WO2021046616A1 - Methods of identifying mhc-bound peptides - Google Patents

Abstract

The present invention relates to a method for characterising major histocompatibility complex-bound peptides via mass spectrometry, wherein sample peptides are labelled with isobaric tags and analysed together with non-sample carrier peptides.

Description

METHODS OF IDENTIFYING MHC-BOUND PEPTIDES

RELATED APPLICATION DATA

This application claims priority from Australian Patent Application No 2019903421 filed on 13 September 2019 and entitled “Methods of Identifying MHC- Bound Peptides. The entire contents of this application is hereby incorporated by reference.

FTETD OF THE INVENTION

The present disclosure relates to methods of identifying one or more major histocompatibility complex (MHC)-bound peptides in a sample. The present disclosure also relates to methods of validating or identifying biomarkers, diagnosis, and therapy.

BACKGROUND OF THE INVENTION

Peptides presented by major histocompatibility complex (MHC) class I and II molecules, such as human leukocyte antigens (HLA), form an important component of the adaptive immune response against viruses, bacteria and tumours. Identifying MHC- bound peptides is therefore crucial to understand the specificity of T cell responses in cancer and infectious disease.

Due to sample limitations, it is often difficult or impossible to confirm epitope presentation on clinical biopsy material. To develop effective and robust personalised medicines, target epitopes need to be directly biochemically identified or validated in patient samples.

There is therefore a need for new methods which can be used to accurately detect and identify MHC-bound peptides from limited amounts of starting material.

SUMMARY OF THE INVENTION

The present inventors have surprisingly developed a mass spectrometry-based platform for identification of HLA-bound peptides isolated from small samples. The platform uses carrier peptides to enable analysis of MHC-bound peptides isolated from low numbers of mammalian cells. The platform advantageously allows for identification of HLA-bound peptides from patient derived samples, enabling detection of low- abundance peptides by mass spectrometry which would otherwise be difficult or impossible to detect. Thus, in an aspect, the present disclosure provides a method of identifying one or more major histocompatibility complex (MHC)-bound peptides in a sample, the method comprising: a) obtaining sample peptides isolated from MHC molecules in the sample, b) labelling the sample peptides with an isobaric label; c) mixing the labelled sample peptides with labelled carrier peptides to form a mixture, wherein the carrier peptides are labelled with a different isobaric label to the sample peptides; and d) performing mass spectrometry on the mixture to identify one or more of the sample peptides.

Advantageously, the method benefits from the additive mass spectrometry (MS) signal of isobaric tagged peptides from the sample and carrier peptides when they are combined in a mixture. In this regard, use of carrier peptides provides a sufficient amount of ions in mass spectrometer for accurate sequence identification of low abundance sample peptides, such as peptides isolated from MHC molecules. Sample-specific information can then be de-convoluted using reporter ions from the isobaric labels.

In some embodiments, the one or more sample peptides identified are present in the mixture in an amount that would be insufficient for identification by mass spectrometry in the absence of the carrier peptides. In some embodiments, the one or more sample peptides are present in an amount that would be insufficient for identification by mass spectrometry using a high resolution mass spectrometer with either an orbital trap or time of flight mass analyser. In some embodiments, the one or more sample peptides are present in an amount that would be insufficient for identification by mass spectrometry using an Orbitrap Tribrid mass spectrometer (Thermo Scientific) in the absence of the carrier peptides. For example, the method of the disclosure enables detection of MHC -bound peptides isolated from samples containing only a single cell, whereas current targeted MS methods for analysing MHC -bound peptides typically require at least 10⁷ cells. In some embodiments, the sample peptides are isolated from MHC molecules by

(i) first isolating MHC-peptide complexes from the sample and (ii) subsequently separating the sample peptides from the MHC molecules. Alternatively, the sample peptides may be isolated directly from cells (e.g., stripped from the cell surface) in the sample without first isolating MHC-peptide complexes. For example, in some embodiments, the sample peptides are eluted directly from cells in the sample. The sample peptides can be eluted directly from the cells in the sample using a mild acid elution, for example.

In some embodiments, the MHC-peptide complexes are isolated from the sample by immunoprecipitation from a lysate of cells or tissue. Advantageously, compared to other methods, immunoprecipitation optimizes the chance of successful epitope identification due to the additional specificity of an immunoaffmity chromatography step and resulting simplification of the range of cellular peptides isolated. In some embodiments, the MHC molecules in the sample comprise an affinity tag and the MHC-peptide complexes are isolated from the sample by affinity chromatography. In some embodiments, the affinity tag is a myc, avi, FLAG, His, SBP, or Xpress tag. In some embodiments, the MHC-peptide complexes are immunoprecipitated using an anti-MHC antibody that is bound to a solid substrate. Any anti-MHC antibody may be suitable for the methods described herein. For example, monoclonal antibodies with specificity toward whole classes of MHC molecules, families of MHC molecules, individual alleles of MHC molecules, and even subsets of an individual allotype are all commercially available.

In some embodiments, the anti-MHC antibody is an anti-MHC class I antibody or an anti-MHC class II antibody. In some embodiments, the anti-MHC antibody is a pan anti-MHC class I antibody or a pan anti-MHC class II antibody. In some embodiments, the anti-MHC antibody is a MHC serotype-specific antibody. In some embodiments, the anti-MHC antibody is L243, LB3.1, SPV-L3, IVD12, IVA12, B721, MA2.1, BB7.2, ME1, W632, or DT9.

In one embodiment, the MHC serotype-specific antibody is an anti-HLA-A2 antibody, such as BB7.2.

In some embodiments, the anti-MHC antibody is noncovalently bound to the solid substrate. In some embodiments, the anti-MHC antibody is noncovalently bound to the solid substrate via protein A. Advantageously, by not covalently linking the antibody to the solid substrate, when the sample peptides are eluted from the MHC-peptide complexes, antibody is also eluted from the solid substrate. This may prevent loss of the sample peptides in subsequent purification steps. In alternative embodiments, the anti- MHC antibody is covalently linked to the solid support. For example, the antibody may be chemically cross-linked to a protein A resin.

In some embodiments, the solid substrate is magnetic. In some embodiments, the solid substrate is non-magnetic. In some embodiments, the solid substrate comprises agarose or sepharose. In some embodiments, the solid substrate comprises protein A ligands.

In some embodiments, less than 10 mL, less than 5 mL, less than 2 mL, less than 1 mL, less than 500 pL, or less than 200 pL of solid substrate is used to immunoprecipitate the MHC-peptide complexes. In some embodiments, 50 to 200 pL of solid substrate is used. In some embodiments, the immunoprecipitation is performed in a container having a volume of no more than 5 mL, no more than 3 mL, or no more than 2 mL. For instance, in some embodiments, the container is a microcentrifuge tube. In some embodiments, the container comprises a filter having a pore size which is sufficiently small to prevent flow of the solid substrate through the filter, but which allows flow of proteins and peptides through the filter. In some embodiments, the pore size is in the range of 100 nm to 100 pm. For example, suitable filters are present in MobiSpin™ columns which can be placed inside a 1.5 mL or 2 mL microcentrifuge tube and contain a 10 pm pore filter.

In some embodiments, the immunoprecipitation is performed by contacting the anti-MHC antibody bound to the solid substrate with the lysate for a period of time in the range of 10 min to 24 h. In some embodiments, the immunoprecipitation is performed by contacting the anti-MHC antibody bound to the solid substrate with the lysate at a temperature in the range of 0°C to 25°C. In some embodiments, the immunoprecipitation is performed by contacting the anti-MHC antibody bound to the solid substrate with the lysate at a temperature in the range of 0°C to 10°C.

In some embodiments, the immunoprecipitated MHC -peptide complexes are washed with a wash buffer prior to elution from the solid substrate. In some embodiments, the wash buffer comprises phosphate buffered saline (PBS) or Tris buffered saline (TBS). In some embodiments, the wash buffer has a volume in the range of 100 pL to 2 mL. In some embodiments, the wash buffer has a volume in the range of 250 pL to 750 pL.

In some embodiments, washing is performed by mixing the immunoprecipitated MHC-peptide complexes and solid substrate with the wash buffer and subsequently centrifuging the mixture through a filter having a pore size which is sufficiently small to prevent flow of the solid substrate through the filter, but which allows flow of proteins and peptides through the filter. In some embodiments, two or three washes are performed.

In some embodiments, the immunoprecipitated MHC-peptide complexes are eluted from the solid substrate using an acidic solution, thereby producing an eluate comprising the sample peptides and MHC molecules. In some embodiments, the acidic solution has a pH in the range of about 2 to about 6. In some embodiments, the acidic solution has a pH in the range of about 2 to about 4. In some embodiments, the acidic solution has a pH of less than about 4. In some embodiments, the acidic solution comprises acetic acid, trifluoroacetic acid, or formic acid. In some embodiments, the acidic solution comprises 0.1% to 20% acetic acid, trifluoroacetic acid, or formic acid. In some embodiments, the acidic solution comprises 5 to 15% acetic acid.

In some embodiments, the immunoprecipitated MHC-peptide complexes are eluted from the solid substrate using a basic solution. In some embodiments, the basic solution has a pH of at least about 9. In some embodiments, the basic solution has a pH of at least about 10. In some embodiments, the anti-MHC antibody is eluted from the solid substrate by the acidic solution, thereby producing an eluate comprising the sample peptides, the MHC molecules, and the anti-MHC antibody. For example, if the anti-MHC antibody is not covalently bound to the solid substrate (e.g., via non-covalent interaction with protein A), then the antibody will also elute with the MHC-peptide complexes when the solid substrate is contacted with an acidic solution. Without wishing to be bound by theory, if the antibody is also present in the eluate then it may reduce the amount of sample peptide loss in subsequent purification steps due to contact-surface adsorption with filters, tubes or other equipment. In some embodiments, the method further comprises heating the eluate to a temperature in the range of 40°C to 100°C. In some embodiments, the method further comprises heating the eluate to a temperature in the range of 60°C to 80°C. Advantageously, such heating steps may promote denaturation and dissociation of the MHC-peptide complexes, thereby making it easier to separate the sample peptides from the MHC molecules in subsequent steps.

In some embodiments, the sample peptides are separated from the MHC molecules by ultrafiltration. In other embodiments, the sample peptides are separated from the MHC molecules by reversed-phase chromatography (e.g., reversed-phase high performance liquid chromatography; RP-HPLC) or by precipitation (e.g., acid and/or organic precipitation).

In some embodiments, ultrafiltration is performed using a filter having a molecular weight cut off which permits flow of the sample peptides through the filter but not MHC and antibody polypeptides. Thus, when immunoprecipitation is used the filter used for the ultrafiltration step is different to the filter (if used) for the immunoprecipitation (e.g., wash and elution) step. In some embodiments, ultrafiltration is performed using a filter having a molecular weight cut off in the range of 1 kDa to 10 kDa. In some embodiments, ultrafiltration is performed using a filter having a molecular weight cut off in the range of 2 kDa to 7 kDa. In some embodiments, ultrafiltration is performed using a filter having a molecular weight cut off of 5 kDa. In some embodiments, the above processes for isolating the sample peptides are at least partially automated. For example, in some embodiments, a liquid handling robot is used.

In some embodiments, the isobaric labels are tandem mass tag (TMT) or isobaric tags for absolute and relative quantification (iTRAQ) labels. Alternative isobaric labels include mass differential tags for absolute and relative quantification, dimethyl labelling (e.g., DiLeu), or deuterium isobaric amine reactive tags (DiART).

In some embodiments, the method comprises identifying one or more MHC- bound peptides in multiple samples, wherein sample peptides from each sample are labelled with different isobaric labels, and wherein the mixture comprises sample peptides from each of the samples and the carrier peptides. Thus, advantageously, peptides from multiple different samples can be multiplexed in a single mixture. This improves sample throughput and permits relative quantification of peptides between the samples. In some embodiments, the method comprises identifying one or more MHC -bound peptides from two, or three, or four, or five, or six, or seven, or eight, or nine, or ten different samples. In some embodiments, the method comprises identifying one or more MHC -bound peptides from eleven, or twelve, or thirteen, or fourteen, or fifteen different samples. For example, TMT1 lplex labels may be used to simultaneously analyse peptides from up to 10 different samples (with one TMT channel for the carrier peptides). Similarly, TMT16plex labels may be used to simultaneously analyse peptides from up to 15 different samples (with one TMT channel for the carrier peptides).

In some embodiments, the carrier peptides are: a) isolated from MHC molecules in a patient derived xenograft (PDX) sample; b) isolated from MHC molecules in cultured cells; c) synthetic peptides which are predicted to bind to MHC molecules; or d) synthetic peptides known to bind MHC molecules.

In some embodiments, the carrier peptides are: a) isolated from MHC molecules purified from cultured cells; b) isolated from MHC molecules purified from tissues or organs c) isolated from MHC molecules in a patient derived xenograft (PDX) sample; d) synthetic peptides derived from antigens of interest which are predicted to bind to MHC molecules relevant to the sample being analysed; e) synthetic peptides derived from antigens of interest known to bind MHC molecules relevant to the sample being analysed; f) a synthetic peptide library; or g) a combination of the above sources.

As will be appreciated by those skilled in the art, the choice of suitable carrier peptides will depend on the overall purpose of the method, as described herein.

In some embodiments, the sample peptides and the carrier peptides are obtained from the same subject. In some examples, the carrier peptides are isolated from peripheral blood mononuclear cells (PBMCs).

In some embodiments, the cultured cells are MHC -matched to the sample. In some embodiments, the cultured cells are MHC-matched to the subject from which the sample is obtained. It is advantageous in certain embodiments when the carrier peptides are derived from a source that is closely related to the sample being analysed. Thus, in some embodiments, the sample is obtained from a subject and the cultured cells are cells obtained from the subject and cultured ex vivo.

In some embodiments, the sample is obtained from a subject and the synthetic peptides which are predicted to bind to MHC are derived from proteomics data or translated whole genome sequencing (WGS), whole exome sequencing (WES), or RNA sequencing (RNAseq) data obtained from DNA or RNA from the subject. For example, such carrier peptides could be used when attempting to identify new antigens (e.g., neoantigens) or to characterize as many different sample peptides as possible. Advantageously, such methods provide carrier peptides which are directly relevant to the sample being analysed but without the labour intensive processes required for obtaining such peptides from biological sources.

Suitable carrier peptides may be predicted to bind MHC using any MHC-binding prediction algorithm known in the art, such as those freely available in the Immune Epitope Database (IEDB; www.iedb.org). In some embodiments, the carrier peptides are predicted to bind to MHC using any one or more of the following algorithms: smm, smmpmbec, ann(NetMHC3.4), NetMHC4, Pickpocket, consensus, NetMHCpan2.8, NetMHCpan3, NetMHCpan4, NetMHCcons, mhcflurry, mhcflurry pan, MixMHCpred, NetMHCIIpan , nn align, smm align, comblib, and tepitope.

In some embodiments, the sample is a biopsy and the DNA or RNA is from cells in the biopsy. In this regard, generating synthetic carrier peptides based on sequencing data from DNA or RNA obtained directly from the biopsy may enhance the likelihood of identifying neo antigens in the sample.

In some embodiments, the sample is obtained from a subject and the carrier peptides are isolated from soluble peptide-MHC complexes in serum of the subject. As will be appreciated by those skilled in the art, while the majority of peptide-MHC complexes are expressed on the cell surface, a small amount of peptide-MHC complexes are soluble and found in bodily fluids. Therefore, these soluble MHC-peptide complexes may provide a source of carrier peptides which are relevant to a sample obtained from the subject. In some embodiments, the sample is a biopsy sample, a tissue sample, or cultured cell sample.

In some embodiments, the biopsy sample is a tissue biopsy sample or a liquid biopsy sample. In some embodiments, the biopsy sample is a tumour biopsy. In some embodiments, the biopsy sample is a biopsy of infected tissue (e.g., viral, bacterial, or fungal lesion) or a biopsy of tissue affected by an autoimmune disease or allergy. In some embodiments, the liquid biopsy sample is a blood sample. In some embodiments, the tumour biopsy is a skin, breast, lung, colon, prostate, stomach, liver, cervix, thyroid, bladder, kidney, or pancreas tumour biopsy. Other types of tumour biopsies are also suitable for the methods of the disclosure, provided thatMHC- bound peptides can be isolated therefrom. In some embodiments, the tumour biopsy is a melanoma biopsy.

As described herein, the methods of the disclosure can advantageously be used to identify MHC -bound peptides that are present in low amounts and which would otherwise be difficult to identify by mass spectrometry. For example, MHC-bound peptides that are isolated from small biopsy samples. In this regard, due to their relatively low copy number and difficulty of isolation, MHC-bound peptides are particularly challenging for accurate detection and identification by mass spectrometry.

In some embodiments, the sample contains no more than about 50 mg, no more than about 20 mg, no more than about 10 mg, no more than about 5 mg, or no more than about 2 mg of tissue. In some embodiments, the sample contains no more than about 10⁶, no more than about 10⁵, no more than about 10⁴, no more than about 5000, no more than about 2000 cells, no more than about 1000, no more than about 500, no more than about 200, or no more than about 100. In some embodiments, the sample contains no more than about 100 cells, no more than about 50 cells, no more than about 25 cells, or no more than about 10 cells. In some embodiments, the sample can contain only a single cell. In this regard, the present inventors have surprisingly found that the methods of the disclosure can be used to accurately identify MHC-bound peptides isolated from samples containing only a single cell. For example, as described herein the inventors were able to successfully identify 348 HLA class I bound peptides isolated from a sample containing a single cell. This equated to approximately 50% of the number of peptides identified from a sample containing 10⁷ cells. In some embodiments, the methods of the disclosure enable identification of at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the number of peptides identified from a sample containing 10⁷ cells. The skilled person will appreciate that the percentage of peptides identified correlates with the sample size with a greater percentage of the MHC ligandome (i.e., all the MHC bound peptides present in the sample) more likely to be identified from a larger sample size. Importantly however, the methods of the disclosure are more sensitive than current methods for identifying MHC-bound peptides, identifying a significant proportion of the MHC -ligandome with a smaller sample size. The methods of the disclosure can be used to identify a single peptide of interest in a sample or can be used to identify multiple different peptides. In some embodiments, at least about 10 peptides are identified. In some embodiments, at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 800, or at least about 1000 peptides are identified.

In some embodiments, the sample is a mammalian sample. In one embodiment, the sample is a human sample. In some embodiments, the method further comprises the step of isolating the sample peptides from MHC molecules in the sample. In some embodiments, the sample peptides are isolated using any of the methods described herein.

As described herein, the methods of the disclosure may be used, for example, to identify MHC -bound peptides which are known antigens (e.g., a disease biomarker) and/or may be used to identify neoantigens created by disease-specific mutations (e.g., tumour mutations). Thus, in one embodiment, the one or more sample peptides comprises a neoantigen. In some embodiments, the one or more sample peptides comprises a known disease-associated antigen.

Suitable methods for identifying peptides from mass spectrometry data are known in the art. In some embodiments, identification of the one or more sample peptides comprises comparing the mass spectrometry data with a database of known protein sequences. For instance, comparing the mass spectrometry data with the database may involve searching a fragmentation pattern observed in the mass spectrometry data against theoretical fragmentation patterns of the known protein or peptide sequences to determine the identity of a sample peptide. Suitable software for performing such comparisons are commercially available (e.g., PEAKS DB, http://www.bioinfor.com/peaksdb; Maxquant, https://www.maxquant.org; and Proteome Discoverer, https://www.thermofisher.com).

In some embodiments, the database of known protein sequences comprises amino acid sequences of proteins from a reference proteome. In some embodiments, the reference proteome is a human reference proteome. In some embodiments, the reference proteome is a UniProt reference proteome, such as the UniProt human reference proteome (proteome ID: UP000005640).

In some embodiments, the database of known protein sequences comprises amino acid sequences from proteomics data or translated WGS, WES, or RNAseq data obtained from DNA or RNA from one or more subjects. Such a database may be advantageous in embodiments where neoantigens are to be identified, as the mutated sequence may not be present in a standard reference proteome. In some embodiments, the sample is a biopsy from the subject and the DNA or RNA is from cells in the biopsy.

In some embodiments, identification of the one or more sample peptides comprises de novo sequencing the sample peptides from the mass spectrometry data.

In some embodiments, the method further comprises quantifying the identified one or more sample peptides. In one embodiment, a relative quantification may be obtained. For example, the amount of a peptide present in a sample may be compared to the amount of that same peptide present in the carrier peptides, or within another sample. Such quantification is made possible by the use of isobaric labels, whereby the intensity of the isobaric label reporter ion in a mass spectrum is correlated with the amount of peptide present.

In one embodiment, the mass spectrometry comprises tandem mass spectrometry (MS/MS or “MS2”). In one embodiment, the mass spectrometry comprises three rounds of mass spectrometry analysis (MS3 or “MS/MS/MS”). For example, selected product ions generated in the second round of mass spectrometry analysis (MS2) can be further fragmented to produce another group of product ions for analysis in the third round of mass spectrometry analysis (MS3). This may be advantageous for accurately quantifying, and determining the presence of, sample peptides using reporter ion peaks from the isobaric labels. Thus, in some embodiments, the identified sample peptides are quantified based on the intensity of reporter ion peaks of the isobaric labels in the MS3 spectra. In some embodiments, MS3 is performed using synchronous precursor selection (SPS).

In one embodiment, the mass spectrometry comprises ion mobility-mass spectrometry (IM-MS).

In some embodiments, the mass spectrometry is preceded by liquid chromatography. Thus, LC-MS/MS or LC-MS3are suitable for the methods described herein.

In some embodiments, the mass spectrometry comprises use of an Orbitrap mass analyser. In some embodiments, the he mass spectrometry comprises use of a time of flight (TOF) mass analyser.

A person skilled in the art would appreciate that different mass spectrometers could be used to perform the methods of the invention, including hybrid instruments that combine different mass analysers to manipulate ions and derive peptide sequence and isobaric tag information.

In another aspect, the present disclosure provides a method of validating a candidate peptide antigen or biomarker, the method comprising: a) obtaining a sample from a subject; b) identifying one or more MHC-bound peptides in the sample according to the methods described herein; and c) verifying if the candidate peptide antigen or biomarker is present in the peptide sample. In another aspect, the present disclosure provides a method of identifying a candidate peptide antigen or biomarker, the method comprising: a) obtaining a sample from a subject; b) identifying one or more MHC-bound peptides in the sample according to the methods described herein.

In another aspect, the present disclosure provides a method of detecting the presence or absence of a peptide antigen or biomarker in a sample, the method comprising: a) obtaining a sample from a subject; b) identifying one or more MHC-bound peptides in the sample according to the methods described herein; and b) determining if the peptide antigen or biomarker is present or absent in the peptide sample.

In another aspect, the present disclosure provides a method of diagnosis, prognosis and/or evaluation of treatment efficacy in a subject in need thereof, the method comprising detecting the presence or absence of a peptide antigen or biomarker according to the method described herein. For example, the methods of the disclosure may be used to identify whether a known disease-associated antigen is present within a sample from a subject, which will help determine whether the subject has the disease or to determine if a particular therapy is efficacious. Thus, the carrier peptides may comprise the known disease-associated antigen to determine whether the antigen is present in the sample.

In another aspect, the present disclosure provides a method of selecting a therapy for treatment of a disease or condition in a subject, the method comprising: a) obtaining a sample from the subject; and b) identifying one or more MHC-bound peptides in the sample according to the methods described herein, wherein the therapy is selected based on the one or more sample peptides identified. For example, the therapy may involve targeting (e.g., using immune cells) or administering the identified sample peptides. In this regard, the methods of the disclosure are particularly useful in personalised medicine, especially immunotherapy. Thus, in some embodiments, the therapy is an immunotherapy.

In some embodiments, the immunotherapy comprises: a) a vaccine comprising at least one of the sample peptides identified; b) an antibody which binds to at least one of the sample peptides identified; or c) an immune effector cell which targets at least one of the sample peptides identified.

In some embodiments, the vaccine is a cancer vaccine. In some embodiments, the antibody binds to the sample peptide in an MHC -restricted manner.

In some embodiments, the immune effector cell is a T cell or a natural killer (NK) cell. In some embodiments, the immune effector cell comprises a chimeric antigen receptor (CAR). Thus, in some embodiments, the immune effector cell is a CAR-T cell. In some embodiments, the immune effector cell is a regulatory T cell. In some embodiments, the immune effector cell is a CD8+ T cell. In some embodiments, the immune effector cell is a CD4+ T cell.

In another aspect, the present disclosure provides a method of treatment of a disease or condition in a subject, the method comprising: a) selecting a therapy according to the method described herein, and b) administering to the subject the selected therapy.

In some embodiments, the disease or condition is a cancer, an autoimmune disease, an inflammatory disease, or an infection. In some embodiments, the inflammatory disease is an immune mediated inflammatory disease (IMIDs) or an allergic disease. In some embodiments, the autoimmune disease is rheumatoid arthritis.

In another aspect, the present disclosure provides a method of isolating peptides from MHC molecules in a sample for identification by mass spectrometry, wherein the sample contains no more than 10⁶ cells or no more than 50 mg of tissue, the method comprising: a) obtaining a lysate of cells or tissue in the sample; b) immunoprecipitating MHC-peptide complexes from the lysate with an anti- MHC antibody; and c) separating the peptides from the MHC molecules using ultrafiltration, thereby isolating the peptides from the MHC in the sample.

Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise. For instance, as the skilled person would understand, examples of sample peptide isolation methods outlined above for the methods of identifying one or more MHC -bound peptides equally apply to the methods of isolating peptides from MHC molecules provided herein.

The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS Figure 1 - An exemplary method for identification of MHC -bound peptides from samples using carrier peptides is shown. The samples are lysed and the MHC molecules affinity purified using antibodies coupled to either magnetic beads or agarose beads coupled with protein A (Figure 1 A). The peptides are then separated from HLA molecules and antibodies using ultrafiltration (Figure 1A). The eluted MHC peptides from the sample and the carrier peptides are neutralized and then tagged with a specific TMT label of interest (Figure IB). In this example, eight MHC -bound peptide samples and two carrier peptide samples are multiplexed. Analysis by high resolution mass spectrometry using synchronous precursor scanning (SPS) and MS3 is then performed (Figure 1C). The carrier peptides provide sufficient MS/MS peak intensity for sequence identification (MS2) and the contribution of each channel of TMT at the MS3 level corresponds to quantity of peptide present in each sample.

Figure 2 - Identification of MHC-bound peptides from cell culture. BLCLs (9033 cell line) were titrated from 5e6 to 1000 cells in duplicate and labelled using TMTIOplex reagents. The intensity of the TMT tags is shown for a MHC class I peptide (Figure 2A). The length distribution of the MHC class I (Brown) and class II (Black) fall within typical ranges expected (Figure 2B). Clustering of the peptides can distinguish the HLA class I alleles expressed by this cell line (HLA-A*03:01, -B*07:02 and HLA-DRB 1*04:01) that were isolated using class I -and DR-specific antibodies w6/32 and L3.1 and match the typical binding motif of these alleles (Figure 2C).

Figure 3 - Identification of MHC-bound peptides from patient biopsies. MHC-bound peptides were identified from either 19 mg or 1 mg of Patient biopsy (melanoma) using a patient derived xenograft (PDX) as a source of carrier peptides (Figure 3A). Exome sequencing allowed generation of a patient-specific database to search for mutated MHC- bound peptides to identify candidate neoantigens (Figure 3 A). The MHC class I peptides isolated had a typical length in the range of 8-12 amino acids (Figure 3B). Several known HLA-A2 epitopes from cancer associated proteins were identified in the biopsy (Figure 3C). Use of a patient-specific protein database from exome sequencing helped identify a neo-antigen arising from a N to S mutation (N401S) in “ER Degradation Enhancing Alpha-Mannosidase Like Protein 1”, which was subsequently validated using synthetic peptide (Figure 3D).

Figure 4 - Identification of HLA-bound peptides from single cells and 100 cells. The carrier channel comprised of 10⁷ cells labelled using TMT-131, and the two samples were labelled with either TMT126 (1 cell) or TMT-128 (100 cells). The HLA class I peptides identified at a false discovery rate of 1% were plotted. The heatmap depicts the intensity of TMT reported ions with each peptide represented as a horizontal line, the colour scale corresponding to intensity is depicted to right of the figure.

Figure 5 - Sequence motif clustering of MHC -bound peptides identified from a single cell. The HLA peptides identified were analysed using GIBBS clustering 2.0 to enable unsupervised clustering of the peptides. The anchor residues of the HLA-A3 and B7 match known motif for these HLA molecules (as described in NetMHC 4.0 motif viewer).

Figure 6 - Peptide length distribution of peptides identified from a single cell, 100 cells, and 10⁷ cells. The peptides identified from all three samples had a similar length and have the characteristics of typical HLA class I peptides. Figure 7 - Synovial tissues from seven rheumatoid arthritis patients (TMT 1-7) and partially HLA-matched 9033 B-lymphoblastoid cells (le4, le5, and le6 cells, TMT 8-10 respectively) were subject to immunoaffmity purification of HLA-class I and II molecules. Clustermaps of Log2 transformed intensities (dark grey indicates low intensity; light grey indicates high intensity) TMT labels for HLA-DR, -DQ, -DP and class I peptides identified are presented in A, B, C and D respectively. All the patients were HLA-DRB 1*04:01 positive and of the 316 HLA-DR peptides identified 145 peptides were classified as strong binders ( See Table 1 for detailed list) and 43 as weak binders per netMHCpanll algorithm. KFY TO THF SEQUENCE LISTING

SEQ ID NO: 1 - peptide sequence from PI 1388-2|TOP2A_HUMAN.

SEQ ID NO:2 - peptide sequence from Q14094-2|CCNI_HUMAN.

SEQ ID NO:3 - peptide sequence from Q15019-2|SEPT2_HUMAN.

SEQ ID NO:4 - peptide sequence from Q9NYB9-2|ABI2_HUMAN. SEQ ID NO:5 - peptide sequence from P40126-2|TYRP2_HUMAN.

SEQ ID NO:6 - peptide sequence from P40126-2|TYRP2_HUMAN.

SEQ ID NO:7 - peptide sequence from P43355|MAGA1_HUMAN.

SEQ ID NO:8 - peptide sequence from P51572-2|BAP31_HUMAN.

SEQ ID NO:9 - peptide sequence from P78395|PRAME_HUMAN. SEQ ID NO: 10 - peptide sequence from Q08431 -3 |MF GM HUMAN.

SEQ ID NO: 11 - peptide sequence from DEK M150I.

SEQ ID NO: 12 - peptide sequence from EDEM1 N401S. SEQ ID NO: 13 peptide sequence from METTL9 A70S. SEQ ID NO: 14 peptide sequence from PIK3R1 R46W. SEQ ID NO: 15 peptide sequence from RERE N425S. SEQ ID NO: 16 peptide sequence from ANKRD17_S2010N. SEQ ID NO: 17 peptide sequence from NDN_I118V. SEQ ID NO: 18 peptide sequence from immunoglobulin lambda variable 4-69. SEQ ID NO: 19 peptide sequence from immunoglobulin lambda variable 4-69. SEQ ID NO:20 peptide sequence from immunoglobulin lambda variable 4-69. SEQ ID NO:21 peptide sequence from immunoglobulin lambda variable 4-69. SEQ ID NO:22 peptide sequence from lysosomal alpha-mannosidase. SEQ ID NO:23 peptide sequence from carboxypeptidase D. SEQ ID NO:24 peptide sequence from carboxypeptidase D. SEQ ID NO:25 peptide sequence from ceruloplasmin SEQ ID NO:26 peptide sequence from angiotensinogen. SEQ ID NO:27 peptide sequence from complement C3. SEQ ID NO:28 peptide sequence from T-cell surface glycoprotein CD4. SEQ ID NO:29 peptide sequence from immunoglobulin heavy variable 3-7. SEQ ID NO:30 peptide sequence from immunoglobulin heavy variable 3-7. SEQ ID NO:31 peptide sequence from immunoglobulin heavy variable 3-7. SEQ ID NO:32 peptide sequence from immunoglobulin heavy variable 3-7. SEQ ID NO:33 peptide sequence from immunoglobulin heavy variable 3-7. SEQ ID NO:34 peptide sequence from immunoglobulin heavy variable 3-7. SEQ ID NO:35 peptide sequence from immunoglobulin heavy variable 3-7. SEQ ID NO:36 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO:37 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO:38 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO:39 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO:40 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO:41 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO:42 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO:43 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO:44 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO:45 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO:46 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO:47 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO:48 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO:49 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO:50 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO:51 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO: 52 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO:53 - peptide sequence from HLA class I histocompatibility antigen, B-7 alpha chain. SEQ ID NO: 54 peptide sequence from fibrinogen alpha chain. SEQ ID NO:55 peptide sequence from fibrinogen alpha chain. SEQ ID NO:56 peptide sequence from fibrinogen beta chain. SEQ ID NO:57 peptide sequence from fibrinogen gamma chain. SEQ ID NO:58 peptide sequence from fibrinogen gamma chain. SEQ ID NO:59 peptide sequence from fibrinogen gamma chain. SEQ ID NO: 60 peptide sequence from fibrinogen gamma chain. SEQ ID NO:61 peptide sequence from fibrinogen gamma chain. SEQ ID NO: 62 peptide sequence from complement Clq subcomponent subunit B. SEQ ID NO: 63 peptide sequence from complement Clq subcomponent subunit B. SEQ ID NO: 64 peptide sequence from complement Clq subcomponent subunit B. SEQ ID NO: 65 peptide sequence from complement Clq subcomponent subunit B. SEQ ID NO: 66 peptide sequence from complement Clq subcomponent subunit B. SEQ ID NO: 67 peptide sequence from alpha-2-HS-glycoprotein. SEQ ID NO: 68 peptide sequence from alpha-2-HS-glycoprotein. SEQ ID NO: 69 - peptide sequence from alpha-2-HS-gly coprotein.

SEQ ID NO:70 - peptide sequence from transferrin receptor protein 1.

SEQ ID NO:71 - peptide sequence from transferrin receptor protein 1.

SEQ ID NO:72 - peptide sequence from apolipoprotein B-100. SEQ ID NO:73 - peptide sequence from apolipoprotein B-100.

SEQ ID NO:74 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain.

SEQ ID NO:75 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain. SEQ ID NO:76 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain.

SEQ ID NO:77 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain.

SEQ ID NO:78 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain.

SEQ ID NO:79 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain.

SEQ ID NO:80 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain. SEQ ID NO:81 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain.

SEQ ID NO:82 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain.

SEQ ID NO:83 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain.

SEQ ID NO:84 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain.

SEQ ID NO:85 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain. SEQ ID NO:86 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain.

SEQ ID NO:87 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain.

SEQ ID NO:88 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain.

SEQ ID NO:89 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain. SEQ ID NO:90 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain.

SEQ ID NO:91 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain. SEQ ID NO:92 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain.

SEQ ID NO:93 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain.

SEQ ID NO:94 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain.

SEQ ID NO:95 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain.

SEQ ID NO:96 - peptide sequence from HLA class I histocompatibility antigen, A-3 alpha chain. SEQ ID NO:97 - peptide sequence from gelsolin.

SEQ ID NO:98 - peptide sequence from gelsolin.

SEQ ID NO:99 - peptide sequence from cathepsin D.

SEQ ID NO: 100 - peptide sequence from cathepsin B.

SEQ ID NO: 101 - peptide sequence from collagen alpha-2. SEQ ID NO: 102 - peptide sequence from 72 kDa type IV collagenase.

SEQ ID NO: 103 - peptide sequence from glutathione S-transferase P.

SEQ ID NO: 104 - peptide sequence from complement C4-B.

SEQ ID NO: 105 - peptide sequence from HLA class I histocompatibility antigen, Cw-7 alpha chain. SEQ ID NO: 106 - peptide sequence from HLA class I histocompatibility antigen, Cw-7 alpha chain.

SEQ ID NO: 107 - peptide sequence from HLA class I histocompatibility antigen, Cw-7 alpha chain.

SEQ ID NO: 108 - peptide sequence from HLA class I histocompatibility antigen, Cw-7 alpha chain.

SEQ ID NO: 109 - peptide sequence from HLA class I histocompatibility antigen, Cw-7 alpha chain,

SEQ ID NO: 110 - peptide sequence from HLA class I histocompatibility antigen, Cw-7 alpha chain. SEQ ID NO: 111 - peptide sequence from HLA class I histocompatibility antigen, Cw-7 alpha chain. SEQ ID NO: 112 - peptide sequence from HLA class I histocompatibility antigen, Cw-7 alpha chain.

SEQ ID NO: 113 - peptide sequence from HLA class I histocompatibility antigen, Cw-7 alpha chain. SEQ ID NO: 114 - peptide sequence from HLA class I histocompatibility antigen, Cw-7 alpha chain.

SEQ ID NO: 115 - peptide sequence from HLA class I histocompatibility antigen, Cw-7 alpha chain.

SEQ ID NO: 116 - peptide sequence from HLA class I histocompatibility antigen, Cw-7 alpha chain.

SEQ ID NO: 117 - peptide sequence from HLA class I histocompatibility antigen, Cw-7 alpha chain.

SEQ ID NO: 117 - peptide sequence from clusterin.

SEQ ID NO: 118 - peptide sequence from clusterin. SEQ ID NO: 119 - peptide sequence from heat shock cognate 71 kDa protein.

SEQ ID NO: 120 - peptide sequence from heat shock cognate 71 kDa protein.

SEQ ID NO: 121 - peptide sequence from heat shock cognate 71 kDa protein.

SEQ ID NO: 122 - peptide sequence from HLA class I histocompatibility antigen, alpha chain E. SEQ ID NO: 123 - peptide sequence from HLA class I histocompatibility antigen, alpha chain E.

SEQ ID NO: 124 - peptide sequence from HLA class I histocompatibility antigen, alpha chain E.

SEQ ID NO: 125 - peptide sequence from HLA class I histocompatibility antigen, alpha chain E.

SEQ ID NO: 126 - peptide sequence from macrophage migration inhibitory factor.

SEQ ID NO: 127 - peptide sequence from beta-l,4-galactosyltransferase 1.

SEQ ID NO: 128 - peptide sequence from ganglioside GM2 activator.

SEQ ID NO: 129 - peptide sequence from ganglioside GM2 activator. SEQ ID NO: 130 - peptide sequence from ganglioside GM2 activator.

SEQ ID NO: 131 - peptide sequence from HLA class II histocompatibility antigen, DP alpha 1 chain.

SEQ ID NO: 132 - peptide sequence from biglycan.

SEQ ID NO: 133 - peptide sequence from cathepsin S. SEQ ID NO: 134 - peptide sequence from lumican.

SEQ ID NO: 135 - peptide sequence from lumican.

SEQ ID NO: 136 - peptide sequence from lumican. SEQ ID NO: 137 - peptide sequence from lumican.

SEQ ID NO: 138 - peptide sequence from lumican.

SEQ ID NO: 139 - peptide sequence from dipeptidyl peptidase 1.

SEQ ID NO: 140 - peptide sequence from gamma-aminobutyric acid receptor-associated protein-like 2.

SEQ ID NO: 141 - peptide sequence from beta-2-microglobulin.

SEQ ID NO: 142 - peptide sequence from beta-2-microglobulin.

SEQ ID NO: 143 - peptide sequence from beta-2-microglobulin.

SEQ ID NO: 144 - peptide sequence from beta-2-microglobulin. SEQ ID NO: 145 - peptide sequence from beta-2-microglobulin.

SEQ ID NO: 146 - peptide sequence from beta-2-microglobulin.

SEQ ID NO: 147 - peptide sequence from beta-2-microglobulin.

SEQ ID NO: 148 - peptide sequence from beta-2-microglobulin.

SEQ ID NO: 149 - peptide sequence from interferon-induced transmembrane protein 3. SEQ ID NO: 150 - peptide sequence from interferon-induced transmembrane protein 3. SEQ ID NO: 151 - peptide sequence from galectin-3 -binding protein.

SEQ ID NO: 152 - peptide sequence from lysosomal-associated transmembrane protein 5.

SEQ ID NO: 153 - peptide sequence from lysosomal-associated transmembrane protein 5.

SEQ ID NO: 154 - peptide sequence from lysosomal-associated transmembrane protein 5.

SEQ ID NO: 155 - peptide sequence from lysosomal-associated transmembrane protein 5. SEQ ID NO: 156 - peptide sequence from lysosomal-associated transmembrane protein 5.

SEQ ID NO: 157 - peptide sequence from lysosomal-associated transmembrane protein 5.

SEQ ID NO: 158 - peptide sequence from lysosomal-associated transmembrane protein 5.

SEQ ID NO: 159 - peptide sequence from desmoglein-2.

SEQ ID NO: 160 - peptide sequence from ceramide glucosyltransferase.

SEQ ID NO: 161 - peptide sequence from plexin domain-containing protein 2.

SEQ ID NO: 162 - peptide sequence from NEDD4 family-interacting protein 1. PET ATT, ED DESCRIPTION OF TUI INVENTION

General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in immunology, molecular biology, cancer therapy, pharmacology, protein chemistry, and biochemistry).

Unless otherwise indicated, the techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al., (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

As used herein, the term “about”, unless stated to the contrary, refers to +/- 10%, more preferably +/- 5%, of the designated value.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

As used in this application, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or”. That is, unless specified otherwise, or clear from context, “X employs A or B” is intended to mean any of the natural inclusive permutations. That is, if X employs A; X employs B; or X employs both A and B, then “X employs A or B” is satisfied under any of the foregoing instances. Further, at least one of A and B and/or the like generally means A or B or both A and B. In addition, the articles “a” and “an” as used in this application and the appended claims may generally be construed to mean “one or more” unless specified otherwise or clear from context to be directed to a singular form.

The term “amino acid” as used herein includes both L- and D-isomers of the naturally occurring amino acids as well as other amino acids (e.g., naturally-occurring amino acids, non-naturally-occurring amino acids, amino acids which are not encoded by nucleic acid sequences, etc.) used in peptide chemistry to prepare synthetic analogs of peptides. Examples of naturally occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, threonine, etc.

The term "peptide" is used herein to designate a series of amino acid residues, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids. The length of the peptides identified by the disclosure is not critical, as long as the correct epitope or epitopes are maintained therein. Typically, MHC -bound peptides are less than 30 amino acids in length.

The term “polypeptide” or “protein” as used herein, refer to a polymer of amino acids generally greater than about 30 amino acids in length.

The term “nucleic acid” refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5’ end of another nucleotide. The nucleic acid is not limited by length, and thus the nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

“Isolated”, as used herein, refers to a peptide, complex, or other molecule separated from other components (or a particular component) that are present in the natural source of the molecule (other nucleic acids, proteins, lipids, sugars, etc.).

“Synthetic”, as used herein, refers to a peptide or nucleic molecule that is not isolated from its natural (e.g., biological sources) sources, e.g., which is produced through recombinant technology or using chemical synthesis.

As used herein, the term “binds” is in reference to a detectable interaction between two molecules, for example, between an MHC molecule and a peptide.

As used herein, the term “subject” can be any animal. In one embodiment, the animal is a vertebrate. For example, the animal can be a mammal, avian, chordate, amphibian or reptile. Exemplary subjects include but are not limited to human, primate, livestock (e.g. sheep, cow, chicken, horse, donkey, pig), companion animals (e.g. dogs, cats), laboratory test animals (e.g. mice, rabbits, rats, guinea pigs, hamsters), captive wild animal (e.g. fox, deer). In one embodiment, the mammal is a human. The terms “treating” or “treatment” as used herein, refer to both direct treatment of a subject by a medical professional (e.g., by administering a therapeutic agent to the subject), or indirect treatment, effected, by at least one party, (e.g., a medical doctor, a nurse, a pharmacist, or a pharmaceutical sales representative) by providing instructions, in any form, that (i) instruct a subject to self-treat according to a claimed method (e.g., self-administer a drug) or (ii) instruct a third party to treat a subj ect according to a claimed method. Also encompassed within the meaning of the term “treating” or “treatment” are prevention or reduction of the disease to be treated, e.g., by administering a therapeutic at a sufficiently early phase of disease to prevent or slow its progression.

The terms "co-administration" or “administered in combination” or the like, as used herein, are meant to encompass administration of the selected therapeutic agents to a single subject, and are intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different time.

MHC -bound peptides The human major histocompatibility complex (MHC) is located on the short arm of chromosome 6 and encompasses approximately 4 Mb, or 0.1%, of the genome. More than 220 genes have been identified in this region and at least 10% of these genes have a direct function in immune responses.

The term "major histocompatibility complex (MHC)", "MHC molecules", or "MHC proteins" refers to proteins capable of binding peptides resulting from the proteolytic cleavage of protein antigens and representing potential T-cell epitopes, transporting them to the cell surface and presenting them there to specific cells, e.g., to cytotoxic T-lymphocytes or T-helper cells. The human MHC is also called the HLA complex. Thus, the term "human leukocyte antigen (HLA) system", "HLA molecules" or "HLA proteins" refers to a gene complex encoding the MHC proteins in humans. The term MHC is referred as the "H-2" complex in murine species. Those of ordinary skill in the art will recognize that the terms " major histocompatibility complex (MHC)", "MHC molecules", "MHC proteins" and "human leukocyte antigen (HLA) system", "HLA molecules", "HLA proteins" are used interchangeably herein. The human MHC can be divided into three regions that encode the class I, class

II, and class III human leukocyte antigen (HLA) gene products. These HLA molecules demonstrate tremendous polymorphism, which reflects the natural evolution of these genes in response to various microbial pathogens in different ethnic populations. HLA class I molecules are expressed on all nucleated cells and associate with short peptides (8-11 amino acids in length) derived from both self and foreign antigens. These peptide ligands are primarily generated in or transported into the cytoplasm and subsequently translocated into the endoplasmic reticulum (ER) where they assemble with nascent MHC class I molecules. These mature, peptide-loaded, complexes are then transported to the cell surface where they are scrutinized by CD8+ cytotoxic T lymphocytes (CTL). Should the peptide ligand be derived from a pathogen and be recognized as foreign in an immunocompetent host, the cell is killed via the cytotoxic armory of the CTL. The expression of HLA class II molecules is confined to a small subset of highly specialized cells called antigen-presenting cells (APCs). The class II molecules associate with longer peptides (9-25 amino acids in length) than class I molecules and this association occurs in late endosomal compartments, a distinct and separate cellular compartment to the ER-Golgi route inhabited by assembling MHC class I molecules. Class II molecules are recognized by CD4+ T helper cells and functional recognition of these complexes is intimately involved in both the humoral and cellular immune response. MHC class I and class II molecules form membrane-distal structures that comprise a cleft in which the antigenic peptide ligands reside. The T-cell receptor (TCR) on CD4+ or CD8+ T cells recognizes MHC molecules in the context of both the class I or class II molecule and the peptide antigen presented in the antigen binding groove of these cell surface molecules.

HLA alleles are expressed in codominant fashion, meaning that the alleles (variants) inherited from both parents are expressed equally. For example, each person carries 2 alleles of each of the 3 class I genes, (HLA- A, HLA-B and HLA-C), and so can express six different types of class II HLA. In the class II HLA locus, each person inherits a pair of HLA-DP genes (DPA1 and DPB1, which encode a and b chains), a couple of genes HLA-DQ (DQA1 and DQB1, for a and b chains), one gene HLA-DRa (DRA1), and one or more genes HLA-DRp (DRBl and DRB3, -4 or -5). That means that one heterozygous individual can inherit six or eight functioning class II HLA alleles, three or more from each parent. Thus, the HLA genes are highly polymorphic; many different alleles exist in the different individuals inside a population. Genes encoding HLA proteins have many possible variations, allowing each person's immune system to react to a wide range of foreign invaders. Some HLA genes have hundreds of identified versions (alleles), each of which is given a particular number. In some embodiments, the class I HLA alleles are HLA-A*02:01, HLA-B* 14:02, HLA-A*23:01, HLA-E*01:01 (non- classical). In some embodiments, class II HLA alleles are HLA-DRB*01:01, HLA- DRB*01:02, HLA-DRB*1 1:01, HLA-DRB*15:01, and HLA-DRB *07:01.

Peptide antigens attach themselves to MHC molecules by competitive affinity binding within the endoplasmic reticulum, before they are presented on the cell surface. Here, the affinity of an individual peptide antigen is directly linked to its amino acid sequence and the presence of specific binding motifs in defined positions within the amino acid sequence. If the sequence of such a peptide is known, it is possible to manipulate the immune system against diseased cells using, for example, peptide vaccines.

MHC-peptide-binding rules have been studied extensively for a subset of HLA alleles and encoded in advanced neural network-based algorithms that predict binding (Hoof et al., 2009 Immunogenetics 61:1-13; Lundegaard et al., 2008 Nucleic Acids Res 36:W509-12). However, several factors limit the power to predict peptides presented on HLA alleles to accurately determine the identity of MHC -bound peptides in a given sample. First, the provenance of peptide data upon which these algorithms are trained is diverse, ranging from peptide library screens to Edman degradation and mass spectrometry-based sequencing of endogenously processed and presented peptides. Mass spectrometry -based peptide identifications make up around 30% of the total identification in IEDB. Second, many existing prediction algorithms have focused on predicting binding but may not fully take into account endogenous processes that generate and transport peptides prior to binding (Larsen et al., 2007 BMC Bioinformatics 8:424). Third, the number of binding peptides for many HLA alleles is too small to develop a reliable predictor.

Therefore, it is preferred to use methods that directly identify the MHC -bound peptides in a sample, for example using mass spectrometry. Until now, however, the generation of high-quality resource datasets has been hampered by inefficient protocols for isolating MHC -bound peptides that necessitate prohibitively large amounts of input cellular material and a lack of database search tools for HLA-peptide sequencing. Unlike standard proteomic workflows, in which peptides generated from deliberate proteolytic digestion of proteinaceous samples are identified by mass spectrometry, studies ofMHC- bound peptides (immunopeptidomics) are considerably more challenging for a number of reasons: first, the precise proteolytic mechanism for generating the mature MHC-peptide complex is unknown and complex (many proteases and peptidases can be involved in liberating the peptide from the mature antigen complex); second, the peptides that bind to MHC molecules are more similar to each other in terms of length and sequence conservation as compared with enzyme-digested protein material; and, finally, the relatively low abundance of MHC -bound peptides, as compared with that of the intact source antigen, makes their detection more difficult.

Methods that allow the direct isolation and identification of MHC -bound peptides, such as those provided by the present disclosure, can be used to identify naturally processed and presented antigens derived from infectious micro-organisms as well as self-peptides associated with autoimmune disorders and cancers. Such methods can be used to design personalised medicines, for example, and are particularly useful in the context of neoantigens.

As used herein the term “MHC -bound peptide” refers to a peptide antigen that binds to molecules encoded by the MHC. In one embodiment, MHC -bound peptides are expressed on the surface of a cell in a sample. Such peptides are also referred to herein as “peptide antigens”. For the avoidance of any doubt, the term “MHC -bound peptide” refers to the peptide itself, whereas the term “MHC -peptide complex” refers to the whole complex formed by the peptide and the MHC molecule. MHC -bound peptides that can be identified using the methods of the disclosure include neoantigens.

As used herein, the term "neoantigen" (also referred to as a “neoepitope”) is an antigen that has at least one alteration that makes it distinct from the corresponding wild- type, parental antigen, e.g., via mutation in a tumour cell or post-translational modification specific to a tumour cell. A neoantigen can include a polypeptide sequence or a nucleotide sequence. A mutation can include a frameshift or nonframeshift indel, missense or nonsense substitution, splice site alteration, genomic rearrangement or gene fusion, or any genomic or expression alteration giving rise to a neoORF. A mutation can also include a splice variant. Post-translational modifications specific to a tumour cell can include aberrant phosphorylation. Post-translational modifications specific to a tumour cell can also include a proteasome-generated spliced antigen (see e.g., Liepe et al., Science. 2016; 354:354-358).

Verifying identified sample peptides

In some embodiments, the methods of the disclosure further comprise verifying the one or more identified sample peptides. As used herein, “verifying” refers to a process of determining whether the identified one or more sample peptides are true MHC -bound peptides (e.g., that they are capable of binding to MHC and are thus not false positives). The peptide sequences identified by the methods of the disclosure may be verified by one of several criteria, comprising MHC binding motif, MHC binding capacity and recognition by CD4+ T lymphocytes.

MHC binding motifs are common structural characteristics of peptides associated to a particular MHC molecule (allelic variant) which are necessary to form stable complexes with MHC molecules. In the case of MHC class I molecules, the typical peptide length varies from about 8 to 12 amino acids. In general, the peptide N and C termini are located in pockets abutting the ends of the cleft, where they are involved in conserved H-bonding interactions with conserved residues in the cleft. These H-bonding interactions are important in stabilizing the MHC -peptide complex, as demonstrated by studies substituting either the amino or carboxyl terminus of a peptide bound to HLA-A2 with a methyl group, thereby abrogating the H-bonding interactions. Peptides with both termini modified simultaneously did not promote stable binding with HLA-A2, suggesting that at least one peptide terminus must be bound within the peptide binding groove via the conserved array of H-bonding interactions for a stable complex to be formed. In the case of MHC class II molecules, the peptide length varies from 12 to 18 amino acids and even longer peptides can bind since both ends of the peptide binding groove are open. Most HLA class II molecules accommodate up to 4 residues relevant for binding, denoted as “anchor residues”, at relative positions PI, P4, P6 and P9 contained in a nonameric core region. This core region, however, can have variable distance from the N-terminus of the peptide. In the majority of cases, 2-4 N-terminal residues precede the core region. Hence, the PI anchor residues is located at positions 3, 4 or 5 in most HLA class II associated peptides. Peptides eluted from HLA-DR class II molecules share a big hydrophobic PI anchor, represented by tyrosine, phenylalanine, tryptophan, methionine, leucine, isoleucine or valine.

The position and the exact type of anchor residues constitute the peptide binding motif which is known for most of the frequently occurring MHC alleles. A computer algorithm allowing motif validation in peptide sequences is “Tepitope”, available by www.vaccinome.com.

The MHC binding capacity of the peptides identified by the methods of the present disclosure may be tested by methods known in the art using, for example, isolated MHC molecules and synthetic peptides with amino acid sequences identical to those identified by the method of the disclosure (Kropshofer et ah, J. Exp. Med. 1992; 175:1799-1803; Vogt et ah, J. Immunol. 1994; 153:1665-1673; Sloan et ah, Nature 1995; 375:802-806). Alternatively, a cellular binding assay using MHC expressing cell lines and biotinylated peptides can be used to verify the identified peptide (Arndt et ah, EMBO J., 2000; 19:1241-1251).

In both assays, the relative binding capacity of a peptide is measured by determining the concentration necessary to reduce binding of a labelled reporter peptide by 50% (IC50). Peptide binding with a reasonable affinity to the relevant MHC molecules attains IC50 values not exceeding 10-fold the IC50 of established reference peptides. The capacity to prime CD4+ T cells is another method for verifying identified sample peptides. This procedure involves testing of peptides identified by the methods of the disclosure for their ability to activate CD4+ T cell populations. Peptides with amino acid sequences either identical to those identified by the methods of the disclosure or corresponding to a core sequence derived from a nested group of peptides identified by the methods of the disclosure are synthesized. The synthetic peptides are then tested for their ability to activate CD4+ in the context of autologous dendritic cells, expressing the MHC molecule of interest.

CD4+ or CD8+ T cell responses can be measured by a variety of in vitro methods known in the art. For example, whole peripheral blood mononuclear cells (PBMC) can be cultured with and without a candidate synthetic peptide and their proliferative responses measured by, e.g., incorporation of [3H]-thymidine into their DNA. That the proliferating T cells are CD4+ or CD8+ T cells can be tested by either eliminating CD4+ or CD8+ T cells from the PBMC prior to assay or by adding inhibitory antibodies that bind to the CD4+ or CD8+ molecule on the T cells, thereby inhibiting proliferation of the latter. In both cases, the proliferative response will be inhibited only if CD4+ or CD8+ T cells are the proliferating cells. Alternatively, CD4+ or CD8+ T cells can be purified from PBMC and tested for proliferative responses to the peptides in the presence of APC expressing the appropriate MHC molecule. Such APCs can be B-lymphocytes, monocytes, macrophages, or dendritic cells, or whole PBMC. APCs can also be immortalized cell lines derived from B -lymphocytes, monocytes, macrophages, or dendritic cells. The APCs can endogenously express the MHC molecule of interest or they can express transfected polynucleotides encoding such molecules. In all cases the APCs can, prior to the assay, be rendered non-proliferative by treatment with, e.g., ionizing radiation or mitomycin-C.

As an alternative to measuring cell proliferation, cytokine production by the CD4+ T cells can be measured by procedures known to those in art. Cytokines include, without limitation, interleukin-2 (IL-2), interferon-gamma (IFN-gamma), interleukin-4 (IL-4), TNF-alpha, interleukin-6 (IL-6), interleukin- 10 (IL-10), interleukin- 12 (IL-12) or TGF- beta. Assays to measure them include, without limitation, ELISA, ELISPOT and bio assays in which cells responsive to the relevant cytokine are tested for responsiveness (e.g., proliferation) in the presence of a test sample.

Methods of isolating MHC -bound peptides

MHC-bound peptides suitable for identification by the methods of the disclosure may be isolated from a variety of sample types. In various embodiments, the peptides are isolated from a source that contains cells expressing MHC class I molecules or class II molecules, including a tissue or body fluid from a subject, such as blood, serum, immune cells (e.g., lymphocytes), blood cells (e.g., PBMCs or a subset thereof), tissues, or a cell line derived from primary cells.

In an embodiment, the sample is a blood cell sample, for example a PBMC sample, or a cell line derived from blood cells such as PBMCs (e.g., an immortalized cell line). Methods for generating a cell line from primary cells, or for immortalizing primary cells, are known in the art and include, for example, immortalization of primary cells by recombinant expression of human telomerase reverse transcriptase (TERT) (Barsov, Curr Protoc Immunol . 2011 7:Unit 7.2 IB), immortalization by recombinant expression of viral genes such as Simian virus 40 (SV40) T antigen, adenovirus E1A and E1B, human papillomavirus (HPV) E6 and E7 and Epstein-Barr Virus (EBV), as well as inactivation of tumour suppression genes such as p53 or Rb. Methods for immortalization of B lymphocytes by EBV are disclosed in Tosato and Cohen Cuff Protoc Immunol. 2007 7:Unit 7.22. Products/reagents for immortalizing mammalian cells are commercially available, for example from ATCC. In an embodiment, the sample is an immortalized cell line derived from primary cells obtained from the subject, in a further embodiment an immortalized B cell line, such as an EBV-transformed B lymphoblastoid cell line (B- LCL).

In other embodiments, the sample is a biopsy sample or a tissue sample. The biopsy sample may be a tissue biopsy sample or a liquid biopsy sample. In some embodiments, the biopsy sample is a tumour biopsy. In some embodiments, the liquid biopsy sample is a blood sample. In some embodiments, the tumour biopsy is a skin, breast, lung, colon, prostate, stomach, liver, cervix, thyroid, bladder, kidney, or pancreas tumour biopsy. Other types of tumour biopsies are also suitable for the methods of the disclosure, provided that MHC -bound peptides can be isolated therefrom.

Conventional methods for isolating MHC-bound peptides from a sample are well known in the art. One of the most commonly used techniques is mild acid elution (MAE) of MHC-bound peptides from intact cells, as described in Fortier et al. (2008) J. Exp. Med. 205:595-610; Storkus et al., (1993) J. Immunother. 14:94-103; and Storkus et al., (1993) J. Immunol. 151:3719-3727. Another suitable technique is immunoprecipitation or affinity purification of MHC -peptide complexes followed by peptide elution (see, e.g., Gebreselassie etal., Hum Immunol. 2006; 67:894-906; Falk et al Nature. 1991; 351:290- 296; and Rammensee et al., Annu. Rev. Immunol. 1993; 11 :213-244).

As used herein, the term “immunoprecipitation” refers to a process of separating out MHC-peptide complexes from a solution by contacting the solution with a binding molecule (e.g., an antibody) which specifically binds to the complexes and which is bound to a solid support (e.g., agarose beads).

Several high-throughput strategies for affinity purification have been described. For example transfection of cell lines with expression vectors coding soluble secreted MHCs (lacking a functional transmembrane domain) and elution of peptides associated with secreted MHCs (Barnea et al., Eur J Immunol. 2002; 32:213-22; and Hickman et al., J Immunol. 2004; 172:2944-52) or chemical/metabolic labelling to provide quantitative profiles of MHC-associated peptides (Weinzierl et al., Mo/ Cell Proteomics. 2007; 6:102- 13; Lemmel et al., Nat Biotechnol. 2004; 22:450-4; Milner, Mol Cell Proteomics. 2006; 5:357-65).

In some embodiments, the sample peptides are isolated from MHC molecules by first isolating MHC-peptide complexes from the sample. For the purification of such complexes from cells, the membranes of the cells have to be solubilized/lysed. Cell lysis may be carried out with methods known in the art, e.g. freeze-and-thaw cycles and the use of detergents, and combinations thereof. Preferred lysis methods are solubilization using detergents, preferably TX-100, NP40, n-octylglucoside, Zwittergent, Lubrol, CHAPS, most preferably TX-100 or Zwittergent 3-12. Cell debris and nuclei have to be removed from cell lysates containing the solubilized receptor-peptide complexes by centrifugation. Therefore, in one further embodiment, the MHC-peptide complexes are isolated from the cells using methods comprising solubilization with a detergent.

Closely allied to the choice of cell line, and its impact upon the complexity of the total pool of bound peptide ligands, is the specificity and efficacy of the monoclonal antibody/antibodies used in the immunoprecipitation of MHC-peptide complexes. Monoclonal antibodies with specificity toward classes of MHC molecule, families of MHC molecule, individual alleles of MHC molecules, and even subsets of molecules of an individual allotype have been generated over the years and are hybridomas are readily accessible commercially through bodies such as the ATCC (http://www.atcc.org).

For immunoprecipitation, antibodies specific for MHC class I or class II molecules (and subsets thereof) are suitable. The specific antibodies are preferably monoclonal antibodies, and are covalently or non-covalently e.g. via Protein A, coupled to beads, e.g. sepharose or agarose beads. Examples of anti-HLA antibodies comprise: anti-HLA-DR antibodies such as L243, TU36, and DA6.147; anti-HLA-DQ antibodies such as SPVL3, TU22, TUI 69; and anti-HLA-DP antibody B7/21, among others known to one of ordinary skill in the art. In some embodiments, the antibody used for immunoprecipitation is L243, LB3.1, SPV-L3, IVD12, IVA12, B721, MA2.1, BB7.2, ME1, W632, DT9, or M5/114.15.2.

Monoclonal antibodies specific for different MHC molecules may be commercially obtained (e.g. Pharmingen, Dianova) or purified from the supernatant of the respective hybridoma cells using Protein A- or Protein G-affmity chromatography. Purified monoclonal antibodies may be coupled to a solid support by various methods known in the art, for example by covalently coupling antibody amino groups to CNBr- activated sepharose. Preferably, the antibody is non-covalently bound to the solid support.

Alternatively, MHC-peptide complexes may be isolated from the sample using an affinity tag. For example, the MHC molecules may be genetically engineered to comprise the affinity tag and the MHC-peptide complexes isolated using the affinity tag’ s binding partner. Suitable affinity tags include a biotin acceptor peptide (BAP), poly histidine tag, poly-histidine-glycine tag, poly-arginine tag, poly- aspartate tag, poly cysteine tag, poly-phenylalanine, c-myc tag, Herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene 10 protein peptide tag, streptavidin tag, streptavidin binding peptide (SPB) tag, Strep-tag, Strep-tag II, albumin binding protein (ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag), calmodulin binding peptide (CBP) tag, chloramphenicol acetyl transferase (CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD) tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP), glutathione-S- transferase (GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, E-tag, HSV tag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag, NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag, Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, Spy Tag, SnoopTag, Profmity eXact tag, Protein C tag, SI -tag, S-tag, biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, or a combination thereof.

Other protein affinity purification options involve the use of proteins that are known to bind HLA, these include; CD8, which binds to the a3 domain of all HLA class I proteins; CD4 which binds to all HLA class II proteins; autologous T-cell receptors; and antigenic peptides which bind HLA with high affinity (computer modelling algorithms can be used to predict peptide/HLA binding characteristics). Any of these high HLA affinity protein options can be immobilized onto an insoluble solid support to prepare an affinity matrix which can be used to capture the MHC-peptide complexes from a sample. Appropriate elution conditions will result in the concentration and purification (isolation) of the sample's MHC-peptide complexes.

Immunoprecipitation or affinity purification of MHC-peptide complexes may be performed by incubating the antibody-bound solid substrate with the cell lysate under rotation/agitation for several hours or chromatographically by pumping the cell lysate through a micro-column. Washing of the antibody-beads may be performed in eppendorf tubes or in a microcolumn. The efficacy of the immunoprecipitation may be analysed by SDS-PAGE and western blotting using antibodies recognizing denatured MHC molecules (e.g., anti-HLA-DRalpha: 1B5).

Subsequent to immunoprecipitation, the MHC-peptide complexes are eluted from the antibody-bound solid substrate. Such processes may also involve breaking apart the MHC-peptide complex. After elution, the sample peptides can be separated from the MHC molecules, fractionated and subjected to sequence analysis by mass spectrometry.

The MHC-peptide complexes may be eluted by a variety of methods known to one of ordinary skill in the art. Such methods include using diluted acid, e.g., diluted acetonitrile (Jardetzky et ak, Nature 1991 353:326-329), diluted acetic acid (Rudensky et al., Nature 1991, 353:622-626; Chicz et ak, Nature 1992, 358:764-768) or diluted trifluoro acetic acid (Kropshofer et ak, J Exp Med 1992, 175:1799-1803). In some embodiments, the MHC-peptide complexes are eluted from the antibody-bound solid substrate using an acidic solution comprising acetic acid. These methods are also suitable for eluting the peptides from the MHC molecules.

In some embodiments, the immunoprecipitated MHC-peptide complexes are washed with water or low salt buffer before elution in order to remove residual detergent contaminants and non-specifically bound proteins. The low salt buffer may be a Tris, phosphate or acetate buffer in a concentration range of 0.5-100 mM, and may comprise salts such as sodium chloride. In one embodiment, the MHC-peptide complexes are washed with ultrapure water (sequencing grade) conventionally used for HPLC analysis.

The peptides can then be separated from the MHC molecules using any method known in the art. In some embodiments, the peptides are separated from the MHC molecules using ultrafiltration. The ultrafiltration may be carried using an ultrafiltration filter with a cut-off of, for example, 30 kD, 20 kD, 10 kD or 5 kD. The ultrafiltration may conveniently be performed in a microcentrifuge tube, e.g., having a tube volume of 0.5- 2.0 ml (such as “Ultrafree” tubes available from Millipore). The above mentioned washing and/or elution steps may be performed in a suitable ultrafiltration tube. The washing in the ultrafiltration tube may be carried out multiple times. The eluted peptides may be separated from the remaining antigen presenting receptor molecules using the same ultrafiltration tube. The eluted peptides may then be lyophilized for storage, if required. Isolated peptides may be subjected to any further purification/enrichment steps, including size exclusion chromatography or ultrafiltration (using a filter with a cut-off of about 5000 Da, for example about 3000 Da), and/or ion exchange chromatography (e.g., cation exchange chromatography), prior to labelling and further analysis by mass spectrometry. Classical protein separation (purification) techniques are based on; size differences (ultrafiltration, gel filtration, or size exclusion chromatography); charge differences (pi) (anion/cation exchange chromatography, or hydrophobic interaction chromatography); and combinations of size and charge differences (ID or 2D electrophoresis). Carrier peptides

As described herein, the methods of the present disclosure include the use of carrier peptides for identifying MHC -bound peptides present in a sample by analysing a mixture containing isobaric labelled carrier and sample peptides by mass spectrometry. As used herein, the term “carrier peptide” refers to a peptide which has the same amino acid sequence of a MHC -bound peptide in the sample, or predicted to be in the sample, and which is present in the mixture in an amount that is sufficient for identification by mass spectrometry (e.g., tandem mass spectrometry). In this regard, by having the same amino acid sequence, the carrier peptide provides an abundant source of fragment ions for accurate sequence identification of the sample peptide by mass spectrometry. Furthermore, the presence of the sample peptide in the mixture can be confirmed by analysing the intensity of the sample peptide’s isobaric label reporter ion in the mass spectra.

As will be appreciated by those skilled in the art, the choice of suitable carrier peptides will depend on the overall purpose of the method. For example, a selection of synthetic peptides, each having a sequence of a known disease antigen (e.g., known tumour associated antigens), may be used when the methods of the disclosure are used to determine the presence (or absence) of those known disease antigens in the sample. Such a method may be used to diagnose a subject, when the presence or absence of the known antigen is associated with a disease, for example. Alternatively, other types of carrier peptides (e.g., isolated from cultured cells, tissues, or a PDX) may be used when the methods of the disclosure are being used to identify new antigens and/or identify as many as possible different antigens present in the sample. In some embodiments, the carrier peptides include at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1000, or at least 2000 different peptides. The precise number of different carrier peptides used will depend on the number of sample peptides which are aimed to be identified and whether the carrier peptides are synthetic or isolated from natural sources (e.g., isolated from cultured cells or from a biopsy).

In some embodiments, the carrier peptides are isolated from a biological source (i.e., from cultured cells or a PDX sample). In other embodiments, the carrier peptides are synthetic (i.e., synthesized chemically).

Examples of carrier peptides isolated from biological sources include those isolated from MHC molecules in cultured cells or isolated from MHC molecules in a patient derived xenograft (PDX) sample. As is known in the art, creation of a PDX involves transplanting tissue or cells from a patient's tumour into an immunodeficient or humanized mouse to monitor tumour growth. PDX models are thus used to create an environment that allows for the natural growth of cancer cells to provide a sufficient source of carrier peptides. For example, MHC -bound peptides can be isolated from a PDX using the methods described herein (or any other method known in the art) to provide a source of carrier peptides for use in identifying MHC -bound peptides present in the original tumour from which the PDX was derived. Thus, the PDX can serve as a means for producing enough peptides for accurate mass spectrometry identification of low abundance peptides in the original sample.

Examples of synthetic carrier peptides are those which are predicted to bind to MHC molecules or which are known to bind MHC molecules. Binding to MHC molecules may be a predicted using tools such as the NetMHCcons software version 1.0 (http://www.cbs.dtu.dk/services/NetMHCcons/). Other suitable MHC prediction algorithms suitable for the methods described herein include smm, smmpmbec, ann(NetMHC3.4), NetMHC4, Pickpocket, consensus, NetMHCpan2.8, NetMHCpan3, NetMHCpan4, NetMHCcons, mhcflurry, mhcflurry pan, MixMHCpred, NetMHCIIpan , nn align, smm align, comblib, and tepitope. An overview of the various available MHC binding prediction tools is provided in Peters et al., PLoS Comput Biol 2006, 2:e65; Trost et al., Immunome Res 2007, 3:5; and Lin et al., BMC Immunology 2008, 9:8). Such MHC binding prediction tools can be accessed online using the IEDB at http://tools.immuneepitope.org/main/tcell/.

In an embodiment, the carrier peptides are predicted to bind to MHC with a predicted affinity of below 50 nM or below 500 nM.

The binding of a peptide to a MHC molecule may be determined using other known methods, for example a T2 Peptide Binding Assay. T2 cell lines are deficient in TAP but still express low amounts of MHC class I on the surface of the cells. The T2 binding assay is based upon the ability of peptides to stabilize the MHC class I complex on the surface of the T2 cell line. T2 cells are incubated with a specific peptide (e.g., a candidate carrier peptide), stabilized MHC class I complexes are detected using a pan- HLA class I antibody, an analysis is carried out (by flow cytometry, for example) and binding is assessed in relation to a non-binding negative control. The presence of stabilized peptide/MHC class I complexes at the surface is indicative that the peptide (e.g., candidate MiHA) binds to MHC class I molecules.

Binding of a peptide of interest (e.g., a candidate carrier peptide) to MHC may also be assessed based on its ability to inhibit the binding of a radiolabelled probe peptide to MHC molecules. For example, MHC molecules are solubilized with detergents and purified by affinity chromatography. They are then incubated for 2 days at room temperature with the inhibitor peptide (e.g., candidate carrier peptide) and an excess of a radiolabelled probe peptide, in the presence of a cocktail of protease inhibitors. At the end of the incubation period, MHC-peptide complexes are separated from unbound radiolabelled peptide by size-exclusion gel -filtration chromatography, and the percent bound radioactivity is determined. The binding affinity of a particular peptide for an MHC molecule may be determined by co-incubation of various doses of unlabelled competitor peptide with the MHC molecules and labelled probe peptide. The concentration of unlabelled peptide required to inhibit the binding of the labelled peptide by 50% (IC50) can be determined by plotting dose versus % inhibition. Binding of a peptide to a MHC molecule may also be determined using an epitope discovery system, such as the Prolmmune REVEAL & ProVE® epitope discovery system.

In some embodiments, the sample is obtained from a subject and the synthetic carrier peptides which are predicted to bind to MHC are derived from proteomics data or translated whole genome sequencing (WGS), whole exome sequencing (WES), or RNA sequencing (RNAseq) data obtained from DNA or RNA from the subject.

Any cell type or tissue can be utilized to obtain nucleic acid samples for use in sequencing. For example, a DNA or RNA sample can be obtained from a tumour or a bodily fluid, e.g., blood, obtained by known techniques (e.g. venipuncture) or saliva. Alternatively, nucleic acid tests can be performed on dry samples (e.g. hair or skin). In addition, a sample can be obtained for sequencing from a tumour and another sample can be obtained from normal tissue for sequencing where the normal tissue is of the same tissue type as the tumour. A sample can be obtained for sequencing from a tumour and another sample can be obtained from normal tissue for sequencing where the normal tissue is of a distinct tissue type relative to the tumour.

Tumours can include one or more of lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, and T cell lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer.

Once the DNA or RNA is obtained from the subject, transcriptome, exome, and/or genome libraries can be created for next generation sequencing. Transcriptome libraries can be generated from the RNA obtained from the sample. Transcriptome library construction may include one or more of the following steps: poly-A mRNA enrichment/purification; RNA fragmentation and priming for cDNA synthesis; reverse transcription (RT) (using random primers); second round of RT to generate a double- stranded cDNA, cDNA purification; end repair of fragmented cDNA, adenylation of the 3' ends, ligation of adaptors and enrichment of DNA fragments containing adapter molecules. Kits suitable for transcriptome library construction are commercially available, for example from Life Technologies (Ambion® RNA-Seq Library Construction Kit), Applied Biosystems (AB Library Builder™ Whole Transcriptome Core Kit), Qiagen (QuantiTect™ Whole Transcriptome Kit) and Sigma-Aldrich (TransPlex® Complete Whole Transcriptome Amplification Kit). Genomic libraries can be generated/constructed from the genomic DNA obtained from the sample. Genomic library construction may include one or more of the following steps: DNA shearing, DNA end repair, 3' ends adenylation, ligation of adaptors, purification of ligation products and amplification (e.g., PCR) to enrich DNA fragments that have adapter molecules. Kits suitable for genomic library construction are commercially available, for example from Illumina (TruSeq™ DNA Sample Preparation Kit (v2) (Cat. No. FC-930-1021), Life Technologies (SOLiD® Fragment Library Construction Kit and New England BioLabs (NEBNext® DNA Library Preparation).

The genomic (DNA-Seq) libraries may be subjected to an enrichment step to sequence only the coding portion (exome) of the human genome. Kits suitable for exome enrichment are commercially available, for example from Illumina (TruSeq™ exome enrichment kit, FC-930-1012), Life Technologies (TargetSeq™ Exome and Custom Enrichment System, A14060-A14063), FlexGen (FleXome whole exome enrichment kit v2), Roche NimbleGen (SeqCap EZ Human Exome Library v2.0) and Agilent Technologies (SureSelect All Exon kits)

Methods to perform whole transcriptome or exome sequencing (RNA-Seq) are known in the art (see, e.g., Wang et al., 2009 Nature Reviews Genetics 10:57-63) and are described herein. Various platforms for performing whole transcriptome/exome sequencing exist, such as the Illumina Genome Analyser platform, the Applied Biosystems (ABI) Solid™ Sequencing platform or Life Science's 454 Sequencing platform (Roche). Real-time single molecule sequencing-by-synthesis technologies rely on the detection of fluorescent nucleotides as they are incorporated into a nascent strand of DNA that is complementary to the template being sequenced. In one method, oligonucleotides 30-50 bases in length are covalently anchored at the 5' end to glass cover slips. These anchored strands perform two functions. First, they act as capture sites for the target template strands if the templates are configured with capture tails complementary to the surface-bound oligonucleotides. They also act as primers for the template directed primer extension that forms the basis of the sequence reading. The capture primers function as a fixed position site for sequence determination using multiple cycles of synthesis, detection, and chemical cleavage of the dye-linker to remove the dye. Each cycle consists of adding the polymerase/labelled nucleotide mixture, rinsing, imaging and cleavage of dye. In an alternative method, polymerase is modified with a fluorescent donor molecule and immobilized on a glass slide, while each nucleotide is color-coded with an acceptor fluorescent moiety attached to a gamma-phosphate. The system detects the interaction between a fluorescently-tagged polymerase and a fluorescently modified nucleotide as the nucleotide becomes incorporated into the de novo chain. Other sequencing-by-synthesis technologies also exist. Any suitable sequencing-by-synthesis platform can be used. As described above, four major sequencing-by-synthesis platforms are currently available: the Genome Sequencers from Roche/454 Life Sciences, the 1G Analyser from Illumina/Solexa, the SOLiD system from Applied BioSystems, and the Heliscope system from Helicos Biosciences. Sequencing- by-synthesis platforms have also been described by Pacific BioSciences and VisiGen Biotechnologies.

Sequencing can also include other massively parallel sequencing or next generation sequencing (NGS) techniques and platforms. Additional examples of massively parallel sequencing techniques and platforms are the Illumina HiSeq or MiSeq, Thermo PGM or Proton, the Pac Bio RS II or Sequel, Qiagen's Gene Reader, and the Oxford Nanopore MinlON. Additional similar current massively parallel sequencing technologies can be used, as well as future generations of these technologies.

Once the DNA or RNA sequenced, the coding regions are translated to provide protein sequences from which carrier peptides can be derived (e.g., by MHC -binding prediction). In silico translation of nucleic acid sequences to protein sequences may be performed using any suitable softwares or tools, including the ExPASy Translate tool, Vector NTI™ (Life Technologies), pyGeno (Granados et al., 2012), Virtual Ribosome (CBS, University of Denmark), etc.

In some embodiments, the carrier peptides are obtained from cells which are MHC-matched to the subject. Subject-specific MHC alleles or MHC genotype of a subject can be determined by any method known in the art. In some embodiments, MHC genotypes are determined by any method described in International Patent Application number PCT/US2014/068746, published June 11, 2015 as W02015085147. Briefly, the methods include determining polymorphic gene types that can comprise generating an alignment of reads extracted from a sequencing data set to a gene reference set comprising allele variants of the polymorphic gene, determining a first posterior probability or a posterior probability derived score for each allele variant in the alignment, identifying the allele variant with a maximum first posterior probability or posterior probability derived score as a first allele variant, identifying one or more overlapping reads that aligned with the first allele variant and one or more other allele variants, determining a second posterior probability or posterior probability derived score for the one or more other allele variants using a weighting factor, identifying a second allele variant by selecting the allele variant with a maximum second posterior probability or posterior probability derived score, the first and second allele variant defining the gene type for the polymorphic gene, and providing an output of the first and second allele variant.

Mass spectrometry

According to the methods provided herein, MHC -bound peptides are identified using mass spectrometry. As used herein, the term “mass spectrometry” refers to an analytical technique that measures the mass-to-charge ratio of ions present in a mixture. The results are typically presented as a mass spectrum, which is a plot of intensity as a function of the mass-to-charge ratio of each ion in the mixture.

As is known in the art, in a typical mass spectrometry procedure, peptides in a sample are first ionized, for example by bombarding them with electrons. This may cause some of the peptides to break into charged fragments or simply become charged without fragmenting. These ions are then separated according to their mass-to-charge (m/z) ratio, for example by accelerating them and subjecting them to an electric or magnetic field. The ions are detected by a mechanism capable of detecting charged particles, such as an electron multiplier, and the results are presented in a mass spectrum. The peptides in the sample can be identified by correlating known masses of peptides or fragments thereof to the observed masses in the mass spectrum or via de novo sequencing the peptide from its characteristic fragmentation pattern.

In some embodiments, the mass spectrometry comprises tandem mass spectrometry (i.e., MS/MS). A tandem mass spectrometer is one capable of multiple rounds of mass spectrometry, usually separated by some form of peptide fragmentation. For example, a first mass analyser can isolate one peptide from many entering a mass spectrometer. A second mass analyser then stabilizes the peptide ions while they collide with a gas, causing them to fragment by collision-induced dissociation (CID). A third mass analyser then sorts the fragments produced from the peptides according to their m/z ratio. Tandem MS can also be done in a single mass analyser over time, as in a quadrupole ion trap. There are various methods for fragmenting molecules for tandem MS, including collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD), blackbody infrared radiative dissociation (BIRD), electron-detachment dissociation (EDD) and surface-induced dissociation (SID).

Typically, mass spectrometry is combined with a preceding separation step to enhance the mass resolving and determining capabilities of the technique. For example, the mass spectrometry step may be preceded by a liquid chromatography, gas chromatography, capillary electrophoresis, or ion mobility separation step. The peptide mixture may be fractionated prior to mass spectrometry by one of a variety of possible chromatographic methods, e.g. by reversed phase, anion exchange, cation exchange chromatography or a combination thereof. In some embodiments, the separation is performed by Cl 8-reverse phase chromatography or by reversed- phase/cation exchange two-dimensional HPLC, for example using MudPit (Washburn et al., 2001 Nat Biotechnol 19:242-247).

The fractionation may be done in a HPLC mode utilizing fused-silica micro capillary columns which are either connected to a nano-flow electrospray source of a mass spectrometer or to a micro-fractionation device which spots the fractions onto a plate for MALDI analysis.

The fractionation of samples can also be accomplished by nanoliquid chromatography (nLC). To reduce losses due to peptides adhering to the large surface area of nLC columns, low-input samples can also be separated by capillary electrophoresis (Lombard-Banek et al., 2016 Angew Chem Int 55:2454-8). Advantageously, by mixing labelled sample peptides with labelled carrier peptides many of the peptides lost due to surface adhesion will be carrier peptides rather than single-cell peptides, as they will be in higher abundance in the mixture. A variety of mass spectrometric techniques are suitable, including MALDI-post source decay (PSD) MS or electrospray ionization tandem mass spectrometry (ESI-MS), and ion-trap ESI-MS. Such techniques are suitable for identifying MHC -bound peptides using the methods described herein.

In some embodiments, the mass spectrometry comprises at least three rounds of mass spectrometry analysis coupled together (e.g., “MS3” or “MS/MS/MS”). For example, a typical MS3 experiment would involve ionisation of peptides in a sample, detection of those ions in a first MS step (MSI), then ions of a specific mass-to-charge ratio (m/z) are selected and fragmented to generate a product ions for detection in a second MS step (MS2), selected product ions generated in MS2 can then be further fragmented to produce another group of product ions for a third MS step (MS3). The use of MS3 can increase the isobaric label reporter ion signal intensity and improves the ratio accuracy, due to improved counting statistics, leading to an increase in the number of quantified peptides. The MS3 step may be performed using synchronous precursor selection (SPS) which is used to select MS2 fragment ions that are likely to retain the intact isobaric tag for further fragmentation and analysis. This is particularly advantageous for identifying low abundance peptides in a sample, such as MHC -bound peptides.

As used herein, the term “identifying”, in the context of identification of a sample peptide, refers to a process of determining the amino acid sequence of the sample peptide and establishing from which protein or polypeptide the peptide is derived. Sequences of individual peptides can be determined by means known to one of ordinary skill in the art. For example, sequence analysis is performed by fragmentation of the peptides and computer-assisted interpretation of the fragment spectra using algorithms, e.g. MASCOT, SEQUEST, PEAKS, Maxquant, or Proteome Discoverer, to search against theoretical fragment spectra for known protein sequences in a database, such as a reference proteome. Both computer algorithms use protein and nucleotide sequence databases to perform cross-correlation analyses of experimental and theoretically generated tandem mass spectra. This allows automated high through-put sequence analysis of the peptides in the mixture.

The above described sequencing methods are well-known to a skilled person and are reviewed in Medzihradszky and Chalkley, 2015 Mass Spectrom Rev 34:43-63. Many proteins are present at over 50,000 copies per cell, whereas a particular

MHC -bound peptide may only be present at about 5,000 copies per cell on average (Hassan et ah, Mol Cell Proteomics. 2013 12:1829-1843), with some MHC-peptides being present in at only one copy per cell. Significantly, only one copy per cell can be effective in mediating an immune response (Croft et ah, PLoS Pathog. 2013 9(1): el003129). This low copy number and the difficulty of isolating such MHC -bound peptides means that mass spectrometry identification of such peptides is typically limited to large sample sizes. Thus, the methods described herein attempt to overcome this problem by providing enough peptide ions for mass spectrometry, such that they may be identified and quantified accurately from small clinically relevant samples. This is achieved, in part, due to the use of isobaric labelled carrier peptides.

As used herein, the term “isobaric labels” refers to chemical compounds that can be attached to a peptide and which have the same mass (i.e., they are “isobaric”), but vary in the distribution of heavy isotopes in their chemical structure. Isobaric labels are designed so that the tag is cleaved at a specific linker region upon high-energy CID (HCD) during tandem mass spectrometry, yielding reporter ions of different masses for each different isobaric label in the mixture. As used herein, the term “different isobaric labels” refers to two or more isobaric labels which have the same mass but different distribution of heavy isotopes in their chemical structure such that they can be distinguished using their reporter ions upon fragmentation in a mass spectrometer. For example, two different TMT “channels” within an isobaric label reagent set e.g., TMT¹⁰-127N and TMT¹⁰-128N within the TMTllplex (and TMTIOplex) reagent set (Thermo Scientific cat. no. A34808), are considered “different isobaric labels” within the context of the present disclosure. By way of explanation, the carrier peptides could be labelled with TMT10- 127N and the sample peptides labelled with TMT¹⁰-128N prior to mixing the two sets of peptides and performing mass spectrometry, for example.

The most common isobaric tags are amine-reactive tags (Bantscheff et ah,

2012 Analytical and Bioanalytical Chemistry 404:939-965). However, tags that react with cysteine residues and carbonyl groups have also been described (Yates, 2014 Journal of Proteome Research 13:5293-5309). These amine-reactive groups go through N-hydroxysuccinimide (NHS) reactions, which are based around three types of functional groups (Yates, 2014 Journal of Proteome Research 13:5293-5309). Isobaric labelling methods include tandem mass tags (TMT), isobaric tags for absolute and relative quantification (iTRAQ), mass differential tags for absolute and relative quantification, dimethyl labelling (e.g., DiLeu, described in Frost et al., 2015 Anal Chem 87:1646-54), or deuterium isobaric amine reactive tags (e.g., DiART, described in Zhang et al., 2010 Anal Chem. 82:7588-95). TMTs and iTRAQ methods are the most common and are commercially available. TMTs, for example have a mass reporter region (forming the reporter ion upon mass spectrometry fragmentation), a cleavable linker region, a mass normalization region, and a protein reactive group and have the same total mass.

Isobaric tandem mass tags (TMTs) are described in Sinitcyn et al. Annu Rev BiomedData Sci. 2018; 1:207-34; Thompson etal ,AnalChem. 2003; 75:1895-904; Ross et al., Mol Cell Proteomics. 2004; 3:1154-69). Such isobaric labels are typically used to label samples of roughly equal total protein amount to obtain a relative quantification of the peptides between the different samples. The present disclosure deviates from such standard use of isobaric labels in that it involves the use of carrier peptides which may be present in a much higher total abundance than the sample peptides for identification the sample peptides. Suitable TMT tags are commercially available from Thermo Scientific and include the TMTduplex, TMTsixplex, TMTIOplex, TMT 11 pi ex, and TMT16plex reagent sets (e.g., cat no. A44520, A34808, A37725, 90110, 90111, 90406, 90061, 90066, 90068, 90062, or 90065). Typical protocols for mass spectrometry identification of peptides begin by lysing the cells with detergents or urea (e.g., Dhabaria et al., 2015 J Proteome Res 14:3403-8). Since these chemicals are incompatible with mass spectrometry, they must be removed by cleanup procedures. These cleanup procedures can result in substantial losses of protein, and methods have been developed to minimize these losses (see e.g., , Hughes et al., 2014 Mol SystBiol 10:757; Kulak et al., 201 Nat Methods 11:319; Sielaff et al., 2017 J Proteome Res 16:4060-72).

Once injected into an MS instrument, peptide ions may need at least two rounds of MS analysis for confident sequence identification (Sinitcyn et al., 2018 Annu Rev Biomed Data Sci 1 :207-34; Cox et al., 2008 Nat Biotechnol 26: 1367-72; Eng et al., 1994 J Am Soc Mass Spectrom 5:976-89). For example, the first MS scan (MSI) determines the mass over charge ratio (m/z) for ions that entered the instrument. Then, selected ions are accumulated and fragmented, and their fragments are analysed by a second (or more) MS2 scan (Sinitcyn et al., 2018 Annu Rev Biomed Data Sc 1:207-34; Aebersold et al., 2003 Nature 422:198-207). The most commonly used fragmentation methods are described herein and break peptides at the peptide bonds with efficiency that varies much from bond to bond. Since some fragments are produced with low efficiency, they will not be detected if the peptide ions have low abundance in the mixture; and thus if not enough fragments are detected, the peptide cannot be identified. As described herein, the present disclosure alleviates this limitation by using isobaric labelled carrier peptide ions having the same m/z (and thus the same sequence as) as the sample peptides so that a larger number of peptide ions are fragmented and used for sequence identification.

Therapeutic methods

As described herein, the methods of the present disclosure can be used for diagnosing, treating, and selecting therapies for diseases in a subject. In some embodiments, the therapy is an immunotherapy. As used herein, the term “immunotherapy” refers to therapy that involves administration of a therapeutic agent that modulates (e.g., stimulates) an immune response. Suitable diseases to be treated and/or diagnosed include cancers, autoimmune diseases (e.g., rheumatoid arthritis), inflammatory diseases (e.g., an immune mediated inflammatory disease), and infections.

The methods of the present disclosure are particularly useful for selecting therapies for personalised medicine. For example, based on the MHC -bound peptides identified in the sample, a person skilled in the art will be able to select a suitable personalised immunotherapy for a subject, which may comprise: a) a vaccine comprising at least one of the sample peptides identified; b) an antibody which binds to at least one of the sample peptides identified; or c) an immune effector cell which targets at least one of the sample peptides identified.

Personalised cancer vaccines using tumour-specific peptides are described in Ott et al., Hematol. Oncol. Clin. N. Am. 2014, 28:559-569, for example. Typical methods for choosing which particular peptides to utilize as a vaccine requires the ability to predict which tumour-specific peptides would efficiently bind to the HLA alleles present in a subject. One of the critical barriers to developing effective and disease-specific immunotherapy is the identification and selection of highly specific and restricted tumour antigens to avoid autoimmunity. For example, tumour neoantigens, which arise as a result of genetic change (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.) within malignant cells, represent the most tumour-specific class of antigens. Neoantigens have rarely been used in cancer vaccine or immunogenic compositions due to technical difficulties in identifying them, selecting optimized antigens, and producing neoantigens for use in a vaccine or immunogenic composition. These problems are addressed by the present disclosure because suitable peptides for use in a vaccine, or for designing antibodies or cell therapies targeting the peptides, can be identified from small clinical samples, such as biopsies. Vaccines

The methods of the disclosure can be used to identify and validate potential peptides for use as a vaccine for treatment or prevention of a disease in a subject. Advantageously, the methods of the disclosure can be used to design vaccine compositions that are specific for a particular subject and thus are particularly well suited to personalised medicine. Vaccine compositions may comprise a single or a plurality of peptides identified or validated using a method described herein. Vaccine compositions can also be referred to as “vaccines”. A vaccine may contain between 1 and 30 such peptides, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 different peptides, 6, 7, 8, 9, 10 11, 12, 13, or 14 different peptides, or 12, 13 or 14 different peptides. Peptides can include post-translational modifications.

In one embodiment, different peptides in the vaccine or nucleotide sequences encoding them are selected so that the peptides are capable of associating with different MHC molecules, such as different MHC class I molecules and/or different MHC class II molecules. In some aspects, one vaccine composition comprises coding sequence for peptides and/or polypeptides capable of associating with the most frequently occurring MHC class I molecules and/or MHC class II molecules. Hence, vaccine compositions can comprise different fragments capable of associating with at least 2 preferred, at least 3 preferred, or at least 4 preferred MHC class I molecules and/or MHC class II molecules. The vaccine composition can preferably be capable of raising a specific cytotoxic

T-cells response and/or a specific helper T-cell response. A vaccine composition can further comprise an adjuvant and/or a carrier. Examples of useful adjuvants and carriers are given herein below. A vaccine can be associated with a carrier such as e.g. a protein or an antigen-presenting cell such as e.g. a dendritic ceil (DC) capable of presenting the peptide to a T-cell. Adjuvants are any substance whose admixture into a vaccine composition increases or otherwise modifies the immune response to the peptide. Carriers can be scaffold structures, for example a polypeptide or a polysaccharide, to which a peptide, is capable of being associated. Optionally, adjuvants are conjugated covalently or noncovalently. The ability of an adjuvant to increase an immune response to an antigen is typically manifested by a significant or substantial increase in an immune-mediated reaction, or reduction in disease symptoms. For example, an increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies raised to the antigen, and an increase in T-cell activity is typically manifested in increased cell proliferation, or cellular cytotoxicity, or cytokine secretion. An adjuvant may also alter an immune response, for example, by changing a primarily humoral or Th response into a primarily cellular, or Th response. Suitable adjuvants include, but are not limited to 1018 ISS, alum, aluminium salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact I-1P321, IS Patch, ISS, ISCOMATRIX, Juvimmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-J, ONTAJZ, PepTel vector system, PLG microparticles, resiquimod, SRL1 72, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon which is derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox or Superfos. Adjuvants such as incomplete Freund's or GM-CSF are useful in some embodiments. Several immunological adjuvants (e.g., MF59) specific for dendritic cells and their preparation have been described previously (Dupuis, et al., Cell Immunol 1998; 186:18-27; Allison, Dev Biol Stand. 1998; 92:3-11). Also cytokines can be used as adjuvants. Several cytokines have been directly linked to influencing dendritic cell migration to lymphoid tissues (e.g., TN'F-alpha), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for Tlymphocytes (e.g., GM-CSF, IL-1 and IL-4) (see eg., US 5,849,589) and acting as immunoadjuvants (e.g., IL-12) (Gabrilovich, et al., J Immunother Emphasis Tumour Immunol . 1996 6:414-418).

CpG immunostimulatmy oligonucleotides have also been reported to enhance the effects of adjuvants in a vaccine setting. Other TLR binding molecules such as RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used. Other examples of useful adjuvants include, but are not limited to, chemically modified CpGs (e.g. CpR, Idem), Poly(l:C)(e.g. polyi:CI2U), non-CpG bacterial DNA or RNA as well as immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafmib, XL- 999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which may act therapeutically and/or as an adjuvant. The amounts and concentrations of adjuvants and additives can readily be determined by the skilled artisan without undue experimentation. Additional adjuvants include colony-stimulating factors, such as Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim).

A vaccine composition can comprise more than one different adjuvant. Furthermore, a therapeutic composition can comprise any adjuvant substance including any of the above or combinations thereof. It is also contemplated that a vaccine and an adjuvant can be administered together or separately in any appropriate sequence. A carrier (or excipient) can be present independently of an adjuvant. The function of a carrier can for example be to increase the molecular weight of in particular mutant to increase activity or immunogenicity, to confer stability or solubility, to increase the biological activity, or to increase serum half-life. Furthermore, a carrier can aid presenting peptides to T-cells. A carrier can be any suitable carrier known to the person skilled in the art, for example a protein or an antigen presenting cell. A carrier protein could be but is not limited to keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid. For immunization of humans, the carrier is generally a physiologically acceptable carrier acceptable to humans and safe. Alternatively, the carrier can be dextrans for example sepharose.

Cytotoxic T-cells (CTLs) recognize an antigen in the form of a peptide bound to an MHC molecule rather than the intact foreign antigen itself. The MHC molecule itself is located at the cell surface of an antigen presenting cell Thus, an activation of CTLs is possible if a trimeric complex of peptide antigen, MHC molecule, and APC is present. Correspondingly, it may enhance the immune response if not only the peptide is used for activation of CTLs, but if additionally APCs with the respective MHC molecule are added. Therefore, in some embodiments a vaccine composition additionally contains at least one antigen presenting cell or MHC molecule. Peptides can also be included in viral vector-based vaccine platforms, such as vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (see e.g., Tatsis et al., Molecular Therapy 2004; 10:616-629), or lentivirus, including but not limited to second, third or hybrid second/third generation lentivirus and recombinant lentivirus of any generation designed to target specific cell types or receptors (Hu et al., Immunol Rev. 2011, 239: 45-61; Sakuma et al., Biochem J 2012, 443:603-18; Cooper et al., Nucl. Acids- Res. 2015, 43:682-690; and Zufferey et al, J Viral. 1998, 72:9873-9880). Dependent on the packaging capacity of the above mentioned viral vector-based vaccine platforms, this approach can deliver one or more nucleotide sequences that encode one or more peptides. Upon introduction of the vector into a host, infected cells express the peptides, and thereby elicit a host immune (e.g., CTL) response against the peptide(s). Vaccinia vectors and methods useful in imnmnization protocols are described in, e.g., US 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. Nature 351:456- 460 (1991). A wide variety of other vaccine vectors useful for therapeutic administration or immunization of peptide vaccines (or nucleic acids encoding them), e.g., Salmonella typhi vectors, and the like will be apparent to those skilled in the art from the description herein.

The peptides in the vaccine be further modified to contain additional chemical moieties not normally part of the peptide. Those derivatized moieties can improve the solubility, the biological half-life, absorption of the peptide, or binding affinity. The moieties can also reduce or eliminate any desirable side effects of the proteins and the like. An overview for those moieties can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000). For example, antigenic peptides having the desired activity can be modified as necessary to provide certain desired attributes, e.g. improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide to bind the desired MHC molecule and activate the appropriate T cell. For instance, the antigenic peptide and polypeptides can be subject to various changes, such as substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use, such as improved MHC binding. Such conservative substitutions can encompass replacing an amino acid residue with another amino acid residue that is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another. The effect of single amino acid substitutions can also be probed using D- amino acids. Such modifications can be made using well known peptide synthesis procedures, as described in e.g., Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides , Gross & Meienhofer, eds. (N.Y., Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, III, Pierce), 2d Ed. (1984).

One of skill in the art from this disclosure and the knowledge in the art will appreciate that there are a variety of ways in which to produce such peptides for use in a vaccine. In general, suitable peptides can be produced either in vitro or in vivo and can then be formulated into a vaccine or immunogenic composition and administered to a subject. Such in vitro production can occur by a variety of methods known to one of skill in the art such as, for example, peptide synthesis or expression of a peptide from a DNA or RNA molecule in any of a variety of bacterial, eukaryotic, or viral recombinant expression systems, followed by purification of the expressed peptide. Alternatively, peptides can be produced in vivo by introducing nucleic acid molecules (e.g., DNA, RNA, viral expression systems, and the like) that encode the peptides into a subject, whereupon the encoded disease specific antigens are expressed. Antibodies

The term "antibody" or "antibodies" is used herein in a broad sense and includes both polyclonal, monoclonal antibodies, and fragments thereof. In addition to intact or "full" immunoglobulin molecules, also included in the term "antibodies" are fragments (e.g. CDRs, Fv, scFv, Fab and Fc fragments) or polymers of those immunoglobulin molecules and humanized versions of immunoglobulin molecules, as long as they exhibit any of the desired properties, e.g., specific binding of the MHC -bound peptide, delivery of an agent to a cell expressing the peptide, and/or inhibiting the activity of a disease associated with expressing the peptide. In some embodiments, the antibody is a polyclonal antibody, monoclonal antibody, bi-specific antibody and/or a chimeric antibody.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e.; the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity (US 4,816,567, which is hereby incorporated in its entirety). The antibodies described herein may bind to a MHC molecule in complex with its bound peptide antigen. In this regard, an antibody may bind to the peptide in an MHC- restricted manner. Methods for generating antibodies that bound to MHC-peptide complexes include: immunizing a genetically engineered non-human mammal (e.g., comprising cells expressing said human MHC class I or II molecules) with a soluble form of a MHC-peptide complex; isolating mRNA molecules from antibody producing cells of said non-human mammal; producing a phage display library displaying protein molecules encoded by said mRNA molecules; and isolating at least one phage from said phage display library, wherein at least one phage displaying the antibody specifically binds to the human MHC-peptide complex. Respective methods for producing such antibodies and MHC-peptide complexes, as well as other tools for the production of these antibodies are disclosed in WO 03/068201 , WO 2004/084798, WO 01/72768, and WO 03/070752.

One of skill in the art will realize that the generation of two or more different sets of monoclonal or polyclonal antibodies maximizes the likelihood of obtaining an antibody with the specificity and affinity required for its intended use (e.g., ELISA, immunohistochemistry, in vivo imaging, immunotoxin therapy). The antibodies are tested for their desired activity by known methods, in accordance with the purpose for which the antibodies are to be used (e.g., ELISA, immunohistochemistry, immunotherapy, etc.; for further guidance on the generation and testing of antibodies, see, e.g., Greenfield, 2014 Antibodies: A Laboratory Manual, Second Edition Chapter 7). For example, the antibodies may be tested in ELISA assays or, Western blots, immunohistochemical staining of formalin- fixed cancers or frozen tissue sections. After their initial in vitro characterization, antibodies intended for therapeutic or in vivo diagnostic use are tested according to known clinical testing methods.

Monoclonal antibodies of the disclosure may be prepared using hybridoma methods. In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in US 4,816,567. DNA encoding the monoclonal antibodies of the disclosure can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 and US 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a F(ab')2 fragment and a pFc' fragment.

The antibody fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody fragment must possess a bioactive property, such as binding activity, regulation of binding at the binding domain, etc. Functional or active regions of the antibody may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the disclosure may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab' or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (US 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production can be employed. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. Human antibodies can also be produced in phage display libraries.

Immune effector cells

The methods described herein may also be used to identify potential therapies comprising immune effector cells that target an MHC -bound peptide. The term “immune effector cell” as used herein refers to any leukocyte involved in defending the body against infectious disease and foreign materials. For example, the immune effector cells can comprise lymphocytes, monocytes, macrophages, dentritic cells, mast cells, neutrophils, basophils, eosinophils, or any combinations thereof.

Immune effector cells are preferably obtained from the subject to be treated (i.e. are autologous). However, in some embodiments, immune effector cell lines or donor effector cells (allogeneic) are used. Immune effector cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumours. Immune effector cells can be obtained from blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. For example, cells from the circulating blood of an individual may be obtained by apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of immune effector cells can be further isolated by positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies directed to surface markers unique to the positively selected cells, e.g., by incubation with antibody-conjugated beads for a time period sufficient for positive selection of the desired immune effector cells. Alternatively, enrichment of immune effector cells population can be accomplished by negative selection using a combination of antibodies directed to surface markers unique to the negatively selected cells.

In some embodiments, the immune effector cell is a T cell (i.e., a T lymphocyte). The term "T cell" includes CD4+ T cells and CD8+ T cells. The term T cell also includes cytotoxic T cells, T helper 1 type T cells and T helper 2 type T cells, and regulatory T cells. T cells as used herein are generally classified by function and cell surface antigens (cluster differentiation antigens, or CDs)Two major classes of T cells involved in adaptive immunity are helper T (TH) cells and cytotoxic T- lymphocytes (CTLs).

Mature helper T (TH) cells express the surface protein CD4 and are referred as CD4+ T cells. Following T cell development, matured, naive T cells leave the thymus and begin to spread throughout the body, including the lymph nodes. Naive T cells are those T cells that have never been exposed to the antigen that they are programmed to respond to. Like all T cells, they express the T cell receptor-CD3 complex. The T cell receptor (TCR) consists of both constant and variable regions. The variable region determines what antigen the T cell can respond to. CD4+ T cells have TCRs with an affinity for Class II MHC, and CD4 is involved in determining MHC affinity during maturation in the thymus. Class II MHC proteins are generally only found on the surface of specialized antigen- presenting cells (APCs). Specialized antigen presenting cells (APCs) are primarily dendritic cells, macrophages and B cells, although dendritic cells are the only cell group that expresses MHC Class II constitutively (at all times). Some APCs also bind native (or unprocessed) antigens to their surface, such as follicular dendritic cells, but unprocessed antigens do not interact with T cells and are not involved in their activation. The peptide antigens that bind to MHC class I proteins are typically shorter than peptide antigens that bind to MHC class II proteins.

Cytotoxic T-lymphocytes (CTLs), also known as cytotoxic T cells, cytolytic T cells, CD8+ T cells, or killer T cells, refer to lymphocytes which induce apoptosis in targeted cells. CTLs form antigen-specific conjugates with target cells via interaction of TCRs with processed antigen (Ag) on target cell surfaces, resulting in apoptosis of the targeted cell. Apoptotic bodies are eliminated by macrophages. The term "CTL response" is used to refer to the primary immune response mediated by CTL cells. Cytotoxic T-lymphocytes have both T-cell receptors (TCR) and CD8 molecules on their surface. T cell receptors are capable of recognizing and binding peptides complexed with the molecules of HLA class I. Each cytotoxic T-lymphocyte expresses a unique T- cell receptor which is capable of binding specific MHC-peptide complexes. Most cytotoxic T cells express T-cell receptors (TCRs) that can recognize a specific antigen. In order for the TCR to bind to the class I MHC molecule, the former must be accompanied by a glycoprotein called CD8, which binds to the constant portion of the class I MHC molecule. Therefore, these T cells are called CD8+ T cells. The affinity between CD8 and the MHC molecule keeps the T cell and the target cell bound closely together during antigen-specific activation. CD8+ T cells are recognized as T cells once they become activated and are generally classified as having a pre-defmed cytotoxic role within the immune system. However, CD8+ T cells also have the ability to make some cytokines.

In some embodiments, the immune effector cell is a regulatory T cell. As used herein, "regulatory T cells" or "Treg cells" refer to T cells (T lymphocytes) that regulate the activity of other T cell(s) and/or other immune cells, usually by suppressing their activity. Regulatory T cells are typically defined by the presence of the cell surface markers CD4, CD25, FOXP3, GITR and CTLA4. In one embodiment, the Treg cells are CD4⁺, CD25⁺, FoxP3⁺ T-cells but it will be appreciated by persons skilled in the art that regulatory T cells are not fully restricted to this phenotype.

In some embodiments, the immune effector cell is a natural killer cell. Natural- killer (NK) cells are CD56 CD3 large granular lymphocytes that can kill infected and transformed cells, and constitute a critical cellular subset of the innate immune system. Unlike cytotoxic CD8+ T lymphocytes, NK cells launch cytotoxicity against tumour cells without the requirement for prior sensitization, and can also eradicate MHC-I-negative cells. NK cells are safer effector cells, as they may avoid the potentially lethal complications of cytokine storms, tumour lysis syndrome, and on-target, off-tumour effects.

In some embodiments, the immune effector cell comprises an engineered receptor. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), a T-cell receptor (TCR), or a B-cell receptor (BCR), an adoptive T cell therapy (ACT), or a derivative thereof.

Chimeric antigen receptors (CARs) can be used to generate immunoresponsive cells, such as T cells, specific for selected targets, such a MHC -bound peptides identified using the method described herein. Suitable constructs for generating CARs are described in US 5,843,728; US 5,851,828; US 5,912, 170; US 6,004,811; US 6,284,240; US 6,392,013; US 6,410,014; US 6,753, 162; US 8,211,422; and W09215322). Alternative CAR constructs can be characterized as belonging to successive generations. First- generation CARs typically consist of a single-chain variable fragment of an antibody specific for an antigen, for example comprising a VL linked to a VH of a specific antibody, linked by a flexible linker, for example by a CD8a hinge domain and a CD8a transmembrane domain, to the transmembrane and intracellular signalling domains of either CD3C or FcRy or scFv-FcRy (see, e.g., US 7,741,465; US 5,912,172; and US 5,906,936). Second-generation CARs incorporate the intracellular domains of one or more costimulatory molecules, such as CD28, 0X40 (CD134), or 4-1BB (CD137) within the endodomain, e.g., scFv-CD28/OX40/4 BB-CD3 (see, e.g., US 8,911,993; US 8,916,381; US 8,975,071; US 9,101,584; US 9,102,760; US 9,102,761). Third-generation CARs include a combination of costimulatory endodomains, such a CD3C-chain, CD97, GDI la-CD18, CD2, ICOS, CD27, CD 154, CDS, 0X40, 4- IBB, or CD28 signalling domains, e.g., scFv-CD28-4 BB-CD3C or scFv-CD28- OX40-CD3Q (see, e.g., US 8,906,682; US 8,399,645; US 5,686,281; WO2014134165; and W02012079000). In some embodiments, costimulation can be coordinated by expressing CARs in antigen- specific T cells, chosen so as to be activated and expanded following, for example, interaction with antigen on professional antigen-presenting cells, with costimulation. Additional engineered receptors can be provided on the immunoresponsive cells, e.g., to improve targeting of a T-cell attack and/or minimize side effects.

In some embodiments, immune effector cells, e.g., from a subject with a disease or condition, can be expanded. For example, expanded T cells that express TCRs specific to an MHC-bound peptide identified using the method described herein can be administered back to a subject. In some embodiments, suitable cells, e.g., PBMCs, are transduced or transfected with polynucleotides for expression of TCRs specific to the MHC -bound peptide and administered to a subject.

The methods described herein can involve adoptive transfer of immune effector, such as T cells, specific for selected MHC -bound peptides, such as tumour or pathogen associated antigens. Various strategies can be employed to genetically modify T cells by altering the specificity of the T cell receptor (TCR) for example by introducing new TCR a and b chains with specificity to a specific immunogenic antigen peptide identified using the method described herein (see, e.g., US 8,697,854; W02003020763, W02004033685, W 02004044004, W02005114215, W02006000830, W02008038002, W02008039818, W02004074322, W02005113595, WO2006125962, WO2013166321, WO2013039889,

WO2014018863, WO2014083173; and US 8,088,379).

Alternative techniques can be used to transform immune effector cells, such as protoplast fusion, lipofection, transfection or electroporation. A wide variety of vectors can be used, such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno- associated viral vectors, plasmids or transposons, such as a Sleeping Beauty transposon (see US 6,489,458; US 7,148,203; US 7,160,682; US 7,985,739; and US 8,227,432), can be used to introduce CARs, for example using 2nd generation antigen-specific CARs signalling through Oϋ3z and either CD28 or CD137. Viral vectors can for example include vectors based on HIV, SV40, EBV, HSV or BPV Immune effector cells that are targeted for transformation can for example include T helper cells, Natural Killer (NK) cells, cytotoxic T lymphocytes (CTL), or tumour-infiltrating lymphocytes (TIL). T cells expressing a desired receptr can for example be selected through co-culture with g-irradiated activating and propagating cells (APC), which co-express the MHC-bound peptide that is targeted by the receptor and co- stimulatory molecules.

Approaches such as the foregoing can be adapted to provide methods of treating and/or increasing survival of a subject having a disease, such as a cancer or pathogenic infection, for example by administering an effective amount of an immune effector cell comprising an receptor that binds to a MHC-bound peptide identified using the methods of the disclosure, wherein the binding activates the immune effector cell.

As described herein, suitable immune effector cell therapy methods can involve ex-vivo activation and expansion of the cells. In some embodiments, immune effector cells are activated before administering them to a subject in need thereof. Examples of these type of treatments include the use tumour infiltrating lymphocyte (TIL) cells (see US 5,126,132), cytotoxic T-cells (see US 6,255,073; and US 5,846,827), expanded tumour draining lymph node cells (see US 6,251,385), and various other lymphocyte preparations (see US 6,194,207; US 5,443,983; US 6,040,177; and US 5,766,920). Allogeneic cells may also be used in the preparation of immune effector cells and a method is described in detail in WO 97/26328. For example, in addition to Drosophila cells and T2 cells, other cells may be used to present antigens such as CHO cells, baculovirus-infected insect cells, bacteria, yeast, and vaccinia- infected target cells. In addition, plant viruses may be used.

In some embodiments, the immune effector cell recognizes a target cell by interacting through its TCR with the HLA/peptide-comples. Thus immune effector are useful in a method of killing target cells in a patient whose target cells aberrantly express a peptide identified using the methods of the disclosure, wherein the patient is administered an effective number of the immune effector cells. The immune effector cells that are administered to the patient may be derived from the patient and activated as described above (i.e. autologous T cells). Alternatively, the immune effector cells are not from the patient but are from another individual.

By "aberrantly expressed" it is meant that the peptide is over-expressed in diseased tissues compared to levels of expression in normal tissues or that the gene is silent in the tissue from which a tumour is derived but in the tumour it is expressed. By "over expressed" the inventors mean that the polypeptide is present at a level at least 1.2-fold of that present in normal tissue; preferably at least 2-fold, and more preferably at least 4- fold or 6-fold the level present in normal tissue. Immune effector cells may be obtained by methods known in the art, e.g. those described above. For example, protocols for adoptive transfer of T cells are well known in the art. Reviews can be found in: Gattioni et al. and Morgan et al. (Gattinoni et ak, 2006; Morgan et al., 2006). Dosages and administration

As will be appreciated by those skilled in the art, the above described therapies will be administered to a subject in a therapeutically effective amount. The terms "effective amount" or "therapeutically effective amount” as used herein, refer to a sufficient amount of a therapeutic agent being administered which will relieve to some extent or prevent worsening of one or more of the symptoms of the disease or condition being treated. The result can be reduction or a prevention of progression of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic uses is the amount of therapeutic agent required to provide a clinically significant decrease in disease symptoms without undue adverse side effects.

The term "therapeutically effective amount" includes, for example, a prophylactically effective amount. An "effective amount" of a therapeutic agent is an amount effective to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects. It is understood that "an effective amount" or "a therapeutically effective amount" can vary from subject to subject, due to variation in metabolism of the compound of any of age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.

It is considered well within the skill of the art for one to determine such therapeutically effective amounts by routine experimentation (including, but not limited to, a dose escalation clinical trial). An appropriate "effective amount" in any individual case may be determined using techniques, such as a dose escalation study.

Where more than one therapeutic agent is used in combination, a “therapeutically effective amount” of each therapeutic agent can refer to an amount of the therapeutic agent that would be therapeutically effective when used on its own, or may refer to a reduced amount that is therapeutically effective by virtue of its combination with one or more additional therapeutic agents.

Peptide vaccines can be administered in an amount sufficient to induce a CTL response, for example. An antigenic peptide or vaccine composition can be administered alone or in combination with other therapeutic agents. Exemplary therapeutic agents include, but are not limited to, a chemotherapeutic or biotherapeutic agent, radiation, or immunotherapy. Any suitable therapeutic treatment for a particular disease can be administered. Examples of chemotherapeutic and biotherapeutic agents include, but are not limited to, aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin alpha, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol®), pilocarpine, prochloroperazine, rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine and vinorelbine tartrate. In addition, the subject can be further administered an anti -immunosuppressive or immunostimulatory agent. For example, the subject can be further administered an anti-CTLA antibody or anti-PD-1 or anti-PD-Ll.

The amount of each peptide to be included in a vaccine composition and the dosing regimen can be determined by one skilled in the art. For example, a peptide or its variant can be prepared for intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, intramuscular (i.m.) injection. Exemplary methods of peptide injection include s.c, i.d., i.p., i.m., and i.v. Exemplary methods of DNA injection include i.d., i.m., s.c, i.p. and i.v. Other methods of administration of the vaccine composition are known to those skilled in the art. A pharmaceutical composition can be compiled such that the selection, number and/or amount of peptides present in the composition is/are disease and/or patient-specific. For example, the exact selection of peptides can be guided by expression patterns of the parent proteins in a given tissue to avoid side effects. The selection can be dependent on the specific type of disease, the status of the disease, earlier treatment regimens, the immune status of the patient, and the HLA-haplotype of the patient. Furthermore, the vaccine according to the present disclosure can contain individualized components, according to personal needs of the particular patient. Examples include varying the amounts of peptides according to the expression of the related antigen in the particular patient, unwanted side-effects due to personal allergies or other treatments, and adjustments for secondary treatments following a first round or scheme of treatment.

Antibodies are preferably administered to a subject in a pharmaceutically acceptable carrier. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of antibody being administered. The antibodies can be administered to the subject, patient, or cell by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular), or by other methods such as infusion that ensure its delivery to the bloodstream in an effective form. The antibodies may also be administered by intratumoural or peritumoural routes, to exert local as well as systemic therapeutic effects. Local or intravenous injection is preferred. Effective dosages and schedules for administering the antibodies may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage of antibodies that must be administered will vary depending on, for example, the subject that will receive the antibody, the route of administration, the particular type of antibody used and other drugs being administered. A typical daily dosage of the antibody used alone might range from about 1 pg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above. Following administration of an antibody, preferably for treating lung cancer (including NSCLC and SCLC), the efficacy of the therapeutic antibody can be assessed in various ways well known to the skilled practitioner. For instance, the size, number, and/or distribution of cancer in a subject receiving treatment may be monitored using standard tumour imaging techniques. A therapeutically-administered antibody that arrests tumour growth, results in tumour shrinkage, and/or prevents the development of new tumours, compared to the disease course that would occurs in the absence of antibody administration, is an efficacious antibody for treatment of cancer.

Immune effector cells may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL- 15, or other cytokines or cell populations. Briefly, pharmaceutical compositions may comprise immune effector cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions for use in the disclosed methods are in some embodimetns formulated for intravenous administration. Pharmaceutical compositions may be administered in any manner appropriate treat MM. The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

A pharmaceutical composition comprising the immune effector cells may be administered at a dosage of 10⁴ to 10⁹ cells/kg body weight, such as 10⁵ to 10⁶ cells/kg body weight, including all integer values within those ranges. Immune effector cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et ah, New Eng. J of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

In certain embodiments, it may be desired to administer activated immune effector cells to a subject and then subsequently re-draw blood (or have an apheresis performed), activate and expand the immune effector cells therefrom, and reinfuse the patient with these activated and expanded cells. This process can be carried out multiple times every few weeks. In certain embodiments, Immune effector cells can be activated from blood draws of from 10 cc to 400 cc. In certain embodiments, immune effector cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Using this multiple blood draw/multiple reinfusion protocol may serve to select out certain populations of immune effector cells.

The administration of any of the disclosed therapeutic agents may be carried out in any convenient manner, including by injection, transfusion, or implantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumourally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In some embodiments, the disclosed agents are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by i.v. injection. The compositions may also be injected directly into a tumour, lymph node, or site of infection.

In certain embodiments, CAR-modified immune effector cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to thalidomide, dexamethasone, bortezomib, and lenalidomide. In further embodiments, the CAR-modified immune effector cells may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. In some embodiments, the CAR-modified immune effector cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as 0KT3 or CAMPATH. In another embodiment, the cell compositions of the present disclosure are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in some embodiments, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the present disclosure. In an additional embodiment, expanded cells are administered before or following surgery. EXAMPLES

Example 1 - Materials and methods

PDX, Cell lines and Biopsy

For patient derived xenograft (PDX) tumour generation, de-identified fresh patient tumour specimens were obtained through the Victorian Cancer Biobank. The use of all human specimens was performed with ethical approval from the Peter MacCallum Cancer Center Human Research Ethics Committee (HREC) and the Alfred Hospital HREC. Freshly isolated or DMSO frozen patient melanoma cells were mixed with growth factor reduced Matrigel (Corning, NY, USA) in a 1:1 ratio and injected subcutaneously into NOD.Cg-Prkdcscid I12rgtmlWjl/SzJ (NSG) mice. NSG mice were obtained from Jackson Laboratory and both male and female mice were used. All mouse experiments were performed under protocols approved by the Alfred Research Alliance Animal Ethics Committee. PDX tumours were resected, weighed and snap frozen in liquid nitrogen. IHW9033 cells were obtained from ATCC and maintained in RPMI (ThermoFisher) media supplemented with 10% FBS in the absence of antibiotics. Cells were counted and subjected to lysis and affinity purification on the same day.

Affinity purification of HLA-peptide complexes

HLA-complexes from BLCLs (IHW9033), PDX and Biopsy material were affinity captured by immunoaffmity resin (Agarose-Protein A) containing w6/32 antibody following lysis in buffer containing 0.5% IGEPAL, 50 mM TIS pH 8.0, 150 mM NaCl, lx Protease inhibitor cocktail). The affinity capture resin was the transferred to a mobi-spin column and washed three times with 500 uL IX PBS by centrifugation. The HLA-peptide complexes were eluted using 10% acetic acid and heated to 70 C for lOmin. The mixture containing HLA heavy chains, antibody and peptides were subjected to a 5 KDa molecular weight cut off filter (MWCO) to separate peptides from HLA molecules and antibodies. The peptides were then neutralised using 1 M triethylammonium bicarbonate (TEAB) buffer to bring pH to 8 to enable TMT labelling. Individual samples were labelled following manufacturer’s instructions and quenched using 5% hydroxylamine. The samples were then combined and concentrated using Cl 8 column prior to centrifugal evaporation to reduce volume and ACN concentration. The peptides from this combined sample were reconstituted in 2% ACN, 0.1% formic acid prior to analysis by mass spectrometry. Mass spectrometry

TMT-labelled peptides were analysed using Orbitrap Tribrid Fusion mass spectrometer (Thermo Scientific) coupled with a RSLC nano-HPLC (Ultimate 3000, Thermo Scientific). Samples were loaded on to a 100 uM, 2 cm PepMaplOO trap column in 2% ACN, 0.1% formic acid at a flow rate of 15 ul/min. Peptides were then eluted at a flow rate of 250 ul/min with starting conditions of 98% Buffer A (0.1% formic acid) and 2% Buffer B (80% ACN, 0.1% formic acid) for 2 minutes; Buffer B was then elevated from 2% to 7.5% B over 1 min, followed by linear gradient from 7.5% to 37.5% B over 120 min, increasing to 42.5% B over 3 min, an additional increase to 99% B at the end of gradient for 6 min followed by reduction to 2% B to allow re-equilibration. The Orbitrap Fusion instrument was operated in a data-dependent acquisition mode utilising synchronous precursor selection (SPS) as described previously with modifications to allow efficient detection of HLA peptides: Survey full scan spectra ( m/z 380-1580) were acquired in the Orbitrap at 120,000 resolution at m/z 200 after accumulation of ions to a 4e5 target value with maximum injection time of 50 ms. Dynamic exclusion was set to 15 s. Ions with 2+ to 6 + charge states were selected for msms fragmentation and to enable collection of singly charged species of interest while eliminating noise, a decision tree was included to only fragment 1+ species above 800 m/z. In both cases, msms fragments were collected in orbitrap at 60,000 resolution with first mass set to 100 m/z, target of 2e5 ions, with maximum injection time of 120 ms. To quantitate the TMT-labelled peptides confidently and to reduce interference, synchronous precursor scans (10 scans) were performed on each msms spectra to select 10 peaks for further MS3 fragmentation and analysed in Orbitrap at 60,000 resolution.

Data analysis

TMT-MS3 LC-MS data was analysed using Peaks X (BSI) against human proteome (uniprot v03_2019) or translated exome sequencing data using PEAKS with the following search parameters: parent mass error tolerance for parent and fragment mass were set to 10 ppm and 0.02 Da respectively; digestion mode was set to unspecific, with TMT-10plex as fixed modification and Oxidation(M), Acetylation (N-term and K), as variable modifications (a maximum of 3 per sequence) with False Discovery Rate (FDR) of 1%. To enable assignment of MS3 reporter ion data to respective samples, PeaksQ quantitation was used with a tight quantification mass tolerance of 3.0 ppm utilising peptides that matched the FDR threshold of 1%. Reporter ion intensities were corrected for isotopic impurities according to lot-specific manufacturer specifications. Peptides and proteins identified were annotated with information from T-Antigen database (Olsen et al., 2017 Cancer Immunol Immunother 66:731-735)., CTA (Almeida et ah, 2009 Nucleic Acids Res 37:D816-9) and uniprot (UniProt, 2019 Nucleic Acids Res 47:D506-D515) to identify known antigens and epitopes. Example 2 - Identification of HLA-bound peptides from cell culture

To identify the level of sensitivity that could be achieved using the methods of the disclosure, varying amounts of IHW9033 B-Lymphoblastoid cells ranging from 5 x 10⁶ to 1000 cells were processed. The cell pellets were lysed, followed by micro-scale immunoaffmity purification of HLA molecules using antibodies specific for HLA-class I (w6/32 pan human class I specificity) and HLA-DR (LB3.1 with pan HLA-DR specificity). This modified HLA-peptide isolation method utilised small columns that were centrifuged to achieve rapid sample loading, washing and elution of the HLA- peptide complexes. The peptides from each sample were then separated from the HLA molecules and antibodies by ultrafiltration (5KDa MWCO filters), adjusted to pH 8 prior to being labelled using a single channel of TMT and the reaction subsequently quenched. The samples were then combined, reversed phase purified and then analysed on a Fusion Tribrid mass spectrometer using a method that takes advantage of synchronous precursor selection combined with MS3 (SPS-MS3) to reduce co-isolation and compression of TMT tag ratios (McAlister et al., 2014 Anal Chem 86:7150-8). A schematic illustrating the steps in this method is shown in Figure 1.

The TMT reporter ion intensity rapidly dropped as the cellular input decreased, however reliable signal for many peptides were still evident even with as few as 1000 cells (Figure 2A). With this level of cellular input, 691 HLA-bound peptides were identified at a false discovery rate (FDR) of 1%. These 691 peptides represent around 80% of the peptides that could be identified in the carrier peptide sample (derived from 5 x 10⁶ cells) which had 861 peptides that satisfied the 1% FDR cut-off criteria. Thus, the use of carrier peptides enabled identification of over 429 peptides that bound to HLA class I and 257 peptides bound to HLA-DR from just 1000 cells. The distribution of length was typical of HLA peptides identified and peptide sequences contain the expected binding motif of HLA molecules expressed in these cells (Figure 2B and Figure 2C).

Example 3 - Identification of HLA-bound peptides from a biopsy

To more accurately reflect a clinical situation, a biopsy from a HLA-A2+ melanoma patient was obtained along with a corresponding patient-derived xenograft (PDX; Figure 3A). The PDX was developed and expanded in nude mice to isolate the MHC -bound peptides to act as a source of carrier peptides for identifying MHC -bound peptides in the biopsy sample. About 400, 150 and 50 mg of PDX were used as carrier channels to determine HLA class I peptides from 19 mg or 1 mg of biopsy sample and each analysis performed in duplicate. The samples were then processed and labelled as described in Example 1 and analysed using mass spectrometry. Over 1257 HLA class I bound peptides, immunoprecipitated with the pan HLA class I mAh W6/32, could be identified in the lmg biopsy. This number of peptides compared well with the 1718 peptides contained in the carrier peptidome (derived from 400 mg PDX). The majority of peptides were 8-12 mers (Figure 3B). The peptides identified have the expected binding motif of the HLA molecules of the patient demonstrating the specificity of the protocol. Analysis of the overall intensities of the TMT label reporter ions from each channel show the expected distribution of intensities with the 400 mg of PDX carrier peptides yielding highest amount of peptide and the 1 mg biopsy the lowest (Fig 3C). Of note, some of the known epitopes were highly abundant in the biopsy, disproportional to the amount of material used when compared to the carrier PDX. This suggests that there may well be considerable differences in epitope densities on the tumour in situ compared to cell lines or even PDX-derived material. Moreover, while the majority of the peptides (80%) had HLA-binding rank (NetMHCpan4.0) of 1 or less, there were several ligands eluted directly from the patient that were within the percentile rank range of 2-5 that would have been missed if prediction software were used to shortlist candidate epitopes.

Analysis of the peptides identified in biopsy has shown expression of 10 known HLA-A2 restricted tumour epitopes listed in the T-Antigen database (Table 1) and an additional 70 peptides from proteins associated with melanoma and various types of cancer including Melanoma-associated antigens 1-3 (MAGE), L-dopachrome tautom erase (TYRP2), Preferentially expressed antigen of Melanoma (PRAME), Protein S100-A1/B (S10A1, S100B), Integrin alpha-5 (ITA5), and beta-catenin (CTNNB1). Additionally, 68 peptides from 46 proteins with keywords associated to melanoma in uniprot database were also identified in the biopsy.

Table 1 List of known tumour antigens identified in the melanoma biopsy

Example 4 - Identification of neoantigens

The transcriptome of the melanoma biopsy described above was studied using Next Generation sequencing (NGS; i.e., whole exome sequencing) to generate a reference proteome specific for the tumour containing variants and mutations. Searching the biopsy HLA-bound peptide mass spectrometry data against the exome sequencing protein database revealed presence of 11 neoantigen peptides spanning 7 mutations (some post- translationally modified; Table 2) including in EDEM1 (N401 S) which was verified using a synthetic peptide (Figure 3D). Identification of such neoantigens from high-resolution data can directly inform the generation of highly relevant targets for immunotherapy and highlights the utility of this approach for directly identifying neoantigens from small biopsies.

Table 2- Neoantigens identified in the melanoma biopsy

In summary, the method described herein was used to identify HLA-class I- and Il-bound peptides from scarce materials including clinical biopsy samples. With the ability to simultaneously analyse up to 10 (TMTllplex) and potentially 15 channels (TMT16 plex), rapid neoantigen discovery can be performed in a translational setting. By using a source of carrier peptides such as a HLA-matched PDX, patient derived cell lines or relevant labelled synthetic peptides, the procedure can be completed in two to three days enabling rapid and sensitive identification of epitopes derived from tumour associated antigens. In addition to relying on MHC -binding prediction alone, the experimental data obtained using the method described above, in combination with NGS, can directly pinpoint patient-specific neoantigens in order to develop precise personalised therapies.

Moreover, it is predicted that the method will be useful for studying peptidomes for other clinical samples where the amount of material is limiting including other types of biopsies taken for autoimmune indications or infection, or for rare cell types such as different APC subsets isolated directly ex vivo.

Example 5 - Identification of MHC -bound peptides isolated from a single cell

To determine the sensitivity of the methods described herein, MHC -bound peptides were isolated from a single cell, 100 cells and 10⁷ cells and were each labelled with a different TMT isobaric tag. In this case, the peptides isolated from the 10⁷ cell sample provided carrier peptides to determine if MHC -bound peptides isolated from a single cell or 100 cells could be identified. The carrier channel was labelled using TMT- 131, and the two samples were labelled with either TMT-126 (1 cell) or TMT-128 (100 cells). The mixture was subjected to mass spectrometry as described in Example 1 and the resulting data was analysed using Peaks X software to quantitate the amount of TMT tags. Analysis of HLA class I data successfully identified 348 peptides from a single cell, compared with 599 peptides from 100 cells and around 700 peptides in the carrier channel, at a false discovery rate of 1%. Of note, 332 peptides of the 348 peptides identified from the single cell have already been described in literature (Source: Immune Epitope Database; IEDB), which validates the identified peptides.

The HLA class I peptides identified were plotted on a heatmap (Figure 4), which depicts the intensity of TMT reporter ions with each peptide represented as a horizontal line, the colour scale corresponding to reporter ion intensity is depicted to right of the figure.

The HLA peptides identified were analysed using GIBBS clustering 2.0 to enable unsupervised clustering of the peptides. The anchor residues of the HLA-A3 and B7 match known motif for these HLA molecules (as described in NetMHC 4.0 motif viewer), further validating the identified peptides (Figure 5). Furthermore, the length distribution of the peptides from all three channels were similar and matched the expected length of HLA class I peptides (Figure 6). Example 6 - Identification of MHC -bound peptides isolated from synovial tissues from rheumatoid arthritis patients

Synovial tissues (individual core biopsy samples with wet weights of between 10- 70 mg) or 9033 B-lymphoblastoid cell pellets (le4 - le6 cells, used as a source of carrier peptides) were subjected to cryomilling, followed by lysis in lysis buffer (0.5% IGEPAL, 150 mM NaCl and 50 mM Tris). The tissue and cell lysates were subject to a pre-column containing protein-A resin to remove non-specific binders. The clarified lysates were then incubated serially with resin coupled to antibodies specific for HLA-DR (LB3.1), -DQ (SPVL-3), -DP (B721) or pan Class I (w6/32). After washing with PBS to remove unbound material, the antibody coupled resin was treated with 10% acetic acid to elute the HLA-peptide complexes. The peptides were separated from HLA molecules using molecular weight cut off filters and subject to Cl 8 reversed phase concentration prior to labelling with respective TMTs. The label was then quenched and removed using CIS- based tips. The samples were then pooled, subjected to centrifugal evaporation and reconstituted for Mass Spectrometric analysis as described herein. Data was collected on Thermo Scientific Orbitrap Fusion Mass spectrometer and analysed using PeaksX software.

All the samples were collected from rheumatoid arthritis patients (HLA- DRB1*04:01 positive) and HLA-DR peptides were of primary interest due to their disease association. Figure 7 shows clustermaps of all the peptides isolated from HLA- DR (Figure 7A), -DQ (Figure 7B), -DP (Figure 7C) or pan Class I (Figure 7D). The mass spectrometry data revealed the presence of 316 HLA-DR-bound peptides (Figure 7 A) of which 145 peptides were classified as strong binders (Table 3) and 43 as weak binders to HLA-DRB 1 *04:01 per netMHCpanll algorithm. In addition to HLA-DR peptides, HLA- DQ, -DP and class I bound peptides were identified in parallel (Figure 7 B, C, D).

Table 3- HLA-DR bound peptides identified from RA synovial tissue that were classified as strong binders ofDRBl *04:01 by netMHCIIpan (EL Rank <2% )

Peptide Protein names %Rank_EL SEQ ID NO

AERYLTISSLQSEDEA Immunoglobulin lambda variable 4- 0.07 18

69

GAERYLT1SSLQSEDEA Immunoglobulin lambda variable 4- 0.1 19

69

AERYLT1SSLQSEDE Immunoglobulin lambda variable 4- 0.04 20

69

GAERYLT1SSLQSEDE Immunoglobulin lambda variable 4- 0.05 21

69

VDQYFYG1KND1QHAG Lysosomal alpha-mannosidase 0.14 22

VPGTYKITASARGYNPV Carboxypeptidase D 0.02 23 Peptide Protein names %Rank_EL SEQ ID NO

EPGEPEFKYIGNMHGNEV Carboxypeptidase D 0.2 24

VG

IRMFTTAPDQ VDKED Ceruloplasmin 0.55 25

NSIFFELEADEREPT Angiotensinogen 1.72 26

ISKYELDKAFSDRN Complement C3 0.38 27

SNQIKILGNQGSFL T-cell surface glycoprotein CD4 0.79 28

NSLYLQMNSLRAEDT Immunoglobulin heavy variable 3-7 0.06 29

DNAKNSLYLQMNSLRAED Immunoglobulin heavy variable 3-7 0.85 30

TA

KNSLYLQMNSLRAEDT Immunoglobulin heavy variable 3-7 0.07 31 KNSL YLQMNSLRAEDTA Immunoglobulin heavy variable 3-7 0.13 32 NSLYLQMNSLRAEDTA Immunoglobulin heavy variable 3-7 0.1 33 SLYLQMNSLRAEDT Immunoglobulin heavy variable 3-7 0.15 34 NSLYLQMNSLRAED Immunoglobulin heavy variable 3-7 0.04 35

VDDTQFVRFDSDAASPRE HLA class I histocompatibility 0.07 36

EPRAP antigen, B-7 alpha chain

DDTQFVRFDSDAASPREE HLA class I histocompatibility 0.02 37

P antigen, B-7 alpha chain

DTQFVRFDSDAASPREEP HLA class I histocompatibility 0 38 antigen, B-7 alpha chain

VDDTQFVRFDSDAASPRE HLA class I histocompatibility 0.03 39

EP antigen, B-7 alpha chain

VDDTQFVRFDSDAASPRE HLA class I histocompatibility 0.08 40

EPR antigen, B-7 alpha chain

DTQFVRFDSDAASPREEP HLA class I histocompatibility 0.03 41

R antigen, B-7 alpha chain

DDTQFVRFDSDAASPREE HLA class I histocompatibility 0.05 42

PR antigen, B-7 alpha chain

VDDTQFVRFDSDAASPRE HLA class I histocompatibility 0 43

E antigen, B-7 alpha chain

DTQFVRFDSDAASPREE HLA class I histocompatibility 0 44 antigen, B-7 alpha chain

DDTQFVRFDSDAASPRE HLA class I histocompatibility 0 45 antigen, B-7 alpha chain

TQFVRFDSDAASPREEPR HLA class I histocompatibility 0.06 46 antigen, B-7 alpha chain

TQFVRFDSDAASPREEP HLA class I histocompatibility 0.02 47 antigen, B-7 alpha chain

DTQFVRFDSDAASPRE HLA class I histocompatibility 0 48 antigen, B-7 alpha chain

VDDTQFVRFDSDAASPRE HLA class I histocompatibility 0.06 49

EPRAPWIE antigen, B-7 alpha chain

ALANIA VDKANLEIMT HLA class II histocompatibility 0.56 50 antigen, DR alpha chain

LANIA VDKANLEIM HLA class II histocompatibility 0.47 51 antigen, DR alpha chain

LANIA VDKANLEIMT HLA class II histocompatibility 0.54 52 antigen, DR alpha chain

ANIA VDKANLEIMT HLA class II histocompatibility 0.58 53 antigen, DR alpha chain

VPPEWKALTDMPQMR Fibrinogen alpha chain 0.2 54

VPPEWKALTDMPQ Fibrinogen alpha chain 1.78 55 Peptide Protein names %Rank_EL SEQ ID NO

GNEKIHLISTQSAIPY Fibrinogen gamma chain 0.25 57

GNEK1HLISTQSAIPYA Fibrinogen gamma chain 0.45 58

EKIHLISTQSAIPY Fibrinogen gamma chain 0.58 59

WLGNEK1HLISTQSAIPYA Fibrinogen gamma chain 1.06 60

NEKIHLISTQSAIPY Fibrinogen gamma chain 0.22 61

FDHVITNMNNNYEPR Complement Clq subcomponent 0.59 62 subunit B

FDHVITNMNNNYEPRS Complement Clq subcomponent 0.6 63 subunit B

DHVITNMNNNYEPR Complement Clq subcomponent 0.58 64 subunit B

HVITNMNNNYEPR Complement Clq subcomponent 1.44 65 subunit B

RFDHVITNMNNNYEPR Complement Clq subcomponent 0.99 66 subunit B ^.

YDLRHTFMGWSLGSPSG Alpha-2-HS-glycoprotein 2 67

LRHTFMGWSLGSPS Alpha-2-HS-glycoprotein 0.99 68

DLRHTFMGWSLGSPS Alpha-2 -HS-glycoprotein 1.65 69

HPVTGQFLYQDSNWASKV Transferrin receptor protein 1 0.25 70

E

TGQFLYQDSNWASKVE Transferrin receptor protein 1 0.06 71

LQTFLDDASPGDKR ApolipoproteinB-100 1.24 72

SASYKADTVAKVQG ApolipoproteinB-100 0.03 73

VDDTQFVRFDSDAASQRM HLA class I histocompatibility 0.15 74

EPR antigen, A-3 alpha chain

DTQFVRFDSDAASQRMEP HLA class I histocompatibility 0.02 75 antigen, A-3 alpha chain

VDDTQFVRFDSDAASQRM HLA class I histocompatibility 0.13 76

EPRAP antigen, A-3 alpha chain

DDTQFVRFDSDAASQRME HLA class I histocompatibility 0.04 77

P antigen, A-3 alpha chain

VDDTQFVRFDSDAASQRM HLA class I histocompatibility 0.02 78

E antigen, A-3 alpha chain

VDDTQFVRFDSDAASQRM HLA class I histocompatibility 0.07 79

EP antigen, A-3 alpha chain

VDDTQFVRFDSDAASQR HLA class I histocompatibility 0 80 antigen, A-3 alpha chain

DDTQFVRFDSDAASQRME HLA class I histocompatibility 0 81 antigen, A-3 alpha chain

DTQFVRFDSDAASQRMEP HLA class I histocompatibility 0.4 82

RAP antigen, A-3 alpha chain

DTQFVRFDSDAASQRM HLA class I histocompatibility 0 83 antigen, A-3 alpha chain

DTQFVRFDSDAASQR I II. L class I histocompatibility 0 84 antigen, A-3 alpha chain

DDTQFVRFDSDAASQR HLA class I histocompatibility 0 85 antigen, A-3 alpha chain

DTQFVRFDSDAASQRME HLA class I histocompatibility 0 86 antigen, A-3 alpha chain

TQFVRFDSDAASQRMEP HLA class I histocompatibility 0.04 87 antigen, A-3 alpha chain

TQFVRFDSDAASQRME HLA class I histocompatibility 0.01 88 antigen, A-3 alpha chain

VDDTQFVRFDSDAASQRM HLA class I histocompatibility 0 89 antigen, A-3 alpha chain

TQFVRFDSDAASQR HLA class I histocompatibility 0 90 antigen, A-3 alpha chain

DTQFVRFDSDAASQRMEP HLA class I histocompatibility 0.07 91

R antigen, A-3 alpha chain

TQFVRFDSDAASQRM HLA class I histocompatibility 0 92 antigen, A-3 alpha chain

DTQFVRFDSDAASQ HLA class I histocompatibility 0 93 antigen, A-3 alpha chain

TQFVRFDSDAASQ HLA class I histocompatibility 0 94 antigen, A-3 alpha chain

DDTQFVRFDSDAASQRME HLA class I histocompatibility 0.1 95

PR antigen, A-3 alpha chain

VDDTQFVRFDSDAASQRM HLA class I histocompatibility 0.11 96

EPRAPWIE antigen, A-3 alpha chain

KIGRFVIEEVPGELM Gelsolin 0.69 97

IGRFVIEEVPGELM Gelsolin 0.5 98 NIFSFYLSRDPDA QPGGE CathepsinD 1.42 99 YNSYSVSNSEKDIMA Cathepsin B 1.78 100 RPKDYEVDA TLKSLNN Collagen alpha-2 0 101 IADA WNAIPDNLDA 72 kDa type IV collagenase 1.33 102 LPGQLKPFETLLSQNQ Glutathione S-transferase P 0.84 103 HREEL VYELNPLDHRGR Complement C4-B 1.02 104

VDDTQFVRFDSDAASPRG HLA class I histocompatibility 0.07 105

EPRAP antigen, Cw-7 alpha chain

VDDTQFVRFDSDAASPRG HLA class I histocompatibility 0.08 106

EPR antigen, Cw-7 alpha chain

DDTQFVRFDSDAASPRGE HLA class I histocompatibility 0.05 107

PR antigen, Cw-7 alpha chain

DTQFVRFDSDAASPRGEP HLA class I histocompatibility 0.03 108

R antigen, Cw-7 alpha chain

DTQFVRFDSDAASPRGEP HLA class I histocompatibility 0 109 antigen, Cw-7 alpha chain

VDDTQFVRFDSDAASPRG HLA class I histocompatibility 0.03 110

EP antigen, Cw-7 alpha chain

DDTQFVRFDSDAASPRGE HLA class I histocompatibility 0.01 111

P antigen, Cw-7 alpha chain

DTQFVRFDSDAASPRG HLA class I histocompatibility 0 112 antigen, Cw-7 alpha chain

VDDTQFVRFDSDAASPRG HLA class I histocompatibility 0.06 113

EPRAPWVE antigen, Cw-7 alpha chain

DDTQFVRFDSDAASPRGE HLA class I histocompatibility 0.11 114

PRAP antigen, Cw-7 alpha chain

DTQFVRFDSDAASPRGEP HLA class I histocompatibility 0.24 115

RAP antigen, Cw-7 alpha chain

TQFVRFDSDAASPRGEPR HLA class I histocompatibility 0.06 116 antigen, Cw-7 alpha chain

SDNELQEMSNQGSK Clusterin 0.72 117

SDNELQEMSNQGSKY Clusterin 0.47 118 NVLRIINEPTAAAIA YG Heat shock cognate 71 kDa protein 0.13 119 VLR1INEPTAAAIA YG Heat shock cognate 71 kDa protein 0.1 120 ERAMTKDNNLLGKFE Heat shock cognate 71 kDa protein 0.07 121 VDDTQFVRFDNDAASPR HLA class I histocompatibility 0 122 antigen, alpha chain E

DTQFVRFDNDAASPR HLA class I histocompatibility 0 123 antigen, alpha chain E

VDDTQFVRFDNDAASPRM HLA class I histocompatibility 0.13 124

VPR antigen, alpha chain E

DLRSWTA VDTAAQISE HLA class I histocompatibility 0.04 125 antigen, alpha chain E

KPPQYIA VHWPDQ Macrophage migration inhibitory 0.53 126 factor

SDVDLIPMNDHNA YR Beta-l,4-galactosyltransferase 1 0.56 127

TTGNYR1ESVLSSSGKR Ganglioside GM2 activator 0.04 128

TTGNYRIESVLSSSGK Ganglioside GM2 activator 0.02 129

TGNYR1ESVLSSSGK Ganglioside GM2 activator 0.02 130

LANIAILNNNLNTL HLA class II histocompatibility 1.39 131 antigen, DP alpha 1 chain

VPKEISPDTTLLDLQN Biglycan 0.93 132

TTAFQYIIDNKGIDSD Cathepsin S 0.03 133

ENLENYYLEVNQLEKFD Lumican 0.79 134

NLENYYLEVNQLEKFD Lumican 0.51 135

LENYYLEVNQLEK Lumican 0.56 136

LENYYLEVNQLEKFD Lumican 0.42 137

ENLENYYLEVNQLEK Lumican 1.33 138

YDHNFVKAINAIQKSW Dipeptidyl peptidase 1 0.05 139

AIFLFVDKTVPQSSL Gamma-aminobutyric acid receptor- 0.13 140 associated protein-like 2

YLL YYTEFTPTEKDE Beta-2-microglobulin 0.86 141 LLYYTEFTPTEKDE Beta-2 -microglobulin 1.21 142 YLL YYTEFTPTEKDEY Beta-2-microglobulin 1.39 143 YLL YYTEFTPTEKD Beta-2 -microglobulin 1.09 144 FYLLYYTEFTPTEKDE Beta-2 -microglobulin 1.04 145 LLYYTEFTPTEKD Beta-2-microglobulin 1.51 146 LL YYTEFTPTEKDEY Beta-2 -microglobulin 1.82 147 YLLYYTEFTPTEK Beta-2 -microglobulin 1.93 148 VIHIRSETSVPDH Interferon-induced transmembrane 0.89 149 protein 3

TVIHIRSETSVPDH interferon-induced transmembrane 0.98 150 protein 3

EGLFVADVTDFEG Galectin-3 -binding protein 0.86 151

WLPSYEEALSLPSKTPEG Lysosomal-associated 0.9 152 transmembrane protein 5

LPSYEEALSLPSKTPEG Lysosomal-associated 0.52 153 transmembrane protein 5 Peptide Protein names %Rank_EL SEQ ID NO

. transmembrane protein 5

VLPSYEEALSLPSKTPE Lysosomal-associated 0.31 155 transmembrane protein 5

WLPSYEEALSLPSKTPE Lysosomal-associated 0.46 156 transmembrane protein 5

VLPSYEEALSLPSKTPEG Lysosomal-associated 0.6 157 transmembrane protein 5

LPSYEEALSLPSKTP Lysosomal-associated 0.14 158 transmembrane protein 5

VTQEIVTERSVSSRQAQ Desmoglein-2 0.14 159 SEfPR YYISANVTGFK Ceramide glucosyltransferase 0.06 160 LDFLKA VDTNRASVG Plexin domain-containing protein 2 0.13 161 FPKPPSYNVA TTLPSYDE NEDD4 family -interacting protein 1 0 162

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the disclosure as shown in the specific embodiments without departing from the spirit or scope of the disclosure as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

All publications cited herein are hereby incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

Claims

1. A method of identifying one or more major histocompatibility complex (MHC)- bound peptides in a sample, the method comprising: a) obtaining sample peptides isolated from MHC molecules in the sample, b) labelling the sample peptides with an isobaric label; c) mixing the labelled sample peptides with labelled carrier peptides to form a mixture, wherein the carrier peptides are labelled with a different isobaric label to the sample peptides; and d) performing mass spectrometry on the mixture to identify one or more of the sample peptides.

2. The method of claim 1, wherein the sample peptides are isolated from MHC molecules by (i) first isolating MHC-peptide complexes from the sample and (ii) subsequently separating the sample peptides from the MHC molecules.

3. The method of claim 2, wherein the MHC-peptide complexes are isolated from the sample by immunoprecipitation from a lysate of cells or tissue.

4. The method of claim 3, wherein the MHC-peptide complexes are immunoprecipitated using an anti-MHC antibody that is bound to a solid substrate.

5. The method of claim 4, wherein the anti-MHC antibody is noncovalently bound to the solid substrate.

6. The method of claim 4 or claim 5, wherein the anti-MHC antibody is a pan anti- MHC class I antibody or a pan anti-MHC class II antibody.

7. The method of claim 5 or claim 6, wherein the immunoprecipitated MHC- peptide complexes are eluted from the solid substrate using an acidic solution, thereby producing an eluate comprising the sample peptides and MHC molecules.

8. The method of claim 7, wherein the anti-MHC antibody is eluted from the solid substrate by the acidic solution, thereby producing an eluate comprising the sample peptides, the MHC molecules, and the anti-MHC antibody.

9. The method of claim 7 or claim 8, further comprising heating the eluate to a temperature in the range of 40°C to 100°C.

10. The method of any one of claims 2 to 9, wherein the sample peptides are separated from the MHC molecules by ultrafiltration.

11. The method of claim 10, wherein ultrafiltration is performed using a filter having a molecular weight cut off in the range of 1 kDa to 10 kDa.

12. The method of any one of claims 1 to 11, wherein the isobaric labels are tandem mass tag (TMT) or isobaric tags for absolute and relative quantification (iTRAQ) labels.

13. The method of any one of claims 1 to 12, wherein the method comprises identifying one or more MHC -bound peptides in multiple samples, wherein sample peptides from each sample are labelled with different isobaric labels, and wherein the mixture comprises sample peptides from each of the samples and the carrier peptides.

14. The method of any one of claims 1 to 13, wherein, the carrier peptides are: a) isolated from MHC molecules in a patient derived xenograft (PDX) sample; b) isolated from MHC molecules in cultured cells; c) synthetic peptides which are predicted to bind to MHC molecules; or d) synthetic peptides known to bind MHC molecules.

15. The method of claim 14, wherein the cultured cells are MHC-matched to the sample.

16. The method of claim 14 or claim 15, wherein the sample is obtained from a subject and the cultured cells are cells obtained from the subject and cultured ex vivo.

17. The method of claim 14, wherein the sample is obtained from a subject and the synthetic peptides which are predicted to bind to MHC are derived from proteomics data or translated whole genome sequencing (WGS), whole exome sequencing (WES), or RNA sequencing (RNAseq) data obtained from DNA or RNA from the subject.

18. The method of claim 17, wherein the sample is a biopsy and the DNA or RNA is from cells in the biopsy.

19. The method of claim 14, wherein the sample is obtained from a subject and the carrier peptides are isolated from soluble peptide-MHC complexes in serum of the subject.

20. The method of any one of claims 1 to 19, wherein the sample is a biopsy sample, a tissue sample, or cultured cell sample.

21. The method of claim 20, wherein the biopsy sample is a tissue biopsy sample or a liquid biopsy sample.

22. The method of claim 20 or claim 21, wherein the biopsy sample is a tumour biopsy.

23. The method of claim 22, wherein the tumour biopsy is a melanoma biopsy.

24. The method of any one of claims 1 to 23, wherein the sample contains no more than about 50 mg, no more than about 20 mg, no more than about 10 mg, no more than about 5 mg, or no more than about 2 mg of tissue.

25. The method of any one of claims 1 to 24, wherein the sample contains no more than about 10⁶, no more than about 10⁵, no more than about 10⁴, no more than about 5000, no more than about 2000, no more than about 1000, no more than about 500, no more than about 200, or no more than about 100 cells.

26. The method of any one of claims 1 to 25, further comprising the step of isolating the sample peptides from MHC molecules in the sample.

27. The method of claim 26, wherein the method comprises isolating the sample peptides as described in any one of claims 2 to 11.

28. The method of any one of claims 1 to 27, wherein the one or more sample peptides comprises a neoantigen.

29. The method of any one of claims 1 to 28, wherein identification of the one or more sample peptides comprises comparing the mass spectrometry data with a database of known protein sequences or de novo sequencing the sample peptides from the mass spectrometry data.

30. The method of claim 29, wherein the database of known protein sequences comprises amino acid sequences of proteins from a reference proteome.

31. The method of claim 29, wherein the database of known protein sequences comprises amino acid sequences from proteomics data or translated WGS, WES, or RNAseq data obtained from DNA or RNA from one or more subjects.

32. The method of claim 31, wherein the sample is a biopsy from the subject and the DNA or RNA is from cells in the biopsy.

33. The method of anyone of claims 1 to 32, further comprising quantifying the identified one or more sample peptides.

34. The method of any one of claims 1 to 33, wherein the mass spectrometry comprises three rounds of mass spectrometry analysis (MS3).

35. The method of claim 34, wherein MS3 is performed using synchronous precursor selection (SPS).

36. The method of claim 34 or claim 35, wherein the identified sample peptides are quantified based on the intensity of reporter ion peaks of the isobaric labels in the MS3 spectra.

37. The method of any one of claims 1 to 36, wherein the mass spectrometry is preceded by liquid chromatography.

38. A method of validating a candidate peptide antigen or biomarker, the method comprising: a) obtaining a sample from a subject; b) identifying one or more MHC -bound peptides in the sample according to the method of any one of claims 1 to 37; and c) verifying if the candidate peptide antigen or biomarker is present in the peptide sample.

39. A method of identifying a candidate peptide antigen or biomarker, the method comprising: a) obtaining a sample from a subject; b) identifying one or more MHC -bound peptides in the sample according to the method of any one of claims 1 to 37.

40. A method of detecting the presence or absence of a peptide antigen or biomarker in a sample, the method comprising: a) obtaining a sample from a subject; b) identifying one or more MHC -bound peptides in the sample according to the method of any one of claims 1 to 37; and b) determining if the peptide antigen or biomarker is present or absent in the peptide sample.

41. A method of diagnosis, prognosis and/or evaluation of treatment efficacy in a subject in need thereof, the method comprising detecting the presence or absence of a peptide antigen or biomarker according to the method of claim 40.

42. A method of selecting a therapy for treatment of a disease or condition in a subject, the method comprising: a) obtaining a sample from the subject; and b) identifying one or more MHC -bound peptides in the sample according to the method of any one of claims 1 to 37, wherein the therapy is selected based on the one or more sample peptides identified.

43. The method of claim 42, wherein the therapy is an immunotherapy.

44. The method of claim 43, wherein the immunotherapy comprises: a) a vaccine comprising at least one of the sample peptides identified; b) an antibody which binds to at least one of the sample peptides identified; or c) an immune effector cell which targets at least one of the sample peptides identified.

45. A method of treatment of a disease or condition in a subject, the method comprising: a) selecting a therapy according to the method of any one of claims 42 to 44, and b) administering to the subject the selected therapy.

46. The method of claim 45, wherein the disease or condition is a cancer, an autoimmune disease, an inflammatory disease, or an infection.

47. A method of isolating peptides from MHC molecules in a sample for identification by mass spectrometry, wherein the sample contains no more than 10⁶ cells or no more than 50 mg of tissue, the method comprising: a) obtaining a lysate of cells or tissue in the sample; b) immunoprecipitating MHC -peptide complexes from the lysate with an anti-

MHC antibody; and c) separating the peptides from the MHC molecules using ultrafiltration, thereby isolating the peptides from the MHC in the sample.