Patent Description:
We have discovered a new class of T-cells effective for treating cancer.

It is established thinking that T-cells recognise individual cancer peptides through their cognate T-cell receptor. Thus, it has been thought that a single TCR recognises a single cancer antigenic peptide typically when presented at the cell surface in the context of human leukocyte antigen (HLA) class I or class II molecule.

This new work presented herein remarkably and significantly shows some T-cells recognise different cancer antigenic peptides (of distinct sequence) using the same T-cell receptor (TCR) thus indicating that a single TCR has the ability to recognise multiple and distinct cancer antigens. This is a unique finding that goes against conventional wisdom and has significantly beneficial implications in the treatment of cancer which is thought to be a multifaceted disease.

Our work shows these T-cells can recognise multiple, distinct peptides that are derived from different cancer antigens when presented at the cell surface in the context of the same human leukocyte antigen (HLA) class I molecule. In most cases the peptides are presented at the surface of the same cancer cell, which has not been described before.

It therefore appears that some rare T-cells are capable of recognising a range of individual cancer antigenic peptides through their cognate T-cell receptor. This novel type of T-cell utilises an identical T-cell receptor (TCR) to recognise cancer cells via multiple different cancer peptides. We have termed these T cells "multipronged T-cells" which, using their cognate TCR, can recognise and attack cancer cells via more than one antigen and thereby vastly reduce the chances of immune escape by cancer cells.

In <NUM> about <NUM> million people had cancer. About <NUM> million new cases occur a year (not including skin cancer other than melanoma). It causes about <NUM> million deaths (<NUM>%) of human deaths. The most common types of cancer in males are lung cancer, prostate cancer, colorectal cancer and stomach cancer. In females, the most common types of cancer are breast cancer, colorectal cancer, lung cancer and cervical cancer. If skin cancer, other than melanoma, were included in total new cancers each year it would account for around <NUM>% of cases. In children, acute lymphoblastic leukaemia and brain tumours are most common except in Africa where non-Hodgkin lymphoma occurs more often. In <NUM>, about <NUM>,<NUM> children under <NUM> years of age were diagnosed with cancer. The risk of cancer increases significantly with age and many cancers occur more commonly in developed countries. Rates are increasing as more people live to an old age and as lifestyle changes occur in the developing world. The financial costs of cancer were estimated at $<NUM> trillion USD per year as of <NUM>. It follows that there is a need to provide better and safer ways of treating or eradicating this disease. An immunotherapy that uses the body's natural defence systems to kill aberrant tissue is acknowledged to be safer than chemical intervention but, to be effective, the immunotherapy must be able to clear the disease. Moreover, the discovery of an immunotherapy that is effective against any type of cancer or a number of cancers would be extremely beneficial as not only could it be administered to individuals suffering from many different types of cancer (i.e. it would have pan-population application) but it could also be administered to a single individual suffering from more than one type of cancer.

The T-cells and their receptors we have identified herein have the afore advantageous characteristics in that they are effective against more than one type of cancer thus safeguarding against a cancer evading the effectiveness of the immune system. Further, the production of these advantageous T cells and their receptors can be brought about by the use of the new anti-cancer peptides described herein.

Prior art references are discussed below:
<CIT>) discloses a pharmaceutical composition comprising a plurality of neoantigenic peptides and a pharmaceutically acceptable carrier, each neoantigenic peptide comprising a tumor-specific neoepitope capable of binding to an HLA protein in a subject, each tumor-specific neoepitope comprising a tumor-specific mutation present in a tumor.

Wooldridge et al. [<NUM>] discloses a comprehensive experimental and mathematical analysis to reveal that a single patient-derived autoimmune CD8+ T cell clone of pathogenic relevance in human type I diabetes recognizes >one million distinct decamer peptides in the context of a single MHC class I molecule.

Knutson et al. [<NUM>] discloses whether HER-<NUM>/neu peptide-specific CD8+ T-cell immunity could be elicited using an immunodominant HER-<NUM>/neu-derived HLA-A2 peptide alone in the absence of exogenous help.

Gagnon et al. [<NUM>] discloses the analysis of human TCR cross-reactivity to eight structurally distinct haptens that are coupled to the HLA-A2-binding Tax peptide with a lysine substitution at position <NUM> (Tax-<NUM>, LLFG[K-hapten]PVYV).

Zhao et al. [<NUM>] discloses that the interaction of TCRs with MHC peptide ligands can be highly flexible, so that many different peptides are recognized by the same TCR in the context of a single restriction element, and a quantative description of such interactions, which allows the identification of T cell epitopes and molecular mimics.

According to a first aspect of the invention there is provided an anti-cancer peptide able to elicit anti-cancer T-cells having a sequence which (i) has at least <NUM>% sequence identity to a sequence selected from the group consisting of:.

(ii) comprises a sequence which has at least <NUM>% sequence identity to a sequence selected from the group consisting of:.

According to a further aspect of the invention there is provided a vector encoding the anti-cancer peptide of the invention.

According to a further aspect of the invention there is provided a vaccine comprising the anti-cancer peptide of the invention.

According to a further aspect of the invention there is provided a pharmaceutical composition or immunogenic agent or bispecific comprising the anti-cancer peptide or the vector of the invention.

According to a further aspect of the invention there is provided a combination therapeutic for use in the treatment of cancer comprising:
the anti-cancer peptide or vaccine or vector or pharmaceutical composition or vaccine of the invention.

According to the disclosure there is described an isolated anti-cancer T-cell receptor (TCR), or a fragment thereof, that recognises a plurality of cancer peptide antigens when said antigens are presented at a cell surface by human leukocyte antigen (HLA) class I molecule and wherein said antigens are distinct from each other and are representative of more than one type of cancer.

According to a further aspect of the disclosure there is described an anti-cancer TCR or a cancer specific TCR, or a fragment thereof, that recognises a plurality of cancer antigens wherein said TCR has a complementarity-determining region selected from the group comprising or consisting of:.

In a further aspect of the disclosure said complementarity-determining region has at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>% identity with any one or more of the afore complementarity-determining regions.

In a further aspect of the disclosure said plurality of antigens are presented at the cell surface in the context of human leukocyte antigen (HLA) class I molecule and, more preferably still, said recognition occurs or is shown to occur by any one or more of, including any combination of, the following activities:.

In a further aspect of the disclosure said TCR has a complementarity-determining region selected from the group comprising or consisting of:.

In a further aspect of the disclosure said more than one types of cancer are selected from the group comprising or consisting of: nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia/lymphoma, tonsil, spleen, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, glioma, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumour, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, muscle cancer, Paget's disease, cervical cancer, ovarian, blood, colon cancer, rectal cancer, oesophagus cancer, gall bladder cancer, cholangioma cancer, head cancer, eye cancer, nasopharynx cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, mesothelioma, myeloma, multiple myeloma, ovarian, endocrine, glucagonoma, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.

In a further aspect of the disclosure said more than one types of cancer are selected from the group comprising or consisting of: pancreatic, blood, ovarian, skin, breast, cervical, prostate, bone, lung, liver, colon and kidney.

Reference herein to cancer antigens that are distinct from each other is reference to cancer antigens that are representative of different types of cancer and so reference to antigens that are distinctly different in terms of their sequence structure or the molecule, typically protein, from which they are derived.

Nevertheless, despite this difference in antigen sequence the TCR of the disclosure is able to recognise a plurality of these distinct or different cancer antigens. Those skilled in the art will appreciate, it would be extremely difficult for cancer cells to escape from T-cells that were targeting them through more than one different cancer antigen as escape would require simultaneous mutation of all targets that lowered or ablated presentation of all cognate peptides.

In a preferred embodiment of the invention said human leukocyte antigen (HLA) class I molecule is MHC class I (A, B, or C). More specifically, said HLA is HLA A2 or HLA A24 or HLA A1 or HLA A3.

MHC class I present peptides from inside the cell. For example, in the context of a cancer cell, the HLA system brings fragments or peptides of the cancer-expressed protein to the surface of the cell so that the cell can be recognised as cancerous and destroyed by the immune system. These peptides are produced from digested proteins that are broken down in the proteasomes. In general, these particular peptides are small polymers, about <NUM>-<NUM>, typically but not exclusively <NUM> or <NUM> amino acids in length. Oncogenic antigens presented by MHC class I system attract killer T-cells (also called CD8 positive- or cytotoxic T-cells) that destroy the cancer cells.

In a further aspect of the disclosure said TCR is an alpha beta (αβ) TCR.

In a further aspect of the disclosure said TCR is a soluble TCR (sTCR) and so lacks the transmembrane and, ideally also, intracellular domains.

In a further aspect of the disclosure said TCR is part of a chimeric receptor having the functionality described herein. Ideally, said TCR is fused to a TCR constant domain or a TCR signalling domain.

In the alternative, there is described a fragment of said TCR such as a monomeric part thereof, ideally a single chain form of the TCR.

In a further alternative, there is described a fragment of said TCR such as the complementarity determining region thereof.

According to a further aspect of the disclosure there is described a T-cell expressing said TCR of the disclosure, ideally, in either a soluble form or membrane compatible form i.e. having a transmembrane region and intracellular region.

According to a further aspect of the disclosure there is described a T-cell clone expressing said TCR of the disclosure, ideally, in either a soluble form and so lacks a transmembrane domain and, ideally also, an intracellular domain or a membrane compatible form i.e. having a transmembrane region and, ideally also, an intracellular domain.

According to a further aspect of the disclosure said clone is a T-cell clone CR24, GD1, GD2, VB6G4. <NUM>, CR1 or VB10 as described herein.

According to a further aspect of the disclosure said clone is CR24 which recognises multiple antigenic cancer peptides, most preferably clone CR24 recognises a plurality of said peptides selected from the group comprising or consisting of: EAAGIGILTV (SEQ ID NO: <NUM>) from Melan A (residues <NUM>-<NUM>), LLLGIGILVL (SEQ ID NO: <NUM>) from BST2 (residues <NUM>-<NUM>) and NLSALGIFST (SEQ ID NO: <NUM>) from IMP2 (residues <NUM>-<NUM>). Preferably, this recognition is in the context of HLA A2 presentation.

According to a further aspect of the disclosure said clone GD1 or GD2 recognises multiple antigenic cancer peptides, most preferably clone GD1 or GD2 recognises the following peptides: RLVDDFLLV (SEQ ID NO: <NUM>) from human telomerase reverse transcriptase (hTERT) (residues <NUM>-<NUM>) and ALKDVEERV (SEQ ID NO: <NUM>) from melanoma associated antigen C2 (MAGE C2) (residues <NUM>-<NUM>). Clone GD1 was able to kill breast, blood and melanoma cancer cell lines.

According to a further aspect of the disclosure said clones VB6G4. <NUM>, CR1 and VB10 recognise the Melan A peptide (EAAGIGILTV (SEQ ID NO: <NUM>)) but not BST2 (LLLGIGILVL (SEQ ID NO: <NUM>) or IMP2 (NLSALGIFST (SEQ ID NO: <NUM>)) peptides (neither as exogenous peptide nor from transduced protein expressed by MOLT3s). Since the CDR3 sequence of the beta TCR chain from VB6G4. <NUM> appeared in clonotyping data for all ten cancer cell lines in <FIG>, this clone responds to multiple cancer cells lines but not by recognition of the IMP2 or BST2 peptides.

According to a further aspect of the disclosure there is described a vector encoding said TCR of the disclosure.

According to a further aspect of the disclosure there is described a pharmaceutical composition or immunogenic agent or bispecific or vaccine comprising said TCR or T-cell or T-cell clone or vector of the disclosure.

According to a further aspect of the disclosure said pharmaceutical composition or immunogenic agent or bispecific or vaccine is for use in the treatment of cancer.

According to a further aspect of the disclosure there is described the TCR or T-cell or T-cell clone or vector as disclosed herein for use in the treatment of cancer.

According to an aspect of the disclosure said cancer is of any type. According to a further aspect of the disclosure, said cancer is selected from the group comprising or consisting of: nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia/lymphoma, tonsil, spleen, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, glioma, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumour, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, muscle cancer, Paget's disease, cervical cancer, ovarian, blood, colon cancer, rectal cancer, oesophagus cancer, gall bladder cancer, cholangioma cancer, head cancer, eye cancer, nasopharynx cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, mesothelioma, myeloma, multiple myeloma, ovarian, endocrine, glucagonoma, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.

According to a further aspect of the disclosure said cancer is pancreatic, blood, ovarian, skin, breast, bone, kidney, colon, cervical, liver, prostate or lung cancer.

Reference herein to a bispecific is reference to a bispecific monoclonal antibody (BsMAb, BsAb) which is an artificial protein that can simultaneously bind to two different types of antigen.

Alternatively still, said TCR may form part of a Bispecific antibody wherein said bispecific includes said TCR, for the purpose of binding to its ligand on a cancer cell, and also an immune cell activating component or ligand that binds and so activates an immune cell such as a Killer T-cell.

According to a yet further aspect of the disclosure there is described a combination therapeutic for use in the treatment of cancer comprising:.

According to a further aspect of the invention there is provided an anti-cancer peptide or peptide antigen able to elicit anti-cancer T-cells, which, ideally but not exclusively, recognises said TCR of the disclosure, or a part thereof, and which when administered to a subject primes the production of: anti-cancer T-cells that act as effector T-cells and/or T-cells that recognise a plurality of cancer antigens when said peptide antigens are presented at a cell surface by human leukocyte antigen (HLA) class I molecule and wherein said cancer antigens are distinct from each other and are representative of more than one type of cancer, which has at least <NUM>% sequence identity to a sequence selected from the group consisting of:.

According to a further aspect or in a preferred embodiment an/said anti-cancer peptide is selected from the group comprising or consisting of:.

Most ideally, said anti-cancer peptide is MTSAIGILPV. More ideally still said peptide has at least <NUM>% identity with one of the afore peptides and so includes one or two substitutions, deletions or additions.

According to a further aspect of the invention there is provided a vaccine comprising said anti-cancer peptide.

According to a further aspect of the invention there is provided a pharmaceutical composition or immunogenic agent or bispecific comprising said anti-cancer peptide.

According to a further aspect of the invention there is provided a an anti-cancer peptide, in its native form or as a vaccine, pharmaceutical composition, immunogenic agent or bispecific, for use in a method of treating cancer comprising administering said anti-cancer peptide, pharmaceutical composition, immunogenic agent or bispecific to a subject.

According to a further aspect of the invention there is provided an anti-cancer peptide for use in treating cancer.

In a preferred embodiment of the invention said cancer is selected from those disclosed herein, especially skin cancer or melanoma.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprises", or variations such as "comprises" or "comprising" is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art. Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.

An embodiment of the present invention will now be described by way of example only with reference to the following wherein:.

RMPI-<NUM> with <NUM> L-glutamine, <NUM> U/mL penicillin and <NUM>µg/mL streptomycin (termed R0) was supplemented with either <NUM>% (R5) or <NUM>% (R10) foetal calf serum. T-cell media was R10 with added <NUM> HEPES buffer, <NUM>. 5X non-essential amino acids, <NUM> sodium pyruvate, <NUM>-<NUM> IU/mL of IL-<NUM> (Aldesleukin, Proleukin, Prometheus, San Diego, CA, USA) and <NUM> ng/mL of IL-<NUM> (Peprotech, Rocky Hill, NJ, USA). D10-F12 media was made as for R10 using DMEM-F12. Unless otherwise stated tissue culture reagents were from Life Technologies (Carlsband, CA, USA). Cell lines C1R, T2 and IM9 were cultured as suspension cells in R10. Malignant melanoma cell lines Mel-<NUM>, Mel-<NUM>, FM-<NUM>, FM-<NUM>, SK-MEL-<NUM> and A-<NUM> were cultured as adherent cells in R10. Melanoma MM909. <NUM> and renal cell carcinoma RCC17 were obtained from patients treated at the CCIT and cultured as suspension cells in R10 and D10-F12 respectively. Other cancer cell lines were maintained as described by the ATCC; breast adenocarcinoma MDA-MB-<NUM> (ATCC® HTB-<NUM>™) and MCF-<NUM> (ATCC® HTB-<NUM>™); prostate adenocarcinoma LnCAP (ATCC® CRL-<NUM>™); colorectal carcinomas COLO <NUM> (ATCC® CCL-<NUM>™) and HCT116 (ATCC® CCL-<NUM>™); lung carcinoma H69 (ATCC® HTB-<NUM>™); liver hepatocellular carcinoma HepG2 (ATCC® HB-<NUM>™); cervical carcinoma MS751 (ATCC® HTB-34TM); acute lymphoblastic leukaemia MOLT3 (ATCC® CRL-<NUM>™); chronic myeloid leukaemia K562 (ATCC® CRL-<NUM>™); myeloma/plasmacytoma U266 (ATCC® TIB-<NUM>™) osteosarcomas U-<NUM> OS (ATCC® HTB-<NUM>™) Saos-<NUM> (ATCC® HTB-<NUM>™) and TK143 (ATCC® CRL-<NUM>™); HEK293T embryonic kidney cell (ATCC® CRL-<NUM>™); acute monocytic leukaemia THP-<NUM> (ATCC® TIB-<NUM>™); and kidney carcinoma A-<NUM> (ATCC® HTB-<NUM>™).

Stage IV metastatic melanoma patient MM909. <NUM> underwent rapid tumour infiltrating therapy for at the Centre for Cancer Immunotherapy (CCIT), Herlev Hospital, Copenhagen [<NUM>]. To date, this patient has experienced lasting remission. Chromium release cytotoxicity assay was used to assess reactivity towards cancer cell lines: autologous melanoma (MM909. <NUM>), MDA-MB-<NUM>, MCF-<NUM>, LnCAP and RCC17. Cell lines (<NUM> ×<NUM><NUM> cells) were labelled for <NUM> with <NUM>µCi of sodium chromate (51Cr) (Perkin Elmer, Waltham, MA, USA), leached for <NUM>, then cultured with TILs overnight. A <NUM>:<NUM> TIL to target cell (<NUM> cells per well) ratio was used. After overnight incubation supernatants were harvested, mixed with scintillant and read using a microbeta counter and specific lysis calculated [<NUM>]. Further cancer cell lines were tested using a TNF processing inhibitor-<NUM> (TAPI-<NUM>) assay [<NUM>]; TILs were harvested from culture washed with RO and rested overnight in R5 media. On the day of the activation assay, cells were harvested then counted and <NUM>,<NUM> incubated with <NUM> TAPI-<NUM> (Sigma-Aldrich) anti-TNF-PE-Vio770TM (clone cA2, Miltenyi Biotech) and anti-CD107a-PE (clone H4A3, BD Biosciences) antibodies in wells of a <NUM> U well plate. Cancer cell lines were added to give a TIL to target cell ratio of <NUM>:<NUM>. In addition to the cancer cell lines above the following were also used; COLO <NUM>, H69, HepG2, MS751 and Saos-<NUM>. The cells were incubated for <NUM>-<NUM> at <NUM> then stained at RT for <NUM> with <NUM>µL of LIVE/DEAD fixable dead cell stain ViVid (Life Technologies) that had been diluted <NUM>:<NUM> using PBS. Antibodies to detect surface markers were added directly to each sample without washing; anti-CD8-APC (clone BW135/<NUM>, Miltenyi Biotech) and anti-CD3-peridinin chlorophyll (PerCP) (clone BW264/<NUM>, Miltenyi Biotech). Data was acquired on a BD FACS Canto II (BD Biosciences) and analysed with FlowJo software (TreeStar Inc. , Ashland, OR, USA). Activated TILs (CD107a+ and/or TNF+) were sorted on a BD FACS Aria (BD Biosciences, San Jose, CA, USA) and used for next generation sequencing of the T-cell receptor (TCR) chains as previously described [<NUM>].

T-cell clones of unknown peptide specificity (termed orphan clones) were generated by culturing <NUM> cells/well in of <NUM> U well plates in T-cell media with <NUM>,<NUM> irradiated (<NUM>-<NUM> cGy) allogenic peripheral blood mononuclear cells (PBMCs) from three donors and <NUM>-<NUM>µg/mL of phytohaemagglutinin (PHA). PBMCs were separated from blood by standard density gradient centrifugation. If needed, red blood cells were lysed using ammonium chloride solution. Blood was procured as 'buffy coats' from the Welsh Blood Service (Pontyclun, Wales, UK). All human tissue was obtained and handled in accordance with Cardiff University's guidelines to comply with the UK Human Tissue Act <NUM>. T-cell clones were screened against autologous melanoma (MM909. <NUM>) and in some case cancer cell lines of different tissue origin. Clones of interest were grown to large number in T25 flasks using the PBMC and PHA method as above. Combinatorial peptide library (CPL) and cancer antigen database screening was performed to find peptides recognized by orphan clones. Combinatorial peptide libraries were synthesized and used as previously described [<NUM>,<NUM>]. Briefly, long-term storage was at -<NUM> as <NUM> DMSO stocks with <NUM> working dilutions made in sealable (silicone sealing mat, AxyGen® AxyMatTM, Corning, New York, US) <NUM> deep round-well plates (AxyGen®, Corning) with R0 (as for R10 but with no serum), which were stored at <NUM>, then vortexed (MixMate®, Eppendorf®, Hamburg, Germany) at <NUM> rpm for <NUM>, then centrifuged (<NUM>, <NUM> mins) before use. Each sub-library was used at a concentration of <NUM> with respect to total peptide concentration. The CPL data was run via a database, which contains the amino acid sequences of proteins expressed by cancers (manuscript in preparation). The cancer antigen database will be available online as part of the PI CPL (peptide identification combinatorial peptide library) webtool hosted by Warwick University's Systems Biology Centre (http://wsbc. uk/wsbcToolsWebpage/user_cases. Candidate peptides from the database were automatically ranked based on their likelihood of being recognised by a clone, with the top <NUM> being tested in peptide titration assays.

HLA A2+ Melanomas, MM909. <NUM> (autologous), Mel-<NUM>, Mel-<NUM>, and HLA A2+ non-melanomas, CIR-HLAA2, MDA-MB-<NUM>, Saos-<NUM>, U2OS, A498, TK143, HEK293T, COLO <NUM>, HCT116, HeLa, HepG2 and THP1 were used as target cells in a TAPI-<NUM> assay, which is described above. HLA A2neg melanomas FM-<NUM> and FM-<NUM>, and wild-type C1Rs (HLA A2neg) were used as controls.

CR24 was rested overnight in R0 then <NUM>,<NUM> used per well of the decamer CPL screen (details above). The peptide length preference of CR24 had previously been established using sizing scan assays [<NUM>] (data not shown). T2 cells (<NUM>,<NUM> per well) were used as antigen presenting cells. The assay was performed in R5 and supernatants harvested for MIP-1β enzyme linked immunosorbent assay (ELISA) according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA).

CR24 was cultured overnight in R5, then <NUM>,<NUM> used per well of a <NUM> U well plate with decreasing concentrations of peptides. After overnight incubation supernatants were used MIP-1β ELISA according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). For tetramer analysis CR24 (<NUM>,<NUM>-<NUM>,<NUM> per sample) was stained in <NUM> polypropylene tubes suitable for flow cytometry. Cells were treated in <NUM>µL of FACS buffer (PBS + <NUM>% FBS) with <NUM> Dasatinib (a protein kinase inhibitor) for <NUM> at <NUM> and phycoerythrin (PE) conjugated tetramer (<NUM>µg) added directly to the sample before being moved to ice for a further <NUM> [<NUM>]. Tetramer was washed with <NUM> of FACS buffer (<NUM>, <NUM>) then labelled with <NUM>µg (<NUM>µg/mL) of mouse anti-PE unconjugated antibody (clone PE001, BioLegend, London, UK) for a further <NUM> on ice [<NUM>]. To test if CR24 could recognise endogenously express antigen MOLT3 cells were used to express various proteins. Codon optimised full-length human HLA A2 (IMGT/HLA Acc No: HLA00005), MLANA (Melan A) (UniProtKB Q16655), BST2 (UniProtKB Q10589), IGF2BP2 (IMP2) (UniProtKB Q9Y6M1), COL6A2 (α2 subunit of collagen type VI) (UniProtKB P12110) and Zika virus (Rio-U1) ancC (GenBank KU926309. <NUM>) genes were synthesized (Genewiz, South Plainfield, NJ, USA) and cloned into the 3rd generation lentiviral transfer vector pELNS (kindly provided by Dr. James Riley, University of Pennsylvania, PA, USA). The pELNS vector contains a rat CD2 (rCD2) marker gene separated from the gene of interest by a self-cleaving 2A sequence. Lentiviral particle production, calcium chloride transfection and rCD2-based purification of cells were performed as previously described [<NUM>].

To demonstrate that CR24 can target autologous melanoma through multiple antigens, guide RNAs to ablate Melan A expression using CRISPR/Cas9 were designed using the cripsr. edu webtool, applied and the Melan A gene sequenced to confirm disruption (data not shown). Intracellular staining for Melan A was performed using Cytofix/Cytoperm™ reagents according to manufacturer's instructions (BD Biosciences). A primary unconjugated rabbit anti-Melan A antibody (clone EP1422Y) (Abcam, Cambridge, UK) was used with a secondary PE conjugated goat anti-rabbit antibody. Wild type and Melan A KO MM909. <NUM> melanomas were used TAPI-<NUM> assays, as described above, with both TILs and CR24.

To generate T-cell peptide lines, CD8 T-cells were purified from the PBMCs of HLA A2+ donors using CD8 microbeads according to the manufacturer's instructions (Miltenyi Biotech, Bergisch Gladbach, Germany). Purified CD8 cells (<NUM> ×<NUM><NUM>) were co-incubated with autologous CD8neg cells (<NUM>-<NUM> ×<NUM><NUM>) in <NUM> well plates in <NUM> of T-cell media, but with no IL-<NUM>. <NUM> of each peptide was used. The cultures had <NUM>% of the media changed thrice weekly. Tetramer staining was performed as above, using <NUM>,<NUM> cells per tube. Each T-cell line was used in an IFNγ enzyme linked immunosorbent spot (ELISpot) assay with cell lines MDA-MB-<NUM>, melanoma MM909. <NUM> and Saos-<NUM>. <NUM>,<NUM> T-cells and <NUM>,<NUM> cancer cells were used per well. Incubation was performed for <NUM>, and the assay developed according the manufacturer's instructions (Mabtech, Nacka Strand, Sweden).

CPL assay of CR24 was performed as described above. Candidate peptide agonists were designed using the CR24 CPL and an online algorithm (http://wsbc. uk/wsbcToolsWebpage/user_cases. Priming of CD8 T-cells from healthy donors, tetramer staining and chromium release cytotoxicity assays were performed as described above.

TAPI-<NUM> and activation assays (ELISA) were performed for VB6G4. <NUM>, CR1 and VB10, as described above for CR24. The data was summarised in tabular from.

Clones GD1 and GD2 were grown from the peripheral blood of different HLA A2+ healthy donors. The clones were used in overnight activation assays with decreasing concentrations of respective peptides, and supernatants used for MIP-1β ELISA, as described above. An overnight activation was performed with GD1 and target cells; K562, K562 HLA A2, CIR, CIR HLA A2, HEK 293T, MCF-<NUM>, COLO <NUM>, U266, HCT116, Mel-<NUM>, Mel-<NUM>, SK-MEL-<NUM>, A375, IM9 and LnCAP. Supernatants were harvested and used for MIP-1β ELISA. A chromium release cytotoxicity assay was performed, as above, with cell lines MCF-<NUM>, U266 and Mel-<NUM>. Incubation times of <NUM> and overnight, with varying T-cell to target cell ratios were used.

Claim 1:
An anti-cancer peptide able to elicit anti-cancer T-cells having a sequence which (i) has at least <NUM>% sequence identity to a sequence selected from the group consisting of:
MTSAIGILPV (SEQ ID NO: <NUM>)
ITSAIGILPV (SEQ ID NO: <NUM>)
ITSAIGVLPV (SEQ ID NO: <NUM>)
MTSAIGVLPV (SEQ ID NO: <NUM>)
LTSAIGVLPV (SEQ ID NO: <NUM>)
ITSGIGVLPV (SEQ ID NO: <NUM>)
QTSAIGILPV (SEQ ID NO: <NUM>) and
ITSAIGVLFV (SEQ ID NO: <NUM>); or
(ii) comprises a sequence which has at least <NUM>% sequence identity to a sequence selected from the group consisting of:
MTSAIGILPV (SEQ ID NO: <NUM>)
ITSAIGILPV (SEQ ID NO: <NUM>)
ITSAIGVLPV (SEQ ID NO: <NUM>)
MTSAIGVLPV (SEQ ID NO: <NUM>)
LTSAIGVLPV (SEQ ID NO: <NUM>)
ITSGIGVLPV (SEQ ID NO: <NUM>)
QTSAIGILPV (SEQ ID NO: <NUM>) and
ITSAIGVLFV (SEQ ID NO: <NUM>).