derived peptides that are presented on the cell surface in complex with a given MHC, only a small number of such peptides, covering a limited number of cancer indications, have been identified to date.
  • TAAs suitable for the development of immunotherapeutic reagents must have limited or no expression in normal cells, to prevent off target activity. It is therefore desirable to provide additional tumour-specific TAA-derived peptides and MHC complexes thereof that can be used for the development of new cancer therapies.
  • FCRLA also known as Fc receptor-like A or Fc receptor homolog expressed in B-cells or Fc receptor-related protein X or Fc receptor-like protein and having Uniprot accession number Q7L513). Expression of FCRLA is associated with cancer (Inozume et al. Int J Cancer. 2005 Mar 20; 1 14(2):283-90). FCRLA is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from FCRLA that are presented on the cell surface in complex with MHC. These peptides are particularly useful for the development of reagents that can targets cells expressing FCRLA and for the treatment of cancers, including cutaneous melanoma and B- cell malignancy.
  • the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: J 1 -J 19, or
  • polypeptide forms a complex with a Major Histocompatibility Complex (MHC) molecule.
  • MHC Major Histocompatibility Complex
  • polypeptides of the invention are presented by MHC on the surface of tumour cells. Accordingly, the polypeptides of the invention, as well as moieties that bind the polypeptide-MHC complexes, can be used to develop therapeutic reagents.
  • the skilled person can determine whether or not a given polypeptide forms a complex with an MHC molecule by determining whether the MHC can be refolded in the presence of the polypeptide using the process set out in Example J2. If the polypeptide does not form a complex with MHC then MHC will not refold. Refolding is commonly confirmed using an antibody that recognises MHC in a folded state only. Further details can be found in Garboczi et al., Proc Natl Acad Sci
  • polypeptides of the invention are from about 8 to about 16 amino acids in length, and are most preferably 8, 9, or 10 or 11 amino acids in length.
  • polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: J1-J19.
  • amino acid residues comprising the polypeptides of the invention may be chemically modified.
  • chemical modifications include those corresponding to post translational modifications for example phosphorylation, acetylation and deamidation (Engelhard et al., Curr Opin Immunol. 2006 Feb;18(1 ):92-7).
  • Amino acid substitution means that an amino acid residue is substituted for a replacement amino acid residue at the same position.
  • Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are immediately adjacent one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent another inserted amino acid residue.
  • One, two or three amino acids may be deleted from the sequence of SEQ NOs: J1-J19. Each deletion can take place at any position of SEQ NOs: J1-J19.
  • the polypeptide of the invention may comprise one, two or three additional amino acids at the C-terminal end and/or at the N-terminal end of the sequence of SEQ NOs: J1- J19.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: J 1 -J 19 with the exception of one amino acid substitution and one amino acid insertion, one amino acid substitution and one amino acid deletion, or one amino acid insertion and one amino acid deletion.
  • a polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: J1-J19, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.
  • Inserted amino acids and replacement amino acids may be naturally occurring amino acids or may be non-naturally occurring amino acids and, for example, may contain a non-natural side chain.
  • Such altered peptide ligands are discussed further in Douat-Casassus et a/., J. Med. Chem, 2007 Apr 5;50(7): 1598-609 and Hoppes et al., J. Immunol 2014 Nov 15;193(10):4803-13 and references therein). If more than one amino acid residue is substituted and/or inserted, the

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Abstract

La présente invention concerne de nouveaux peptides dérivés de la protéine centrosomale C10orf90 (C10orf90) ainsi que d'autres protéines, des complexes comprenant de tels peptides liés à des molécules recombinées du CMH, et des cellules présentant ledit peptide dans un complexe avec des molécules du CMH. L'invention concerne également des fractions de liaison qui se lient aux peptides et/ou aux complexes de l'invention. De telles fractions sont utiles dans le développement de réactifs immunothérapeutiques pour le traitement de maladies telles que le cancer.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019211633A1 (fr) * 2018-05-04 2019-11-07 Oxford University Innovation Limited Procédé de diagnostic et thérapie
US20190389930A1 (en) * 2016-04-21 2019-12-26 Immatics Biotechnologies Gmbh Immunotherapy against melanoma and other cancers
DE102018115865A1 (de) * 2018-06-29 2020-02-20 Immatics Biotechnologies Gmbh A*03-restringierte Peptide zur Verwendung in der Immuntherapie gegen Krebs und verwandte Verfahren
US10828357B2 (en) 2018-06-29 2020-11-10 Immatics Biotechnologies Gmbh A*03 restricted peptides for use in immunotherapy against cancers and related methods
WO2021092223A1 (fr) * 2019-11-05 2021-05-14 Board Of Regents, The University Of Texas System Récepteurs des lymphocytes t de hormad1 restreints aux hla et leurs utilisations
US20220265795A1 (en) * 2015-12-11 2022-08-25 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against various cancers

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