WO2019008365A1

WO2019008365A1 - Method for treating cancer by targeting a frameshift indel neoantigen

Info

Publication number: WO2019008365A1
Application number: PCT/GB2018/051893
Authority: WO
Inventors: Charles Swanton; Samra TURAJLIC; Kevin LITCHFIELD
Original assignee: The Francis Crick Institute Limited
Priority date: 2017-07-05
Filing date: 2018-07-04
Publication date: 2019-01-10
Also published as: GB201710812D0

Abstract

The present invention relates to amethod of treating or preventing cancer in a subject, comprising targeting an expressed frameshift expressed frameshift indel neo-antigen.an expressed frameshift indel mutation.

Description

METHOD FOR TREATING CANCER BY TARGETING A FRAMESHIFT INDEL NEOANTIGEN

FIELD OF THE INVENTION

The present invention relates to a method for treating cancer in a subject, comprising targeting a neoantigen that is the result of an expressed frameshift indel mutation.

BACKGROUND

The present invention is in the field of cancer immunotherapy. Cancer immunotherapy involves the use of a subject's own immune system to treat or prevent cancer. Immunotherapies exploit the fact that cancer cells often have subtly different molecules on their surface that can be detected by the immune system. These molecules, or cancer antigens, are most commonly proteins, but also include molecules such as carbohydrates. Immunotherapy thus involves provocation of the immune system into attacking tumor cells via these target antigens.

SUMMARY OF THE INVENTION

The present inventors have found that, surprisingly, indel mutations that lead to neoantigens may represent useful targets when designing immunotherapies for the treatment of cancer.

As described in the present Examples, the present inventions have found that frame shift insertion/deletions (fs-indels) represent an infrequent (pan-cancer median = 4 per tumor) but a highly immunogenic subset of somatic variants. Fs-indels can produce an increased abundance of tumor specific neoantigens with greater mutant-binding specificity. However, fs-indels cause premature termination codons (PTCs) and are susceptible to degradation at the messenger RNA level through the process of non-sense mediated decay (NMD). NMD normally functions as a surveillance pathway to protect eukaryotic cells from the toxic accumulation of truncated proteins. The present inventors have found that a subset of fs- indels escape NMD degradation, which when translated contribute substantially to directing anti-tumor immunity, and therefore represent a target for design of immunotherapies.

An "indel mutation" as referred to herein refers to an insertion and/or deletion of bases in a nucleotide sequence (e.g. DNA or RNA) of an organism. Typically, the indel mutation occurs in the DNA, preferably the genomic DNA, of an organism. Suitably, the indel mutation occurs in the genomic DNA of a tumor cell in the subject. Suitably, the indel may be an l insertion mutation. Suitably, the indel may be a deletion mutation. In a preferred aspect of the invention as described herein the indel mutation is a frameshift indel mutation.

Suitably, the indel may be from 1 to 100 bases, for example 1 to 90, 1 to 50, 1 to 23 or 1 to 10 bases.

The invention provides an agent which targets a neoantigen resulting from an expressed frameshift indel mutation for use in the treatment or prevention of cancer in a subject.

The invention also provides other aspects related to this utility, as discussed herein. DESCRIPTION OF THE FIGURES

Figure 1 : (a) Kidney cancers have the highest pan-can indel proportion. Plotted is the proporption of mutations which are indels (i.e. # indels / (#indels + #SNVs), across 19 solid tumor types form TCGA. The last two boxplots are additional independent renal cell carcinoma replication datasets. Statistical association is calculated based on the KIRC cohort compared to all other non-kidney TCGA samples, (b) Kidney cancers have the highest pan-can indel count. Plotted is the absolute count of indel mutations across 19 solid tumor types form TCGA. The last two boxplots are additional independent renal cell carcinoma replication datasets. Statistical association is calculated based on the KIRC cohort compared to all other non-kidney TCGA samples.

Figure 2: Recurrent genes with frameshift indel neoantigens, across the all patients in TCGA pan-cancer cohort. Ploted on the X-axis are the number of unique samples containing a frameshift indel neoantigen, and on the Y-axis are the number of unique neoantigens (i.e. each mutation can generate multiple neoantigens). Marked are genes either mutated in > 30 samples or with >80 neoantigens.

Figure 3: Tumor specific neoantigen counts by cancer type. The first panel plots the count of snv derived neoantigens, second panel is the count of frameshift indel derived neoantigens, third is the count of mutant only neoantigen binders, fourth is the proportion of neantigens derived from SNVs/lndels, fifth is the proportion of neoantigens where mutant allele only binds and last are pie charts presenting the proportion of samples with more or less than 5 mutant only neoantigen binders. The first 3 panels are ordered by median value, from lowest (left) to highest (right). Panels four and five are ordered the same as panel three. Figure 4: (a) Non-synonymous SNV mutation burden (first), in-frame indelburden (second) and frameshift indel burden (third) are split by response to checkpoint inhibitor therapy across Hugo et al., Snyder et al., and Van Allen et al. melanoma cohorts, (b) Checkpoint inhibitor patient response rates based on non-synonymous SNV mutation burden (top), in- frame indel burden (middle) and frameshift inde burden (bottom). Patients are split into high (upper quartile) and low (bottom 3 quartiles) groups for each measure. Analysis presented for Hugo et al., Snyder et al., and Van Allen et al. melanoma cohorts.

Figure 5: Immune gene signatures were compared in ccRCCpatients based on i) frameshift indel neoantigen count (fs-indel-NeoAtgs), ii) in-frame indel mutation count (if-indel- mutations) and iii) nonsynonymousSNV neoantigen count (ns-snv-NeoAtg). Left: Percentage change in median signature expression (FPKM-Upper Quartile normalised) is shown, between high and low groups, for i), ii) and iii). Several pathways were found to be exclusively up-regulated in the high fs-indel-NeoAtggroup. Right: Correlation analysis within the high fs-indel-NeoAtggroup demonstrated the CD8+ T Cell signature was strong correlated with both MHC Class I antigen presentation genes and Cytolytic activity.

Figure 6: Non-synonymous SNV mutation burden (first), in-frame indel burden (second), frameshift indel burden (third) and clonal frameshift indel burden (fourth) are split by response to checkpoint inhibitor therapy in the Snyder et al., melanoma cohort.

Figure 7: Panel A shows an overview of study design and methodological approach. The left hand side of the panel shows a fs-indel triggered premature termination codon, which falls in a middle exon of the gene, a position associated with efficient non-sense mediated decay (NMD). The right hand side of the panel shows a fs-indel triggered premature termination codon, which falls in the last exon of the gene, a position associated with bypassing NMD. Panel B shows the odds ratio (OR), between expressed fs-indels and non-expressed fs- indels, for falling into either first, middle, penultimate or last exon positions. Odds ratios and associated p-values were calculated using Fisher's Exact Test. Coloring is used arbitrarily to distinguish groups. Error bars denote 95% confidence intervals of OR estimates. Panel C shows variant allele frequencies for expressed fs-indels by exon group position. Kruskal- Wallis test was used to test for a difference in distribution between groups. Panel D shows protein expression levels for non-expressed, versus expressed, fs-indel mutations. Two- sided Mann Whitney U test was used to assess for a difference between groups. Figure 8: Panel A shows three melanoma checkpoint inhibitor (CPI) treated cohorts, split into groups based on "no-clinical benefit" or "clinical benefit" to therapy. Three metrics are displayed per cohort: (top row) TMB non-synonymous SNV count, (middle row) frameshift indel count and (bottom row) NMD-escape mutation count. In the first column is the Van Allen et al. anti-CTLA4 cohort, middle column is the Snyder et al. et al. anti-CTLA4 cohort, and the last column is the Hugo et al. anti-PD1 cohort. Far right are meta-analysis p-values, for each metric across the three cohorts, showing the association with clinical benefit from CPI treatment. Two-sided Mann Whitney U test was used to assess for a difference between groups. Meta-analysis of results across cohorts was conducted using the Fisher method of combining P values from independent tests. Panel B shows the % of patient with clinical benefit from CPI therapy, for patients with => 1 NMD-escape mutation and zero NMD- escape mutations. Panel C shows the same three metrics, compared in an adoptive cell therapy treated cohort.

Figure 9: Shows the exonic positions of fs-indels, experimentally tested for T cell reactivity in personalized vaccine and CPI studies, which were found to either be a) T cell reactive (left hand column) or b) T cell non-reactive (right hand column). Where the fs-indel mutation fell into an exonic position (first, penultimate or last) associated with NMD-escape the transcript was colored dark blue; where the fs-indel fell in an exonic position (middle) associated with NMD-competence the transcript was coloured light blue. In grey line bars the overall proportion of fs-indels falling into an NMD-escape exon position, for T cell reactive and T cell non-reactive groups, is shown. P-value is calculated using a Fisher's Exact Test.

Figure 10: Panel A shows selection analysis for fs-indels, as benchmarked against functionally equivalent SNV stop-gain mutations. The odds ratio for a fs-indel (compared to SNV stop-gains), to fall into each exon position group is shown. Odds ratios and associated p-values were calculated using Fisher's Exact Test. Coloring is used arbitrarily to distinguish groups. Error bars denote 95% confidence intervals of OR estimates. Panel B shows overall survival Kaplan-Meir plots are shown for TCGA SKCM (left) and MSI (right) cohorts. Overall survival analysis was conducted using a Cox proportional hazards model.

Figure 11 : Data shows three melanoma checkpoint inhibitor (CPI) treated cohorts, split into groups based on "no-clinical benefit" (light blue) or "clinical benefit" (dark blue) to therapy, with expressed nsSNV mutation count (detected using allele specific RNAseq) tested for association. In the first column is the Van Allen et al. anti-CTLA4 cohort, middle column is the Snyder et al. et al. anti-CTLA4 cohort, and the last column is the Hugo et al. anti-PD1 cohort.

DETAILED DESCRIPTION

It is proposed that indel mutations result in mutagenic antigenic peptides that may be highly distinct from self antigens, and hence be a source of T cell reactive tumor specific neoantigens, due to both an increased number of mutant peptides and reduced susceptibility to self-tolerance mechanisms. In addition, indel mutations - particularly frameshift mutations - generate an increased number of neoantigens per mutation compared to SNV mutations. The present Examples show that indel mutations are associated with anti-tumor immunogenic responses. The present invention relates to the concept of targeting indel mutations, for example when designing immunotherapies for the treatment of cancer.

IDENTIFYING A NEOANTIGEN RESULTING FROM AN EXPRESSED FRAMESHIFT INDEL MUTATION

In one aspect the present invention provides a method for identifying in a tumor from a subject a neoantigen resulting from an expressed frameshift indel mutation, said method comprising the following steps: i) determining mutations present in a sample isolated from the tumor; and

ii) identifying an expressed frameshift indel mutation; and

iii) identifying an expressed frameshift expressed frameshift indel neo-antigen, which is an antigen encoded by a sequence which comprises the expressed frameshift indel mutation.

In one aspect, mutations may be identified in more than one sample. The presence of mutations in cancer cells from one or more tumor regions isolated from a tumor may be determined.

For example, the method may comprise determining the mutations present in at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten or more biopsies isolated from a tumor. The method can also be used to determine mutations in one biopsy.

The individual tumor samples may be isolated from different regions located throughout a tumor within a primary site or between primary and metastases or within a metastasis or between metastases. For example, determining the mutations present in tumors which are known to display morphological disparate histology in different regions may involve determining the mutations present in a number of individual samples isolated from morphologically disparate regions.

A "mutation" refers to a difference in a nucleotide sequence (e.g. DNA or RNA) in a tumor cell compared to a healthy cell from the same individual. The difference in the nucleotide sequence can result in the expression of a protein which is not expressed by a healthy cell (e.g. a non-cancer cell) from the same individual and/or the presentation of 'non-self peptides by MHC class I molecules expressed by the tumor cell.

Indel mutations may be identified by Exome sequencing, RNA-seq, whole genome sequencing and/or targeted gene panel sequencing and or routine Sanger sequencing of single genes. Suitable methods are known in the art.

Descriptions of Exome sequencing and RNA-seq are provided by Boa et al. (Cancer Informatics. 2014; 13(Suppl 2):67-82.) and Ares et al. (Cold Spring Harb Protoc. 2014 Nov 3;2014(1 1):1 139-48); respectively. Descriptions of targeted gene panel sequencing can be found in, for example, Kammermeier et al. (J Med Genet. 2014 Nov; 51 (1 1):748-55) and Yap KL et al. (Clin Cancer Res. 2014. 20:6605). See also Meyerson et al., Nat. Rev. Genetics, 2010 and Mardis, Annu Rev Anal Chem, 2013. Targeted gene sequencing panels are also commercially available (e.g. as summarised by Biocompare ((http://www.biocompare.com/ Editorial-Articles/161 194-Build-Your-Own-Gene-Panels-with-These-Custom-NGS-Targeting- Tools/)).

Suitable sequencing methods include, but are not limited to, high throughput sequencing techniques such as Next Generation Sequencing (lllumina, Roche Sequencer, Life Technologies SOLID™), Single Molecule Real Time Sequencing (Pacific Biosciences), True Single Molecule Sequencing (Helicos), or sequencing methods using no light emitting technologies but other physical methods to detect the sequencing reaction or the sequencing product, like Ion Torrent (Life Technologies).

Sequence alignment to identify indels in DNA and/or RNA from a tumor sample compared to DNA and/or RNA from a non-tumor sample may be performed using methods which are known in the art. For example, nucleotide differences compared to a reference sample may be performed using the method as described in the present examples and by Koboldt DC, Zhang Q, Larson DE, Shen D, McLellan MD, Lin L, et al. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome research. 2012;22(3):568-76. Nucleotide differences compared to a reference sample may be performed using the methods described in the present Examples. Suitably, the reference sample may be the germline DNA and/or RNA sequence.

According to the present invention, the indel mutation is a frameshift indel mutation. A frameshift indel mutation is a change in the reading frame of the nucleotide sequence caused by an insertion or deletion of one or more nucleotides. Such frameshift indel mutations may generate a novel open-reading frame which is typically highly distinct from the polypeptide encoded by the non-mutated DNA/RNA in a corresponding healthy cell in the subject.

As described above, frameshift mutations typically introduce premature termination codons (PTCs) into the open reading frame and the resultant mRNAs are targeted for nonsense mediated decay (NMD). The present inventors have determined that distinct open-reading frames generated by frameshift indel mutations are able to escape NMD and undergo productive translation to generate polypeptide sequences. Without wishing to be bound by theory, indel frameshift mutations which are not typically targeted for NMD, and will thus generate peptides which can be presented by MHC class I molecules in tumor cells, may provide an effective target for T cell mediated immune responses.

In one aspect, indel frameshift mutations may be targeted for NMD. As used herein, the term "expressed indel" is intended to be equivalent to an indel that escapes NMD (and is therefore expressed). As such, an "expressed frameshift indel" is equivalent to a frameshift indel which has escaped NMD. SAMPLE

Isolation of biopsies and samples from tumors is common practice in the art and may be performed according to any suitable method, and such methods will be known to one skilled in the art.

The sample may be a tumor sample, blood sample, tumor-associated lymph node sample or sample from a metastatic site, or tissue sample, or be peripheral blood mononuclear cells from the subject.

In certain embodiments that sample is a tumor-associated body fluid or tissue. The tumor may be a solid tumor or a non-solid tumor.

The sample may be a blood sample. The sample may contain a blood fraction (e.g a serum sample or a plasma sample) or may be whole blood. Techniques for collecting samples from a subject are well known in the art.

Suitably, the sample may be circulating tumor DNA, circulating tumor cells or exosomes comprising tumor DNA. The circulating tumor DNA, circulating tumor cells or exosomes comprising tumor DNA may be isolated from a blood sample obtained from the subject using methods which are known in the art.

Tumor samples and non-cancerous tissue samples can be obtained according to any method known in the art. For example, tumor and non-cancerous samples can be obtained from cancer patients that have undergone resection, or they can be obtained by extraction using a hypodermic needle, by microdissection, or by laser capture. Control (non-cancerous) samples can be obtained, for example, from a cadaveric donor or from a healthy donor. ctDNA and circulating tumor cells may be isolated from blood samples according to e.g. Nature. 2017 Apr 26;545(7655):446-451 or Nat Med. 2017 Jan;23(1): 114-119.

DNA and/or RNA suitable for downstream sequencing can be isolated from a sample using methods which are known in the art. For example DNA and/or RNA isolation may be performed using phenol-based extraction. Phenol-based reagents contain a combination of denaturants and RNase inhibitors for cell and tissue disruption and subsequent separation of DNA or RNA from contaminants. For example, extraction procedures such as those using DNAzol™, TRIZOL™ or TRI REAGENT™ may be used. DNA and/or RNA may further be isolated using solid phase extraction methods (e.g. spin columns) such as PureLink™ Genomic DNA Mini Kit or QIAGEN RNeasy™ methods. Isolated RNA may be converted to cDNA for downstream sequencing using methods which are known in the art (RT-PCR).

TARGETING A NEOANTIGEN RESULTING FROM AN EXPRESSED FRAMESHIFT INDEL MUTATION

Once identified, neoantigens resulting from an expressed frameshift indel mutation (also referred to herein as "expressed frameshift indel neoantigens") may represent a target for therapeutic or prophylactic intervention in the treatment or prevention of cancer in a subject.

References herein to "expressed frameshift indel neoantigens" are intended to include also peptides derived from expressed frameshift indel neoantigens.

The methods of the invention may be used in vitro or in vivo, for example either for in situ treatment or for ex vivo treatment followed by the administration of the treated cells to the subject.

By "targeting a neoantigen resulting from an expressed frameshift indel mutation" is meant that a therapeutic or prophylactic intervention is based on such a neoantigen.

This is discussed in further detail below, but in brief may comprise an active immunotherapy approach, such as administering an immunogenic composition or vaccine comprising an expressed frameshift indel neoantigen to a subject. Alternatively, a passive immunotherapy approach may be taken, for example adoptive T cell transfer or B cell transfer, wherein a T or B cell or T and B cells which recognise an expressed frameshift indel neoantigen are isolated from tumors, or other bodily tissues (including but not limited to lymph node, blood or ascites), expanded ex vivo or in vitro and readministered to a subject.

In a further alternative an antibody which recognises an expressed frameshift indel neoantigen may be administered to a subject. One skilled in the art will appreciate that if the expressed frameshift indel neoantigen is a cell surface antigen, an antibody as referred to herein will recognise the expressed frameshift indel neoantigen. Where the expressed frameshift indel neoantigen is an intracellular antigen, the antibody will recognise the expressed frameshift indel neoantigen peptide:MHC complex. As referred to here in, an antibody which "recognises" an expressed frameshift indel neoantigen encompasses both of these possibilities.

As such, in one aspect the invention is directed to a method of treating or preventing cancer in a subject, comprising administering to said subject:

(i) an expressed frameshift indel neoantigen;

(ii) an immune cell which recognises an expressed frameshift indel neoantigen; or

(iii) an antibody which recognises an expressed frameshift indel neoantigen.

In another aspect the invention provides an expressed frameshift indel neoantigen for use in the treatment or prevention of cancer in a subject. Alternatively put, the invention provides the use of an expressed frameshift indel neoantigen in the manufacture of a medicament for use in the treatment or prevention of cancer in a subject. In a further alternative the invention provides the use of an expressed frameshift indel neoantigen in treating or preventing cancer in a subject.

In a further aspect the invention provides an immune cell, preferably a T cell which recognises an expressed frameshift indel neoantigen for use in the treatment or prevention of cancer in a subject. Alternatively put, the invention provides the use of an immune cell, preferably a T cell, which recognises an expressed frameshift indel neoantigen in the manufacture of a medicament for use in the treatment or prevention of cancer in a subject. In a further alternative the invention provides the use of an immune cell, preferably a T cell, which recognises an expressed frameshift indel neoantigen in treating or preventing cancer in a subject.

References to "an immune cell" are intended to encompass cells of the immune system, for example T cells, NK cells, NKT cells, B cells and dendritic cells. In a preferred embodiment the immune cell is a T cell, as discussed herein.

In a further aspect the invention provides an antibody which recognises an expressed frameshift indel neoantigen for use in the treatment or prevention of cancer in a subject.

Alternatively put, the invention provides the use of an antibody which recognises an expressed frameshift indel neoantigen in the manufacture of a medicament for use in the treatment or prevention of cancer in a subject. In a further alternative the invention provides the use of an antibody which recognises an expressed frameshift indel neoantigen in treating or preventing cancer in a subject.

NEOANTIGENS

Suitably, the expressed frameshift indel mutation generates a neoantigen.

As described herein, the expressed frameshift indel mutation according to the invention generates a frameshift neoantigen. That is, the invention as described herein may be directed to targeting a neoantigen generated by an expressed frameshift indel mutation. This may be referred to herein as "an expressed frameshift indel neoantigen".

A neoantigen is a tumor-specific antigen which arises as a consequence of a mutation within a cancer cell. Thus, a neoantigen is not expressed by healthy (i.e. non-tumor cells). As described herein, a neoantigen may be processed to generate distinct peptides which can be recognised by T cells when presented in the context of MHC molecules.

CLONAL NEOANTIGEN

In one aspect the expressed frameshift indel neoantigen is a clonal expressed frameshift neoantigen. That is, the expressed frameshift indel mutation generates a clonal expressed frameshift indel neoantigen.

A "clonal" neoantigen is a neoantigen which is expressed effectively throughout a tumor and encoded within essentially every tumor cell. A "sub-clonal" neoantigen' is a neoantigen which is expressed in a subset or a proportion of cells or regions in a tumor.

'Present throughout a tumor', 'expressed effectively throughout a tumor' and 'encoded within essentially every tumor cell' may mean that the clonal neoantigen is expressed in all regions of the tumor from which samples are analysed. It will be appreciated that a determination that a mutation is 'encoded within essentially every tumor cell' refers to a statistical calculation and is therefore subject to statistical analysis and thresholds.

Likewise, a determination that a clonal neoantigen is 'expressed effectively throughout a tumor' refers to a statistical calculation and is therefore subject to statistical analysis and thresholds.

Expressed effectively in essentially every tumor cell or essentially all tumor cells means that the mutation is present in all tumor cells analysed in a sample, as determined using appropriate statistical methods.

By way of the example, the cancer cell fraction (CCF), describing the proportion of cancer cells that harbour a mutation may be used to determine whether mutations are clonal or sub- clonal. For example, the cancer cell fraction may be determined by integrating variant allele frequencies with copy numbers and purity estimates as described by Landau et al. (Cell. 2013 Feb 14; 152(4):714-26).

Suitably, CCF values may be calculated for all mutations identified within each and every tumor region analysed. If only one region is used (i.e. only a single sample), only one set of CCF values will be obtained. This will provide information as to which mutations are present in all tumor cells within that tumor region, and will thereby provide an indication if the mutation is clonal or sub-clonal.

As stated, determining a clonal mutation is subject to statistical analysis and threshold. As such, a mutation may be identified as clonal if it is determined to have a CCF 95% confidence interval >= 0.75, for example 0.80, 0.85, 0.90, 0.95, 1.00 or >1.00. Conversely, a mutation may be identified as sub-clonal if it is determined to have a CCF 95% confidence interval <= 0.75, for example 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10, 0.05, 0.01 in any sample analysed.

It will be appreciated that the accuracy of a method for identifying clonal mutations is increased by identifying clonal mutations for more than one sample isolated from the tumor. In one embodiment the methods may involve identifying a plurality i.e. more than one clonal neo-antigen.

In one embodiment the number of clonal neo-antigens is 2-1000. For example, the number of clonal neo-antigens may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000, for example the number of clonal neo-antigens may be from 2 to 100.

In one aspect, the methods as described herein may provide a plurality or population, i.e. more than one, of T cells wherein the plurality of T cells comprises a T cell which recognises a clonal expressed frameshift expressed frameshift indel neo-antigen and a T cell which recognises a different clonal expressed frameshift expressed frameshift indel neo-antigen. As such, the method provides a plurality of T cells which recognise different clonal expressed frameshift expressed frameshift indel neo-antigens.

In a preferred embodiment the number of clonal expressed frameshift expressed frameshift indel neo-antigens recognised by the plurality of T cells is 2-1000. For example, the number of clonal expressed frameshift expressed frameshift indel neo-antigens recognised may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000, for example the number of clonal expressed frameshift expressed frameshift indel neo-antigens recognised may be from 2 to 100.

In one aspect a plurality of T cells recognises the same clonal expressed frameshift expressed frameshift indel neo-antigen.

TUMOR SUPRESSORS

In one aspect, the expressed frameshift indel mutation may be in a tumor suppressor gene.

A tumor suppressor gene may be defined as a gene that protects a cell from developing to a tumor/cancer cell. Mutations which cause a loss or reduction in function of the protein encoded by a tumor suppressor gene can therefore contribute to the cell progressing to cancer, usually in combination with other genetic changes. Tumor suppressor genes may be grouped into categories including caretaker genes, gatekeeper genes, and landscaper genes. Proteins encoded by tumor suppressor genes typically have a damping or repressive effect on the regulation of the cell cycle and/or promote apoptosis.

Examples of tumor suppressor genes include, but are not limited to, retinoblastoma (RB), TP53, ARID 1 A, PTEN, MLL2/MLL3, APC, VHL, CD95, ST5, YPEL3, ST7, ST14 and genes encoding components of the SWI/SNF chromatin remodeling complex.

EXPRESSED FRAMESHIFT INDEL NEOANTIGEN PEPTIDES

The term "expressed frameshift indel neoantigen" as used herein is intended to encompass any part of a neoantigen that is immunological. This may include peptides derived from an expressed frameshift indel neoantigen. An "antigenic" molecule as referred to herein is a molecule which itself, or a part thereof, is capable of stimulating an immune response, when presented to the immune system or immune cells in an appropriate manner.

Expressed frameshift indel neoantigen peptides may be synthesised using methods which are known in the art.

The term "peptide" is used in the normal sense to mean a series of residues, typically L- amino acids, connected one to the other typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acids. The term includes modified peptides and synthetic peptide analogues.

The peptide may be made using chemical methods (Peptide Chemistry, A practical Textbook. Mikos Bodansky, Springer-Verlag, Berlin.). For example, peptides can be synthesized by solid phase techniques (Roberge JY et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY). Automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.

The peptide may alternatively be made by recombinant means, or by cleavage from the polypeptide which is or comprises the neoantigen. The composition of a peptide may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure). The neoantigen peptide may comprise the cancer cell specific mutation (e.g. the non-silent amino acid substitution encoded by a SNV) at any residue position within the peptide. By way of example, a peptide which is capable of binding to an MHC class I molecule is typically 7 to 13 amino acids in length. As such, the amino acid substitution may be present at position 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or 13 in a peptide comprising thirteen amino acids.

In a further aspect, longer peptides, for example 27-31 mers, may be used, and the mutation may be at any position, for example at the centre of the peptide, e.g. at positions 13, 14, 15 or 16 can also be used to stimulate both CD4 and CD8 cells to recognise indel neoantigens.

T CELL AND T CELL POPULATION

As discussed herein, the present invention encompasses therapeutic utilities of a T cell which recognises an expressed frameshift indel neoantigen, and also for methods for providing such a T cell or population thereof

The invention encompasses a method for providing an expressed frameshift indel neoantigen specific T cell which comprises the following steps:

i) determining mutations present in a sample isolated from the tumor; and

ii) identifying an expressed frameshift indel mutation; and

iii) identifying an expressed frameshift indel neoantigen, which is an antigen encoded by a sequence which comprises the expressed frameshift indel mutation; and iv) providing a T cell or population of T cells which recognises said expressed frameshift indel neoantigen.

In one aspect of the invention as described herein mutations may be determined in a plurality of samples isolated from the tumor.

The T cell population may be expanded in order to increase the number of T cells which recognise or target expressed frameshift indel neoantigens. Expansion of T cells may be performed using methods which are known in the art.

For example, T cells may be expanded by ex vivo culture in conditions which are known to provide mitogenic stimuli for T cells. By way of example, the T cells may be cultured with cytokines such as IL-2 or with mitogenic antibodies such as anti-CD3 and/or CD28. The T cells may be co-cultured with antigen-presenting cells (APCs), which may have been irradiated. The APCs may be dendritic cells. The dendritic cells may have been pulsed with peptides containing the identified indel mutations as single stimulants or as pools of stimulating indel neoantigen peptides. Expansion of T cells may be performed using methods which are known in the art, including for example the use of artificial antigen presenting cells (aAPCs), which provide additional co-stimulatory signals, and autologous PBMCs which present appropriate peptides. Autologous PBMCs may be pulsed with peptides containing indel mutations as discussed herein as single stimulants, or alternatively as pools of stimulating indel neoantigens.

In one aspect the invention provides a method for expanding a T cell population for use in the treatment of cancer in a subject, wherein the T cell population targets an expressed frameshift indel neo-antigen, the method comprising the steps of:

a) providing a T cell population comprising a T cell which is capable of specifically recognising said expressed frameshift expressed frameshift indel neo-antigen; and

b) co-culturing the T cell population with a composition comprising the expressed frameshift expressed frameshift indel neo-antigen;

wherein the neo-antigen has been identified in a sample of a tumor from said subject as being an expressed frameshift indel neo-antigen.

In one aspect expansion may be performed by co-culture of a T cell with an expressed frameshift indel neoantigen and an antigen presenting cell. The antigen presenting cell may be a dendritic cell. The expressed frameshift indel neo-antigen may be a clonal neo-antigen. The expansion may be a selective expansion of T cells which are specific for the indel neoantigen.

The invention provides a method for producing a composition comprising an antigen presenting cell and an expressed frameshift indel neo-antigen or expressed frameshift indel neo-antigen peptide. The expressed frameshift indel neo-antigen may be identified according to methods of the present invention. In one embodiment said method comprises the following steps:

(a) identifying an expressed frameshift indel neo-antigen in a tumor from a subject which comprises the steps of:

i) determining mutations present in a sample isolated from the tumor; and ii) identifying an expressed frameshift indel mutation; and

iii) identifying an expressed frameshift indel neo-antigen, which is an antigen encoded by a sequence which comprises the indel mutation; and

b) producing a composition comprising said expressed frameshift indel neo-antigen or expressed frameshift indel neo-antigen peptide and an antigen presenting cell.

The invention also provides a composition comprising an antigen presenting cell, e.g. a dendritic cell, and an expressed frameshift indel neo-antigen or expressed frameshift indel neo-antigen peptide. The expressed frameshift indel neo-antigen may be identified according to the methods of the invention as discussed herein.

The composition may be produced according to a method as described herein. The composition may also be used in the methods of the invention described herein, for example in methods of producing a T cell or T cell population or composition as discussed herein

Compositions as described herein may be a pharmaceutical composition which additionally comprises a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion.

In one aspect, expansion may involve culturing the T cell population with IL-2 or an anti-CD3 and/or an CD28 antibody.

In one aspect of the invention as described herein the T cell population is isolated from the patient to be treated, for example from a tumor sample obtained from said patient.

The T cell population may comprise tumor infiltrating lymphocytes (TILs).

A T cell composition is provided in which said T cell population is enriched with an increased number of T cells which target expressed frameshift indel neo-antigens compared with the initial T cell population isolated from the subject. Also provided is a T cell composition useful for the treatment of a cancer in a subject which comprises T cells selectively expanded to target expressed frameshift indel neo-antigens characteristic of the subject's cancer.

A T cell composition as described herein may be enriched with T cells which are specific to expressed frameshift indel neo-antigens.

In a T cell composition as described herein the expanded population of expressed frameshift indel neo-antigen-reactive T cells may have a higher activity than the population of T cells which have not been expanded, as measured by the response of the T cell population to restimulation with an expressed frameshift indel neo-antigen peptide. Activity may be measured by cytokine production, and wherein a higher activity is a 5-10 fold or greater increase in activity.

A T cell, T cell population or T cell composition as described herein may be obtained or obtainable by any of the methods as described herein.

A T cell, T cell population or T cell composition as described herein may be used in the treatment of cancer.

The invention encompasses a method for treating cancer in a subject comprising administering a T cell composition as described herein to the subject. The invention also encompasses a T cell composition as described herein for use in the manufacture of a medicament for the treatment of cancer.

The method may comprise the following steps:

(i) isolation of a T cell population from a sample from the subject;

(ii) expansion of the T cell population which targets an expressed frameshift indel neo- antigen; and

(iii) administering the T cell population from (ii) to the subject.

The method may comprise the following steps:

(i) isolation of a T cell from a sample from the subject; (ii) engineering the T cell to express a CAR or TCR which recognises said expressed frameshift indel neo-antigen as described herein to provide a T cell population which targets the indel neo-antigen; and

(iii) administering the T cell population from (ii) to the subject.

In one aspect said T cells are selectively expanded using a plurality of expressed frameshift indel neo-antigens, wherein each of said peptides comprises a different indel mutation. Said plurality may be from 2 to 100, from 5 to 75 or from 10 to 50 neo-antigens.

A method of the invention may comprise firstly identifying an expressed frameshift indel neoantigen, and then expanding a T cell population to target the indel neoantigen.

Thus, in one aspect the invention provides a method for providing a T cell population which targets an expressed frameshift indel neoantigen, said method comprising the steps of:

(a) identifying an expressed frameshift indel neoantigen in a tumor from a subject by a method which comprises the steps of:

i) determining mutations present in a sample isolated from the tumor;

ii) identifying an expressed frameshift indel mutation; and

iii) identifying an expressed frameshift indel neo-antigen, which is an antigen encoded by a sequence which comprises the indel mutation: and

(b) expanding a population of T cells to provide a T cell population that targets the indel neoantigen.

Mutations may be determined in a plurality of samples isolated from said tumor.

Following expansion, the resulting T cell population is enriched with an increased number of T cells which target indel neoantigens (for example, compared with the sample isolated from the subject).

Thus, in one aspect the invention provides a T cell which recognises an expressed frameshift indel neoantigen. In a further aspect the invention relates to a population of T cells which recognise an expressed frameshift indel neoantigen or a population of T cells as described herein. In a preferred embodiment the invention provides a plurality or population, i.e. more than one, of T cells wherein the plurality of T cells comprises a T cell which recognises an expressed frameshift indel neoantigen and a T cell which recognises a different indel neoantigen. As such, the invention provides a plurality of T cells which recognise different expressed frameshift indel neoantigens. Different T cells in the plurality or population may have different TCRs which recognise the same indel neoantigen.

In a preferred embodiment the number of expressed frameshift indel neoantigens recognised by the plurality of T cells is 2-1000. For example, the number of expressed frameshift indel neoantigens recognised may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000, preferably 2 to 100. There may be a plurality of T cells with different TCRs but which recognise the same expressed frameshift indel neoantigen.

The T cell population may be all or primarily composed of CD8+ T cells, or all or primarily composed of a mixture of CD8+ T cells and CD4+ T cells or all or primarily composed of CD4+ T cells.

In particular embodiments, the T cell population is generated from T cells isolated from a subject with a tumor.

For example, the T cell population may be generated from T cells in a sample isolated from a subject with a tumor. The sample may be a tumor sample, a peripheral blood sample or a sample from other tissues of the subject.

In a particular embodiment the T cell population is generated from a sample from the tumor in which the expressed frameshift indel neo-antigen is identified. In other words, the T cell population is isolated from a sample derived from the tumor of a patient to be treated.

In one embodiment the T cell population comprises tumor infiltrating lymphocytes (TILs).

T cells may be isolated using methods which are well known in the art. For example, T cells may be purified from single cell suspensions generated from samples on the basis of expression of CD3, CD4 or CD8. T cells may be enriched from samples by passage through a Ficoll-Paque gradient. The present invention also provides a method for providing a T cell population which targets an expressed frameshift indel neoantigen in a tumor from a subject which comprises the steps of:

i) isolating a T cell or population of T cells from a sample isolated from the subject; and ii) expanding the T cell or population of T cells to increase the number or relative proportion of T cells that target expressed frameshift indel neoantigens.

The T cell population that is produced in accordance with the present invention will have an increased number or proportion of T cells that target one or more expressed frameshift indel neoantigens. For example, the T cell population of the invention will have an increased number of T cells that target an expressed frameshift indel neoantigen compared with the T cells in the sample isolated from the subject. That is to say, the composition of the T cell population will differ from that of a "native" T cell population (i.e. a population that has not undergone the expansion steps discussed herein), in that the percentage or proportion of T cells that target an expressed frameshift indel neoantigen will be increased, and the ratio of T cells in the population that target indel neoantigens to T cells that do not target indel neoantigens will be higher in favour of the T cells that target expressed frameshift indel neoantigens.

The T cell population according to the invention may have at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% T cells that target an expressed frameshift indel neoantigen. For example, the T cell population may have about 0.2%-5%, 5%-10%, 10-20%, 20-30%, 30-40%, 40-50 %, 50-70% or 70-100% T cells that target an expressed frameshift indel neoantigen. In one aspect the T cell population has at least about 1 , 2, 3, 4 or 5% T cells that target an expressed frameshift indel neoantigen, for example at least about 2% or at least 2% T cells that target an expressed frameshift indel neoantigen.

Alternatively put, the T cell population may have not more than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.1 , 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8% T cells that do not target an expressed frameshift indel neo-antigen. For example, the T cell population may have not more than about 95%-99.8%, 90%-95%, 80-90%, 70-80%, 60-70%, 50-60 %, 30-50% or 0-30% T cells that do not target an expressed frameshift indel neo-antigen. In one aspect the T cell population has not more than about 99, 98, 97, 96 or 95% T cells that do not target an expressed frameshift indel neo-antigen, for example not more than about 98% or 95% T cells that do not target an expressed frameshift indel neo-antigen.

An expanded population of expressed frameshift indel neoantigen-reactive T cells may have a higher activity than a population of T cells not expanded, for example, using an expressed frameshift indel neoantigen peptide. Reference to "activity" may represent the response of the T cell population to restimulation with an expressed frameshift indel neoantigen peptide, e.g. a peptide corresponding to the peptide used for expansion, or a mix of indel neoantigen peptides. Suitable methods for assaying the response are known in the art. For example, cytokine production may be measured (e.g. IL2 or IFNy production may be measured). The reference to a "higher activity" includes, for example, a 1 -5, 5-10, 10-20, 20-50, 50-100, 100- 500, 500-1000-fold increase in activity. In one aspect the activity may be more than 1000- fold higher.

The population of T cells may comprise CD8⁺ T cells, CD4⁺ T cells or CD8⁺ and CD4⁺ T cells.

Helper T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells become activated when they are presented with peptide antigens by MHC class II molecules on the surface of antigen presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1 , TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to facilitate different types of immune responses.

Cytotoxic T cells (TC cells, or CTLs) destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. CTLs express the CD8 at their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated, which prevents autoimmune diseases.

A T cell as described herein may be an engineered T cell. The expressed frameshift indel neoantigen specific T cell may express a chimeric antigen receptor (CAR) or a T cell receptor (TCR) which specifically binds an expressed frameshift indel neoantigen or an expressed frameshift indel neoantigen peptide, or an affinity- enhanced T cell receptor (TCR) which specifically binds an expressed frameshift indel neoantigen or an expressed frameshift indel neoantigen peptide (as discussed further herein below). For example, the T cell may express a chimeric antigen receptor (CAR) or a T cell receptor (TCR) which specifically binds to an expressed frameshift indel neo-antigen or an expressed frameshift indel neo-antigen peptide (for example an affinity enhanced T cell receptor (TCR) which specifically binds to an expressed frameshift indel neo-antigen or an expressed frameshift indel neo-antigen peptide).

CARs are proteins which, in their usual format, graft the specificity of a monoclonal antibody (mAb) to the effector function of a T-cell. Their usual form is that of a type I transmembrane domain protein with an antigen recognizing amino terminus, a spacer, a transmembrane domain all connected to a compound endodomain which transmits T-cell survival and activation signals.

The most common form of these molecules use single-chain variable fragments (scFv) derived from monoclonal antibodies to recognize a target antigen. The scFv is fused via a spacer and a transmembrane domain to a signaling endodomain. Such molecules result in activation of the T-cell in response to recognition by the scFv of its target. When T cells express such a CAR, they recognize and kill target cells that express the target antigen. Several CARs have been developed against tumor associated antigens, and adoptive transfer approaches using such CAR-expressing T cells are currently in clinical trial for the treatment of various cancers.

Methods for generating TCRs and affinity enhanced TCRs are known in the art. Affinity enhanced TCRs are TCRs with enhanced affinity for a peptide-MHC complex (including e.g. the isolation of TCR genes that encode TCRs from patient samples (e.g. patient peripheral blood or TILs) and the improvement of TCR affinity for a peptide-MHC complex via modification of TCR sequences (e.g. by in vitro mutagenesis and selection of enhanced affinity (or affinity matured) TCRs). Methods of introducing such TCR genes into T cells are known in the art. Methods of identifying optimal-affinity TCRs involving the immunisation of antigen-negative humanised transgenic mice which have a diverse human TCR repertoire (e.g. TCR/MHC humanised mice such as ABabDII mice) with antigen, and isolation of antigen-specific TCRs from such immunised transgenic mice are also known in the art (see e.g. Obenaus M et al., Nat Biotechnol. 33(4):402-7, 2015.

T cells may bear high affinity TCRs, and hence affinity enhancement may not be necessary. High affinity TCRs may be isolated from T cells from a subject and may not require affinity enhancement.

Candidate T cell clones capable of binding an expressed frameshift indel neo-antigen peptide may be identified using MHC multimers comprising the expressed frameshift indel neo-antigen peptide, for example.

Identified TCRs and/or CARs which specifically target an expressed frameshift indel neo- antigen peptide or expressed frameshift indel neo-antigen may be expressed in autologous T cells from a subject using methods which are known in the art, for example by introducing DNA or RNA coding for the TCR or CAR by one of many means including transduction with a viral vector, transfection with DNA or RNA.

The invention encompasses a T cell as described herein, for example an engineered T cell.

In certain aspects according to the invention as described herein the T cell or T cell population is reinfused into a subject, for example following T cell isolation and expansion as described herein. Suitable methods to achieve this will be known to one skilled in the art. For example, methods for generating, selecting and expanding T cells are known in the art, see e.g. Dudley J Immunother. 2003; 26(4): 332-342, and Rosenberg et al. 2011 Clin Cancer Res:17(13):4550-7. Methods for reinfusing T cells are described in Dudley et al. Clin Cancer Res. 2010 Dec 15; 16(24): 6122-6131 , and Rooney et al. Blood. 1998 Sep 1 ;92(5): 1549-55. The T cell, T cell population or T cell composition according to the invention can be used in the treatment or prevention of cancer according the invention as described herein.

In one aspect the present invention relates to a method for treating cancer in a subject which comprises administering a T cell or T cell population according to the invention to the subject.

The method may comprise the following steps: (i) isolation of a T cell-containing sample from the subject;

(ii) expansion of a T cell population which targets an expressed frameshift indel neoantigen; and

(iii) administering the cells from (ii) to the subject.

In one aspect the T cell may be engineered to express a CAR or affinity-enhanced TCR as described herein.

The invention also provides a method of treating a patient who has cancer comprising administering to said patient a T cell or T cell population as defined herein.

The indel neoantigen, T cell or T cell population may have been identified or produced according to any of the aspects of the invention as described herein.

The expansion may be ex vivo or in vitro, and may be performed by methods known in the art.

The invention also provides a composition comprising an antigen presenting cell, and an expressed frameshift indel neoantigen or expressed frameshift indel neoantigen peptide. The expressed frameshift indel neoantigen may be identified according to the methods of the invention as discussed herein.

In one aspect the antigen presenting cells have been pulsed or loaded with said peptide.

The invention also provides a T cell composition which comprises a population of expressed frameshift indel neo-antigen-specific T cells, wherein said population of expressed frameshift indel neo-antigen-specific T cells are produced by co-culturing T cells with antigen presenting cells which present expressed frameshift indel neo-antigen peptides.

Compositions as described herein may be a pharmaceutical composition which additionally comprises a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion. In one aspect the antigen presenting cell is a dendritic cell. In one aspect the antigen presenting cell is irradiated. In one aspect the antigen presenting cell is a cell capable of presenting the relevant peptide, for example in the correct HLA context. Such a cell may be an autologous activated PBMC expressing an autologous HLA molecule, or a non- autologous cell expressing an array of matched HLAs. In one aspect the artificial antigen presenting cell is irradiated.

T cells may also be enriched by initial stimulation of TILs with indel neoantigens in the presence or absence of exogenous APCs followed by polyclonal stimulation and expansion with cytokines such as IL-2 or with mitogenic antibodies such as anti-CD3 and/or CD28. Such methods are known in the art. For example, see Forget et al. J Immunother. 2014 Nov- Dec;37(9):448-60, Donia et al. Cytotherapy. 2014 Aug;16(8):1 117-20, Donia et al. Scand J Immunol. 2012 Feb;75(2): 157-67 and Ye et al. J Transl Med. 2011 Aug 9;9: 131.

MHC MULTIMERS

Identification of expressed frameshift indel neoantigen-specific T cells in a mixed starting population of T cells may be performed using methods which are known in the art. For example, such T cells may be identified using MHC multimers comprising an expressed frameshift indel neo-antigen peptide identified by the method of the present invention.

MHC multimers are oligomeric forms of MHC molecules, designed to identify and isolate T- cells with high affinity to specific antigens amid a large group of unrelated T-cells. Multimers may be used to display class 1 MHC, class 2 MHC, or nonclassical molecules (e.g. CD1d).

The most commonly used MHC multimers are tetramers. These are typically produced by biotinylating soluble MHC monomers, which are typically produced recombinantly in eukaryotic or bacterial cells. These monomers then bind to a backbone, such as streptavidin or avidin, creating a tetravalent structure. These backbones are conjugated with fluorochromes to subsequently isolate bound T-cells via flow cytometry, for example.

The invention provides an MHC multimer comprising an expressed frameshift indel neo- antigen peptide. The expressed frameshift indel neo-antigen may be identified by a method according to the invention as described herein. In one aspect, the present invention provides a method for producing an MHC multimer which may be used according to the invention as described herein. Said method comprises the steps of:

i) determining mutations present in a sample isolated from the tumor; and

ii) identifying an expressed frameshift indel mutation; and

(b) producing an expressed frameshift indel neo-antigen peptide from said expressed frameshift indel neo-antigen; and

(c) producing an MHC multimer comprising said expressed frameshift indel neo-antigen peptide.

MHC multimers according to the invention may be used in methods for identifying, isolating, expanding or otherwise producing a T cell, T cell population or composition as described herein, expressed frameshift indel neo-antigen peptides may be synthesised using methods which are known in the art.

HLA ALLELES

Antigens are presented to T cells in the context of antigen-derived peptides bound by major histocompatibility molecules (MHC).

Thus expressed frameshift indel neo-antigen may be recognised by a T cell as an expressed frameshift indel neo-antigen derived peptide. All references herein to "expressed frameshift indel neoantigen" are intended to encompass peptides derived from said expressed frameshift indel neoantigen.

An expressed frameshift indel neo-antigen peptide is a peptide which is derived from the region of a polypeptide which comprises a cancer cell specific mutation. As such expressed frameshift indel neo-antigen peptides should not be derived from polypeptides encoded by the genome of healthy cells. MHC class I proteins form a functional receptor on most nucleated cells of the body. There are 3 major MHC class I genes in HLA: HLA-A, HLA-B, HLA-C and three minor genes HLA-E, HLA-F and HLA-G. 2-microglobulin binds with major and minor gene subunits to produce a heterodimer.

Peptides that bind to MHC class I molecules are typically 7 to 13, more usually 8 to 1 1 amino acids in length. The binding of the peptide is stabilised at its two ends by contacts between atoms in the main chain of the peptide and invariant sites in the peptide-binding groove of all MHC class I molecules. There are invariant sites at both ends of the groove which bind the amino and carboxy termini of the peptide. Variations in peptide length are accommodated by a kinking in the peptide backbone, often at proline or glycine residues that allow the required flexibility.

There are 3 major and 2 minor MHC class II proteins encoded by the HLA. The genes of the class II combine to form heterodimeric (αβ) protein receptors that are typically expressed on the surface of antigen-presenting cells.

Peptides which bind to MHC class II molecules are typically between 8 and 20 amino acids in length, more usually between 10 and 17 amino acids in length, and can be longer (for example up to 40 amino acids). These peptides lie in an extended conformation along the MHC II peptide-binding groove which (unlike the MHC class I peptide-binding groove) is open at both ends. The peptide is held in place mainly by main-chain atom contacts with conserved residues that line the peptide-binding groove.

The methods of the present invention may involve the step of assessing a subject's HLA alleles to determine if an expressed frameshift indel neo-antigen peptide will bind to an MHC molecule expressed by the subject.

The HLA allele profile of an individual may be determined by methods which are known in the art. For example, the HLA profile of an individual may be determined by HLA-serotyping and/or HLA gene sequencing. HLA-phenotyping with single specific primer-PCR (SSP-PCR) is an alternative strategy for determining the HLA profile of an individual. In the present examples, the HLA profile of an individual is determined by sequencing of the HLA locus and processing using the Optitype prediction algorithm to determine the HLA type for each individual (Szolek et al.; Bioinformatics; 2014; 30(23): 3310-3316).

The binding of a peptide to a particular MHC molecule may be predicted using methods which are known in the art. Examples of methods for predicting MHC binding include those described by Lundegaard et al. (Nucleic Acids Res. 2008:W509-12.2008 & Bioinformatics. 2008 Jun 1 ;24(1 1): 1397-8) and Shen et al. (Proteome Sci. 2013 Nov 7;1 1 (Suppl 1):S15).

The methods of the present invention may comprise determining an expressed frameshift indel neo-antigen peptide which is predicted to bind to an MHC molecule expressed by the subject. In particular, the methods may comprise the step of determining and selecting an expressed frameshift indel neo-antigen peptide which is predicted to bind strongly to an MHC molecule expressed by the subject. The exact definition of 'binding strongly' will depend on the method used to predict the MHC binding interaction (see Lundegaard et al. (as above), for example). However, in all cases the expressed frameshift indel neo-antigen peptide selected will be predicted to be capable of binding to, and being presented in the context of, an MHC molecule expressed by the subject.

The binding affinity to an expressed frameshift indel neo-antigen peptide may be below 500nM. By "high affinity" may mean 0 to 50nM binding affinity. In other embodiments the expressed frameshift indel neo-antigen peptide may bind the MHC molecule with an intermediate affinity of 50 to150nM binding affinity, or low affinity of 150 to 500nM binding affinity.

In certain embodiments, the expressed frameshift indel neo-antigen peptide may be predicted to bind to the MHC molecule with a high affinity whilst a corresponding wild-type peptide (e.g. an equivalent peptide derived from the same region of the corresponding wild- type polypeptide) is predicted to bind to the same MHC molecule with low affinity.

COMPOSITION

The present invention further provides a composition which comprises an expressed frameshift indel neoantigen or peptide, indel neoantigen specific T cell, or an antibody which recognises an expressed frameshift indel neoantigen. Compositions as described herein may be a pharmaceutical composition additionally comprising a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion.

ANTIBODY

In one aspect of the invention an antibody which recognises an expressed frameshift indel neoantigen is provided.

Once an expressed frameshift indel neoantigen has been identified, for example by a method according to the invention, methods known in the art can be used to generate an antibody.

"Antibody" (Ab) includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity. The term "immunoglobulin" (Ig) may be used interchangeably with "antibody".

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The "Fc" fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

The antibody may be a human antibody. A "human antibody" refers to an antibody naturally existing in humans, a functional fragment thereof, or a humanized antibody, i.e., a genetically engineered antibody a portion of which (e.g., Fc region) derives from a naturally- occurring human antibody. A "humanized antibody" is generally considered to be a human antibody that has one or more amino acid residues introduced into it from a source that 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, by substituting import hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. Furthermore, chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.

In a preferred aspect the antibody is a monoclonal antibody. As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies may be prepared by the hybridoma methodology (Kohler et al., Nature, 256:495 (1975)), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells. Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581 -597 (1991), for example.

Monoclonal antibodies may also be produced by recombinant DNA methods that are known in the art. DNA encoding suitable monoclonal antibodies may be 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. 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. Monoclonal antibodies may 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 same biological activity.

Antibody fragments may also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the binding 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 will be known to one skilled in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody fragment.

Antibodies may be 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 sub-sequences 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 antibody 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 may comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

Methods for humanizing non-human antibodies are 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. As such, "humanized" antibodies are chimeric antibodies 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) may be used to produce a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. 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.

"Synthetic antibody" as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

IMMUNOGENIC COMPOSITION

The present invention provides an immunogenic composition, or vaccine, comprising an expressed frameshift indel neoantigen or expressed frameshift indel neoantigen peptide as defined herein. For example, the expressed frameshift indel neoantigen may be identified by a method of the present invention described herein. The immunogenic composition or vaccine may be used in any method of treating or preventing cancer according to the invention. As such, the invention encompasses a method of treating or preventing cancer in a subject comprising administering to the subject an immunogenic composition or vaccine according to the invention.

By "immunogenic composition" is meant a composition that is capable of inducing an immune response in a subject. The immunogenic composition may be a vaccine composition. By "vaccine composition" is meant a composition that is capable of inducing an immune response in a subject that has a therapeutic or prophylactic effect on the condition to be treated.

The immunogenic composition or vaccine may comprise more than one expressed frameshift indel neoantigen or expressed frameshift indel neoantigen peptide.

In one aspect the immunogenic composition or vaccine may comprise more than one different expressed frameshift indel neoantigen or expressed frameshift indel neoantigen peptide, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 different neoantigens or neoantigen peptides. The expressed frameshift indel neo-antigen may also be in the form of a protein.

In one embodiment the immunogenic composition or vaccine may comprise a polypeptide which comprises an expressed frameshift indel neo-antigen as defined herein. In one embodiment of the invention the immunogenic composition or vaccine may comprise more than one different polypeptide each comprising an expressed frameshift indel neo-antigen, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 different polypeptides.

The immunogenic composition or vaccine may be a pharmaceutical composition which additionally comprises a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion. See, for example, Butterfield, BMJ. 2015 22;350 for a discussion of cancer vaccines.

In particular, the immunogenic composition or vaccine may additionally comprise an adjuvant. Examples of adjuvants include but are not limited to aluminium salts, oil emulsions and bacterial components (e.g. LPS and liposomes). In one embodiment the adjuvant may be poly ICLC, which is a synthetic complex of carboxymethylcellulose, polyinosinic- polycytidylic acid, and poly-L-lysine double-stranded RNA.

Suitable doses of peptides may be determined by one skilled in the art. The dose may depend on the peptide which is to be used. For in vivo use of a peptide an in vivo dose of 0.1-4000μ9, e.g. 0.1 -2000μ9, 0.1 -1000 or 0.1 -500 μg, for example 0.1 -100Mg, may be employed.

The immunogenic composition or vaccine according to the invention as discussed herein may lead to generation of an immune response in the subject. An "immune response" which may be generated may be humoral and/or cell-mediated immunity, for example the stimulation of antibody production, or the stimulation of cytotoxic or killer cells, which may recognise and destroy (or otherwise eliminate) cells expressing antigens corresponding to the antigens in the vaccine on their surface. The term "stimulating an immune response" thus includes all types of immune responses and mechanisms for stimulating them and encompasses stimulating CTLs which forms a preferred aspect of the invention. Preferably the immune response which is stimulated is cytotoxic CD8+ T cells and helper CD4+T Cells. The extent of an immune response may be assessed by markers of an immune response, e.g. secreted molecules such as IL-2 or IFNy or the production of antigen specific T cells.

In addition, an expressed frameshift indel neo-antigen may be delivered in the form of a cell, such as an antigen presenting cell, for example a dendritic cell. The antigen presenting cell such as a dendritic cell may be pulsed or loaded with the expressed frameshift indel neo- antigen or expressed frameshift indel neo-antigen peptide or genetically modified (via DNA or RNA transfer) to express one, two or more expressed frameshift indel neo-antigens or expressed frameshift indel neo-antigen peptides (see e.g. Butterfield 2015 supra; Palucka 2013 supra), for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 expressed frameshift indel neo-antigens or expressed frameshift indel neo-antigen peptides. Methods of preparing dendritic cell immunogenic compositions or vaccines are known in the art.

Alternatively, DNA or RNA encoding one or more expressed frameshift indel neo-antigen, or peptide or protein derived therefrom may be used in the immunogenic composition or vaccine, for example by direct injection to a subject. For example, DNA or RNA encoding 2, 3, 4, 5, 6, 7, 8, 9 or 10 expressed frameshift indel neo-antigens, or peptide or protein derived therefrom. The one or more expressed frameshift indel neo-antigen or expressed frameshift indel neo-antigen peptide may be delivered via a bacterial or viral vector containing DNA or RNA sequences which encode one or more expressed frameshift indel neo-antigens or expressed frameshift indel neo-antigen peptides.

Immunogenic compositions or vaccines as described herein may be administered in any suitable way. For example, they may be delivered by any suitable delivery mechanism as known in the art. The composition may involve the use of a vector delivery system, or a vector delivery system may not be necessary. Vectors may be viral or bacterial. Suitable viral vectors may be derived from retroviruses adenoviruses, lentiviruses or pox viruses. Liposomes may be used as a delivery system. Listeria vaccines or electroporation may also be used.

Cell-based immunogenic compositions or vaccines may be prepared ex vivo and then administered to the subject.

The invention further provides a cell expressing an expressed frameshift indel neoantigen, or a part thereof, on its surface, or a population thereof, which cell is obtainable (or obtained) by any of the methods herein. Such a cell may be used for treating or preventing cancer.

The invention therefore further provides a cell expressing an expressed frameshift indel neoantigen or expressed frameshift indel neo-antigen peptide on its surface (or intracellular^), or a population of such cells, which cell or population is obtainable (or obtained) by methods as defined herein. In a preferred embodiment the cell is an antigen presenting cell such as a dendritic cell.

For in vivo administration of cells as described herein, any mode of administration of the cell population which is common or standard in the art may be used, e.g. injection or infusion, by an appropriate route. In one aspect 1x10⁴ to 1x10⁸ cells are administered per kg of subject (e.g. 1.4x10⁴ to 2.8x10⁶ per kg in human). In one aspect about or not more than 10⁷ cells per kg of subject are administered. Thus, for example, in a human, a dose of 0.1 -20x10⁷ cells per kg of subject may be administered in a dose, i.e. per dose, for example as a dose of T cells or a vaccination dose. In one aspect, between 1x10⁴ to 1x10⁵ cells, between 1x10⁵ to 1x10⁶ cells, between 1x10⁶ to 1x10⁷ cells or between 1x10⁷ to 1x10⁸ cells per kg of subject are administered. For vaccination applications, 1-20x10⁶ cells per dose may be used. The dose can be repeated at later times if necessary. The immunogenic composition or vaccine according to the invention may be used in the treatment of cancer.

The invention also provides a method for treating cancer in a subject comprising administering an immunogenic composition or vaccine as described herein to said subject. The method may additionally comprise the step of identifying a subject who has cancer.

In a further aspect the invention provides a method for producing an immunogenic composition or vaccine comprising an expressed frameshift indel neo-antigen peptide or expressed frameshift indel neo-antigen, said method comprising the steps of:

i) determining mutations present in a sample isolated from the tumor; and

ii) identifying an expressed frameshift indel mutation; and

(b) producing an expressed frameshift indel neo-antigen peptide or expressed frameshift indel neo-antigen from said expressed frameshift indel neo-antigen; and

(c) producing an immunogenic composition or vaccine with said expressed frameshift indel neo-antigen peptide or expressed frameshift indel neo-antigen protein.

In aspect of the invention producing the vaccine involves preparing a dendritic cell vaccine, wherein said dendritic cell presents an expressed frameshift indel neoantigen or indel neoantigen peptide.

An expressed frameshift indel neo-antigen protein may also be used in the immunogenic compositions or vaccines and methods relating to vaccination according to the invention.

In a further aspect the invention provides a method for producing an immunogenic composition or vaccine comprising a DNA or RNA molecule encoding an expressed frameshift indel neo-antigen peptide or expressed frameshift indel neo-antigen, said method comprising the steps of:

(b) producing a DNA or RNA molecule encoding the expressed frameshift indel neo-antigen peptide or expressed frameshift indel neo-antigen; and

(c) producing an immunogenic composition or vaccine with said DNA or RNA molecule.

The immunogenic composition or vaccine may be delivered by suitable methods as described hereinbefore.

In one aspect the vaccination is therapeutic vaccination. In this aspect the immunogenic composition or vaccine is administered to a subject who has cancer to treat the cancer.

In a further aspect the vaccination is prophylactic vaccination. In this aspect the immunogenic composition or vaccine is administered to a subject who may be at risk of developing cancer.

In one aspect the immunogenic composition or vaccine is administered to a subject who has previously had cancer and in whom there is a risk of the cancer recurring.

An immunogenic composition or vaccine may also be in the form of DNA or RNA coding for one or several of the expressed frameshift indel neo-antigenic peptides or proteins and delivered by additional methods including but not limited to viral vectors, antigen presenting cells and electroporation.

SUBJECT

In a preferred embodiment of the present invention, the subject is a mammal, preferably a cat, dog, horse, donkey, sheep, pig, goat, cow, mouse, rat, rabbit or guinea pig, but most preferably the subject is a human.

As defined herein "treatment" refers to reducing, alleviating or eliminating one or more symptoms of the disease which is being treated, relative to the symptoms prior to treatment.

"Prevention" (or prophylaxis) refers to delaying or preventing the onset of the symptoms of the disease. Prevention may be absolute (such that no disease occurs) or may be effective only in some individuals or for a limited amount of time. CANCER

Suitably, the cancer may be ovarian cancer, breast cancer, endometrial cancer, kidney cancer (renal cell), lung cancer (small cell, non-small cell and mesothelioma), brain cancer (gliomas, astrocytomas, glioblastomas), melanoma, Merkel cell carcinoma, clear cell renal cell carcinoma (ccRCC), lymphoma, small bowel cancers (duodenal and jejunal), leukaemia, pancreatic cancer, hepatobiliary tumors, germ cell cancers, prostate cancer, head and neck cancers, thyroid cancer and sarcomas.

In one embodiment the cancer may have a mutation in a DNA-repair pathway.

In one embodiment, the cancer is melanoma. In one embodiment, the cancer is kidney cancer (renal cell cancer). In one embodiment the cancer may be selected from melanoma, merkel cell carcinoma, renal cancer, non-small cell lung cancer (NSCLC), urothelial carcinoma of the bladder (BLAC) and head and neck squamous cell carcinoma (HNSC) and microsatellite instability (MSI)-high cancers.

In one embodiment the cancer may be an MSI-high cancer.

Treatment using the compositions and methods of the present invention may also encompass targeting circulating tumor cells and/or metastases derived from the tumor.

Treatment according to the present invention targeting one or more expressed frameshift indel neo-antigens may help prevent the evolution of therapy resistant tumor cells which may occur with standard approaches.

COMBINATION WITH OTHER CANCER TREATMENTS

The methods and uses for treating cancer according to the present invention may be performed in combination with additional cancer therapies. In particular, the T cell compositions according to the present invention may be administered in combination with immune checkpoint intervention, co-stimulatory antibodies, chemotherapy and/or radiotherapy, targeted therapy or monoclonal antibody therapy.

Immune checkpoint molecules include both inhibitory and activatory molecules, and interventions may apply to either or both types of molecule. Immune checkpoint inhibitors include, but are not limited to, PD-1 inhibitors, PD-L1 inhibitors, Lag-3 inhibitors, Tim-3 inhibitors, TIGIT inhibitors, BTLA inhibitors and CTLA-4 inhibitors, for example. Co- stimulatory antibodies deliver positive signals through immune-regulatory receptors including but not limited to ICOS, CD137, CD27 OX-40 and GITR. In a preferred embodiment the checkpoint inhibitor is a CTLA-4 inhibitor.

Examples of suitable immune checkpoint interventions which prevent, reduce or minimize the inhibition of immune cell activity include pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, tremielimumab and ipilimumab.

A chemotherapeutic entity as used herein refers to an entity which is destructive to a cell, that is the entity reduces the viability of the cell. The chemotherapeutic entity may be a cytotoxic drug. A chemotherapeutic agent contemplated includes, without limitation, alkylating agents, anthracyclines, epothilones, nitrosoureas, ethylenimines/methylmelamine, alkyl sulfonates, alkylating agents, antimetabolites, pyrimidine analogs, epipodophylotoxins, enzymes such as L-asparaginase; biological response modifiers such as IFNa, IL-2, G-CSF and GM-CSF; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin, anthracenediones, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (ο,ρ'-DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide.

'In combination' may refer to administration of the additional therapy before, at the same time as or after administration of the T cell composition according to the present invention.

In addition or as an alternative to the combination with checkpoint blockade, the T cell composition of the present invention may also be genetically modified to render them resistant to immune-checkpoints using gene-editing technologies including but not limited to TALEN and Crispr/Cas. Such methods are known in the art, see e.g. US20140120622. Gene editing technologies may be used to prevent the expression of immune checkpoints expressed by T cells including but not limited to PD-1 , Lag-3, Tim-3, TIGIT, BTLA CTLA-4 and combinations of these. The T cell as discussed here may be modified by any of these methods.

The T cell according to the present invention may also be genetically modified to express molecules increasing homing into tumors and or to deliver inflammatory mediators into the tumor microenvironment, including but not limited to cytokines, soluble immune-regulatory receptors and/or ligands.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with a general dictionary of many of the terms used in this disclosure.

This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspects or embodiments of this disclosure which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation.

The term "protein", as used herein, includes proteins, polypeptides, and peptides.

Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to understand that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

The terms "comprising", "comprises" and "comprised of as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of also include the term "consisting of.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

The invention will now be described, by way of example only, with reference to the following Examples.

EXAMPLES

The pattern of indel mutations on a pan-cancer basis, and their association with anti-tumor immune response and outcome following checkpoint blockade, was determined. Results

Indel frequencies were compared on a pan-cancer basis, across 19 solid tumor types, utilising 5,777 samples from the cancer genome atlas (TCGA). The contribution of indels was analysed as a proportion of the total mutational count per sample (indel proportion) and the absolute number of indels per sample (indel count) and observed median values of 0.05 and 4 respectively, cohort-wide. Across all tumor types, clear cell renal cell carcinoma (ccRCC) was found to have the highest proportion of coding indels, 0.12 (P=2.2x10^"16, figure 2), a 2.4-fold increased as compared to the pan-cancer average. This result was replicated in two further independent studies, with median observed indel proportions of 0.10 and 0.12, respectively (1 , 2) (Figure 1). Papillary renal cell carcinoma (pRCC) and chromophobe renal cell carcinoma (chrRCC) had the second/third highest indel proportion, suggesting a possible tissue specific mutational process contributing the acquisition of indels in renal cancers. pRCC, chrRCC and ccRCC also had the highest absolute indel count across all tumor types, with a median indel number of 10, 8, and 7, respectively. ccRCC is characterised by loss of function (LoF) mutations in one or more tumor suppressor genes: VHL, PBRM1, SETD2, BAP1 and KDM5C (1 1), which can be inactivated by nsSNV or indel mutations. To exclude the possibility these hallmark mutations were distorting the results, ccRCC indel proportion was recalculated excluding VHL, PBRM1, SETD2, BAP1 and KDM5C; the revised indel proportion remained at 0.12. Utilising previously published multi- region whole exome sequencing data from ten ccRCC cases (2) the clonal nature of indel mutations was assessed, revealing 48% of frameshifting indels to be clonal in nature (present in all tumor regions).

For frameshift neoantigens to contribute to anti-tumor immunity the mutant peptides must be expressed. Frameshifts cause premature termination codons (PTCs) and the resultant mRNAs are targeted for nonsense mediated decay (NMD). Published analyses of germline samples show that PTCs frequently lead to the loss of expression of the variant allele, but that some mutant transcripts escape NMD based on the exact location of the frameshift within a gene (16). Combined analyses of mutational and expression data from over 10,000 cancer samples showed that NMD is triggered with variable efficacy, and even when effective might not alter expression levels due factors such as short mRNA half-life (17). Using the TCGA ccRCC data, the gene expression levels were compared in the samples harbouring a mutation in the given gene, to that in non-mutated samples. This analysis was performed for both indel and SNV mutations, with the latter included as a benchmark comparator. The overall impact of NMD on the expression level of indel mutated genes was estimated to be 14%, markedly below what would be expected under fully operational NMD, pointing to the existence of NMD-evading PTCs.

The potential immunogenicity of nsSNV and indel mutations was determined through analysis of MHC Class l-associated tumor specific neoantigen binding predictions in the pan- cancer TCGA cohort. Across all samples, HLA-specific neoantigen predictions were performed on 335,594 nsSNV mutations, resulting in a total of 214,882 high affinity binders (defined as epitopes with predicted IC50 < 50 nM), equating to a rate of 0.64 neoantigens per nsSNV mutation (snv-neoantigens). In a similar manner predictions were made on 19,849 frameshift indel mutations, resulting in 39,768 high affinity binders with a rate of 2.00 neoantigens per frameshift mutation (frameshift-neoantigens). Thus on a per mutation basis, frameshift indels could generate -three-fold more high affinity neoantigen binders (Table 1), consistent with the prediction in a recent analyses of a colorectal cancer cohort (18). When both wild type and mutant peptides are predicted to bind central immune tolerance mechanisms may delete cells with the reactive T-cell receptor. Therefore a pan-cancer analyses was repeated, restricting the neoantigens to mutant specific binders (i.e. where the wild-type peptide is not predicted to bind), and demonstrated that frameshift indels were nine-fold enriched for mutant-allele only binders (Table 1).

Table 1 - Neoantigens per variant class

Variant No. of No. of No. per No. of mutant specific No. per

Class mutations neoantigens* mutation neoantigens** mutation ns-SNVs 335,594 214,882 0.64 75,224 0.22 fs-lndels 19,849 39,768 2.00 39,768 2.00

Enrichment 3.13 8.94

* Strong binders (<50nM affinity)

** Wildtype allele non-binding (>500nM affinity)

Of particular interest were genes that are frequently altered via frameshift mutations and with high propensity for MHC binding. In a pan-cancer analysis they were enriched for classic tumor suppressor genes including TP53, ARID1A, PTEN, MLL2/MLL3, APC and VHL (figure 2). Collectively the top 15 genes with highest number of frameshifts mutations were mutated in >500 samples (-10% of the cohort) with >2,400 high affinity neoantigens predicted. Tumor suppressor genes have been a previously intractable mutational target, but they may be targetable as potent neoantigens. Furthermore, by virtue of being founder events many alterations in tumor suppressor genes are clonal, present in all cancer cells, rendering them compelling targets for the immune system.

The clinical impact of indel mutations was considered by assessing the relationship between neoantigen enrichment and therapeutic benefit. To date, CPIs have been approved for the treatment of six solid tumor types: melanoma (anti-PD1/CTLA-4), merkel cell carcinoma (anti-PDI), ccRCC (anti-PD1), NSCLC (anti-PD1), BLAC (anti-PD-L1) and HNSC (anti-PD1). Consistent with a potential role of frameshifts in the generation of neoantigens, the CPI approved tumor types were all found to harbour an above average number of frameshift neoantigens, despite dramatic differences in the total SNV/indel mutational burden, i.e. ccRCC (figure 3). Overall, the number of frameshift neoantigens was considerably higher in the CPI-approved tumor types versus those that are not CPI-approved to date (P=2.2x10^"16). The impact of frameshift neoantigens on CPI efficacy was assessed using exome sequencing results from a recent anti-PD-1 study in melanoma (n=38 patients) (3). Three classes of mutation were defined: (i) non-synonymous SNVs, (ii) in-frame (3n) indels and (iii) frameshift (non-3n) indels, and each tested for an association with response to treatment. While class (i) and (ii) mutations showed a non-significant trend (P=0.26, P=0.22)), class (iii) framehshift indel mutations were significantly associated with anti-PD-1 response, with P=0.02 (figure 4a). The upper quartile of patients, with the highest burden of class (iii) frameshift indels, had an 88% response rate (RR) to anti-PD-1 therapy, compared to 43% for the lower three quartiles (figure 4b). To confirm the reproducibility of this association CPI response data were obtained from two additional melanoma cohorts with genomic profiling: Snyder et al. (n=62, anti-CTLA-4 treated) (4) and Van Allen et al. (n=100, anti-CTLA-4 treated) (5). The same analyses were conducted in each cohort and frameshift indel burden was significantly associated with CPI response in both additional datasets, with P=0.0074 and P=0.024 respectively (figure 4a). An overall meta-analysis across the three cohorts confirmed frameshift indel count to be significantly associated with CPI response (P=3.8x10^" ⁴), and with stronger association than nsSNV count (P=3.5x10^"3). In addition an improved overall survival was observed in the class (iii) frameshift indel group (Supplementary Figure 3). Finally, to assess the relationship between frameshift indel load and CPI response in another tumor type, a small cohort of 31 non-small cell lung cancer patients treated with anti- PDI therapy was obtained from Rizvi et al. (6). Although non-significant, a trend of higher frameshift indel load in CPI responders (P=0.2) was observed. Finally, while genomic data are not available to correlate with CPI response in ccRCC, the relationship between frameshift-neoantigen load and immune responses within the tumor was analysed using RNAseq gene expression data. Patients were split into groups based on the burden of frameshift-neoantignes (high defined as >10 frameshifts/case) versus snv- neoantigens (high defined as >17 nsSNVs/case, with this threshold set to ensure matched patient sample sizes). A high load of frameshift-neoantigens was associated with up- regulation of immune signatures classically linked to immune activation, including: MHC Class I antigen presentation, CD8+ T cell activation and increased cytolytic activity, a pattern not observed in the high snv-neoantigen group (figure 5). Furthermore, correlation analysis within the high frameshift-neoantigen group demonstrated that CD8+ T Cell signature was strongly correlated with both MHC Class I antigen presentation genes and cytolytic activity (p=0.78 and p=0.83 respectively) (Figure 5).

Methods

Study design and patients

Pan-cancer somatic mutational data were obtained from the cancer genome atlas (TCGA), for 5,777 available patients who had undergone whole exome sequencing, across 19 different solid tumor types: Bladder urothelial carcinoma (BLCA), Breast invasive carcinoma (BRCA), Cervical and endocervical cancers (CESC), Colorectal adenocarcinoma (COADREAD), Glioma (GMBLGG), Head and Neck squamous cell carcinoma (HNSC), Kidney Chromophobe (KICH), Kidney renal clear cell carcinoma (KIRC), Kidney renal papillary cell carcinoma (KIRP), Liver hepatocellular carcinoma (LIHC), Lung adenocarcinoma (LUAD), Lung squamous cell carcinoma (LUSC), Ovarian serous cystadenocarcinoma (OV), Pancreatic adenocarcinoma (PAAD), Prostate adenocarcinoma (PRAD), Skin Cutaneous Melanoma (SKCM), Stomach adenocarcinoma (STAD), Thyroid carcinoma (THCA) and Uterine Carcinosarcoma (UCS). Patient level mutation annotation files were extracted from the Broad Institute TCGA GDAC Firehose repository (https://gdac.broadinstitute.org/), which had been previously curated by TCGA analysis working group experts to ensure strict quality control. Replication analysis was conducted in two additional ccRCC patient cohorts: i) a whole exome sequencing study of 106 ccRCCs reported by Sato et al (1) ii) a whole exome sequencing study of 10 ccRCCs reported by Gerlinger et al (2). Final post quality control (QC) patient level mutation annotation files were obtained for each study. In order to test for an association between non-synonymous SNVs/indel loads and patient response to checkpoint inhibitor (CPI) therapy further four patient cohorts were utilised. The first dataset consisted of 38 melanoma patients treated with anti-PD-1 therapy, as reported by Hugo et al. (3). Final post-QC mutation annotation files and clinical outcome data were obtained, and 32 patients were retained for analysis after excluding cases where DNA had been extracted from patient derived cell lines and patients where tissue samples were obtained after CPI therapy. This later exclusion was of particular importance, given the fact CPI therapy itself is likely to alter mutational frequencies through possible elimination of immunogenic tumor clones. The second CPI cohort comprised 62 melanoma patients treated with anti-CTLA-4 therapy, as reported by Snyder et al. (4). All patients samples were taken pre-CPI treatment from fresh snap frozen tumor tissue, so accordingly all 62 cases were retained for analysis. The third CPI cohort comprised 100 melanoma patients treated with anti-CTLA-4 therapy, as reported by Van Allen et al. (5), again all patients were eligible for inclusion using the same criteria as above. The final CPI cohort comprised 31 non-small cell lung cancer patients treated with anti-PD1 therapy, as reported by Rizvi et al.(6), again all patients were eligible for inclusion. For the Snyder et al. , Van Allen et al. and Rizvi et al. cohorts, final mutation annotation files including indel mutations were not available, so raw BAM files were obtained and variant calling was conducted using a standardized bioinformatics pipeline as described below.

Whole exome sequencing variant calling

BAM files representing both the germline and tumor regions from Snyder et al., Van Allen et al. and Rizvi et al. cohorts were obtained and converted to FASTQ format using picard tools (1.107) SamToFastq. Raw paired end reads (100bp) in FastQ format were aligned to the full hg19 genomic assembly (including unknown contigs) obtained from GATK bundle 2.8 (7), using bwa mem (bwa-0.7.7) (8). Picard tools v1.107 was used to clean, sort and merge files from the same patient region and to remove duplicate reads (http://broadinstitute.github.io/picard). Picard tools (1.107), GATK (2.8.1) and FastQC (0.10.1) (http://www.bioinformatics.babraham.ac.uk/proiects/fastqc/) were used to produce quality control metrics. SAMtools mpileup (0.1.19) (9) was used to locate non-reference positions in tumor and germline samples. Bases with a phred score of <20 or reads with a mapping-quality <20 were omitted. BAQ computation was disabled and the coefficient for downgrading mapping quality was set to 50. VarScan2 somatic (v2.3.6) (58) utilized output from SAMtools mpileup in order to identify somatic variants between tumor and matched germline samples. Default parameters were used with the exception of minimum coverage for the germline sample that was set to 10 and minimum variant frequency was changed to 0.01. VarScan2 processSomatic was used to extract the somatic variants. The resulting single nucleotide variant (SNV) calls were filtered for false positives using Varscan2's associated fpfilter.pl script, initially with default settings then repeated with again with min- var-frac = 0.02, having first run the data through bam-readcount (0.5.1) (https://github.com/genome/bam-readcount). Only INDEL calls classed as 'high confidence' by VarScan2 processSomatic were kept for further analysis, with somatic_p_value scores < 5x10^"4. MuTect (1.1.4) (10) was also used to detect SNVs utilising annotation files contained in GATK bundle 2.8. Following completion, variants called by MuTect were filtered according to the filter parameter 'PASS'.

Pan-cancer insertion/deletion analysis

In the pan-cancer cohort SNV and insertion/deletion (indel) mutation counts were computed per case, considering all variant types. Across all 5,777 samples a total of 1 ,227,075 SNVs and 54,207 indels were observed. Dinucleotide and trinucleotide substitutions were not considered. The metric "indel burden" was simply defined as the absolute indel count per case and "indel proportion" was defined as: # indels / (# indels + # SNVs). The same analysis was repeated in the two ccRCC replication cohorts.

Non-sense mediated decay analysis

Non-sense mediated decay (NMD) efficiency was estimated using RNAseq expression data (as measured in TPM), obtained from the TCGA GDAC Firehose repository https://gdac.broadinstitute.org/). The extent of NMD was estimated for all indel and SNV mutations by comparing the mRNA expression level in samples with a mutation to the median mRNA expression level of the same transcript across all other tumor samples where the mutation was absent. Specifically, the mRNA expression level of every mutation-bearing transcript was divided by the median mRNA expression level of that transcript in non- mutated samples, to give an NMD index. The overall NMD index values observed were 0.93 (indels) and 1.00 (SNVs), suggesting an overall 0.07 reduction in expression in indel mutated transcripts. Tumor purity in the KIRC cohort is reported to be 0.54 (1 1), and assuming constant expression levels in the remaining 0.46 normal cellular content, that would yield an adjusted 0.136 drop in expression in indel mutation bearing cancer cells. Assuming tumor mutations are clonal, of heterozygote genotype, in a diploid genomic region and wild-type allele expression in mutated cancer cells remains constant, a purity adjusted reduction of 0.5 would be expected under a model of fully effective NMD. Hence this data suggests NMD operates with reduced efficiency in the KIRC cohort, however we acknowledge the above assumptions will have some impact. These data are presented as a global approximation of NMD efficiency, utilizing methodology in line with previous publications (12).

Tumor specific neoantigen analysis

For a subset of patients from the TCGA cohort (n=4,592), tumor specific neoantigen binding affinity prediction data was also available and obtained from Rooney et al. (60). In brief, the 4-digit HLA type for each sample, along with mutations in class I HLA genes, were determined using POLYSOLVER (POLYmorphic loci reSOLVER). Somatic mutations were determined using Mutect (14) and Strelka tools. All possible 9 and 10-mer mutant peptides were computed, based on the detected somatic snv and indel mutation across the cohort. Binding affinities of mutant and corresponding wildtype peptides, relevant to the corresponding POLYSOLVER-inferred HLA alleles, were predicted using NetMHCpan (v2.4). Strong affinity binders were defined as IC50<50 nM. Wildtype allele non-binding was defined as IC50 >500nM. We excluded (from the pan-cancer neoantigen analyses) cancers that are associated with a high level of viral genome integration including cervical (>80% rate of HPV integration), hepatocellular carcinoma (>50% rate of HepB integration ), but not HNCC (<15% rate of HPV integration). There was no TCGA dataset available for Merkel cell carcinoma.

Immune signatures RNAseq analysis

Immune gene signature data was obtained from Rooney et al. (15) with gene sets defined as per supplementary table 1. Immune signature scores were calculated as the geometric mean of genes within the set, based on RNAseq Transcripts Per Kilobase Million (TPM) expression levels per sample. Analysis was conducted for ccRCC TCGA (KIRC) patients, where both RNAseq and neoantigen data was available (n=392). A high burden of frameshift indel strong affinity neoatigens was defined as >10 per case (n=32), and the percentage difference in expression was compared between the high indel neoatigen group and all other patients, across each immune signature. Immune signatures with minimal expression (<0.5 TPM) in all groups were excluded. The same analysis was repeated for a high burden of snv derived strong affinity neoantigens, with a threshold of >17 snv neoantigens selected in order to size match the high burden groups (equal number of patients, n=32 across all high load groups) across mutational types. The percentage differences in expression were plotted in heatmap format. Correlation analysis was conducted within the high frameshift indel neoatigen group (n=32 ccRCC patients).

Checkpoint inhibitor (CPI) response analysis

Across the four CPI treated patient cohorts (i) non-synonymous SNV, (ii) all coding indel and (iii) frameshift indel variant counts were tested for an association with patient response to therapy. For each measure (i), (ii) and (iii) high and low groups were defined as the top quartile (high) and bottom-three quartiles (low). The same criteria was used across all four datasets, and the proportion of patients responding to therapy (response rate) in high and low groups was compared. Measures of patient response were defined in each study as follows:

Snyder et al. (4)

• Long-term clinical benefit (LB): (i) radio- graphic evidence of freedom from disease or (ii) evidence of a stable disease or (iii) decreased volume of disease; for more than 6 months.

• Lack of long-term clinical benefit (NB): (i)tumor growth on every CT scan after the initiation of treatment (no benefit) or (ii) a clinical benefit lasting 6 months or less (minimal benefit).

Hugo et al.(3):

• Responding tumors: complete response (CR), partial response (PR) and stable disease (SD).

• Non-responding tumors: disease progression (PD)

VanAllen et al. (5):

• Clinical Benefit:CR/PR/SD

• No Clinical benefit: PD or SD with OS<1 year

Rizvi et al. (6):

• Durable clinical benefit (DCB): PR or SD lasting longer than 6 months

• No durable benefit (NDB): PD <6 months from beginning of therapy

Statistical analysis

Indel burden and proportion measures were compared between ccRCC and all other non- kidney cancers using a two-sided Mann Whitney test. In the CPI response analysis, non- synonymous SNV, exonic indel and frameshift indel counts were each compared to patient response outcome using a two-sided Mann Whitney test. Meta-analysis of results across the four CPI datasets was conducted using the Fisher method of combining P values from independent tests. Immune signature correlation analysis was conducted using a spearman's rank correlation coefficient. Statistical analyses were carried out using R3.0.2 (http://www.r-project.org/). A P value of 0.05 (two sided) was considered as being statistically significant. Clonality

The impact of clonality was additionally assessed, and clonal frameshift indels were found to have a further predictive advantage beyond all frameshift indels (clonal and subclonal). See Figure 6 in this regard.

References

1. Sato Y, Yoshizato T, Shiraishi Y, Maekawa S, Okuno Y, Kamura T, et al. Integrated molecular analysis of clear-cell renal cell carcinoma. Nat Genet. 2013;45(8):860-7.

2. Gerlinger M, Horswell S, Larkin J, Rowan AJ, Salm MP, Varela I, et al. Genomic architecture and evolution of clear cell renal cell carcinomas defined by multiregion sequencing. Nat Genet. 2014;46(3):225-33.

3. Hugo W, Zaretsky JM, Sun L, Song C, Moreno BH, Hu-Lieskovan S, et al. Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma. Cell. 2017; 168(3):542.

4. Snyder A, Makarov V, Merghoub T, Yuan J, Zaretsky JM, Desrichard A, et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N Engl J Med. 2014;371 (23):2189-99.

5. Van Allen EM, Miao D, Schilling B, Shukla SA, Blank C, Zimmer L, et al. Genomic correlates of response to CTLA-4 blockade in metastatic melanoma. Science. 2015;350(6257):207-11.

6. Rizvi NA, Hellmann MD, Snyder A, Kvistborg P, Makarov V, Havel JJ, et al. Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science. 2015;348(6230): 124-8.

7. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome research. 2010;20(9): 1297-303.

8. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25(14): 1754-60.

9. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25(16):2078-9

10. Cibulskis K, Lawrence MS, Carter SL, Sivachenko A, Jaffe D, Sougnez C, et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nature biotechnology. 2013;31 (3):213-9.

1 1. Cancer Genome Atlas Research N. Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature. 2013;499(7456):43-9.

12. Lindeboom RG, Supek F, Lehner B. The rules and impact of nonsense-mediated mRNA decay in human cancers. Nat Genet. 2016;48(10):1 112-8.

13. Rooney MS, Shukla SA, Wu CJ, Getz G, Hacohen N. Molecular and genetic properties of tumors associated with local immune cytolytic activity. Cell. 2015; 160(1-2):48-61.

14. Cibulskis K, Lawrence MS, Carter SL, Sivachenko A, Jaffe D, Sougnez C, et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nature biotechnology. 2013;31 (3):213-9.

15. Jamal-Hanjani M, Wilson GA, McGranahan N, Birkbak NJ, Watkins TBK, Veeriah S, et al. Tracking the Evolution of Non-Small-Cell Lung Cancer. N Engl J Med. 2017.

16. Lappalainen T, Sammeth M, Friedlander MR, t Hoen PA, Monlong J, Rivas MA, et al. Transcriptome and genome sequencing uncovers functional variation in humans. Nature. 2013;501 (7468):506-11. 17. Lindeboom RG, Supek F, Lehner B. The rules and impact of nonsense-mediated mRNA decay in human cancers. Nat Genet. 2016;48(10): 11 12-8.

18. Giannakis M, Mu XJ, Shukla SA, Qian ZR, Cohen O, Nishihara R, et al. Genomic Correlates of Immune-Cell Infiltrates in Colorectal Carcinoma. Cell Rep. 2016;17(4): 1206.

Example 2

It was determined in Example 1 that fs-indels are associated with improved response to checkpoint inhibitor therapy. The effects of non-sense mediated decay were then investigated.

Materials and methods

Study cohorts

Matched DNA/RNA sequencing analysis was conducted in the following cohorts all treated with immunotherapy:

• Van Allen et al. (8), an advanced melanoma checkpoint inhibitor (CPI) (anti-CTLA-4) treated cohort. Cases with both RNA sequencing and whole exome (DNA) sequencing data were utilised (n=33).

• Snyder et al. (7), an advanced melanoma CPI (anti-CTLA-4) treated cohort. Cases with both RNA sequencing and whole exome (DNA) sequencing data were utilised (n=21).

• Hugo et al. (4), an advanced melanoma CPI (anti-PD-1) treated cohort. Cases with both RNA sequencing and whole exome (DNA) sequencing data were utilised (n=24).

• Lauss et al. (10), an advanced melanoma adoptive cell therapy treated cohort. Cases with both RNA sequencing and whole exome (DNA) sequencing data were utilised (n=22).

• Snyder et al. (18), a metastatic urothelial cancer CPI (anti-PD-L1) treated cohort.

Cases with both RNA sequencing and whole exome (DNA) sequencing data were utilised (n=23).

Matched DNA/RNA sequencing analysis was conducted in the following cohorts (not specifically treated with immunotherapy):

• Skin cutaneous melanoma (SKCM) tumors, obtained from the cancer genome atlas (TCGA) project. Cases with paired end RNA sequencing data and curated variant calls from TCGA GDAC Firehose (2016_01_28 release) were utilised (n=368).

• Microsatellite instable (MSI) tumors, across all histological subtypes from TCGA project. MSI cases IDs were identified based on classification from Cortes-Ciriano et al. (19). Cases with paired end RNA sequencing data and curated variant calls from TCGA GDAC Firehose (2016_01_28 release) were utilised (n=96).

Prediction of NMD-escape features (based on DNA exonic mutation position only, rather than matched DNA/RNA sequencing analysis) was conducted in the following immunotherapy treated cohorts:

• Ott et al. (22), an advanced melanoma personalized vaccine treated cohort (n=6 cases).

• Rahma et al. (23), a metastatic renal cell carcinoma personalized vaccine treated cohort (n=6 cases).

• Le et al. (24), an advanced mismatch repair-deficient cohort, across cancers across 12 different tumor types, treated with anti-PD-1 blockade (n=86 cases, functional neoantigen reactivity T cell work only conducted in n=1 case).

Whole exome sequencing (DNA) variant calling

For Van Allen et al. (8), Snyder et al. (7) and Snyder et al. (18) cohorts, we obtained germline/tumor BAM files from the original authors and reverted these back to FASTQ format using Picard tools (version 1.107) SamToFastq. Raw paired-end reads in FastQ format were aligned to the full hg19 genomic assembly (including unknown contigs) obtained from GATK bundle (version 2.8), using bwa mem (bwa-0.7.7). We used Picard tools to clean, sort and to remove duplicate reads. GATK (version 2.8) was used for local indel realignment. We used Picard tools, GATK (version 2.8), and FastQC (version 0.10.1) to produce quality control metrics. SAMtools mpileup (version 0.1.19) was used to locate non- reference positions in tumor and germline samples. Bases with a Phred score of less than 20 or reads with a mapping quality less than 20 were omitted. VarScan2 somatic (version 2.3.6) used output from SAMtools mpileup to identify somatic variants between tumor and matched germline samples. Default parameters were used with the exception of minimum coverage for the germline sample, which was set to 10, and minimum variant frequency was changed to 0 01. VarScan2 processSomatic was used to extract the somatic variants. Single nucleotide variant (SNV) calls were filtered for false positives with the associated fpfilter.pl script in Varscan2, initially with default settings then repeated with min-var-frac=0 02, having first run the data through bam-readcount (version 0.5.1). MuTect (version 1.1.4) was also used to detect SNVs, and results were filtered according to the filter parameter PASS. In final QC filtering, an SNV was considered a true positive if the variant allele frequency (VAF) was greater than 2% and the mutation was called by both VarScan2, with a somatic p-value <=0.01 , and MuTect. Alternatively, a frequency of 5% was required if only called in VarScan2, again with a somatic p-value <=0.01. For small scale insertion/deletions (INDELs), only calls classed as high confidence by VarScan2 processSomatic were kept for further analysis, with somatic_p_value scores less than 5 ^χ 10^"4. Variant annotation was performed using Annovar (version 2016Feb01). Variants in either the first, penultimate or last exon, of the relevant transcript as annotated first (default) by Annovar, were considered to be mutations in exonic positions associated with NMD-escape. Middle exon mutations were considered to be all those not in first, penultimate or last exon positions. For the Hugo et al. (4) cohort, we obtained final post-quality control mutation annotation files generated as previously described (4). Briefly, SNVs were detected using MuTect, VarScan2 and the GATK Unified Genotyper, while INDELs were detected using VarScan2, IndelLocator and GATK-UGF. Mutations that were called by at least two of the three SNV/INDEL callers were retained as high confidence calls. For the Lauss et al. (10) cohort, SNVs and INDELs were called as described previously (10). Briefly, SNVs were detected using the intersection of MuTect and VarScan2 variants, while INDELs were detected using VarScan2 only. For VarScan2, high confidence calls at a VAF greater than 10% were retained.

Whole transcriptome sequencing (RNA) variant calling

RNAseq data was obtained in BAM format for all studies, and reverted back to FASTQ format using bam2fastq (v1.1.0). Insertion/deletion mutations were called from raw paired end FASTQ files, using mapsplice (v2.2.0), with sequence reads aligned to hg19 genomic assembly (using bowtie pre-built index). Minimum QC thresholds were set to retain variants with => 5 alternative reads, and variant allele frequency => 0.05. Insertions and deletions called in both RNA and DNA sequencing assays were intersected, and designated as expressed indels, with a +/- 10bp padding interval included to allow for minor alignment mismatches. SNVs in RNA sequencing data were called directly from the hg19 realigned BAM files, using Rsamtools to extract read counts per allele for each genomic position where a SNV had already called in DNA sequencing analysis. Similarly, minimum QC thresholds of => 5 alternative reads, and variant allele frequency => 0.05, were utilised and variants passing these thresholds were designated as expressed SNVs.

Protein expression analysis

We retrieved Level 4 (L4) normalized protein expression data for 223 proteins, across n=453 TCGA melanoma/MSI tumors (which overlapped with the TCGA cohorts also analysed via DNA/RNA sequencing) from the cancer proteome atlas (http://tcpaportal.org/tcpa/index.html). We filtered the data to sample/protein combinations which also contained an fs-indel mutation (n=136), as called by DNA sequencing. The dataset was then split into two groups, based on the fs-indel being expressed or not (as measured by RNAseq, using the method detailed above). The two groups were compared using a two-sided Mann Whitney test.

Outcome analysis

Across all immunotherapy treated cohorts, measures of patient clinical benefit/no-clinical benefit were kept as consistent with original author's criteria/definitions. For TCGA outcome analysis, overall survival (OS) data was utilized, based on clinical annotation data obtained from TCGA GDAC Firehose repository.

Selection analysis

To test for evidence of selection, fs-indel mutations were compared to stop-gain SNV mutations, in the SKCM TCGA cohort (n=368 cases). Stop-gain SNV mutations were utilised a benchmark comparator, due to their likely equivalent functional impact (i.e. loss of function), equivalent treatment by the NMD pathway (i.e. last exon stop-gain SNVs will still escape NMD and cause truncated protein accumulation) but lack of immunogenic potential (i.e. no mutated peptides are generated). Across all SKCM cases n=1 ,594 fs-indels and n=9,833 stop-gain SNVs were considered. All alterations in each group were annotated for exon position (i.e. first, middle, penultimate or last exon, as defined above). The odds of having an fs-indel in first, middle, penultimate or last exon positions was then benchmarked against the equivalent odds for a stop-gain SNV.

Statistical methods

Odds ratios were calculated using Fisher's Exact Test for Count Data, with each exon position group compared to all others. Kruskal-Wallis test was used to test for a difference in distribution between three or more independent groups. Two-sided Mann Whitney U test was used to assess for a difference in distributions between two population groups. Metaanalysis of results across cohorts was conducted using the Fisher method of combining P values from independent tests. Logistic regression was used to assess multiple variables jointly for independent association with binary outcomes. Overall survival analysis was conducted in the SKCM TCGA cohort using a Cox proportional hazards model, with stage, sex and age included as covariates. Overall survival analysis was conducted in the MSI TCGA cohort using a Cox proportional hazards model, with primary disease site included as a covariate. Statistical analysis were carried out using R3.4.4 (http://www. r-project org/) . We considered a P value of 0.05 (two sided) as being statistically significant.

Results

Detection of NMD-escape mutations

Expressed frameshift indels (fs-indels) were detected using paired DNA and RNA sequencing, with data processed through an allele specific bioinformatics pipeline (Fig. 7A). Across all processed TCGA samples (n=453, see methods for cohort details) a median of 4 fs-indels were detected per tumor (range 0-470), of which mutant allele expression was detected in a median of 1 per tumor (range 0-94). Thus, expressed fs-indel mutations were present at relatively low frequency and abundance. In fact, 49.6% of samples profiled had zero expressed fs-indel mutations detected. Exon positions were annotated for expressed fs- indels (n=1 ,840), and compared to non-expressed fs-indels (i.e. mutant allele present in DNA, but not in RNA) (n=8,691). Expressed fs-indels were enriched for mutations in penultimate (odds ratio versus non expressed fs-indels = 1.80, 95% confidence interval [1.53-2.11], p=3.2x10^"12) and last exon positions (OR=1.80 [1.60-2.04], p<2.2x10^"16), while being depleted in middle exon locations (OR=0.56 [0.51 -0.62], p<2.2x10^"16) (Fig. 7B). These exon positions are consistent with known patterns of NMD-escape, as previously established (14). First exon position mutations were unexpectedly depleted (OR=0.71 [0.55-0.91 , p=0.006), however the absolute number of observed mutations in this group was small (only n=80 expressed fs-indels) and a proportion of them (n=21) were >200nt from the gene start. Next we considered RNA variant allele frequency (VAF) estimates for expressed fs-indels, and found them to be highest for last (median=0.33), penultimate (0.28) and then first (0.26) exon positions, with middle exon alterations having the lowest value (0.19) (Fig. 7C, p<2.2x10^"16). Finally, we obtained protein expression data from the cancer proteome atlas (17), for 223 proteins across 453 tumors, which overlapped with the DNA/RNAseq processed cohort. Intersecting samples with both an fs-indel gene mutation(s), and matched protein expression data, we compared the protein levels of expressed (n=40) versus non expressed fs-indels (n=96). Protein abundance was found to be significantly higher for expressed fs-indels (p=0.018, Fig. 7D). Taken collectively, these results suggest that expressed fs-indels are (at least partially) escaping NMD and being translated to the protein level. Expressed fs-indels are here after referred to as NMD-escape, and non-expressed fs- indels as NMD-competent. NMD-escape mutation burden associates with clinical benefit to immune checkpoint inhibition

To assess the impact of NMD-escape mutations on anti-tumor immune response, we assessed the association between NMD-escape mutation count and CPI clinical benefit in three independent melanoma cohorts with matched DNA and RNA sequencing data: Van Allen et al. (n=33, anti-CTLA-4 treated), Snyder et al. (n=21 , anti-CTLA-4 treated) and Hugo et al. (n=24, anti-PD-1 treated). For each sample, mutation burden was quantified based on the following classifications: i) TMB: all non-synonymous SNVs (nsSNVs), ii) fs-indels, and iii) NMD-escape fs-indels. Each mutation class was tested for an association with clinical benefit (Fig. 8a). In the pooled meta-analysis of the three melanoma cohorts with both WES and RNAseq (total n=78), a trend towards significance was observed for nsSNVs (metaanalysis across all cohorts, P_meta=0.12) and marginal significance for fs-indels (P_meta=0.048), while NMD-escape mutation count had the strongest overall association with clinical benefit (Pmeta=0.0087) (Fig. 8a). For clarity, we note sample sizes utilised here are smaller than previously reported, since only a subset of cases had both matched DNA and RNA sequencing data available, and that nsSNV and fs-indel measures are significant in the full datasets. Patients with one or more NMD-escape mutation had higher rates of clinical benefit to immune checkpoint blockade compared to patients with no NMD-escape mutations: 56% versus 12% (Van Allen et al.), 57% versus 14% (Snyder et al.), and 71 % versus 35% (Hugo et al.) (Fig. 8b). To ensure the NMD-escape group was not simply reflecting the importance of neoantigen expression in general, we examined expressed nsSNVs detected using allele-specific RNAseq analysis and found that the association with clinical benefit remained non-significant (P_meta=0.24, Fig 11). We additionally assessed for evidence of correlation between TMB and nmd-escape metrics, and found only a weak correlation between the two variables (r=0.21 , P=0.06, n=78). And in multivariate logistic regression analysis, we tested both variables together in a joint model to assess for independent significance (n=78, study ID was also included as a model term to control for cohort specific factors), and NMD-escape mutation count was found to independently associate with CPI clinical benefit (P=0.032), whereas TMB did not reach independent significance (P=0.25). Finally to investigate a potential association in other tumor types, NMD-escape analysis was conducted in a CPI treated metastatic urothelial cancer cohort (n=23 cases) (18). Previous analysis in this study found that neither TMB, predicted neoantigen load nor expressed neoantigen load, were associated with CPI clinical benefit (18). Similarly, here we found no evidence of an association between NMD-escape count and clinical benefit (P=1.0), possibly due to small sample size, lower mutational load lower in this cohort (TMB=~0-5 missense SNVs/megabase, as compared to -10.0 in a larger recently published cohort (9)), or lower response rates in general in metastatic urothelial cancer. For completeness, the NMD-escape CPI meta-analysis was repeated to include the above bladder data, together with the three melanoma cohorts, and the association remains significant (P_meta=0.028).

Clinical benefit to adoptive cell therapy (ACT) associates with NMD-escape mutation burden

To further investigate the importance of NMD-escape mutations in directing anti-tumor immune response, we analysed matched DNA and RNA sequencing data from patients with melanoma (n=22) treated with adoptive cell therapy (10). TMB ns-SNVs (P=0.027), fs-indels (P=0.025) and NMD-escape count (P=0.021) were all associated with clinical benefit from therapy (Fig. 8c). All patients with NMD-escape count≥ 1 experienced clinical benefit (n=4, 100%), compared to 33% (6/18) of patients who had no NMD-escape mutations, further highlighting the potential strong immunogenic effect from just a single NMD-escape mutation. As previously reported (10), patients with high nsSNV load (defined as the upper fertile of patients) had improved progression free survival compared to patients with intermediate (middle fertile) or low (bottom fertile) nsSNV count (P=0.0008). We note that of the patients with NMD-escape count≥ 1 , the majority (3 of 4) were in the intermediate (rather than high) fertile nsSNV group, and may have been missed as high likelihood potential responders if TMB alone was used a predictive biomarker. The hazard ratio (HR) per single NMD-escape mutation was 0.28 (95% confidence interval 0.07 - 1.09), equivalent to approximately 845 nsSNV mutations (HR=0.28 (0.08-0.92)) (Table S1).

Table SI. Multivariate analysis

PFS Adjusted hazard ratio (95% CI) P- alue

TMB (per mutation) 0.9985 (0.997 - 0.9999) 0.038

N MD escape (per mutation) 0.2812 (0.073 - 1.0865) 0.0658

PFS Adjusted hazard ratio (95% CI) P- value

TMB (per 845 mutations) 0.2813 (0.079 - 0.919)

N MD escape (per mutation) 0.2812 (0.073 - 1.0865) Table S1

Multivariate progression free survival analysis results are shown for Lauss et al cohort, using a Cox proportional hazards model, with nsSNVs and NMD-escape mutation counts both included in the model as continuous variables. The first table shows the adjusted hazard ratio per single mutation for each measure, and the second table shows the comparable hazard ratio for how many TMB (nsSNVs) mutations are required to equal the same risk reduction as one NMD-escape mutation.

T cell reactivate neoantigens are enriched in genomic positions predicted to escape NMD

While of translational relevance and clinical utility, biomarker associations do not directly isolate specific neoantigens driving anti-tumor immune response. Accordingly, we obtained data from two anti-tumor personalised vaccine studies and one CPI study in which T cell reactivity against specific neopeptides had been established by functional assay of patient T cells. Across these three studies, six fs-indel derived neoantigens were functionally validated as eliciting T cell reactivity: DHX40 p.S754fs, RALGAPB p.H404fs, BTBD7 p.Y324fs, SLC16A4 p.F475fs, DEPDC1 p.K418fs, and VHL p.L116fs (Fig. 9). Thus, at a proof of concept level, the ability of fs-indels to elicit anti-tumor immune response has been previously established. Across these same studies, 12 fs-indel derived neoantigens had also undergone functional screening, but were found to be T cell non-reactive (Fig. 9). Paired DNA and RNA sequencing data were not available for all these cohorts to determine expression, so annotation of exonic position was used to estimate the likelihood of NMD escape. Within the group of fs-indels shown to be T cell reactive, 5 out of 6 were annotated in exon positions with reduced NMD efficiency (i.e. first, penultimate and last exon), compared to only 3 out of 12 for fs-indel peptides screened but with no T cell reactivity found (Fig. 9). While exceptions are observed (i.e. middle exon position mutations eliciting T cell response, and conversely last exon position mutations failing to generate T cell reactivity), an enrichment is observed with T cell reactive fs-indels more likely to occur in NMD-escape exon positions (OR=12.5 [0.9-780.7], P=0.043) (Fig. 9).

NMD-escape mutations show evidence of negative selection

Next, we assessed for evidence of selective pressure against NMD-escape mutations, which may reflect the potential to generate native anti-tumor immunogenicity. In additional to potential immunogenic selective pressure, fs-indels have also previously been reported to be under functional selection (15) due to their loss of protein function effect. To account for this, we used stop-gain SNV mutations as a benchmark comparator, as these variants have equivalent functional impact but no immunogenic potential (i.e. loss of function but no neoantigens generated). Furthermore, the rules of NMD apply equally to both stop-gain SNVs and fs-indels, as both trigger premature termination codons. Using the skin cutaneous melanoma (SKCM) TCGA cohort, we annotated all fs-indel (n=1 ,594) and stop-gain (n=9,883) mutations for exonic position. Penultimate and last exon alterations were found to be significantly depleted in fs-indels compared to stop-gain events (OR=0.58 [0.46-0.71], P=1.5x10^"5 and OR=0.65 [0.55-0.75], P=1.5x10^"7 respectively) (Fig. 10A). By contrast fs- indel mutations were more likely to occur in middle exon positions (OR=1.51 [1.33-1.68], P=1.2x10^"11). First exon mutations were not enriched either way, possibly due to small absolute numbers (only n=69 fs-indels were first exon). This data suggests negative selective immune pressure acts against fs-indel mutations in exonic positions likely to escape NMD (e.g. penultimate and last), leading to cancer cells with middle exon fs-indels being more likely to survive immunoediting.

NMD-escape mutation burden is associated with improved overall survival

Finally, to assess evidence of natural anti-tumor immunogenicity of NMD-escape mutations in melanomas, we examined matched DNA and RNA sequencing data from 368 patients in the TCGA SKCM cohort. Patients with at least one NMD-escape mutation had significantly improved OS (HR=0.69 [0.50-0.96], P=0.03), as compared to those with zero NMD-escape mutations (Fig. 10B). Additionally, using matched DNA and RNA sequencing data from MSI carcinomas (which have high abundance of fs-indel events) identified by Cortes-Ciriano et al. (19) (n=96), a similar but non-significant trend in improved OS was observed among patients with high NMD-escape mutation load (defined as > cohort median value rather than =>1 , due to the high level of indel events) (HR=0.67 [0.31-1.45], P=0.313).

The results presented herein show that expressed fs-indels are highly enriched in genomic positions predicted to escape NMD, and have higher protein-level expression (relative to non-expressed fs-indels). Expressed fs-indels (a.k.a. NMD-escape mutations) also significantly associated with clinical benefit from immunotherapy.

NMD-escape mutation count was found to significantly associate with clinical benefit from immunotherapy, across both CPI and ACT modalities, and with a stronger association than either nsSNVs or fs-indels. CPI clinical benefit rates for patients with≥ one NMD-escape mutation were elevated (range across the cohorts analysed = 0.56-0.71) compared to patients with zero such events (range 0.12-0.35). Furthermore experimental evidence, analyzed from anti-tumor vaccine and CPI studies, demonstrates T cell reactivity against expressed frameshifted neoepitopes directly in human patients. T cell reactive fs-indel neoantigens were enriched in NMD-escape exon positions (OR=12.5 [0.9-780.7], P=0.043, versus experimentally screened, but T cell non-reactive fs-indels.

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All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

Claims

1. A method of treating or preventing cancer in a subject, comprising targeting an expressed frameshift expressed frameshift indel neo-antigen.

2. A method according to claim 1 , comprising administering to said subject:

(i) an expressed frameshift expressed frameshift indel neo-antigen;

(ii) an immune cell which recognises an expressed frameshift expressed frameshift indel neo-antigen; or

(iii) an antibody which recognises an expressed frameshift expressed frameshift indel neo-antigen.

3. The method according to claim 1 or claim 2 wherein the immune cell is a T cell, B cell or dendritic cell.

4. The method according to claim 3 wherein the immune cell is a T cell.

5. The method according to claim 1 or claim 2 wherein the antibody is a monoclonal antibody.

6. The method according to any one of claims 1 to 5 wherein the cancer is selected from bladder cancer, gastric cancer, oesophageal cancer, breast cancer, colorectal cancer, cervical cancer, ovarian cancer, endometrial cancer, kidney cancer (renal cell), lung cancer (small cell, non-small cell and mesothelioma), brain cancer (gliomas, astrocytomas, glioblastomas), melanoma, lymphoma, small bowel cancers (duodenal and jejunal), leukaemia, pancreatic cancer, hepatobiliary tumors, germ cell cancers, prostate cancer, head and neck cancers, thyroid cancer and sarcomas.

7. The method according to claim 6 wherein the cancer is kidney cancer.

8. The method according to claim 6 wherein the cancer is melanoma.

9. The method according to any one of claims 1 to 8 wherein the subject is a mammal, preferably a human, cat, dog, horse, donkey, sheep, pig, goat, cow, mouse, rat, rabbit or guinea pig.

10. The method according to claim 9 wherein the subject is a human.

1 1. A composition which comprises an expressed frameshift indel neoantigen, expressed frameshift expressed frameshift indel neo-antigen specific immune cell, or an antibody specific to an expressed frameshift expressed frameshift indel neo-antigen.

12. The composition according to claim 1 1 wherein said immune cell is a T cell.

13. The composition according to claim 12 wherein said composition is enriched for T cells that target expressed frameshift indel neoantigens.

14. A method for providing a T cell population which targets an expressed frameshift indel neoantigen in a tumor from a subject which comprises the steps of:

i) isolating a population of T cells from a subject; and

ii) expanding the population of T cells to increase the number or relative proportion of T cells that target expressed frameshift indel neoantigens.

15. A composition comprising a population of T cells obtained or obtainable by the method according to claim 14.

16. A immunogenic composition or vaccine comprising an expressed frameshift expressed frameshift indel neo-antigen.

17. The composition or vaccine according to claim 16 wherein the expressed frameshift expressed frameshift indel neo-antigen is in the form of a peptide, a protein, a DNA or RNA molecule which encodes the peptide or protein, or is encompassed in a cell.

18. A cell expressing an expressed frameshift expressed frameshift indel neo-antigenic molecule, or a part thereof, on its surface, or a population thereof.

19. An expressed frameshift expressed frameshift indel neo-antigen, immune cell which recognises an expressed frameshift expressed frameshift indel neo-antigen, or antibody which recognises an expressed frameshift expressed frameshift indel neo-antigen for use in the treatment or prevention of cancer in a subject.

20. Use of an expressed frameshift expressed frameshift indel neo-antigen, immune cell which recognises an expressed frameshift expressed frameshift indel neo-antigen or antibody which recognises an expressed frameshift expressed frameshift indel neo-antigen in the manufacture of a medicament for use in the treatment or prevention of cancer in a subject.