WO2017089786A1

WO2017089786A1 - Peptides

Info

Publication number: WO2017089786A1
Application number: PCT/GB2016/053668
Authority: WO
Inventors: Alex POWLESLAND
Original assignee: Immunocore Ltd; Adaptimmune Ltd
Current assignee: Immunocore Ltd; Adaptimmune Ltd
Priority date: 2015-11-23
Filing date: 2016-11-23
Publication date: 2017-06-01
Anticipated expiration: 2018-05-23

Abstract

The present invention relates to novel peptides derived from Centrosomal protein C10orf90 (C10orf90) as well as from other proteins, complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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.

Description

Peptides

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. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

The identification of particular 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. For example, such reagents may be based, exclusively, or in part, on T cells, or T cell receptors (TCRs), or antibodies. However, because of the inherent difficulties in identifying 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. Furthermore, 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.

In silico algorithms, such as SYFPETHEI (Rammensee, et al., Immunogenetics. 1999 Nov; 50(3- 4):213-9 (access via www.syfpeithi.de) and BIMAS (Parker, et al., J. Immunol. 1994 Jan

1 ;152(1 ):163-75 (access via http://www-bimas.cit.nih.gov/molbio/hla bind/)) are available to predict the amino acid sequences of MHC-presented peptides derived from proteins. However, these methods are known to generate a high proportion of false positives (since they simply define the likelihood of a given peptide being able to bind a given MHC and do not account for intracellular processing). Therefore, it is not possible to accurately predict whether a given peptide-MHC is actually presented by tumour cells. Direct experimental data is typically required.

C10orf90 (also known as centrosomal protein C10orf90 or fragile-site associated tumor suppressor homolog and having Uniprot accession number Q96M02) 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. These peptides are particularly useful for the development of reagents that can targets cells expressing C10orf90 and for the treatment of cancers, including uveal melanoma. In a first aspect, 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

(b) the amino acid sequence of any one of SEQNOs: A1-A9 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

wherein the polypeptide forms a complex with a Major Histocompatibility Complex (MHC) molecule.

The inventors have found that 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

USA. 1992 Apr 15;89(8):3429-33.

Preferably, 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: A1-A9.

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 SEQNOs: A1-A9. Each deletion can take place at any position of SEQNOs: A1-A9. In some embodiments, 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. Preferably, polypeptides of the invention bind to MHC in the peptide binding groove of the MHC molecule. Generally the amino acid modifications described above will not impair the ability of the peptide to bind MHC. In a preferred embodiment, the amino acid modifications improve the ability of the peptide to bind MHC. For example, 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. Alternatively, 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. Alternatively, in vitro cell-free systems may be used. Such systems are known in the art and are commercially available for example from Life

Technologies, Paisley, UK. The 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.

In a second aspect the invention provides a complex of the polypeptide of the first aspect and an MHC molecule. Preferably, 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. 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. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, the term " 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. For example, 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. co// cells or insect cells. A suitable method is provided in Example A2 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. Alternatively, 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. Examples of 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. In a preferred embodiment 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. In general, polypeptide-MHC multimers may be produced using peptide-MHC tagged with a biotin residue and complexed through fluorescent labelled streptavidin. Alternatively, multimeric polypeptide-MHC complexes may be formed by using immunoglobulin as a molecular scaffold. In this system, 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. Thus, 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.

In a third aspect, 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. In a fourth aspect, 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. In one embodiment, 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.

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. For instance, 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.

In the context of the present invention, 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. 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.

In a fifth aspect, 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. In particular, 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. Such binding moieties may be used as immunotherapeutic reagents and may include antibodies and TCRs. In a sixth aspect, the invention provides a binding moiety that binds the polypeptide of the invention. Preferably the binding moiety binds the polypeptide when said polypeptide is in complex with MHC. In the latter instance, 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.

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;

Lefranc, (201 1 ), Cold Spring Harb Protoc 2011(6): 595-603; Lefranc, (2001 ), Curr Protoc Immunol Appendix 1 : Appendix 10; Lefranc, (2003), Leukemia 17(1 ): 260-266, and on the IMGT website (www.IMGT.org) The TCRs of the invention may be in any format known to those in the art. For example, the TCRs may be αβ heterodimers, or they may be in single chain format (such as those described in W09918129). Single chain TCRs include αβ TCR polypeptides of the type: Va-L-νβ, νβ-L-Va, Va- Ca-L-νβ, να-ί-\ β-0β or Va- Ca -ίΛ/β-Οβ, optionally in the reverse orientation, wherein Va and νβ are TCR a and β variable regions respectively, Ca and C are TCR a and β 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. In addition the TRAC/TRBC may contain modifications. For example, 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. Likewise, 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 al., Nat Biotechnol 2005

Mar;23(3):349-54). In addition the use of the stable disulphide linked soluble TCR enables more convenient assessment of binding affinity and binding half-life. Alternative positions for the formation of a non-native disulphide bond are described in WO 03/020763.

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. In multimeric high affinity TCR complexes such as those described in Zhu et ai, J. Immunol. 2006 Mar 1 ;176(5):3223-32, (formed, for example, using biotinylated heterodimers) 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. Thus, 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.

In a further aspect, 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. Also included in the scope of the invention is a cell harbouring a first expression vector which comprises nucleic acid encoding an alpha chain of a TCR of the invention, and a second expression vector which comprises nucleic acid encoding a beta chain of a TCR of the invention. Alternatively, 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. There are a number of methods suitable for the transfection of T cells with DNA or RNA encoding the TCRs of the invention (see for example Robbins et al., J. Immunol. 2008 May 1 ;180(9):6116-31 ). T cells expressing the TCRs of the invention are suitable for use in adoptive therapy-based treatment of diseases such as cancers. As will be known to those skilled in the art there are a number of suitable methods by which 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. As is well known, 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. The term "antibody" as used herein 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. The term "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. 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".

It is possible to take an antibody, for example a monoclonal antibody, and use 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). 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.

It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of 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. 1989 Oct 12;341 (6242):544-6) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al., Science. 1988 Oct 21 ;242(4877):423-6; Huston et al., Proc Natl Acad Sci U S A. 1988 Aug;85(16):5879-83); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94/13804; P. Hollinger et al., Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6444-8). 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. by a peptide linker) but unable to associate with each other to form an antigen binding site: 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). Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Hollinger & Winter, Curr Opin

Biotechnol. 1993 Aug;4(4):446-9), 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. Of particular preference are TCR-mimic antibodies, such as, for example those described in WO2007143104 and Sergeeva ef al., Blood. 201 1 Apr

21 ;1 17(16):4262-72 and/or Dahan and Reiter. Expert Rev Mol Med. 2012 Feb 24;14:e6.

Also encompassed within the present invention are binding moieties based on engineered protein scaffolds. 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. Examples of 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. 2008 Jun;275(1 1 ):2677-83), nanobodies, and DARPins. 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)].

In another aspect, 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.

In a further aspect, the invention provides 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. 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

pharmaceutically acceptable carrier. For example, 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. For example, 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. As will be appreciated, vaccines may take several forms (Schlom, J Natl Cancer Inst. 2012 Apr 18;104(8):599-613). For example, 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. Preferably 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.

For example, 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). 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. Alternatively, 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. In a preferred embodiment, 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. For this purpose 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. The present invention will be further illustrated in the following Examples and Figures which are given for illustration purposes only and are not intended to limit the invention in any way.

Brief description of the Figures 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.

Examples Example A1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from C10orf90 on the surface of tumour cell lines was investigated using mass spectrometry.

Method

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 10⁷ 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. Columns were washed with low salt and high salt buffers and complexes eluted in acid. Eluted peptides were separated from HLA complexes by reversed phase chromatography using a solid phase extraction cartridge (Phenomenex). Bound material was eluted from the column and reduced in volume using a vacuum centrifuge.

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. 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.

For peptide identification the mass spectrometer was operated using an information dependent acquisition (IDA) workflow. 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 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

The following describes a suitable method for the preparation of soluble recombinant HLA loaded with TAA peptide. Class I HLA-A^*02 molecules (HLA-A^*02-heavy chain and HLA light-chain (β2ηι)) 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 β2ιη 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 β2πι 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 μητι 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 A3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

Methods for the isolation of TCRs 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. Other suitable methods include isolation from TCR libraries (as described in WO2005/1 16646 and WO2005/116074 for example).

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction.

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. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

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.

In a first aspect, 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

(b) the amino acid sequence of any one of SEQNOs: B1-B3 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions, wherein the polypeptide forms a complex with a Major Histocompatibility Complex (MHC) molecule. The inventors have found that 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 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

USA. 1992 Apr 15;89(8):3429-33.

Preferably, 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 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.

In some embodiments, 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.

Preferably, polypeptides of the invention bind to MHC in the peptide binding groove of the MHC molecule. Generally the amino acid modifications described above will not impair the ability of the peptide to bind MHC. In a preferred embodiment, the amino acid modifications improve the ability of the peptide to bind MHC. For example, 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.

In a second aspect the invention provides a complex of the polypeptide of the first aspect and an MHC molecule. Preferably, 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. 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. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, 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. For example, 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. 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. Alternatively, 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 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. In general, polypeptide-MHC multimers may be produced using peptide-MHC tagged with a biotin residue and complexed through fluorescent labelled streptavidin. Alternatively, multimeric polypeptide-MHC complexes may be formed by using immunoglobulin as a molecular scaffold. In this system, 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. Thus, 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.

In a third aspect, 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.

In a fourth aspect, 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. In one embodiment, 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.

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. Such binding moieties may be used as immunotherapeutic reagents and may include antibodies and TCRs.

In a sixth aspect, the invention provides a binding moiety that binds the polypeptide of the invention. Preferably the binding moiety binds the polypeptide when said polypeptide is in complex with MHC. In the latter instance, 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. 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;

Lefranc, (2011 ), Cold Spring Harb Protoc 2011(6): 595-603; Lefranc, (2001), Curr Protoc Immunol Appendix 1 : Appendix 10; Lefranc, (2003), Leukemia 17(1 ): 260-266, and on the IMGT website (www.IMGT.org)

The TCRs of the invention may be in any format known to those in the art. For example, the TCRs may be αβ heterodimers, or they may be in single chain format (such as those described in

W09918129). Single chain TCRs include αβ TCR polypeptides of the type: Va-L-νβ, νβ-L-Va, Va- Ca-L-νβ, Va-L-νβ-Οβ or Va- Ca -ίΛ β-Οβ, optionally in the reverse orientation, wherein Va and νβ are TCR a and β variable regions respectively, Ca and C are TCR a and β 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.

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. In multimeric high affinity TCR complexes such as those described in Zhu et al., J. Immunol. 2006 Mar 1 ; 176(5):3223-32, (formed, for example, using biotinylated heterodimers) 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. Thus, 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.

In a further aspect, 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. Also included in the scope of the invention is a cell harbouring a first expression vector which comprises nucleic acid encoding an alpha chain of a TCR of the invention, and a second expression vector which comprises nucleic acid encoding a beta chain of a TCR of the invention. Alternatively, 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. There are a number of methods suitable for the transfection of T cells with DNA or RNA encoding the TCRs of the invention (see for example Robbins et al., J. Immunol. 2008 May 1 ;180(9):61 16-31 ). T cells expressing the TCRs of the invention are suitable for use in adoptive therapy-based treatment of diseases such as cancers. As will be known to those skilled in the art there are a number of suitable methods by which adoptive therapy can be carried out (see for example Rosenberg et al., Nat Rev Cancer. 2008 Apr;8(4):299-308).

It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of 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. 1989 Oct 12;341 (6242):544-6) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al., Science. 1988 Oct 21 ;242(4877):423-6; Huston et al., Proc Natl Acad Sci U S A. 1988 Aug;85(16):5879-83); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94/13804; P. Hollinger et al., Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6444-8). 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. by a peptide linker) but unable to associate with each other to form an antigen binding site: 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). Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Hollinger & Winter, Curr Opin

The binding moiety may be an antibody-like molecule that has been designed to specifically bind a peptide-MHC complex of the invention. Of particular preference are TCR-mimic antibodies, such as, for example those described in WO2007143104 and Sergeeva et al., Blood. 201 1 Apr

In another aspect, 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.

In a further aspect, the invention provides 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. 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

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. For example, 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. As will be appreciated, vaccines may take several forms (Schlom, J Natl Cancer Inst. 2012 Apr 18;104(8):599-613). For example, 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. Preferably 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 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.

For example, 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). 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.

Alternatively, 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. In a preferred embodiment, 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. For this purpose 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.

The present invention will be further illustrated in the following Examples and Figures which are given for illustration purposes only and are not intended to limit the invention in any way.

Brief description of the Figures

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.

Examples

Example B1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from CDK5R2 on the surface of tumour cell lines was investigated using mass spectrometry. Method

For peptide identification the mass spectrometer was operated using an information dependent acquisition (IDA) workflow. 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 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. Amino acid Example cancer

SEQNO HLA antibody

sequence cell line

B1 SGGAPAASSA HLA-BW SUP-M2

B2 VAPPVPGGSP HLA-A*02 SW982

B3 YSYMGNEISY class I IGR37

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

The following describes a suitable method for the preparation of soluble recombinant HLA loaded with TAA peptide. Class I HLA-A*02 molecules (HLA-A*02-heavy chain and HLA light-chain (β2ιη)) 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 β2πι 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 β2πι 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.

Example B3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction. 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. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

The identification of particular 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. For example, such reagents may be based, exclusively, or in part, on T cells, or T cell receptors (TCRs), or antibodies. However, because of the inherent difficulties in identifying 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. Furthermore, 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. In silico algorithms, such as SYFPETHEI (Rammensee, et al., Immunogenetics. 1999 Nov; 50(3- 4):213-9 (access via www.syfpeithi.de) and BIMAS (Parker, et al., J. Immunol. 1994 Jan

1 ;152(1 ):163-75 (access via http://www-bimas.cit.nih.qov/molbio/hla bind/)) are available to predict the amino acid sequences of MHC-presented peptides derived from proteins. However, these methods are known to generate a high proportion of false positives (since they simply define the likelihood of a given peptide being able to bind a given MHC and do not account for intracellular processing). Therefore, it is not possible to accurately predict whether a given peptide-MHC is actually presented by tumour cells. Direct experimental data is typically required.

CNGB1 (also known as cyclic nucleotide-gated cation channel beta-1 or CNG channel 4 or GARP and having Uniprot accession number Q14028) functions as a subunit of cyclic nucleotide-gated (CNG) channels (Chen et al. Nature. 1993 Apr 22;362(6422):764-7). 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.

In a first aspect, 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

(b) the amino acid sequence of any one of SEQ NOs: C1-C9 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions, wherein the polypeptide forms a complex with a Major Histocompatibility Complex (MHC) molecule.

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

USA. 1992 Apr 15;89(8):3429-33. Preferably, 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: C1-C9.

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. In some embodiments, 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

In a second aspect the invention provides a complex of the polypeptide of the first aspect and an MHC molecule. Preferably, 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. 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. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, 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. For example, 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 C2 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. Alternatively, 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.

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;

Lefranc, (201 1 ), Cold Spring Harb Protoc 2011(6): 595-603; Lefranc, (2001 ), Curr Protoc Immunol Appendix 1 : Appendix 10; Lefranc, (2003), Leukemia 17(1 ): 260-266, and on the IMGT website (www.IMGT.org)

The TCRs of the invention may be in any format known to those in the art. For example, the TCRs may be αβ heterodimers, or they may be in single chain format (such as those described in W09918129). Single chain TCRs include αβ TCR polypeptides of the type: Va-L-νβ, νβ-L-Va, Va- Ca-L-νβ, να-ί-\ β-0β or Va- Ca -ίΛ/β-Οβ, optionally in the reverse orientation, wherein Va and νβ are TCR a and β variable regions respectively, Ca and C are TCR a and β 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. In addition the TRAC/TRBC may contain modifications. For example, 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. Likewise, 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 al., Nat Biotechnol 2005

Mar;23(3):349-54). In addition the use of the stable disulphide linked soluble TCR enables more convenient assessment of binding affinity and binding half-life. Alternative positions for the formation of a non-native disulphide bond are described in WO 03/020763. 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. In multimeric high affinity TCR complexes such as those described in Zhu et al., J. Immunol. 2006 Mar 1 ; 176(5):3223-32, (formed, for example, using biotinylated heterodimers) 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 further aspect of the invention provides a cell displaying on its surface a TCR of the invention. The cell may be a T cell. There are a number of methods suitable for the transfection of T cells with DNA or RNA encoding the TCRs of the invention (see for example Robbins et al., J. Immunol. 2008 May 1 ;180(9):61 16-31 ). T cells expressing the TCRs of the invention are suitable for use in adoptive therapy-based treatment of diseases such as cancers. As will be known to those skilled in the art there are a number of suitable methods by which 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. As is well known, 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).

It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of 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. 1989 Oct 12;341 (6242):544-6) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et ai, Science. 1988 Oct 21 ;242(4877):423-6; Huston et al., Proc Natl Acad Sci U S A. 1988 Aug;85(16):5879-83); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94/13804; P. Hollinger et al., Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6444-8). 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. by a peptide linker) but unable to associate with each other to form an antigen binding site: 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). Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Hollinger & Winter, Curr Opin

Biotechnol. 1993 Aug;4(4):446-9), 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. Of particular preference are TCR-mimic antibodies, such as, for example those described in WO2007143104 and Sergeeva ef al., Blood. 201 1 Apr

21 ;1 17(16):4262-72 and/or Dahan and Reiter. Expert Rev Mol Med. 2012 Feb 24;14:e6. Also encompassed within the present invention are binding moieties based on engineered protein scaffolds. 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. Examples of 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. 2008 Jun;275(1 1 ):2677-83), nanobodies, and DARPins. 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)]. In another aspect, 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.

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. For example, 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

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.

Brief description of the Figures

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.

Examples

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.

Method

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 10⁷ 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. Columns were washed with low salt and high salt buffers and complexes eluted in acid. Eluted peptides were separated from HLA complexes by reversed phase chromatography using a solid phase extraction cartridge (Phenomenex). Bound material was eluted from the column and reduced in volume using a vacuum centrifuge.

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. 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.

Results The polypeptides set out in table C1 , corresponding to SEQ NOs: C1 -C9, 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 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.

Example C2 - Preparation of recombinant peptide-HLA complexes

The following describes a suitable method for the preparation of soluble recombinant HLA loaded with TAA peptide.

Class I HLA-A^*02 molecules (HLA-A^*02-heavy chain and HLA light-chain (β2ηι)) 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 β2ιη 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 β2πι 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.

Example C3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction.

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. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

CNTNAP2 (also known as contactin-associated protein-like 2 or cell recognition molecule Caspr2 and having Uniprot accession number Q9UHC6) may play a role in the formation nerve impulses in myelinated nerve fibres. 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.

In a first aspect, 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

(b) the amino acid sequence of SEQ NO: D1 with the exception of 1 , 2 or 3 amino acid

substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions, wherein the polypeptide forms a complex with a Major Histocompatibility Complex (MHC) molecule.

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

USA. 1992 Apr 15;89(8):3429-33. Preferably, 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.

In some embodiments, 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.

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. Preferably, polypeptides of the invention bind to MHC in the peptide binding groove of the MHC molecule. Generally the amino acid modifications described above will not impair the ability of the peptide to bind MHC. In a preferred embodiment, the amino acid modifications improve the ability of the peptide to bind MHC. For example, 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.

Technologies, Paisley, UK. The 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. In a second aspect the invention provides a complex of the polypeptide of the first aspect and an MHC molecule. Preferably, 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. 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. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, 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. For example, 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 D2 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. Alternatively, 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.

21 ;1 17(16):4262-72 and/or Dahan and Reiter. Expert Rev Mol Med. 2012 Feb 24;14:e6. Also encompassed within the present invention are binding moieties based on engineered protein scaffolds. 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. Examples of 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. 2008 Jun;275(1 1 ):2677-83), nanobodies, and DARPins. 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)]. In another aspect, 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.

Brief description of the Figures

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.

Examples

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.

Method

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.

Example D2 - Preparation of recombinant peptide-HLA complexes

Class I HLA-A*02 molecules (HLA-A*02-heavy chain and HLA light-chain (β2ιη)) 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 β2πι 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 β2πι 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 μιτι 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 D3 - identification of TCRs that bind to a peptide-MHC complex of the invention

Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction. 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. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

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.

Expression of CT45A3 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 31 ;102(22):7940-5). 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.

In a first aspect, 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

(b) the amino acid sequence of any one of SEQ NOs: E1-E2 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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

USA. 1992 Apr 15;89(8):3429-33.

Preferably, 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.

In some embodiments, 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.

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. Preferably, polypeptides of the invention bind to MHC in the peptide binding groove of the MHC molecule. Generally the amino acid modifications described above will not impair the ability of the peptide to bind MHC. In a preferred embodiment, the amino acid modifications improve the ability of the peptide to bind MHC. For example, 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).

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 E2 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. Alternatively, 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.

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; Lefranc, (201 1 ), Cold Spring Harb Protoc 2011(6): 595-603; Lefranc, (2001 ), Curr Protoc Immunol Appendix 1 : Appendix 10; Lefranc, (2003), Leukemia 17(1 ): 260-266, and on the IMGT website (www.IMGT.org) The TCRs of the invention may be in any format known to those in the art. For example, the TCRs may be αβ heterodimers, or they may be in single chain format (such as those described in W09918129). Single chain TCRs include αβ TCR polypeptides of the type: Va-L-νβ, νβ-L-Va, Va- Ca-L-νβ, να-ί-\ β-0β or Va- Ca -ίΛ/β-Οβ, optionally in the reverse orientation, wherein Va and νβ are TCR a and β variable regions respectively, Ca and C are TCR a and β 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.

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. In multimeric high affinity TCR complexes such as those described in Zhu et al., J. Immunol. 2006 Mar 1 ; 176(5):3223-32, (formed, for example, using biotinylated heterodimers) 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 further aspect of the invention provides a cell displaying on its surface a TCR of the invention. The cell may be a T cell. There are a number of methods suitable for the transfection of T cells with DNA or RNA encoding the TCRs of the invention (see for example Robbins et al., J. Immunol. 2008 May 1 ;180(9):61 16-31 ). T cells expressing the TCRs of the invention are suitable for use in adoptive therapy-based treatment of diseases such as cancers. As will be known to those skilled in the art there are a number of suitable methods by which adoptive therapy can be carried out (see for example Rosenberg et al., Nat Rev Cancer. 2008 Apr;8(4):299-308).

In another aspect, 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 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. For example, 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

Brief description of the Figures

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.

Examples

Example E1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from CT45A3 on the surface of tumour cell lines was investigated using mass spectrometry. Method

Results

The 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. Amino acid Example cancer

SEQ NO HLA antibody

sequence cell line

E1 SQIDDFTGF class I HT 144

E2 TDKTEKVAVD HLA-A^*02 NCI H1703

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. Example E2 - Preparation of recombinant peptide-HLA complexes

Inclusion bodies of β2πι 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 β2ιη 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.

(buffered to pH 8), 7.5 mM MgCI2, and 5 pg/ml BirA enzyme (purified according to O'Callaghan et a/., (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.

Example E3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction. 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. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

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

31 ;102(22):7940-5). 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.

In a first aspect, 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

(b) the amino acid sequence of any one of SEQ NOs: F1-F2 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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

USA. 1992 Apr 15;89(8):3429-33.

Preferably, 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.

In some embodiments, 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.

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 F2 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. Alternatively, 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.

In another aspect, 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.

Brief description of the Figures

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.

Examples

Example F1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from CT45A5 on the surface of tumour cell lines was investigated using mass spectrometry. Method

Results

The polypeptides set out in table F1 , corresponding to SEQ NOs: F1-F2, 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. Amino acid Example cancer

SEQ NO HLA antibody

sequence cell line

F1 FESIIKEAA Class I NCI-H1975

F2 YEKIFEML Class I HT 144

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. Example F2 - Preparation of recombinant peptide-HLA complexes

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.

Example F3 - identification of TCRs that bind to a peptide-MHC complex of the invention

Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction. 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. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

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. In a first aspect, 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

(b) the amino acid sequence of any one of SEQ NOs: G1-G8 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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

USA. 1992 Apr 15;89(8):3429-33.

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.

In some embodiments, 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.

Technologies, Paisley, UK. The 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. In a second aspect the invention provides a complex of the polypeptide of the first aspect and an MHC molecule. Preferably, 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. 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. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, the term " 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. For example, 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. 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. Alternatively, 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.

In another aspect, 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 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. For example, 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 candidate binding moiety may be a binding moiety of the type already described, such as a TCR or an antibody. For example, 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). 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.

Alternatively, 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. In a preferred embodiment, 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. For this purpose 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.

Brief description of the Figures

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.

Method

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.

Results

The 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.

Class I HLA-A*02 molecules (HLA-A*02-heavy chain and HLA light-chain (β2ιη)) 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 β2πι 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 β2ιη 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 μητι 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.

Example G3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction.

The[LS7] present invention also relates to novel peptides derived from DNA

nucleotidylexotransferase (DNTT), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

DNTT (also known as DNA nucleotidylexotransferase or terminal deoxynucleotidyltransferase or terminal addition enzyme and having Uniprot accession number P04053) 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.

In a first aspect, 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

(b) the amino acid sequence of any one of SEQ NOs: H1-H18 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

H13 LYDKTKRI

H14 MQKAGFLYY

H15 RMQKAGFLYY

H16 VLCPYERRAF

H17 VLNDERYQSF

H18 YYDLVESTF

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 H2. 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 USA. 1992 Apr 15;89(8):3429-33. Preferably, 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: H1-H18.

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.

In some embodiments, 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

Preferably, polypeptides of the invention bind to MHC in the peptide binding groove of the MHC molecule. Generally the amino acid modifications described above will not impair the ability of the peptide to bind MHC. In a preferred embodiment, the amino acid modifications improve the ability of the peptide to bind MHC. For example, 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. Alternatively, 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. Alternatively, in vitro cell-free systems may be used. Such systems are known in the art and are commercially available for example from Life

Technologies, Paisley, UK. The 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. In a second aspect the invention provides a complex of the polypeptide of the first aspect and an MHC molecule. Preferably, 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. 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. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, 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. For example, 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. Alternatively, MHC molecules may be produced synthetically, or using cell free systems.

Polypeptide-MHC complexes of the invention may be provided in soluble form, or may be immobilised by attachment to a suitable solid support. Examples of 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. In a preferred embodiment 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. In general, polypeptide-MHC multimers may be produced using peptide-MHC tagged with a biotin residue and complexed through fluorescent labelled streptavidin. Alternatively, multimeric polypeptide-MHC complexes may be formed by using immunoglobulin as a molecular scaffold. In this system, 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. Thus, 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. In a third aspect, 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.

In a fourth aspect, 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. In one embodiment, 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. 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. For instance, 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 TCRs of the invention may be in any format known to those in the art. For example, the TCRs may be αβ heterodimers, or they may be in single chain format (such as those described in W09918129). Single chain TCRs include αβ TCR polypeptides of the type: Va-L-νβ, νβ-L-Va, Va- Ca-L-νβ, να-ί-\ β-0β or Va- Ca -ίΛ/β-Οβ, optionally in the reverse orientation, wherein Va and νβ are TCR a and β variable regions respectively, Ca and C are TCR a and β 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. In addition the TRAC/TRBC may contain modifications. For example, 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. Likewise, 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). In addition the use of the stable disulphide linked soluble TCR enables more convenient assessment of binding affinity and binding half-life. Alternative positions for the formation of a non-native disulphide bond are described in WO 03/020763. 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. In multimeric high affinity TCR complexes such as those described in Zhu et al., J. Immunol. 2006 Mar 1 ; 176(5):3223-32, (formed, for example, using biotinylated heterodimers) 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 further aspect of the invention provides a cell displaying on its surface a TCR of the invention. The cell may be a T cell. There are a number of methods suitable for the transfection of T cells with DNA or RNA encoding the TCRs of the invention (see for example Robbins et al., J. Immunol. 2008 May 1 ;180(9):61 16-31 ). T cells expressing the TCRs of the invention are suitable for use in adoptive therapy-based treatment of diseases such as cancers. As will be known to those skilled in the art there are a number of suitable methods by which 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. As is well known, 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. The term "antibody" as used herein 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. The term "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. 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".

The binding moiety may be an antibody-like molecule that has been designed to specifically bind a peptide-MHC complex of the invention. Of particular preference are 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.

In another aspect, 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 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. For example, 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 μg/kg. A physician will ultimately determine appropriate dosages to be used.

Brief description of the Figures

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.

Examples

Example H1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from DNTT on the surface of tumour cell lines was investigated using mass spectrometry.

Method

Results

The 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.

Example H2 - Preparation of recombinant peptide-HLA complexes

Class I HLA-A*02 molecules (HLA-A*02-heavy chain and HLA light-chain (β2ιη)) 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 β2ιη 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 β2ιη 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.

Example H3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction.

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. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

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

Aug;1826(1 ):1 -12). 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.

In a first aspect, 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

(b) the amino acid sequence of any one of SEQ NOs: 11-18 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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

USA. 1992 Apr 15;89(8):3429-33. Preferably, 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 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.

In some embodiments, 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.

The complex of the invention may be isolated and/or in a substantially pure form. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, 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. For example, 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. Alternatively, MHC molecules may be produced synthetically, or using cell free systems.

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. In general, polypeptide-MHC multimers may be produced using peptide-MHC tagged with a biotin residue and complexed through fluorescent labelled streptavidin. Alternatively, multimeric polypeptide-MHC complexes may be formed by using immunoglobulin as a molecular scaffold. In this system, 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. Thus, 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. In a third aspect, 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.

In a sixth aspect, the invention provides a binding moiety that binds the polypeptide of the invention. Preferably the binding moiety binds the polypeptide when said polypeptide is in complex with MHC. In the latter instance, 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.

The TCRs of the invention may be in any format known to those in the art. For example, the TCRs may be αβ heterodimers, or they may be in single chain format (such as those described in W09918129). Single chain TCRs include αβ TCR polypeptides of the type: Va-L-νβ, νβ-L-Va, Va- Ca-L-νβ, να-ί-\ β-0β or Va- Ca -ίΛ/β-Οβ, optionally in the reverse orientation, wherein Va and νβ are TCR a and β variable regions respectively, Ca and C are TCR a and β 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.

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. There are a number of methods suitable for the transfection of T cells with DNA or RNA encoding the TCRs of the invention (see for example Robbins et al., J. Immunol. 2008 May 1 ;180(9):6116-31 ). T cells expressing the TCRs of the invention are suitable for use in adoptive therapy-based treatment of diseases such as cancers. As will be known to those skilled in the art there are a number of suitable methods by which 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. As is well known, 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).

21 ;1 17(16):4262-72 and/or Dahan and Reiter. Expert Rev Mol Med. 2012 Feb 24;14:e6. Also encompassed within the present invention are binding moieties based on engineered protein scaffolds. 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. Examples of 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. 2008 Jun;275(1 1 ):2677-83), nanobodies, and DARPins. 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)]. In another aspect, 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. In a further aspect, the invention provides 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. 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

pharmaceutically acceptable carrier. For example, 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.

The present invention will be further illustrated in the following Examples and Figures which are given for illustration purposes only and are not intended to limit the invention in any way. Brief description of the Figures

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.

Examples Example 11 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from ETV4 on the surface of tumour cell lines was investigated using mass spectrometry.

Method

The polypeptides set out in table 11 , corresponding to SEQ NOs: 11 -18, 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

Inclusion bodies of β2πι 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 β2πι 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 μητι 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.

Example I3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction.

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. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

1 ;152(1 ):163-75 (access via http://www-bimas.cit.nih.qov/molbio/hla bind/)) are available to predict the amino acid sequences of MHC-presented peptides derived from proteins. However, these methods are known to generate a high proportion of false positives (since they simply define the likelihood of a given peptide being able to bind a given MHC and do not account for intracellular processing). Therefore, it is not possible to accurately predict whether a given peptide-MHC is actually presented by tumour cells. Direct experimental data is typically required. 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.

In a first aspect, 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

(b) the amino acid sequence of any one of SEQ NOs: J1-J19 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

J15 SEPFHLIVSY

J16 VVAITVQEL

J17 YTFSEPFHL

J18 YTFSEPFHLI

J19 YTFSEPFHLIV

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

The polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: J1-J19.

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.

In some embodiments, 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

Technologies, Paisley, UK. The 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. In a second aspect the invention provides a complex of the polypeptide of the first aspect and an MHC molecule. Preferably, 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. 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 ).

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 J2 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. Alternatively, 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.

In a third aspect, 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.

In another aspect, 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, cutaneous melanoma and B-cell malignancy. In a further aspect, the invention provides 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. 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 pharmaceutically acceptable carrier. For example, 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. For example, 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.

Brief description of the Figures

Figures J1 to J19 show the respective fragmentation spectra for the peptides of SEQ NOs: J1 to J 19, eluted from cells. A table highlighting the matching ions is shown below each spectrum. Examples

Example J1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from FCRLA on the surface of tumour cell lines was investigated using mass spectrometry.

Method

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. 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. For peptide identification the mass spectrometer was operated using an information dependent acquisition (IDA) workflow. 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 polypeptides set out in table J1 , corresponding to SEQ NOs: J1-J19, 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 J 1 -J 19 show representative fragmentation patterns for the peptides of SEQ NOs: J 1 -J 19 respectively. A table highlighting the matching ions is shown below each spectrum. Example J2 - Preparation of recombinant peptide-HLA complexes

Class I HLA-A^*02 molecules (HLA-A^*02-heavy chain and HLA light-chain (β2ηι)) 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 β2ιη 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 β2ιη 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.

Example J3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 J2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction.

The[LSiO] present invention also relates to novel peptides derived from Glypican-2 (GPC2), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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.

1 ;152(1 ):163-75 (access via http://www-bimas.cit.nih.qov/molbio/hla bind/)) are available to predict the amino acid sequences of MHC-presented peptides derived from proteins. However, these methods are known to generate a high proportion of false positives (since they simply define the likelihood of a given peptide being able to bind a given MHC and do not account for intracellular processing). Therefore, it is not possible to accurately predict whether a given peptide-MHC is actually presented by tumour cells. Direct experimental data is typically required. GPC2 (also known as Glypican-2 and having Uniprot accession number Q8N158) is a cell surface proteoglycan. Expression of glypicans is known to play a role in cancer (De Robertis et al.

Oncotarget. 2015 Oct 20. doi: 10.18632/oncotarget.5652). GPC2 is an ideal target for

immunotherapeutic applications. The inventors have found novel peptides derived from GPC2 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 GPC2 and for the treatment of cancers, including small cell lung cancer.

In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: K1 -K4, or

(b) the amino acid sequence of any one of SEQ NOs: K1-K4 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

wherein the polypeptide forms a complex with a Major Histocompatibility Complex (MHC) molecule. The inventors have found that 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 K2. 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

USA. 1992 Apr 15;89(8):3429-33.

Preferably, 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: K1-K4. 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: K1-K4. Each deletion can take place at any position of SEQ NOs: K1-K4.

In some embodiments, 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: K1- K4. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: K1-K4 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: K1-K4, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

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 K2 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. Alternatively, 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.

Brief description of the Figures

Figures K1 to K4 show the respective fragmentation spectra for the peptides of SEQ NOs: K1 to K4, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples

Example K1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from GPC2 on the surface of tumour cell lines was investigated using mass spectrometry. Method

Results

The polypeptides set out in table K1 , corresponding to SEQ NOs: K1-K4, 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. Amino acid Example cancer

SEQ NO HLA antibody

sequence cell line

K1 AQHSLTQLF class I Nalm6

K2 FYGESGEGLD class I NCI H209

K3 LIRETEATF class I Nalm6 ISF

K4 TLADFWAQL class I NCI H209

Figures K1 -K4 show representative fragmentation patterns for the peptides of SEQ NOs: K1-K4 respectively. A table highlighting the matching ions is shown below each spectrum. Example K2 - Preparation of recombinant peptide-HLA complexes

Example K3 - identification of TCRs that bind to a peptide-MHC complex of the invention

Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 K2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction. The[LSi i] present invention also relates to novel peptides derived from Glutamate receptor ionotropic MDA 2D (GRIN2D), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

GRIN2D (also known as Glutamate receptor ionotropic MDA 2D or EB1 1 or Glutamate [NMDA] receptor subunit epsilon-4 or NMDAR2D and having Uniprot accession number 015399) is a glutamate gated ion channel (Hess et al. J Neurochem. 1998 Mar;70(3): 1269-79). GRIN2D is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from GRIN2D 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 GRIN2D and for the treatment of cancers, including colon cancer and oesophageal cancer.

In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: L1 -L28, or

(b) the amino acid sequence of any one of SEQ NOs: L1-L28 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

L13 LGPAVAAAVR

L14 LSAQTSLPI

L15 LVFAWEHLV

L16 MLLVAMGLSL

L17 PAAAATAVGPPL

L18 PFVETGISV

L19 PQLAGGGGSGAPG

L20 PRGAAGRPLSPPA

L21 PRPAPGPAPF

L22 RGAAGRPLSP

L23 SESLGGASL

L24 SPSAFLEPYSPAV

L25 SPVGYN RSL

L26 VIVEPADPIS

L27 VMVARSNGTV

L28 VRPVALVL

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 L2. 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 USA. 1992 Apr 15;89(8):3429-33. Preferably, 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: L1-L28.

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: L1-L28. Each deletion can take place at any position of SEQ NOs: L1 -L28.

In some embodiments, 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: L1- L28. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: L1-L28 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: L1-L28, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

In a second aspect the invention provides a complex of the polypeptide of the first aspect and an MHC molecule. Preferably, 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. 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 ).

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 L2 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. Alternatively, 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.

The TCRs of the invention may be in any format known to those in the art. For example, the TCRs may be αβ heterodimers, or they may be in single chain format (such as those described in W09918129). Single chain TCRs include αβ TCR polypeptides of the type: Va-L-νβ, νβ-L-Va, Va- Ca-L-νβ, να-ί-Ν/β-ΰβ or Va- Ca -ίΛ/β-ΰβ, optionally in the reverse orientation, wherein Va and νβ are TCR a and β variable regions respectively, Ca and Cβ are TCR a and β 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. In addition the TRAC/TRBC may contain modifications. For example, 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. Likewise, 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 al., Nat Biotechnol 2005

It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of 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. 1989 Oct 12;341 (6242):544-6) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al., Science. 1988 Oct 21 ;242(4877):423-6; Huston et al., Proc Natl Acad Sci U S A. 1988 Aug;85(16):5879-83); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94/13804; P. Hollinger et al., Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6444-8). 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. by a peptide linker) but unable to associate with each other to form an antigen binding site: 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). Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Hollinger & Winter, Curr Opin Biotechnol. 1993 Aug;4(4):446-9), 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).

In another aspect, 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, colon cancer and oesophageal cancer. In a further aspect, the invention provides 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. 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

Figures L1 to L28 show the respective fragmentation spectra for the peptides of SEQ NOs: L1 to L28, eluted from cells. A table highlighting the matching ions is shown below each spectrum. Examples

Example L1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from GRIN2D on the surface of tumour cell lines was investigated using mass spectrometry.

Method

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 10⁷ 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 protein A-Sepharose scaffold. Columns were washed with low salt and high salt buffers and complexes eluted in acid. Eluted peptides were separated from HLA complexes by reversed phase chromatography using a solid phase extraction cartridge (Phenomenex). Bound material was eluted from the column and reduced in volume using a vacuum centrifuge.

Results

The polypeptides set out in table L1 , corresponding to SEQ NOs: L1 -L28, 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.

L7 DLPPPAPTS class 1 SW982

L8 FLQPVDDTQHL class 1 NCI H1755

L9 HTIGFSYDL HLA-A*02 AGS

L10 IVDFSVPFV class 1 AGC

Lll KLDAFIYDA HLA-A*02 AGS

L12 LATGKRPGGSTF class 1 NCI H1944

L13 LGPAVAAAVR HLA-A*02 Granta

L14 LSAQTSLPI class 1 MEWO

L15 LVFAWEHLV HLA-A*02 AGS

L16 MLLVAMGLSL HLA-A*02 NCI H1755

L17 PAAAATAVGPPL class 1 NCI H2023

L18 PFVETGISV HLA-A*02 NCI H1944

L19 PQLAGGGGSGAPG class 1 Nalm6 FCS

L20 PRGAAGRPLSPPA HLA-A*02 NCI H1944

L21 PRPAPGPAPF class 1 U266

L22 RGAAGRPLSP class 1 U266

L23 SESLGGASL class 1 NCI H1755

L24 SPSAFLEPYSPAV class 1 NCI H1755

L25 SPVGYNRSL HLA-B*07 Colo205

L26 VIVEPADPIS HLA-A*02 EJM

L27 VMVARSNGTV class 1 NCI H1915

L28 VRPVALVL HLA-A*02 SW982

Figures L1 -L28 show representative fragmentation patterns for the peptides of SEQ NOs: L1-L28 respectively. A table highlighting the matching ions is shown below each spectrum. Example L2 - Preparation of recombinant peptide-HLA complexes

The following describes a suitable method for the preparation of soluble recombinant HLA loaded with TAA peptide. Class I HLA-A*02 molecules (HLA-A*02-heavy chain and HLA light-chain (β2ιη)) 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 β2πι 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 β2ιη 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.

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 L3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 L2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction.

The[LSi2] present invention also relates to novel peptides derived from metabotropic glutamate receptor 4 (GRM4), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

GRM4 (also known as metabotropic glutamate receptor 4 or mGluR4 and having Uniprot accession number Q14833) id a G protein coupled receptor for glutamate. GRM4 has been associated with cancer (Savage et al. Nat Genet. 2013 Jul;45(7):799-803). GRM4 is an ideal target for

immunotherapeutic applications. The inventors have found novel peptides derived from GRM4 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 GRM4 and for the treatment of cancers, including ovarian cancer.

In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: M1 -M3, or

(b) the amino acid sequence of any one of SEQ NOs: M1-M3 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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 M2. 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

USA. 1992 Apr 15;89(8):3429-33.

Preferably, 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: M1-M3. 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: M1-M3. Each deletion can take place at any position of SEQ NOs: M1 -M3.

In some embodiments, 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: M1- M3. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: M1-M3 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: M1-M3, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

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 M2 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. Alternatively, 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.

In another aspect, 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.

Brief description of the Figures

Figures 1 to M3 show the respective fragmentation spectra for the peptides of SEQ NOs: M1 to M3, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples

Example M1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from GRM4 on the surface of tumour cell lines was investigated using mass spectrometry. Method

Results

The polypeptides set out in table M1 , corresponding to SEQ NOs: M1-M3, 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. Amino acid Example cancer

SEQ NO HLA antibody

sequence cell line

M1 AMVDIVRAL HLA-A*02 MDA-MD-453

M2 GMLYMPKVYI HLA-A*02 MDA-MD-453

M3 RTLDPRFAR HLA-A*02 MDA-MD-453

Figures M1 -M3 show representative fragmentation patterns for the peptides of SEQ NOs: M1-M3 respectively. A table highlighting the matching ions is shown below each spectrum. Example M2 - Preparation of recombinant peptide-HLA complexes

Inclusion bodies of P2m 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 β2ιη 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.

Example M3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 M2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction. The[LSi3] present invention also relates to novel peptides derived from HORMA domain-containing protein 1 (HORMAD1 ), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

HORMAD1 (also known as HORMA domain-containing protein 1 or CT46 and having Uniprot accession number Q86X24) belongs to the cancer testis family of germline encoded cancer antigens. Expression of HORMAD1 has been reported in various tumours while expression in normal tissues is restricted to testis (Chen et al. Cancer Immun. 2005 Jul 7;5:9). HORMAD1 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from HORMAD1 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 HORMAD1 and for the treatment of cancers, including breast cancer. In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: N1 -N5, or

(b) the amino acid sequence of any one of SEQ NOs: N1-N5 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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 N2. 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

USA. 1992 Apr 15;89(8):3429-33. Preferably, 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: N1-N5.

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: N1-N5. Each deletion can take place at any position of SEQ NOs: N1 -N5.

In some embodiments, 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: N1- N5. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: N1-N5 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: N1-N5, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

The complex of the invention may be isolated and/or in a substantially pure form. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, 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. For example, 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 N2 herein. Alternatively, MHC molecules may be produced synthetically, or using cell free systems.

21 ;1 17(16):4262-72 and/or Dahan and Reiter. Expert Rev Mol Med. 2012 Feb 24;14:e6. Also encompassed within the present invention are binding moieties based on engineered protein scaffolds. 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. Examples of 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. 2008 Jun;275(1 1 ):2677-83), nanobodies, and DARPins. 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)]. In another aspect, 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. In a further aspect, the invention provides 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

Figures N1 to N5 show the respective fragmentation spectra for the peptides of SEQ NOs: N1 to N5, eluted from cells. A table highlighting the matching ions is shown below each spectrum. Examples

Example N1 - Identification of target-derived peptides by Mass spectrometry Presentation of HLA-restricted peptides derived from HORMAD1 on the surface of tumour cell lines was investigated using mass spectrometry.

Method

Results

The polypeptides set out in table N1 , corresponding to SEQ NOs: N1 -N5, 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 N1 -N5 show representative fragmentation patterns for the peptides of SEQ NOs: N1-N5 respectively. A table highlighting the matching ions is shown below each spectrum.

Example N2 - Preparation of recombinant peptide-HLA complexes The following describes a suitable method for the preparation of soluble recombinant HLA loaded with TAA peptide.

Example N3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 N2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction.

The[LSU] present invention also relates to novel peptides derived from Interphotoreceptor matrix proteoglycan 2 (IMPG2), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

IMPG2 (also known as interphotoreceptor matrix proteoglycan 2 or IPM 200 or spacrcan and having Uniprot accession number Q9BZV3) is a proteoglycan involved in the organization of interphotoreceptor matrix. IMPG2 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from IMPG2 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 IMPG2 and for the treatment of cancers, including ovarian cancer.

In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: 01 -05, or

(b) the amino acid sequence of any one of SEQ NOs: 01-05 with the exception of 1 , 2 or

3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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 02. 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

USA. 1992 Apr 15;89(8):3429-33.

Preferably, 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: 01-05.

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: 01-05. Each deletion can take place at any position of SEQ NOs: 01-05.

In some embodiments, 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: 01- 05. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: 01-05 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: 01-05, 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

The complex of the invention may be isolated and/or in a substantially pure form. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, 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. For example, 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 02 herein. Alternatively, MHC molecules may be produced synthetically, or using cell free systems.

Brief description of the Figures

Figures 01 to 05 show the respective fragmentation spectra for the peptides of SEQ NOs: 01 to 05, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples

Example 01 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from IMPG2 on the surface of tumour cell lines was investigated using mass spectrometry. Method

Results

The polypeptides set out in table 01 , corresponding to SEQ NOs: 01-05, 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. Amino acid Example cancer

SEQ NO HLA antibody

sequence cell line

01 AEAVANHV class I NCI H1975

02 LSSIENAVKY class I Colo205

03 SDVSLTSSPY class I NCI H1975

04 SEVPGVDDY class I NCI H209

05 SSELSSPVPVGDT class I NCI H1975

Figures 01 -05 show representative fragmentation patterns for the peptides of SEQ NOs: 01-05 respectively. A table highlighting the matching ions is shown below each spectrum. Example 02 - Preparation of recombinant peptide-HLA complexes

Example 03 - identification of TCRs that bind to a peptide-MHC complex of the invention

Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 02) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction. The[LSi5] present invention also relates to novel peptides derived from Alpha-internexin (INA), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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.

1 ;152(1 ):163-75 (access via http://www-bimas.cit.nih.qov/molbio/hla bind/)) are available to predict the amino acid sequences of MHC-presented peptides derived from proteins. However, these methods are known to generate a high proportion of false positives (since they simply define the likelihood of a given peptide being able to bind a given MHC and do not account for intracellular processing). Therefore, it is not possible to accurately predict whether a given peptide-MHC is actually presented by tumour cells. Direct experimental data is typically required. INA (also known as alpha-internexin or NF-66 and having Uniprot accession number Q16352) is a neuronal filament involved in the morphogenesis of neurons. Expression of INA has been associated with cancer (Matsumura et al. Brain Tumor Pathol. 2015 Oct;32(4):261-7). INA is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from INA 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 INA and for the treatment of cancers, including small cell lung cancer.

In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: P1 -P20, or

(b) the amino acid sequence of any one of SEQ NOs: P1-P20 with the exception of 1 , 2 or

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 P2. 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

USA. 1992 Apr 15;89(8):3429-33.

The polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: P1-P20. 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: P1-P20. Each deletion can take place at any position of SEQ NOs: P1-P20.

In some embodiments, 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: P1- P20. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: P1-P20 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: P1-P20, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

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. co// cells or insect cells. A suitable method is provided in Example P2 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. Alternatively, 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.

Brief description of the Figures Figures P1 to P20 show the respective fragmentation spectra for the peptides of SEQ NOs: P1 to P20, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples Example P1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from INA on the surface of tumour cell lines was investigated using mass spectrometry.

Method

The polypeptides set out in table P1 , corresponding to SEQ NOs: P1 -P20, 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 P1 -P20 show representative fragmentation patterns for the peptides of SEQ NOs: P1-P20 respectively. A table highlighting the matching ions is shown below each spectrum. Example P2 - Preparation of recombinant peptide-HLA complexes

Class I HLA-A^*02 molecules (HLA-A^*02-heavy chain and HLA light-chain (β2ηι)) were expressed separately in E. col i 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 β2ηι 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 β2ηι 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 (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.

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 P3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 P2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction.

The[LSi6] present invention also relates to novel peptides derived from Potassium voltage-gated channel subfamily H member 5 (KCNH5), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

KCNH5 (also known as Potassium voltage-gated channel subfamily H member 5 or hEAG2 and having Uniprot accession number Q8NCM2) is a pore-forming (alpha) subunit of voltage-gated potassium channel (Ju et al. FEBS Lett. 2002 Jul 31 ;524(1-3):204-10. KCNH5 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from KCNH5 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 KCNH5 and for the treatment of cancers, including oesophageal cancer and small cell lung cancer.

In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of SEQ NO: Q1 , or

(b) the amino acid sequence of SEQ NO: Q1 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions, wherein the polypeptide forms a complex with a Major Histocompatibility Complex (MHC) molecule. The inventors have found that 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 Q2. 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

USA. 1992 Apr 15;89(8):3429-33.

The polypeptides of the invention may consist or consist essentially of the amino acids sequence provided in SEQ NO: Q1. 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: Q1. Each deletion can take place at any position of SEQ NO: Q1.

In some embodiments, 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: Q1. A polypeptide of the invention may comprise the amino acid sequence of SEQ NO: Q1 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: Q1 , with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

Technologies, Paisley, UK. The 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. In a second aspect the invention provides a complex of the polypeptide of the first aspect and an MHC molecule. Preferably, 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. 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/imgt/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. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, 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. For example, 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 Q2 herein. Alternatively, MHC molecules may be produced synthetically, or using cell free systems.

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. In a preferred embodiment 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. In general, polypeptide-MHC multimers may be produced using peptide-MHC tagged with a biotin residue and complexed through fluorescent labelled streptavidin. Alternatively, multimeric polypeptide-MHC complexes may be formed by using immunoglobulin as a molecular scaffold. In this system, 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.

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. For instance, 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. In the context of the present invention, 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. 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.

Lefranc, (2011 ), Cold Spring Harb Protoc 2011(6): 595-603; Lefranc, (2001 ), Curr Protoc Immunol Appendix 1 : Appendix 10; Lefranc, (2003), Leukemia 17(1 ): 260-266, and on the IMGT website (www.IMGT.org) The TCRs of the invention may be in any format known to those in the art. For example, the TCRs may be αβ heterodimers, or they may be in single chain format (such as those described in W09918129). Single chain TCRs include αβ TCR polypeptides of the type: Va-L-νβ, νβ-L-Va, Va- Ca-L-νβ, Va-L-νβ-Οβ or Va- Ca -L-νβ-Οβ, optionally in the reverse orientation, wherein Va and νβ are TCR a and β variable regions respectively, Ca and Οβ are TCR a and β 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. In addition the TRAC/TRBC may contain modifications. For example, 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. Likewise, 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 al., Nat Biotechnol 2005

Mar;23(3):349-54). In addition the use of the stable disulphide linked soluble TCR enables more convenient assessment of binding affinity and binding half-life. Alternative positions for the formation of a non-native disulphide bond are described in WO 03/020763. 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. In multimeric high affinity TCR complexes such as those described in Zhu et al., J. Immunol. 2006 Mar 1 ; 176(5):3223-32, (formed, for example, using biotinylated heterodimers) 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. Thus, 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.

Biotechnol. 1993 Aug;4(4):446-9), 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. Of particular preference are TCR-mimic antibodies, such as, for example those described in WO2007143104 and Sergeeva et al., Blood. 201 1 Apr

21 ;1 17(16):4262-72 and/or Dahan and Reiter. Expert Rev Mol Med. 2012 Feb 24;14:e6. Also encompassed within the present invention are binding moieties based on engineered protein scaffolds. 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. Examples of 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. 2008 Jun;275(1 1 ):2677-83), nanobodies, and DARPins. 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)]. In another aspect, 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, oesophageal cancer and small cell lung cancer.

Alternatively the vaccine composition may take the form of an antigen presenting cell displaying the peptide of the invention in complex with MHC. Preferably 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.

Brief description of the Figures

Figure Q1 shows the fragmentation spectra for the peptide of SEQ NO: Q1 , eluted from cells. A table highlighting the matching ions is shown below the spectrum.

Examples

Example Q1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from KCNH5 on the surface of tumour cell lines was investigated using mass spectrometry. Method

Results

The polypeptides set out in table Q1 , corresponding to SEQ NO: Q1 , 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. Amino acid Example cancer

SEQ NO HLA antibody

sequence cell line

Q1 TIFGNVTTI HLA-B*07 SW982

Figure Q1 shows representative fragmentation patterns for the peptides of SEQ NO: Q1 respectively. A table highlighting the matching ions is shown below each spectrum. Example Q2 - Preparation of recombinant peptide-HLA complexes

(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 Q3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 Q2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction. The[LSi7] present invention also relates to novel peptides derived from Lipocalin-15 (LCN15), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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.

1 ;152(1 ):163-75 (access via http://www-bimas.cit.nih.qov/molbio/hla bind/)) are available to predict the amino acid sequences of MHC-presented peptides derived from proteins. However, these methods are known to generate a high proportion of false positives (since they simply define the likelihood of a given peptide being able to bind a given MHC and do not account for intracellular processing). Therefore, it is not possible to accurately predict whether a given peptide-MHC is actually presented by tumour cells. Direct experimental data is typically required. LCN15 (also known as Lipocalin-15 and having Uniprot accession number Q6UWW0) is a small molecule transporter. LCN15 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from LCN15 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 LCN15 and for the treatment of cancers, including colon cancer and small cell lung cancer.

In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: R1-R3, or

(b) the amino acid sequence of any one of SEQ NOs: R1-R3 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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 R2. 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

USA. 1992 Apr 15;89(8):3429-33.

Preferably, 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: R1-R3.

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: R1-R3. Each deletion can take place at any position of SEQ NOs: R1 -R3.

In some embodiments, 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: R1- R3. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: R1-R3 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: R1-R3, 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

The complex of the invention may be isolated and/or in a substantially pure form. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, 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. For example, 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 R2 herein. Alternatively, MHC molecules may be produced synthetically, or using cell free systems.

In another aspect, 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, colon cancer or small cell lung cancer.

Brief description of the Figures

Figures R1 to R3 show the respective fragmentation spectra for the peptides of SEQ NOs: R1 to R3, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples

Example R1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from LCN15 on the surface of tumour cell lines was investigated using mass spectrometry. Method

Results

The polypeptides set out in table R1 , corresponding to SEQ NOs: R1-R3, 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. Amino acid Example cancer

SEQ NO HLA antibody

sequence cell line

R1 DTDYSSFAVLY class I NCI H508

R2 FLLGAILT HLA-A*02 NCI H508

R3 LLWAPTAQA class I VMRC LCD

Figures R1 -R3 show representative fragmentation patterns for the peptides of SEQ NOs: R1-R3 respectively. A table highlighting the matching ions is shown below each spectrum. Example R2 - Preparation of recombinant peptide-HLA complexes

Example R3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 R2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction. The[LSi8] present invention also relates to novel peptides derived from Neuroblastoma breakpoint family member 4 (NBPF4), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

NBPF4 (also known as Neuroblastoma breakpoint family member 4 and having Uniprot accession number Q96M43) is a protein expressed in testis (Vandepoele et al. Mol Biol Evol. 2005

Nov;22(1 1 ):2265-74). NBPF4 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from NBPF4 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 NBPF4 and for the treatment of cancers, including breast cancer.

In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: S1 -S4, or

(b) the amino acid sequence of any one of SEQ NOs: S1-S4 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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 S2. 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

USA. 1992 Apr 15;89(8):3429-33.

Preferably, 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: S1-S4. 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: S1-S4. Each deletion can take place at any position of SEQ NOs: S1-S4.

In some embodiments, 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: S1- S4. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: S1-S4 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: S1-S4, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

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 S2 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. Alternatively, 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.

In another aspect, 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.

Brief description of the Figures

Figures S1 to S4 show the respective fragmentation spectra for the peptides of SEQ NOs: S1 to S4, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples

Example S1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from NBPF4 on the surface of tumour cell lines was investigated using mass spectrometry. Method

Results

The polypeptides set out in table S1 , corresponding to SEQ NOs: S1-S4, 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. Amino acid Example cancer

SEQ NO HLA antibody

sequence cell line

S1 AEMNILEIN Class I NCI H1975

S2 LEKQSDLEE Class I NCI H1975

S3 RLSQELPEV Class 1 SKMel37

S4 SVNEVYLTPSVHH Class 1 NCI H209

Figures S1 -S4 show representative fragmentation patterns for the peptides of SEQ NOs: S1-S4 respectively. A table highlighting the matching ions is shown below each spectrum. Example S2 - Preparation of recombinant peptide-HLA complexes

Example S3 - identification of TCRs that bind to a peptide-MHC complex of the invention

Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 S2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction. The[LSi9] present invention also relates to novel peptides derived from NACHT, LRR and PYD domains-containing protein 7 (NLRP7), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

1 ;152(1 ):163-75 (access via http://www-bimas.dt.nih.gov/molbio/hla bind/)) are available to predict the amino acid sequences of MHC-presented peptides derived from proteins. However, these methods are known to generate a high proportion of false positives (since they simply define the likelihood of a given peptide being able to bind a given MHC and do not account for intracellular processing). Therefore, it is not possible to accurately predict whether a given peptide-MHC is actually presented by tumour cells. Direct experimental data is typically required.

NLRP7 (also known as NACHT, LRR and PYD domains-containing protein 7 or Nucleotide-binding oligomerization domain protein 12 or PYRIN-containing APAF1 -like protein 3 and having Uniprot accession number Q8WX94) is an ATP binding protein that inhibits CASP1/caspase-1 -dependent IL1 B secretion (Kinoshita et al. J Biol Chem. 2005 Jun 10;280(23):21720-5). NLRP7 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from NLRP7 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 NLRP7 and for the treatment of cancers, including non small cell lung cancer (squamous), head and neck cancer and ovarian cancer.

In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: T1 -T29, or

(b) the amino acid sequence of any one of SEQ NOs: T1-T29 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions, wherein the polypeptide forms a complex with a Major Histocompatibility Complex (MHC) molecule.

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 T2. 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

USA. 1992 Apr 15;89(8):3429-33.

The polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: T1-T29. 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: T1-T29. Each deletion can take place at any position of SEQ NOs: T1-T29.

In some embodiments, 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: T1- T29. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: T1-T29 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: T1-T29, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

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.

In a second aspect the invention provides a complex of the polypeptide of the first aspect and an MHC molecule. Preferably, 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-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. 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 ).

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 T2 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. Alternatively, 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.

In a third aspect, 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. In a fourth aspect, 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. In one embodiment, 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. 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. For instance, 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 TCRs of the invention may be in any format known to those in the art. For example, the TCRs may be αβ heterodimers, or they may be in single chain format (such as those described in W09918129). Single chain TCRs include αβ TCR polypeptides of the type: Va-L-νβ, νβ-L-Va, Va- Ca-L-νβ, Ν/α-ίΛ/β-Οβ or Va- Ca -ίΛ β-Οβ, optionally in the reverse orientation, wherein Va and νβ are TCR a and β variable regions respectively, Ca and Οβ are TCR a and β 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.

Mar;23(3):349-54). In addition the use of the stable disulphide linked soluble TCR enables more convenient assessment of binding affinity and binding half-life. Alternative positions for the formation of a non-native disulphide bond are described in WO 03/020763. 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).

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. Thus, 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. In a further aspect, 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. Also included in the scope of the invention is a cell harbouring a first expression vector which comprises nucleic acid encoding an alpha chain of a TCR of the invention, and a second expression vector which comprises nucleic acid encoding a beta chain of a TCR of the invention. Alternatively, one vector may encode both an alpha and a beta chain of a TCR of the invention.

The binding moiety of the invention may be an antibody. The term "antibody" as used herein 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. The term "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. 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". It is possible to take an antibody, for example a monoclonal antibody, and use 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). 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.

It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of 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. 1989 Oct 12;341 (6242):544-6) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al., Science. 1988 Oct 21 ;242(4877):423-6; Huston et al., Proc Natl Acad Sci U S A. 1988 Aug;85(16):5879-83); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (W094/13804; P. Hollinger et al., Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6444-8). 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. by a peptide linker) but unable to associate with each other to form an antigen binding site: 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). Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Hollinger & Winter, Curr Opin

Biotechnol. 1993 Aug;4(4):446-9), 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. coll. 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. Of particular preference are 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.

In another aspect, 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), head and neck cancer and ovarian cancer.

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. For example, 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 μg/kg. A physician will ultimately determine appropriate dosages to be used.

Figures T1 to T29 show the respective fragmentation spectra for the peptides of SEQ NOs: T1 to T29, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples

Example T1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from NLRP7 on the surface of tumour cell lines was investigated using mass spectrometry.

Method

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. 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. For peptide identification the mass spectrometer was operated using an information dependent acquisition (IDA) workflow. 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 polypeptides set out in table T1 , corresponding to SEQ NOs: T1 -T29, 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.

T25 SEALQEACSL class 1 U266

T26 SLFSSNSNL HLA-A^*02 EJM

T27 SPDIKQEL HLA-B^*07 EJM

T28 YLSEALQEA HLA-A^*02 U266

T29 YPDCKLQTL class 1 EJM

Figures T1 -T29 show representative fragmentation patterns for the peptides of SEQ NOs: T1-T29 respectively. A table highlighting the matching ions is shown below each spectrum. Example T2 - Preparation of recombinant peptide-HLA complexes

Example T3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 T2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction. The[LS20] present invention also relates to novel peptides derived from Melanocyte protein PMEL (PMEL), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

PMEL (also known as melanocyte protein PMEL or Pmel 17 or gp100 or SILV and having Uniprot accession number P40967) plays a central role in the biogenesis of melanosomes. PMEL is normally expressed at low levels in quiescent adult melanocytes but is overexpressed in melanomas (Chen et al. J Biol Chem. 2012 Jul 13;287(29):24082-91 ; W09734613; W09522561 ). PMEL is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from PMEL 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 PMEL and for the treatment of cancers, including uveal melanoma. In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: U1 -U96, or

(b) the amino acid sequence of any one of SEQ NOs: U1-U96 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

U14 DFGDSSGTLIS U62 SAFTITDQV

U15 DGGNKHFL U63 SAFTITDQVPFSV

U16 DQVPFSVSV U64 SCPiGENSP

U17 DQVPFSVSVSQLR U65 SCPiGENSPL

U18 DTNSLAW U66 SIALNFPG

U19 EAPNTTAGQV U67 SLADTNSL

U20 EVISTAPVQM U68 SLADTNSLAVV

U21 EVMGTTLAEM U69 SLKVSNDGPT

U22 FALQLHDPSGYLA U70 SVQVPTTEV

U23 FTITDQVPFSV U71 SVTLDIVQ

U24 GGQVSLKVS U72 SVTLDIVQG

U25 GLGQVPLI U73 SYVPLAHSSSAF

U26 GQEAGLGQVPL U74 TAQVVLQA

U27 GQVPLIVGI U75 TLISRALVV

U28 GQVPTTEVV U76 TTEVISTAPV

U29 GQVSLKVS U77 TWDFGDSSG

U30 GTHTMEVTV U78 TWDFGDSSGT

U31 HDPSGYLA U79 TWDFGDSSGTLIS

U32 HDPSGYLAE U80 VLGGPVSGLSI

U33 HDPSGYLAEADLS U81 VPSGEGDAF

U34 HQILKGGSGT U82 VQVPTTEVI

U35 HQILKGGSGTY U83 VSLKVSND

U36 HSSSAFTITDQVP U84 VSLKVSNDGPT

U37 HT EVTVYH U85 VSLKVSNDGPTLIG

U38 HTYLEPGPVT U86 VSNDGPTL

U39 HTYLEPGPVTA U87 VSTQLIMPV

U40 HTYLEPGPVT AQ U88 VTLDIVQGI

U41 IMPGQEAGLGQV U89 WDFGDSSGT

U42 KQDFSVPQL U90 YLAEADLS

U43 KVSNDGPTL U91 YLEPGPVTAQV

U44 LAVIGALLAVG U92 YRYGSFSVTL

U45 LDGGNKHFL U93 YTWDFGDSSGT

U46 LEPGPVTAQ U94 YTWDFGDSSGTL

U47 LGTHTMEV U95 YTWDFGDSSGTLI

U48 LHDPSGYL U96 YTWDFGDSSGTLIS

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 U2. 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

The polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: U1-U96.

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: U1-U96. Each deletion can take place at any position of SEQ NOs: U1-U96.

In some embodiments, 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: U1- U96. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: U1-U96 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: U1-U96, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

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. co// cells or insect cells. A suitable method is provided in Example U2 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. Alternatively, 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.

Brief description of the Figures Figures U1 to U96 show the respective fragmentation spectra for the peptides of SEQ NOs: U1 to U96, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples Example U1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from PMEL on the surface of tumour cell lines was investigated using mass spectrometry.

Method

The polypeptides set out in table U1 , corresponding to SEQ NOs: U1 -U96, 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.

U34 HQILKGGSGT Class I Mel526

U35 HQILKGGSGTY Class I Mel526

U36 HSSSAFTITDQVP HLA-BW IGR37

U37 HTMEVTVYH Class I Mel526

U38 HTYLEPGPVT HLA-B^*07 Skmel28

U39 HTYLEPGPVTA HLA-B^*07 Skmel28

U40 HTYLEPGPVT AQ HLA-B^*07 Skmel28

U41 IMPGQEAGLGQV HLA-A^*02 IGR37

U42 KQDFSVPQL HLA-A^*02 Mel526

U43 KVSNDGPTL HLA-A^*02 IGR37

U44 LAVIGALLAVG HLA-B^*07 U266

U45 LDGGNKHFL HLA-A^*02 SkMel28

U46 LEPGPVTAQ HLA-A^*02 MEWO

U47 LGTHTMEV HLA-A^*02 KYSE140

U48 LHDPSGYL class I KYSE140

U49 LHDPSGYLA class I IGR37

U50 LHDPSGYLAEADLS HLA-B^*07 IGR37

U51 LIMPGQEAG HLA-A^*02 IGR37

U52 LIMPGQEAGL HLA-A^*02 KYSE140

U53 LIMPVPGILL HLA-A^*02 IGR37

U54 LKVSNDGPT HLA-B^*07 Skmel28

U55 LLDGTATLRLV HLA-A^*02 IGR37

U56 QAAIPLTS HLA-B^*07 IGR37

U57 QEAGLGQVPL class I IGR37

U58 QLHDPSGYLAEA HLA-A^*02 Skmel28

U59 QVLGGPVSGL HLA-A^*02 Mel526

U60 RALDGGNKHFL HLA-A^*02 Skmel28

U61 RLDCWRGGQV HLA-A^*02 KYSE140

U62 SAFTITDQV HLA-A^*02 SkMel28

U63 SAFTITDQVPFSV HLA-A^*02 Skmel28

U64 SCPIGENSP HLA-BW KYSE140

U65 SCPIGENSPL HLA-BW KYSE140

U66 SIALNFPG HLA-B^*07 IGR37

U67 SLADTNSL HLA-A^*02 Skmel28

U68 SLADTNSLAW HLA-A^*02 Mel526

U69 SLKVSNDGPT HLA-B^*07 KYSE140

U70 SVQVPTTEV HLA-A^*02 Skmel28

U71 SVTLDIVQ HLA-B^*07 IGR37

U72 SVTLDIVQG HLA-A^*02 IGR37 U73 SYVPLAHSSSAF class I KYSE140

U74 TAQVVLQA HLA-B^*07 Skmel28

U75 TLISRALVV HLA-A^*02 Skmel28

U76 TTEVISTAPV HLA-A^*02 Skmel28

U77 TWDFGDSSG HLA-B^*07 Skmel28

U78 TWDFGDSSGT HLA-B^*07 Skmel28

U79 TWDFGDSSGTLIS class I IGR37

U80 VLGGPVSGLSI HLA-A^*02 IGR37

U81 VPSGEGDAF class I KYSE140

U82 VQVPTTEVI HLA-A^*02 IGR37

U83 VSLKVSND HLA-A^*02 IGR37

U84 VSLKVSNDGPT HLA-BW Skmel28

U85 VSLKVSNDGPTLIG HLA-B^*07 IGR37

U86 VSNDGPTL Class I Mel526

U87 VSTQLIMPV HLA-A^*02 IGR37

U88 VTLDIVQGI HLA-A^*02 Skmel28

U89 WDFGDSSGT HLA-A^*02 KYSE140

U90 YLAEADLS HLA-A^*02 Skmel28

U91 YLEPGPVTAQV HLA-A^*02 Skmel28

U92 YRYGSFSVTL HLA-A^*02 IGR37

U93 YTWDFGDSSGT HLA-A^*02 Skmel28

U94 YTWDFGDSSGTL class I IGR37

U95 YTWDFGDSSGTLI HLA-A^*02 KYSE140

U96 YTWDFGDSSGTLIS HLA-A^*02 Skmel28

Figures U1 -U96 show representative fragmentation patterns for the peptides of SEQ NOs: U1-U96 respectively. A table highlighting the matching ions is shown below each spectrum. Example U2 - Preparation of recombinant peptide-HLA complexes

The following describes a suitable method for the preparation of soluble recombinant HLA loaded with TAA peptide. Class I HLA-A^*02 molecules (HLA-A^*02-heavy chain and HLA light-chain (β2ηι)) 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 β2ηι 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 β2ιη 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.

Example U3 - identification of TCRs that bind to a peptide-MHC complex of the invention

Method Methods for the isolation of TCRs 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. Other suitable methods include isolation from TCR libraries (as described WO2005/1 16646 and WO2005/116074 for example).

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 U2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction.

The[LS2i] present invention also relates to novel peptides derived from Serine protease 33

(PRSS33), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

PRSS33 (also known as serine protease 33 or serine protease EOS and having Uniprot accession number Q8NF86) is a serine protease that has amidolytic activity (Chen et al. Biochem J. 2003 Aug 15;374(Pt 1 ):97-107). Serine proteases are known to play a role in cancer. PRSS33 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from PRSS33 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 PRSS33 and for the treatment of cancers, including colon cancer.

In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: V1 -V2, or

(b) the amino acid sequence of any one of SEQ NOs: V1-V2 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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 V2. 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

USA. 1992 Apr 15;89(8):3429-33.

The polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: V1-V2. 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: V1-V2. Each deletion can take place at any position of SEQ NOs: V1-V2. In some embodiments, 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: V1- V2. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: V1-V2 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: V1-V2, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

Preferably, polypeptides of the invention bind to MHC in the peptide binding groove of the MHC molecule. Generally the amino acid modifications described above will not impair the ability of the peptide to bind MHC. In a preferred embodiment, the amino acid modifications improve the ability of the peptide to bind MHC. For example, 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. Alternatively, 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. Alternatively, in vitro cell-free systems may be used. Such systems are known in the art and are commercially available for example from Life

In a second aspect the invention provides a complex of the polypeptide of the first aspect and an MHC molecule. Preferably, 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. 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/imgt/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. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, 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. For example, 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 V2 herein. Alternatively, MHC molecules may be produced synthetically, or using cell free systems.

In another aspect, 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, colon cancer.

Brief description of the Figures

Figures V1 to V2 show the respective fragmentation spectra for the peptides of SEQ NOs: V1 to V2, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples

Example V1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from PRSS33 on the surface of tumour cell lines was investigated using mass spectrometry. Method

Results

The polypeptides set out in table V1 , corresponding to SEQ NOs: V1-V2, 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. Amino acid Example cancer

SEQ NO HLA antibody

sequence cell line

V1 ALPN RPGVYTSV class I A375

V2 RTCDGLYHV HLA-A^*02 Skmel28

Figures V1 -V2 show representative fragmentation patterns for the peptides of SEQ NOs: V1-V2 respectively. A table highlighting the matching ions is shown below each spectrum. Example V2 - Preparation of recombinant peptide-HLA complexes

Example V3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 V2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction. The[LS22] present invention also relates to novel peptides derived from Receptor-type tyrosine- protein phosphatase-like (PTPRN), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

PTPRN (also known as receptor-type tyrosine-protein phosphatase-like N or 1CA 512 or islet cell autoantigen 3 or PTP IA-2 and having Uniprot accession number Q16849) is a tyrosine phosphtase involved in vesicle-mediated secretory processes, including insulin secretion. Expression of PTPRN is associated with cancer Xie et al. Cancer Res. 1996 Jun 15;56(12):2742-4). PTPRN is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from PTPRN 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 PTPRN and for the treatment of cancers, including pancreatic cancer. In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: W1 -W27, or

(b) the amino acid sequence of any one of SEQ NOs: W1-W27 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

W14 INISVVGPAL

W15 IPTGSAPAA

W16 ISVVGPAL

W17 KEIDIAATL

W18 KLLEILAEHV

W19 LFQDSGLLY

W20 LQPYLFHQF

W21 QLFQDSGLLYL

W22 SELEAQTGL

W23 SLADVTQQA

W24 SLYHVYEV

W25 TMEGPVEGR

W26 VLAGYGVEL

W27 VVGPALTF

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 W2. 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 USA. 1992 Apr 15;89(8):3429-33. Preferably, 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: W1-W27.

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: W1-W27. Each deletion can take place at any position of SEQ NOs: W1-W27.

In some embodiments, 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: W1- W27. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: W1-W27 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: W1-W27, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

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 W2 herein. Alternatively, MHC molecules may be produced synthetically, or using cell free systems.

In the context of the present invention, 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. 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. In a fifth aspect, 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. In particular, 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.

Lefranc, (201 1 ), Cold Spring Harb Protoc 2011(6): 595-603; Lefranc, (2001 ), Curr Protoc Immunol Appendix 1 : Appendix 10; Lefranc, (2003), Leukemia 17(1 ): 260-266, and on the IMGT website (www.IMGT.org) The TCRs of the invention may be in any format known to those in the art. For example, the TCRs may be αβ heterodimers, or they may be in single chain format (such as those described in W09918129). Single chain TCRs include αβ TCR polypeptides of the type: Va-L-νβ, νβ-L-Va, Va- Ca-L-νβ, να-ί-Ν/β-Οβ or Va- Ca -ίΛ/β-Οβ, optionally in the reverse orientation, wherein Va and νβ are TCR a and β variable regions respectively, Ca and C are TCR a and β 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.

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. Thus, 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 binding moiety may be an antibody-like molecule that has been designed to specifically bind a peptide-MHC complex of the invention. Of particular preference are TCR-mimic antibodies, such as, for example those described in WO2007143104 and Sergeeva et 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.

In another aspect, 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, pancreatic cancer.

compositions are encompassed in the present invention. As will be appreciated, vaccines may take several forms (Schlom, J Natl Cancer Inst. 2012 Apr 18;104(8):599-613). For example, 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. Preferably 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(1 1 ): 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.

Brief description of the Figures Figures W1 to W27 show the respective fragmentation spectra for the peptides of SEQ NOs: W1 to W27, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples Example W1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from PTPRN on the surface of tumour cell lines was investigated using mass spectrometry. Method

Class I HLA complexes were purified by immunoaffinity using commercially available anti-HLA antibodies BB7.1 (anti-HLA-BW), 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 10⁷ 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. Columns were washed with low salt and high salt buffers and complexes eluted in acid. Eluted peptides were separated from HLA complexes by reversed phase chromatography using a solid phase extraction cartridge (Phenomenex). Bound material was eluted from the column and reduced in volume using a vacuum centrifuge. 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.

Results

The polypeptides set out in table W1 , corresponding to SEQ NOs: W1-W27, 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.

W11 GVAGLLVALA HLA-A*02 A375

W12 ILAEHVHMS class I NCI H508

W13 INISVVGPA HLA-B*07 SR

W14 INISVVGPAL HLA-A*02 NCI H1755

W15 IPTGSAPAA HLA-A*02 IGR37

W16 ISVVGPAL class I NCI H1755

W17 KEIDIAATL class I NCI H1755

W18 KLLEILAEHV HLA-A^*02 NCI H1915

W19 LFQDSGLLY HLA-A*02 VMRC LCD

W20 LQPYLFHQF class I NCI H1915

W21 QLFQDSGLLYL HLA-A*02 NCI H1755

W22 SELEAQTGL class I NCI H1755

W23 SLADVTQQA class I NCI H1755

W24 SLYHVYEV HLA-A*02 NCI H1755

W25 TMEGPVEGR HLA-A*02 U937

W26 VLAGYGVEL HLA-A*02 NCI H1915

W27 VVGPALTF class I NCI H1755

Figures W1 -W27 show representative fragmentation patterns for the peptides of SEQ NOs: W1- W27 respectively. A table highlighting the matching ions is shown below each spectrum. Example W2 - Preparation of recombinant peptide-HLA complexes

(buffered to pH 8), 7.5 mM MgCI2, and 5 pg/ml BirA enzyme (purified according to O'Callaghan et a/., (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.

Example W3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

Methods for the isolation of TCRs 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. Other suitable methods include isolation from TCR libraries (as described in WO2005/1 16646 and WO2005/116074 for example). To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 W2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction.

The [LS23]present invention also relates to novel peptides derived from Ropporin-1A (ROPN1 ), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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.

1 ;152(1 ):163-75 (access via http://www-bimas.cit.nih.qov/molbio/hla bind/)) are available to predict the amino acid sequences of MHC-presented peptides derived from proteins. However, these methods are known to generate a high proportion of false positives (since they simply define the likelihood of a given peptide being able to bind a given MHC and do not account for intracellular processing). Therefore, it is not possible to accurately predict whether a given peptide-MHC is actually presented by tumour cells. Direct experimental data is typically required. ROPN1 (also known as ropporin-1 A or Rhophilin-associated protein 1A or CT91 and having

Uniprot accession number Q9HAT0) is involved in GTPase mediated signal transduction. ROPN1 belongs to the cancer/testis family of germline encoded tumour antigens. Expression of ROPN1 has been reported in various tumours, while expression in normal tissue is limited to testis (Li ei a/. Int J Cancer. 2007 Oct 1 ;121 (7):1507-11 ; Chiriva-lnternati et al. J immunother. 201 1 Jul- Aug;34(6):490-9). ROPN1 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from ROPN1 that are presented on the cell surface in complex with HC. These peptides are particularly useful for the development of reagents that can targets cells expressing ROPN1 and for the treatment of cancers, including breast cancer. In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: X1 -X5, or

(b) the amino acid sequence of any one of SEQ NOs: X1-X5 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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 X2. 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

USA. 1992 Apr 15;89(8):3429-33.

The polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: X1-X5.

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: X1-X5. Each deletion can take place at any position of SEQ NOs: X1-X5.

In some embodiments, 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: X1- X5. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: X1-X5 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: X1-X5, 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

In a second aspect the invention provides a complex of the polypeptide of the first aspect and an MHC molecule. Preferably, 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-ET08, 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. 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. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, the term " 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. For example, 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. co// cells or insect cells. A suitable method is provided in Example X2 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. Alternatively, 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. Examples of solid supports include, but are not limited to, a bead, a membrane, sepharose, a magnetic bead, a plate, a tube, a column.

In the context of the present invention, 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.

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 TCRs of the invention may be in any format known to those in the art. For example, the TCRs may be αβ heterodimers, or they may be in single chain format (such as those described in W09918129). Single chain TCRs include αβ TCR polypeptides of the type: Va-L-νβ, νβ-L-Va, Va- Ca-L-νβ, Va-L-νβ-Οβ or Va- Ca -ίΛ β-Οβ, optionally in the reverse orientation, wherein Va and νβ are TCR a and β variable regions respectively, Ca and Οβ are TCR a and β 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.

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. In multimeric high affinity TCR complexes such as those described in Zhu et a/., J. Immunol. 2006 Mar 1 ; 176(5):3223-32, (formed, for example, using biotinylated heterodimers) 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.

Biotechnol. 1993 Aug;4(4):446-9), 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. coll. 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).

21 ;1 17(16):4262-72 and/or Dahan and Reiter. Expert Rev Mol Med. 2012 Feb 24;14:e6. Also encompassed within the present invention are binding moieties based on engineered protein scaffolds. 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. Examples of 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. 2008 Jun;275(1 1 ):2677-83), nanobodies, and DARPins. 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 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. For example, 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.

Brief description of the Figures

Figures X1 to X5 show the respective fragmentation spectra for the peptides of SEQ NOs: X1 to X5, eluted from cells. A table highlighting the matching ions is shown below each spectrum. Examples

Example X1 - Identification of target-derived peptides by Mass spectrometry Presentation of HLA-restricted peptides derived from ROPN1 on the surface of tumour cell lines was investigated using mass spectrometry.

Method

Results

The polypeptides set out in table X1 , corresponding to SEQ NOs: X1 -X5, 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 X1 -X5 show representative fragmentation patterns for the peptides of SEQ NOs: X1 -X5 respectively. A table highlighting the matching ions is shown below each spectrum.

Example X2 - Preparation of recombinant peptide-HLA complexes The following describes a suitable method for the preparation of soluble recombinant HLA loaded with TAA peptide.

Example X3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

Methods for the isolation of TCRs 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. Other suitable methods include isolation from TCR libraries (as described in WO2005/1 16646 and WO2005/116074 for example). To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 X2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction.

The[LS24] present invention also relates to novel peptides derived from Sodium/potassium/calcium exchanger 5 (SLC24A5), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

SLC24A5 (also known as solute carrier family 24 member 5 or Sodium/potassium/calcium exchanger 5 and having Uniprot accession number Q71 RS6) functions as a cation exchanger and is involved in pigmentation in melanocytes. Expression of SLC24A5 has been associated with melanoma (Nan et al. Int J Cancer. 2009 Aug 15;125(4):909-17). SLC24A5 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from SLC24A5 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 SLC24A5 and for the treatment of cancers, including uveal melanoma. In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: Y1 -Y14, or

(b) the amino acid sequence of any one of SEQ NOs: Y1-Y14 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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 Y2. 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

USA. 1992 Apr 15;89(8):3429-33.

The polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: Y1-Y14. 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: Y1-Y14. Each deletion can take place at any position of SEQ NOs: Y1-Y14.

In some embodiments, 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: Y1- Y14. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: Y1-Y14 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: Y1-Y14, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

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. co// cells or insect cells. A suitable method is provided in Example Y2 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. Alternatively, 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.

Brief description of the Figures Figures Y1 to Y14 show the respective fragmentation spectra for the peptides of SEQ NOs: Y1 to Y14, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples Example Y1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from SLC24A5 on the surface of tumour cell lines was investigated using mass spectrometry.

Method

The polypeptides set out in table Y1 , corresponding to SEQ NOs: Y1 -Y14, 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 Y1 -Y14 show representative fragmentation patterns for the peptides of SEQ NOs: Y1-Y14 respectively. A table highlighting the matching ions is shown below each spectrum. Example Y2 - Preparation of recombinant peptide-HLA complexes

The following describes a suitable method for the preparation of soluble recombinant HLA loaded with TAA peptide. Class I HLA-A*02 molecules (HLA-A*02-heavy chain and HLA light-chain (β2ητι)) 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 β2ηι 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 β2ιη 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.

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 a/., (1999) Anal. Biochem. 266: 9-15). The mixture was then allowed to incubate at room temperature overnight.

Example Y3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 Y2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction.

The[LS25] present invention also relates to novel peptides derived from Solute carrier family 35 member D3 (SLC35D3), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

SLC35D3 (also known as solute carrier family 35 member D3 or fringe connection-like protein 1 and having Uniprot accession number Q5M8T2) is a transporter involved in hemostasis (Chintala et al. Blood. 2007 Feb 15; 109(4): 1533-40). The inventors have found novel peptides derived from SLC35D3 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 SLC35D3 and for the treatment of cancers, including colon cancer and oesophageal cancer.

In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: Z1 -Z2, or

(b) the amino acid sequence of any one of SEQ NOs: Z1-Z2 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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 Z2. 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

USA. 1992 Apr 15;89(8):3429-33.

The polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: Z1-Z2. 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: Z1-Z2. Each deletion can take place at any position of SEQ NOs: Z1-Z2. In some embodiments, 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: Z1- Z2. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: Z1-Z2 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: Z1-Z2, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

The complex of the invention may be isolated and/or in a substantially pure form. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, 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. For example, 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 Z2 herein. Alternatively, MHC molecules may be produced synthetically, or using cell free systems.

In another aspect, 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, colon cancer and oesophageal cancer.

Brief description of the Figures

Figures Z1 to Z2 show the respective fragmentation spectra for the peptides of SEQ NOs: Z1 to Z2, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples

Example Z1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from SLC35D3 on the surface of tumour cell lines was investigated using mass spectrometry. Method

Results

The polypeptides set out in table Z1 , corresponding to SEQ NOs: Z1-Z2, 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. Amino acid Example cancer

SEQ NO HLA antibody

sequence cell line

Z1 LARSFAGVAV HLA-A*02 NCI H508

Z2 TLGSIIYCV HLA-A*02 NCI H508

Figures Z1 -Z2 show representative fragmentation patterns for the peptides of SEQ NOs: Z1-Z2 respectively. A table highlighting the matching ions is shown below each spectrum. Example Z2 - Preparation of recombinant peptide-HLA complexes

Example Z3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 Z2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction. The[LS26] present invention also relates to novel peptides derived from Spermatogenesis -and oogenesis-specific basic helix-loop-helix-containing protein 1 (SOHLH1 ), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

SOHLH1 (also known as spermatogenesis- and oogenesis-specific basic helix-loop-helix- containing protein 1 and having Uniprot accession number Q5JUK2) is a transcription factor expressed in undifferentiated spermatogonia (Ballow et al. Dev Biol. 2006 Jun 1 ;294(1 ):161-7). SOHLH1 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from SOHLH1 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 SOHLH1 and for the treatment of cancers, including non small cell lung cancer (squamous) and head and neck cancer. In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: AA1 -AA2, or

(b) the amino acid sequence of any one of SEQ NOs: AA1-AA2 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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 AA2. 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

USA. 1992 Apr 15;89(8):3429-33. Preferably, 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: AA1-AA2.

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: AA1-AA2. Each deletion can take place at any position of SEQ NOs: AA1-AA2.

In some embodiments, 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: AA1- AA2. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: AA1-AA2 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: AA1-AA2, 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

The complex of the invention may be isolated and/or in a substantially pure form. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, 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. For example, 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 AA2 herein. Alternatively, MHC molecules may be produced synthetically, or using cell free systems.

In another aspect, 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 head and neck cancer.

Brief description of the Figures

Figures AA1 to AA2 show the respective fragmentation spectra for the peptides of SEQ NOs: AA1 to AA2, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples

Example AA1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from SOHLH1 on the surface of tumour cell lines was investigated using mass spectrometry. Method

Results

The polypeptides set out in table AA1 , corresponding to SEQ NOs: AA1-AA2, 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. Amino acid Example cancer

SEQ NO HLA antibody

sequence cell line

AA1 LAQEAGSAL class I EJM

AA2 SGPPKAPTV HLA-A*02 EJM

Figures AA1 -AA2 show representative fragmentation patterns for the peptides of SEQ NOs: AA1- AA2 respectively. A table highlighting the matching ions is shown below each spectrum.

Example AA2 - Preparation of recombinant peptide-HLA complexes

Inclusion bodies of β2ηι 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 β2ιη 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 (buffered to pH 8), 7.5 mM MgCI2, and 5 pg/ml BirA enzyme (purified according to O'Callaghan et

All a/., (1999) Anal. Biochem. 266: 9-15). The mixture was then allowed to incubate at room temperature overnight.

Example AA3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 AA2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction. The[LS27] present invention also relates to novel peptides derived from Transcription factor Sp8 (SP8), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

SP8 (also known as transcription factor Sp8 or specificity protein 8 and having Uniprot accession number Q8IXZ3) is a transcription factor that plays a key role in limb development. SP transcription factors are known to play a role in cancer (Safe et al. Eur J Cancer. 2005 Nov;41 (16):2438-48). SP8 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from SP8 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 SP8 and for the treatment of cancers, including non-small cell lung cancer (adenocarcinoma) and prostate cancer.

In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: AB1 -AB8, or

(b) the amino acid sequence of any one of SEQ NOs: AB1-AB8 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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 AB2. 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

USA. 1992 Apr 15;89(8):3429-33. Preferably, 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: AB1-AB8.

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: AB1-AB8. Each deletion can take place at any position of SEQ NOs: AB1-AB8.

In some embodiments, 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: AB1- AB8. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: AB1-AB8 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: AB1-AB8, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

The complex of the invention may be isolated and/or in a substantially pure form. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, 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. For example, 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 AB2 herein. Alternatively, MHC molecules may be produced synthetically, or using cell free systems.

21 ;1 17(16):4262-72 and/or Dahan and Reiter. Expert Rev Mol Med. 2012 Feb 24;14:e6. Also encompassed within the present invention are binding moieties based on engineered protein scaffolds. 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. Examples of 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. 2008 Jun;275(1 1 ):2677-83), nanobodies, and DARPins. 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)]. In another aspect, 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 (adenocarcinoma) and prostate cancer.

Brief description of the Figures

Figures AB1 to AB8 show the respective fragmentation spectra for the peptides of SEQ NOs: AB1 to AB8, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples Example AB1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from SP8 on the surface of tumour cell lines was investigated using mass spectrometry.

Method

The polypeptides set out in table AB1 , corresponding to SEQ NOs: AB1 -AB8, 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 AB1 -AB8 show representative fragmentation patterns for the peptides of SEQ NOs: AB1- AB8 respectively. A table highlighting the matching ions is shown below each spectrum.

Example AB2 - Preparation of recombinant peptide-HLA complexes

Inclusion bodies of β2ιη 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 β2πι 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 μητι 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.

Example AB3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 AB2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction.

The[LS28] present invention also relates to novel peptides derived from Sperm protein associated with the nucleus on the X chromosome B2 (SPANXB2), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

SPANXB2 (also known as sperm protein associated with the nucleus on the X chromosome B1 or CT1 1.2 and having Uniprot accession number Q9NS25) belongs to the cancer testis family of germline encoded tumour antigens. Expression of SPANXB2 has been reported in various tumours while expression in normal tissues is restricted to testis (Almanzar et al. Clin Cancer Res. 2009 Mar 15; 15(6): 1954-63). SPANXB2 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from SPANXB2 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 SPANXB2 and for the treatment of cancers, including head and neck cancer.

In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of SEQ NO: AC1 or

(b) the amino acid sequence of SEQ NO: AC1 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions, wherein the polypeptide forms a complex with a Major Histocompatibility Complex (MHC) molecule.

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 AC2. 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

USA. 1992 Apr 15;89(8):3429-33. Preferably, 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 acid sequence provided in SEQ NO: AC1.

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: AC1. Each deletion can take place at any position of SEQ NO: AC1.

In some embodiments, 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: AC1. A polypeptide of the invention may comprise the amino acid sequence of SEQ NO: AC1 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: AC1 , 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

The complex of the invention may be isolated and/or in a substantially pure form. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, 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. For example, 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 AC2 herein. Alternatively, MHC molecules may be produced synthetically, or using cell free systems.

In another aspect, 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.

Brief description of the Figures

Figure AC1 shows the respective fragmentation spectra for the peptide of SEQ NO: AC1 , eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples

Example AC1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from SPANXB2 on the surface of tumour cell lines was investigated using mass spectrometry. Method

Results

The polypeptide set out in table AC1 , corresponding to SEQ NO: AC1 , 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. Amino acid Example cancer

SEQ NO HLA antibody

sequence cell line

AC1 KTSESSTILW Class I SW982

Figure AC1 shows representative fragmentation pattern for the peptide of SEQ NO: AC1. A table highlighting the matching ions is shown below the spectra. Example AC2 - Preparation of recombinant peptide-HLA complexes

(buffered to pH 8), 7.5 mM MgCI2, and 5 g/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 AC3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 AC2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction. The[LS29] present invention also relates to novel peptides derived from Protein SSX1 (SSX1), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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.

1 ;152(1 ):163-75 (access via http://www-bimas.cit.nih.qov/molbio/hla bind/)) are available to predict the amino acid sequences of MHC-presented peptides derived from proteins. However, these methods are known to generate a high proportion of false positives (since they simply define the likelihood of a given peptide being able to bind a given MHC and do not account for intracellular processing). Therefore, it is not possible to accurately predict whether a given peptide-MHC is actually presented by tumour cells. Direct experimental data is typically required. SSX1 (also known as Synovial sarcoma, X breakpoint 1 or CT5.1 and having Uniprot accession number Q16384) belongs to the cancer testis family of germline encoded tumour antigens.

Expression of SSX1 has been reported in various tumours while expression in normal tissues is restricted to testis (Smith et al. Clin Dev Immunol. 2010;2010:150591 ; Gure et al. Int J Cancer. 1997 Sep 17;72(6):965-71 ). SSX1 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from SSX1 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 SSX1 and for the treatment of cancers, including liver cancer.

In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: AD1 -AD3, or

(b) the amino acid sequence of any one of SEQ NOs: AD1-AD3 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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 AD2. 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

USA. 1992 Apr 15;89(8):3429-33.

Preferably, 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: AD1-AD3.

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: AD1-AD3. Each deletion can take place at any position of SEQ NOs: AD1-AD3.

In some embodiments, 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: AD1- AD3. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: AD1- AD3 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: AD1 -AD3, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

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 AD2 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. Alternatively, 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.

In another aspect, 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, liver cancer.

Brief description of the Figures

Figures AD1 to AD3 show the respective fragmentation spectra for the peptides of SEQ NOs: AD1 to AD3, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples

Example AD1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from SSX1 on the surface of tumour cell lines was investigated using mass spectrometry. Method

Results

The polypeptides set out in table AD1 , corresponding to SEQ NOs: AD1-AD3, 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. Amino acid Example cancer

SEQ NO HLA antibody

sequence cell line

AD1 RIIPKIMPK class I NCI H929

AD2 SEKISYVY class I NCI H929

AD3 VEHPQMTF class I NCI H929

Figures AD1 -AD3 show representative fragmentation patterns for the peptides of SEQ NOs: AD1- AD3 respectively. A table highlighting the matching ions is shown below each spectrum.

Example AD2 - Preparation of recombinant peptide-HLA complexes

Inclusion bodies of β2πι 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 β2ιη 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.

Example AD3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 AD2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction. The[LS30] present invention also relates to novel peptides derived from Protein SSX4 (SSX4), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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.

1 ;152(1 ):163-75 (access via http://www-bimas.cit.nih.qov/molbio/hla bind/)) are available to predict the amino acid sequences of MHC-presented peptides derived from proteins. However, these methods are known to generate a high proportion of false positives (since they simply define the likelihood of a given peptide being able to bind a given MHC and do not account for intracellular processing). Therefore, it is not possible to accurately predict whether a given peptide-MHC is actually presented by tumour cells. Direct experimental data is typically required. SSX4 (also known as Protein SSX4 or CT5.4 and having Uniprot accession number 060224) belongs to the cancer testis family of germline encoded tumour antigens. Expression of SSX4 has been reported in various tumours while expression in normal tissues is restricted to testis (Smith et al. Clin Dev Immunol. 2010;2010:150591 ; Gure et al. Int J Cancer. 1997 Sep 17;72(6):965-71 ). SSX4 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from SSX4 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 SSX4 and for the treatment of cancers, including liver cancer and melanoma.

In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: AE1 -AE2, or

(b) the amino acid sequence of any one of SEQ NOs: AE1-AE2 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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 AE2. 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

USA. 1992 Apr 15;89(8):3429-33.

The polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: AE1-AE2. 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: AE1-AE2. Each deletion can take place at any position of SEQ NOs: AE1-AE2. In some embodiments, 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: AE1- AE2. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: AE1-AE2 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: AE1-AE2, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

The complex of the invention may be isolated and/or in a substantially pure form. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, 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. For example, 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 AE2 herein. Alternatively, MHC molecules may be produced synthetically, or using cell free systems.

In another aspect, 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, liver cancer and melanoma.

Brief description of the Figures

Figures AE1 to AE2 show the respective fragmentation spectra for the peptides of SEQ NOs: AE1 to AE2, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples

Example AE1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from SSX4 on the surface of tumour cell lines was investigated using mass spectrometry. Method

Results

The polypeptides set out in table AE1 , corresponding to SEQ NOs: AE1-AE2, 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. Amino acid Example cancer

SEQ NO HLA antibody

sequence cell line

AE1 MTSNSPRGYD class I Granta

AE2 RIFPKIMPK class I NCI H1944

Figures AE1 -AE2 show representative fragmentation patterns for the peptides of SEQ NOs: AE1- AE2 respectively. A table highlighting the matching ions is shown below each spectrum.

Example AE2 - Preparation of recombinant peptide-HLA complexes

Example AE3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 AE2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction. The[LS3i] present invention also relates to novel peptides derived from Protein Wnt-10a (WNT10A), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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.

1 ;152(1 ):163-75 (access via http://www-bimas.cit.nih.qov/molbio/hla bind/)) are available to predict the amino acid sequences of MHC-presented peptides derived from proteins. However, these methods are known to generate a high proportion of false positives (since they simply define the likelihood of a given peptide being able to bind a given MHC and do not account for intracellular processing). Therefore, it is not possible to accurately predict whether a given peptide-MHC is actually presented by tumour cells. Direct experimental data is typically required. WNT10A (also known as Protein Wnt-10a and having Uniprot accession number Q9GZT5) is a receptor ligand that may be a signalling molecule in CNS development. WNT10A has been associated with cancer (Hsu et al. PLoS One. 2012;7(10):e47649). WNTI OA is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from WNT10A 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 WNT1 OA and for the treatment of cancers, including head and neck cancer.

In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: AF1 -AF13, or

(b) the amino acid sequence of any one of SEQ NOs: AF1-AF13 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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 AF2. 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

USA. 1992 Apr 15;89(8):3429-33.

The polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: AF1-AF13.

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: AF1-AF13. Each deletion can take place at any position of SEQ NOs: AF1-AF13. In some embodiments, 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: AF1- AF13. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: AF1- AF13 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: AF1- AF13, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

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. co// cells or insect cells. A suitable method is provided in Example AF2 herein. Alternatively, MHC molecules may be produced synthetically, or using cell free systems.

Brief description of the Figures

Figures AF1 to AF13 show the respective fragmentation spectra for the peptides of SEQ NOs: AF1 to AF13, eluted from cells. A table highlighting the matching ions is shown below each spectrum. Examples

Example AF1 - Identification of target-derived peptides by Mass spectrometry Presentation of HLA-restricted peptides derived from WNT10A on the surface of tumour cell lines was investigated using mass spectrometry.

Method

Results

The polypeptides set out in table AF1 , corresponding to SEQ NOs: AF1 -AF13, 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 HI_A antibody used for immunoaffinity purification.

Figures AF1 -AF13 show representative fragmentation patterns for the peptides of SEQ NOs: AF1- AF13 respectively. A table highlighting the matching ions is shown below each spectrum.

Example AF2 - Preparation of recombinant peptide-HLA complexes

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 (buffered to pH 8), 7.5 mM MgCI2, and 5 pg/ml BirA enzyme (purified according to O'Callaghan et a/., (1999) Anal. Biochem. 266: 9-15). The mixture was then allowed to incubate at room temperature overnight.

Example AF3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

Methods for the isolation of TCRs 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. Other suitable methods include isolation from TCR libraries (as described in WO2005/1 16646 and WO2005/116074 for example). To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 AF2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction.

The[LS32] present invention also relates to novel peptides derived from Wilms tumor protein (WT1 ), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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.

1 ;152(1 ):163-75 (access via http://www-bimas.cit.nih.qov/molbio/hla bind/)) are available to predict the amino acid sequences of MHC-presented peptides derived from proteins. However, these methods are known to generate a high proportion of false positives (since they simply define the likelihood of a given peptide being able to bind a given MHC and do not account for intracellular processing). Therefore, it is not possible to accurately predict whether a given peptide-MHC is actually presented by tumour cells. Direct experimental data is typically required. WT1 (also known as Wilms tumor protein or WT33 and having Uniprot accession number P19544) is a transcription factor that plays an important role in cellular development and cell survival. WT1 is a well-known oncogene (Sugiyama, Jpn J Clin Oncol. 2010 May;40(5):377-87). WT1 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from WT1 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 WT1 and for the treatment of cancers, including ovarian cancer.

In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: AG1 -AG5, or

(b) the amino acid sequence of any one of SEQ NOs: AG1-AG5 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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 AG2. 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

The polypeptides of the invention may consist or consist essentially of the amino acids sequences provided in SEQ NOs: AG1-AG5.

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: AG1-AG5. Each deletion can take place at any position of SEQ NOs: AG1-AG5. In some embodiments, 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: AG1- AG5. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: AG1- AG5 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: AG1 -AG5, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

The complex of the invention may be isolated and/or in a substantially pure form. For example, the complex may be provided in a form which is substantially free of other polypeptides or proteins. It should be noted that in the context of the present invention, 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. For example, 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 AG2 herein. Alternatively, MHC molecules may be produced synthetically, or using cell free systems.

Figures AG1 to AG5 show the respective fragmentation spectra for the peptides of SEQ NOs: AG1 to AG5, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples Example AG1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from WT1 on the surface of tumour cell lines was investigated using mass spectrometry.

Method

The polypeptides set out in table AG1 , corresponding to SEQ NOs: AG1 -AG5, 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 AG1-AG5 show representative fragmentation patterns for the peptides of SEQ NOs: AG1- AG5 respectively. A table highlighting the matching ions is shown below each spectrum.

Example AG2 - Preparation of recombinant peptide-HLA complexes

Inclusion bodies of β2πι 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 β2πι 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 μητι 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 a/., (1999) Anal. Biochem. 266: 9-15). The mixture was then allowed to incubate at room temperature overnight.

Example AG3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

Methods for the isolation of TCRs 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. Other suitable methods include isolation from TCR libraries (as described in WO2005/1 16646 and WO2005/1 6074 for example). To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 AG2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction.

The present invention also relates to novel peptides derived from Melanoma-associated antigen 8 (MAGEA8), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules. Also provided by the present invention are 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). In humans, 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. 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. However, as a consequence of the selection processes which occur during T cell maturation in the thymus, there is a scarcity of T cells (and TCRs) in the circulating repertoire, which recognise TAA-derived peptides with a sufficiently high level of affinity. Therefore tumour cells often escape detection.

MAGEA8 (also known as Melanoma-associated antigen 8 or CT1.8 and having Uniprot accession number P43361 ) belongs to the MAGE family of germline encoded cancer antigens. Expression of MAGEA8 has been reported in various tumours while expression in normal tissues is restricted to testis (De Plaen et al. Immunogenetics. 1994;40(5):360-9). MAGEA8 is an ideal target for immunotherapeutic applications. The inventors have found novel peptides derived from MAGEA8 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 MAGEA8 and for the treatment of cancers, including non small cell lung cancer (adenocarcinoma) and liver cancer. In a first aspect, the invention provides a polypeptide comprising, consisting essentially of, or consisting of (a) the amino acid sequence of any one of SEQ NOs: AH1 -AH2, or

(b) the amino acid sequence of any one of SEQ NOs: AH1-AH2 with the exception of 1 , 2 or 3 amino acid substitutions, and/or 1 , 2 or 3 amino acid insertions, and/or 1 , 2 or 3 amino acid deletions,

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 AH2. 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

USA. 1992 Apr 15;89(8):3429-33. Preferably, 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: AH1-AH2.

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: AH1-AH2. Each deletion can take place at any position of SEQ NOs: AH1-AH2.

In some embodiments, 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: AH1- AH2. A polypeptide of the invention may comprise the amino acid sequence of SEQ NOs: AH1- AH2 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: AH1 -AH2, with the exception of one amino acid substitution, one amino acid insertion and one amino acid deletion.

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 AH2 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. Alternatively, 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.

In another aspect, 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 (adenocarcinoma) and liver cancer.

Brief description of the Figures

Figures AH1 to AH2 show the respective fragmentation spectra for the peptides of SEQ NOs: AH1 to AH2, eluted from cells. A table highlighting the matching ions is shown below each spectrum.

Examples

Example AH1 - Identification of target-derived peptides by Mass spectrometry

Presentation of HLA-restricted peptides derived from MAGEA8 on the surface of tumour cell lines was investigated using mass spectrometry. Method

Results

The polypeptides set out in table AH1 , corresponding to SEQ ID NOs: AH1-AH2, 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. Amino acid Example cancer

SEQ ID NO HLA antibody

sequence cell line

AH1 EAI WEALS V HLA-A*02 U266

AH2 KEVDPAGHSYIL class I U266

Figures AH1 -AH2 show representative fragmentation patterns for the peptides of SEQ NOs: AH1- AH2 respectively. A table highlighting the matching ions is shown below each spectrum.

Example AH2 - Preparation of recombinant peptide-HLA complexes

Example AH3 - identification of TCRs that bind to a peptide-MHC complex of the invention Method

To confirm that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention isolated 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 AH2) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25°C in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1 :1 interaction.

Claims

1. A polypeptide comprising, consisting essentially of or consisting of

(a) the amino acid sequence of any one of SEQ NOs: A1-A9, SEQ NOs: B1-B3, SEQ NOs:

C1-C9, SEQ NO: D1, SEQ NOs: E1-E2, SEQ NOs: F1-F2, SEQ NOs: G1-G8, SEQ NOs: H1-H18, SEQ NOs: 11-18, SEQ NOs: J1-J19, SEQ NOs: K1-K4, SEQ NOs: L1-L28, SEQ NOs: M1-M3, SEQ NOs: N1-N5, SEQ NOs: 01-05, SEQ NOs: P1-P20, SEQ NO: Q1, SEQ NOs: R1-R3, SEQ NOs: S1-S4, SEQ NOs: T1-T29, SEQ NOs: U1-U96, SEQ NOs: V1-V2, SEQ NOs: W1-W27, SEQ NOs: X1-X5, SEQ NOs: Y1-Y14, SEQ NOs: Z1-Z2, SEQ NOs: AA1-AA2, SEQ NOs: AB1-AB8, SEQ NO: AC1, SEQ NOs: AD1-AD3, SEQ NOs: AE1-AE2, SEQ NOs: AF1-AF13, SEQ NOs: AG 1 -AG 5 or SEQ NOs: AH1-AH2, or

(b) the amino acid sequence of any one of SEQNOs: A1-A9, SEQ NOs: B1-B3, SEQ NOs: C1-C9, SEQ NO: D1, SEQ NOs: E1-E2, SEQ NOs: F1-F2, SEQ NOs: G1-G8, SEQ NOs: H1-H18, SEQ NOs: 11-18, SEQ NOs: J1-J19, SEQ NOs: K1-K4, SEQ NOs: L1-L28, SEQ NOs: M1-M3, SEQ NOs: N1-N5, SEQ NOs: 01-05, SEQ NOs: P1-P20, SEQ NO: Q1, SEQ NOs: R1-R3, SEQ NOs: S1-S4, SEQ NOs: T1-T29, SEQ NOs: U1-U96, SEQ NOs: V1-V2, SEQ NOs: W1-W27, SEQ NOs: X1-X5, SEQ NOs: Y1-Y14, SEQ NOs: Z1-Z2, SEQ NOs: AA1-AA2, SEQ NOs: AB1-AB8, SEQ NO: AC1, SEQ NOs: AD1-AD3, SEQ NOs: AE1-AE2, SEQ NOs: AF1-AF13, SEQ NOs: AG 1 -AG 5 or SEQ NOs: AH1-AH2 with the exception of 1, 2 or 3 amino acid substitutions and/or 1, 2 or 3 amino acid insertions, and/or 1, 2 or 3 amino acid deletions,

2. The polypeptide of claim 1 , wherein the polypeptide consists of from 8 to 16 amino acids.

3. The polypeptide of claim 1 or claim 2, wherein the polypeptide consists of the amino acid sequence of SEQ NOs A1-A9, SEQ NOs: B1-B3, SEQ NOs: C1-C9, SEQ NO: D1, SEQ NOs: E1- E2, SEQ NOs: F1-F2, SEQ NOs: G1-G8, SEQ NOs: H1-H18, SEQ NOs: 11-18, SEQ NOs: J1-J19, SEQ NOs: K1-K4, SEQ NOs: L1-L28, SEQ NOs: M1-M3, SEQ NOs: N1-N5, SEQ NOs: 01-05, SEQ NOs: P1-P20, SEQ NO: Q1, SEQ NOs: R1-R3, SEQ NOs: S1-S4, SEQ NOs: T1-T29, SEQ NOs: U1-U96, SEQ NOs: V1-V2, SEQ NOs: W1-W27, SEQ NOs: X1-X5, SEQ NOs: Y1-Y14, SEQ NOs: Z1-Z2, SEQ NOs: AA1-AA2, SEQ NOs: AB1-AB8, SEQ NO: AC1, SEQ NOs: AD1-AD3, SEQ NOs: AE1-AE2, SEQ NOs: AF1-AF13, SEQ NOs: AG 1 -AG 5 or SEQ NOs: AH1-AH2.

4. A complex of the polypeptide of any preceding claim and a Major Histocompatibility Complex (MHC) molecule.

5. The complex of claim 4, wherein the MHC molecule is MHC class I.

6. A nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide as defined in any one of claims 1-3.

7. A vector comprising a nucleic acid sequence as defined in claim 6.

A cell comprising a vector as claimed in claim 7.

9. A binding moiety that binds the polypeptide of any one of claims 1 -3.

10. The binding moiety of claim 9, which binds the polypeptide when it is in complex with MHC.

11. The binding moiety of claim 10, wherein the binding moiety is a T cell receptor (TCR) or an antibody.

12. The binding moiety of claim 1 1 , wherein the TCR is on the surface of a cell.

13. A polypeptide as defined in any one of claims 1-3, a complex as defined in claim 4 or claim 5, a nucleic acid molecule as defined in claim 6, a vector as defined in claim 7, a cell as defined in claim 8 or a binding moiety as defined in any one of claims 9-12 for use in medicine.

14. The polypeptide, complex, nucleic acid, vector or cell for use as defined in claim 13 for use in treating or preventing cancer.

15. A pharmaceutical composition comprising a polypeptide as defined in any one of claims 1- 3, a complex as defined in claim 4 or claim 5, a nucleic acid molecule as defined in claim 6, a vector as defined in claim 7, a cell as defined in claim 8 or a binding moiety as defined in any one of claims 9-12 together with a pharmaceutically acceptable carrier.

16. A method of identifying a binding moiety that binds a complex as claimed in claim 4 or claim 5, the method comprising contacting a candidate binding moiety with the complex and determining whether the candidate binding moiety binds the complex.