Patent Publication Number: US-2021162032-A1

Title: Cancer vaccines for breast cancer

Description:
FIELD OF THE INVENTION 
     The invention relates to the field of cancer, in particular breast cancer. In particular, it relates to the field of immune system directed approaches for tumor reduction and control. Some aspects of the invention relate to vaccines, vaccinations and other means of stimulating an antigen specific immune response against a tumor in individuals. Such vaccines comprise neoantigens resulting from frameshift mutations that bring out-of-frame sequences of the GATA3, CDH1, MAP3K1, RUNX1, and TP53 genes in-frame. Such vaccines are also useful for ‘off the shelf’ use. 
     BACKGROUND OF THE INVENTION 
     There are a number of different existing cancer therapies, including ablation techniques (e.g., surgical procedures and radiation) and chemical techniques (e.g., pharmaceutical agents and antibodies), and various combinations of such techniques. Despite intensive research such therapies are still frequently associated with serious risk, adverse or toxic side effects, as well as varying efficacy. 
     There is a growing interest in cancer therapies that aim to target cancer cells with a patient&#39;s own immune system (such as cancer vaccines or checkpoint inhibitors, or T-cell based immunotherapy). Such therapies may indeed eliminate some of the known disadvantages of existing therapies, or be used in addition to the existing therapies for additional therapeutic effect. Cancer vaccines or immunogenic compositions intended to treat an existing cancer by strengthening the body&#39;s natural defenses against the cancer and based on tumor-specific neoantigens hold great promise as next-generation of personalized cancer immunotherapy. Evidence shows that such neoantigen-based vaccination can elicit T-cell responses and can cause tumor regression in patients. 
     Typically the immunogenic compositions/vaccines are composed of tumor antigens (antigenic peptides or nucleic acids encoding them) and may include immune stimulatory molecules like cytokines that work together to induce antigen-specific cytotoxic T-cells that target and destroy tumor cells. Vaccines containing tumor-specific and patient-specific neoantigens require the sequencing of the patients&#39; genome and tumor genome in order to determine whether the neoantigen is tumor specific, followed by the production of personalized compositions. Sequencing, identifying the patient&#39;s specific neoantigens and preparing such personalized compositions may require a substantial amount of time, time which may unfortunately not be available to the patient, given that for some tumors the average survival time after diagnosis is short, sometimes around a year or less. 
     Accordingly, there is a need for improved methods and compositions for providing subject-specific immunogenic compositions/cancer vaccines. In particular it would be desirable to have available a vaccine for use in the treatment of cancer, wherein such vaccine is suitable for treatment of a larger number of patients, and can thus be prepared in advance and provided off the shelf. There is a clear need in the art for personalized vaccines which induce an immune response to tumor specific neoantigens. One of the objects of the present disclosure is to provide personalized therapeutic cancer vaccines that can be provided off the shelf. An additional object of the present disclosure is to provide cancer vaccines that can be provided prophylactically. Such vaccines are especially useful for individuals that are at risk of developing cancer. 
     SUMMARY OF THE INVENTION 
     In a preferred embodiment, the disclosure provides a vaccine or collection of vaccines for use in the treatment of breast cancer, said vaccine comprising: 
     (i) a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 1, an amino acid sequence having 90% identity to Sequence 1, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 1; and 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 2, an amino acid sequence having 90% identity to Sequence 2, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 2; preferably also comprising 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 3, an amino acid sequence having 90% identity to Sequence 3, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 3; 
     (ii) a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 17, an amino acid sequence having 90% identity to Sequence 17, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 17; and 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 18, an amino acid sequence having 90% identity to Sequence 18, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 18; preferably also comprising 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 19, an amino acid sequence having 90% identity to Sequence 19, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 19 and/or a peptide having the amino acid sequence selected from Sequence 20, an amino acid sequence having 90% identity to Sequence 20, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 20; and/or a peptide having the amino acid sequence selected from Sequence 21, an amino acid sequence having 90% identity to Sequence 21, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 21 and/or a peptide having the amino acid sequence selected from Sequence 22, an amino acid sequence having 90% identity to Sequence 22, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 22; 
     (iii) a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 45, an amino acid sequence having 90% identity to Sequence 45, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 45; and 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 46, an amino acid sequence having 90% identity to Sequence 46, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 46; 
     (iv) a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 108, an amino acid sequence having 90% identity to Sequence 108, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 108; and 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequences 109-111, an amino acid sequence having 90% identity to Sequences 109-111, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 109-111; 
     and/or 
     (v) at least two peptides, wherein each peptide, or a collection of tiled peptides, comprises a different amino acid sequence selected from Sequences 200-206, an amino acid sequence having 90% identity to Sequences 200-206, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 200-206. 
     In a preferred embodiment, the disclosure provides a collection of frameshift-mutation peptides comprising: 
     (i) a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 1, an amino acid sequence having 90% identity to Sequence 1, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 1; and 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 2, an amino acid sequence having 90% identity to Sequence 2, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 2; preferably also comprising 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 3, an amino acid sequence having 90% identity to Sequence 3, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 3; 
     (ii) a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 17, an amino acid sequence having 90% identity to Sequence 17, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 17; and 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 18, an amino acid sequence having 90% identity to Sequence 18, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 18; preferably also comprising 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 19, an amino acid sequence having 90% identity to Sequence 19, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 19; and/or a peptide having the amino acid sequence selected from Sequence 20, an amino acid sequence having 90% identity to Sequence 20, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 20; and/or a peptide having the amino acid sequence selected from Sequence 21, an amino acid sequence having 90% identity to Sequence 21, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 21 and/or a peptide having the amino acid sequence selected from Sequence 22, an amino acid sequence having 90% identity to Sequence 22, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 22 
     (iii) a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 45, an amino acid sequence having 90% identity to Sequence 45, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 45; and 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 46, an amino acid sequence having 90% identity to Sequence 46, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 46; 
     (iv) a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 108, an amino acid sequence having 90% identity to Sequence 108, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 108; and 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequences 109-111, an amino acid sequence having 90% identity to Sequences 109-111, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 109-111; 
     and/or 
     (v) at least two peptides, wherein each peptide, or a collection of tiled peptides, comprises a different amino acid sequence selected from Sequences 200-206, an amino acid sequence having 90% identity to Sequences 200-206, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 200-206. 
     In a preferred embodiment, a collection of frameshift-mutation peptides comprises a peptide, or a collection of tiled peptides, having the amino acid sequence 17, an amino acid sequence having 90% identity to Sequence 17, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 17; and a peptide, or a collection of tiled peptides, having the amino acid sequence 18 or 19, an amino acid sequence having 90% identity to Sequence 18 or 19, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 18 or 19. Preferably, the collection comprises a peptide having the sequence 17, a peptide having the sequence 18, and a peptide having the sequence 19 (or a collection of titled peptides, or amino acid sequence having 90% identity to said sequences or a fragment thereof comprising at least 10 consecutive amino acids of said sequences). 
     In a preferred embodiment, the disclosure provides a peptide, or a collection of tiled peptides, comprising an amino acid sequence selected from the groups: 
     (i) Sequences 1-16 or 231-234, an amino acid sequence having 90% identity to Sequences 1-16 or 231-234, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 1-16 or 231-234; 
     (ii) Sequences 17-44, an amino acid sequence having 90% identity to Sequences 17-44, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 17-44; 
     (iii) Sequences 45-107, an amino acid sequence having 90% identity to Sequences 45-107, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 45-107; 
     (iv) Sequences 108-199, an amino acid sequence having 90% identity to Sequences 108-199, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 108-199; and 
     (v) Sequences 200-230, an amino acid sequence having 90% identity to Sequences 200-230, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 200-230. 
     Preferred collections comprise Sequences 4, 5, and/or 6. For example, a preferred collection comprises Sequence 4 (an amino acid sequence having 90% identity to Sequence 4, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 4) and Sequence 1 (an amino acid sequence having 90% identity to Sequence 1, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 1). 
     In some embodiments of the disclosure, the peptides are linked, preferably wherein said peptides are comprised within the same polypeptide. 
     In a preferred embodiment, the disclosure provides one more isolated nucleic acid molecules encoding the peptides or collection of peptides as disclosed herein. In a preferred embodiment, the disclosure provides one or more vectors comprising the nucleic acid molecules disclosed herein, preferably wherein the vector is a viral vector. In a preferred embodiment, the disclosure provides a host cell comprising the isolated nucleic acid molecules or the vectors as disclosed herein. 
     In a preferred embodiment, the disclosure provides a binding molecule or a collection of binding molecules that bind the peptide or collection of peptides disclosed herein, where in the binding molecule is an antibody, a T-cell receptor, or an antigen binding fragment thereof. 
     In a preferred embodiment, the disclosure provides a chimeric antigen receptor or collection of chimeric antigen receptors each comprising i) a T cell activation molecule; ii) a transmembrane region; and iii) an antigen recognition moiety; wherein said antigen recognition moieties bind the peptide or collection of peptides disclosed herein. In a preferred embodiment, the disclosure provides a host cell or combination of host cells that express the binding molecule or collection of binding molecules, or the chimeric antigen receptor or collection of chimeric antigen receptors as disclosed herein. 
     In a preferred embodiment, the disclosure provides a vaccine or collection of vaccines comprising the peptide or collection of peptides, the nucleic acid molecules, the vectors, or the host cells as disclosed herein; and a pharmaceutically acceptable excipient and/or adjuvant, preferably an immune-effective amount of adjuvant. 
     In a preferred embodiment, the disclosure provides the vaccines or collection of vaccines as disclosed herein for use in the treatment of breast cancer in an individual. In a preferred embodiment, the disclosure provides the vaccines as disclosed herein for prophylactic use in the prevention of breast cancer in an individual. In a preferred embodiment, the disclosure provides the vaccines as disclosed herein for use in the preparation of a medicament for treatment of breast cancer in an individual or for prophylactic use. In a preferred embodiment, the disclosure provides methods of treating an individual for breast cancer or reducing the risk of developing said cancer, the method comprising administering to the individual in need thereof a therapeutically effective amount of a vaccine as disclosed herein. 
     In a preferred embodiment, the individual has breast cancer and one or more cancer cells of the individual:
     (i) expresses a peptide having the amino acid sequence selected from Sequences 1-234, an amino acid sequence having 90% identity to any one of Sequences 1-234, or a fragment thereof comprising at least 10 consecutive amino acids of amino acid sequence selected from Sequences 1-234;   (ii) or comprises a DNA or RNA sequence encoding an amino acid sequences of (i).   

     In a preferred embodiment, the individual has a germline mutation in the PALB2, CHEK2, BRCA1, and/or BRCA2 gene. In a preferred embodiment, the method, preferably the prophylactic method, further comprises determining whether said individual has a germline mutation in the PALB2, CHEK2, BRCA1, and/or BRCA2 gene. In a preferred embodiment, the individual has a germline mutation in the BRCA1 and/or BRCA2 gene and preferably the individual is provided with a vaccine against TP53, GATA3, and/or RUNX1 novel open reading frames. In some embodiments, the individual prophylactically administered a vaccine as disclosed herein has not been diagnosed with breast cancer. 
     In a preferred embodiment, the disclosure provides a method of stimulating the proliferation of human T-cells, comprising contacting said T-cells with the peptide or collection of peptides, the nucleic acid molecules, the vectors, the host cell, or the vaccine as disclosed herein. 
     In a preferred embodiment, the disclosure provides a storage facility for storing vaccines. Preferably the facility stores at least two different cancer vaccines as disclosed herein. Preferably the storing facility stores:
     a vaccine comprising:   

     (i) a peptide having the amino acid sequence selected from Sequence 1, an amino acid sequence having 90% identity to Sequence 1, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 1; and 
     a peptide having the amino acid sequence selected from Sequence 2, an amino acid sequence having 90% identity to Sequence 2, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 2; preferably also comprising 
     a peptide having the amino acid sequence selected from Sequence 3, an amino acid sequence having 90% identity to Sequence 3, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 3;
     and one or more vaccines selected from:   a vaccine comprising:   

     (i) a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 1, an amino acid sequence having 90% identity to Sequence 1, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 1; and 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 2, an amino acid sequence having 90% identity to Sequence 2, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 2; preferably also comprising 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 3, an amino acid sequence having 90% identity to Sequence 3, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 3;
     and one or more vaccines selected from:   a vaccine comprising:   

     (ii) a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 17, an amino acid sequence having 90% identity to Sequence 17, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 17; and 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 18, an amino acid sequence having 90% identity to Sequence 18, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 18; preferably also comprising 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 19, an amino acid sequence having 90% identity to Sequence 19, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 19; and/or a peptide having the amino acid sequence selected from Sequence 20, an amino acid sequence having 90% identity to Sequence 20, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 20; and/or a peptide having the amino acid sequence selected from Sequence 21, an amino acid sequence having 90% identity to Sequence 21, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 21 and/or a peptide having the amino acid sequence selected from Sequence 22, an amino acid sequence having 90% identity to Sequence 22, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 22
     a vaccine comprising:   

     (iii) a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 45, an amino acid sequence having 90% identity to Sequence 45, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 45; and 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 46, an amino acid sequence having 90% identity to Sequence 46, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 46;
     a vaccine comprising:   

     (iv) a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequence 108, an amino acid sequence having 90% identity to Sequence 108, or a fragment thereof comprising at least 10 consecutive amino acids of Sequence 108; and 
     a peptide, or a collection of tiled peptides, having the amino acid sequence selected from Sequences 109-111, an amino acid sequence having 90% identity to Sequences 109-111, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 109-111; and/or
     a vaccine comprising:   (v) at least two peptides, wherein each peptide, or a collection of tiled peptides, comprises a different amino acid sequence selected from Sequences 200-206, an amino acid sequence having 90% identity to Sequences 200-206, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 200-206.   

     Preferably, the storage facility comprises at least 5, at least 10, or at least 20 vaccines as disclosed herein. 
     In a preferred embodiment, the disclosure provides a method for providing a vaccine for immunizing a patient against a cancer in said patient comprising determining the sequence of GATA3; TP53; CDH1; MAP3K1 or RUNX1 in cancer cells of said cancer and when the determined sequence comprises a frameshift mutation that produces a neoantigen of Sequence 1-234 or a fragment thereof, providing a vaccine comprising said neoantigen or a fragment thereof. Preferably, the vaccine is obtained from a storage facility as disclosed herein. 
     REFERENCE TO A SEQUENCE LISTING 
     The Sequence listing, which is a part of the present disclosure, includes a text file comprising amino acid and/or nucleic acid sequences. The subject matter of the Sequence listing is incorporated herein by reference in its entirety. The information recorded in computer readable form is identical to the written sequence listing. In the event of a discrepancy between the Sequence listing and the description, e.g., in regard to a sequence or sequence numbering, the description (e.g., Table 1) is leading. 
     DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENTS 
     One issue that may arise when considering personalized cancer vaccines is that once a tumor from a patient has been analysed (e.g. by whole genome or exome sequencing), neoantigens need to be selected and made in a vaccine. This may be a time consuming process, while time is something the cancer patient usually lacks as the disease progresses. 
     Somatic mutations in cancer can result in neoantigens against which patients can be vaccinated. Unfortunately, the quest for tumor specific neoantigens has yielded no targets that are common to all tumors, yet foreign to healthy cells. Single base pair substitutions (SNVs) at best can alter 1 amino acid which can result in a neoantigen. However, with the exception of rare site-specific oncogenic driver mutations (such as RAS or BRAF) such mutations are private and thus not generalizable. 
     An “off-the-shelf” solution, where vaccines are available against each potential-neoantigen would be beneficial. The present disclosure is based on the surprising finding that, despite the fact that there are infinite possibilities for frame shift mutations in the human genome, a vaccine can be developed that targets the novel amino acid sequence following a frame shift mutation in a tumor with potential use in a large population of cancer patients. 
     Neoantigens resulting from frame shift mutations have been previously described as potential cancer vaccines. See, for example, WO95/32731, WO2016172722 (Nantomics), WO2016/187508 (Broad), WO2017/173321 (Neon Therapeutics), US2018340944 (University of Connecticut), and WO2019/012082 (Nouscom), as well as Rahma et al. (Journal of Translational Medicine 2010 8:8) which describes peptides resulting from frame shift mutations in the von Hippel-Lindau tumor suppressor gene (VHL) and Rajasagi et al. (Blood 2014 124(3):453-462) which reports the systematic identification of personal tumor specific neoantigens. 
     The present disclosure provides a unique set of sequences resulting from frame shift mutations and that are shared among breast cancer patients. The finding of shared frame shift sequences is used to define an off-the-shelf breast cancer vaccine that can be used for both therapeutic and prophylactic use in a large number of individuals. 
     In the present disclosure we provide a source of common neoantigens induced by frame shift mutations, based on analysis of 986 TCGA breast tumor samples and 568 and 1918 breast tumor samples from other resources (see Priestley et al. 2019 at https://doi.org/10.1101/415133 and Razavi et al. 2018 Cancer Cell 34:427-438, respectively). We find that these frame shift mutations can produce long neoantigens. These neoantigens are typically new to the body, and can be highly immunogenic. The heterogeneity in the mutations that are found in tumors of different organs or tumors from a single organ in different individuals has always hampered the development of specific medicaments directed towards such mutations. The number of possible different tumorigenic mutations, even in a single gene as P53 was regarded prohibitive for the development of specific treatments. In the present disclosure it was found that many of the possible different frame shift mutations in a gene converge to the same small set of 3′ neo open reading frame peptides (neopeptides or NOPs). We find a fixed set of only 1,244 neopeptides in as much as 30% of all TCGA cancer patients. For some tumor classes this is higher; e.g. for colon and cervical cancer, peptides derived from only ten genes (saturated at 90 peptides) can be applied to 39% of all patients. 50% of all TCGA patients can be targeted at saturation (using all those peptides in the library found more than once). A pre-fabricated library of vaccines (peptide, RNA or DNA) based on this set can provide off the shelf, quality certified, ‘personalized’ vaccines within hours, saving months of vaccine preparation. This is important for critically ill cancer patients with short average survival expectancy after diagnosis. 
     The concept of utilizing the immune system to battle cancer is very attractive and studied extensively. Indeed, neoantigens can result from somatic mutations, against which patients can be vaccinated1-11. Recent evidence suggests that frame shift mutations, that result in peptides which are completely new to the body, can be highly immunogenic12-15. The immune response to neoantigen vaccination, including the possible predictive value of epitope selection has been studied in great detail8, 13, 16-21 and WO2007/101227, and there is no doubt about the promise of neoantigen-directed immunotherapy. Some approaches find subject-specific neoantigens based on alternative reading frames caused by errors in translation/transcription (WO2004/111075). Others identify subject specific neoantigens based on mutational analysis of the subjects tumor that is to be treated (WO1999/058552; WO2011/143656; US20140170178; WO2016/187508; WO2017/173321). The quest for common antigens, however, has been disappointing, since virtually all mutations are private. For SNV-derived amino acid changes, one can derive algorithms that predict likely good epitopes, but still every case is different. 
     A change of one amino acid in an otherwise wild-type protein may or may not be immunogenic. The antigenicity depends on a number of factors including the degree of fit of the proteasome-produced peptides in the MHC and ultimately on the repertoire of the finite T-cell system of the patient. In regards to both of these points, novel peptide sequences resulting from a frame shift mutation (referred to herein as novel open reading frames or pNOPs) are a priori expected to score much higher. For example, a fifty amino acid long novel open reading frame sequence is as foreign to the body as a viral antigen. In addition, novel open reading frames can be processed by the proteasome in many ways, thus increasing the chance of producing peptides that bind MHC molecules, and increasing the number of epitopes will be seen by T-cell in the body repertoire. 
     It is has been established that novel proteins/peptides can arise from frameshift mutations 32,36 . Furthermore, tumors with a high load of frameshift mutations (micro-satellite instable tumors) have a high density of tumor infiltrating CD8+ T cells 33 . In fact, it has been shown that neo-antigens derived from frameshift mutations can elicit cytotoxic T cell responses 32,34,33 . A recent study demonstrated that a high load of frameshift indels or other mutation types correlates with response to checkpoint inhibitors 33 . 
     Binding affinity to MHC class-I molecules was systematically predicted for frameshift indel and point mutations derived neoantigens 35 . Based on this analysis, neoantigens derived from frameshifts indels result in 3 times more high-affinity MHC binders compared to point mutation derived neoantigens, consistent with earlier work 31 . Almost all frameshift derived neoantigens are so-called mutant-specific binders, which means that cells with reactive T cell receptors for those frameshift neoantigens are (likely) not cleared by immune tolerance mechanisms 35 . These data are all in favour of neo-peptides from frameshift being superior antigens. 
     Here we report that frame shift mutations, which are also mostly unique among patients and tumors, nevertheless converge to neo open reading frame peptides (NOPs) from their translation products that surprisingly result in common neoantigens in large groups of cancer patients. The disclosure is based, in part, on the identification of common, tumor specific novel open reading frames resulting from frame shift mutations. Accordingly, the present disclosure provides novel tumor neoantigens and vaccines for the treatment of cancer. In some embodiments, multiple neoantigens corresponding to multiple NOPs can be combined, preferably within a single peptide or a nucleic acid molecule encoding such single peptide. This has the advantage that a large percentage of the patients can be treated with a single vaccine. 
     While not wishing to be bound by theory, the surprisingly high number of frame shift induced novel open reading frames shared by cancer patients can be explained, at least in part, as follows. Firstly, on the molecular level, different frame shift mutations can lead to the generation of shared novel open reading frames (or sharing at least part of a novel open reading frame). Secondly, the data presented herein suggests that frame shift mutations are strong loss-of-function mutations. This is illustrated in  FIG. 2A , where it can be seen that the SNVs in the TCGA database are clustered within the p53 gene, presumably because mutations elsewhere in the gene do not inactive gene function. In contrast, frame shift mutations occur throughout the p53 gene ( FIG. 2B ). This suggests that frame shift mutations virtually anywhere in the p53 ORF reduce function (splice variants possibly excluded), while not all point mutations in p53 are expected to reduce function. Finally, the process of tumorigenesis naturally selects for loss of function mutations in genes that may suppress tumorigenesis. Interestingly, the present disclosure identifies frame shift mutations in genes that were not previously known as classic tumor suppressors, or that apparently do so only in some tissue tumor types (see, e.g.,  FIG. 8 ). These three factors are likely to contribute to the surprisingly high number of frame shift induced novel open reading frames shared by cancer patients; in particular, while frame shift mutations generally represent less than 10% of the mutations in cancer cells, their contribution to neoantigens and potential as vaccines is much higher. The high immunogenic potential of peptides resulting from frameshifts is to a large part attributable to their unique sequence, which is not part of any native protein sequence in humans, and would therefore not be recognised as ‘self’ by the immune system, which would lead to immune tolerance effects. The high immunogenic potential of out-of-frame peptides has been demonstrated in several recent papers. 
     Neoantigens are antigens that have at least one alteration that makes them distinct from the corresponding wild-type, parental antigen, e.g., via mutation in a tumor cell. A neoantigen can include a polypeptide sequence or a nucleotide sequence 
     As used herein the term “ORF” refers to an open reading frame. As used herein the term “neoORF” is a tumor-specific ORF (i.e., neoantigen) arising from a frame shift mutation. Peptides arising from such neo ORFs are also referred to herein as neo open reading frame peptides (NOPs) and neoantigens. 
     A “frame shift mutation” is a mutation causing a change in the frame of the protein, for example as the consequence of an insertion or deletion mutation (other than insertion or deletion of 3 nucleotides, or multitudes thereof). Such frameshift mutations result in new amino acid sequences in the C-terminal part of the protein. These new amino acid sequences generally do not exist in the absence of the frameshift mutation and thus only exist in cells having the mutation (e.g., in tumor cells and pre-malignant progenitor cells). 
       FIGS. 3 and 4  and the data discussed above provide the answer to the question: how many cancer patients exhibit in their tumor a frame shift in region x or gene y of the genome. The patterns result from the summation of all cancer patients. The disclosure surprisingly demonstrates that within a single cancer type (i.e. breast cancer), the fraction of patients with a frame shift in a subset of genes is much higher than the fractions identified when looking at all cancer patients. We find that careful analysis of the data shows that frame shift mutations in only five genes together are found in 20% of all breast cancers. 
     Novel 3′ neo open reading frame peptides (i.e., NOPs) of GATA3, CDH1, MAP3K1, RUNX1, and TP53 are depicted in table 1. The NOPs, are defined as the amino acid sequences encoded by the longest neo open reading frame sequence identified. Sequences of these NOPs are represented in table 1 as follows: 
     GATA3: Sequences 1-16, and 231-234; more preferably sequences 1-6. 
     TP53: Sequences 17-44, more preferably sequences 17-30. 
     CDH1: Sequences 45-107, more preferably sequences 45-55. 
     MAP3K1: Sequences 108-199, more preferably sequences 108-117. 
     RUNX1: Sequences 200-230, more preferably sequences 200-212. 
     The most preferred neoantigens are GATA3 frameshift mutation peptides, followed by TP53 frameshift mutation peptides, followed by CDH1 frameshift mutation peptides, followed by MAP3K1 frameshift mutation peptides, followed by RUNX1 frameshift mutation peptides. The preference for individual neoantigens directly correlates with the frequency of their occurrence in breast cancer patients, with GATA3 frameshift mutation peptides covering 9.2% of breast cancer patients, TP53 frameshift mutation peptides covering 5% of breast cancer patients, CDH1 frameshift mutation peptides covering 3.6% of breast cancer patients, MAP3K1 frameshift mutation peptides covering 3% of breast cancer patients, and RUNX1 frameshift mutation peptides covering 1.7% of breast cancer patients. Frameshift mutation peptides in additional genes cover additional breast cancer patients, such as TBX3 (1.6%), ARID1A (1.5%), PTEN (1%). For example, Sequences 1 and 2 or an amino acid sequence having 90% identity to Sequences 1 and 2, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 1 and 2 are highly preferred neoantigens. Specifically, the C-terminal (3′) part of Sequences 1 and 2 are highly preferred neoantigens, covering 7.5% of all breast cancer patients (see  FIG. 10 ). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Library of NOP sequences 
               
               
                 Sequences of NOPs including the percentage of patients identified in the present study with 
               
               
                 each NOP. The sequences referred to herein correspond to the sequence numbering in the 
               
               
                    table below. Different predicted alternative splice forms are indicated as “alt splice x”. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 % Breast 
               
               
                   
                   
                 Length 
                   
                   
                 Cancer 
               
               
                 Sequence 
                 PeptideID 
                 (aa) 
                 PeptideSeq 
                 gene 
                 Patients 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 1 
                 pNOP24385 
                 113 
                 PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHG 
                 GATA3 
                 4.58 
               
               
                   
                   
                   
                 LEPCSMLTGPPARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRS 
                   
                   
               
               
                   
                   
                   
                 SLWCLCSNH 
                   
                   
               
               
                   
               
               
                 2 
                 pNOP146515 
                 48 
                 AQAKAVCSQESRDVLCELSDHHNHTLEEECQWGPCLQCLWALLQASQY 
                 GATA3 
                 2.82 
               
               
                   
               
               
                 3 
                 pNOP111297 
                 57 
                 PRPRRCTRHPACPLDHTTPPAWSPPWVRALLDAHRAPSESPCSPFRLAFLQE 
                 GATA3 
                 0.89 
               
               
                   
                   
                   
                 QYHEA 
                   
                   
               
               
                   
               
               
                 4 
                 pNOP101981 
                 60 
                 TDRTGPSLSPSEGCLQPGEQGRPVRTVRPPQPHSGGGMPMGTLSAMPVGST 
                 GATA3 
                 0.37 
               
               
                   
                   
                   
                 TSFTILTDP 
                   
                   
               
               
                   
               
               
                 5 
                 pNOP481880 
                 18 
                 QTPDYEEGRHPDQKPKNV 
                 GATA3 
                 0.23 
               
               
                   
               
               
                 6 
                 pNOP160582 
                 46 
                 RRKASRPETEKCLANPKSAKKCMTHWRTSPRTARLTRPPSPDTCPP 
                 GATA3 
                 0.12 
               
               
                   
               
               
                 7 
                 pNOP1989 
                 250 
                 HRRSRQPRPALLRKLGQGHGAEVPSDPPREPGVPPASASWIPTLAGRRQSPG 
                 GATA3 
                 0.09 
               
               
                   
                 alt splice a 
                   
                 QPPHRLPLESQPLLQDVHPPRLPGAPLRLPPGLVLLLVGGPRQPAPLHLPAHP 
                   
                   
               
               
                   
                   
                   
                 AEGRLPGPIAVHPRLGRLGPAGRERVPQVPGAPARQHEAGVVPLPWQHDRP 
                   
                   
               
               
                   
                   
                   
                 GWSLLVDPPPHHHLPALRARVQLRTLPPQQPAGRLPHRLRMQVQAQGPVQ 
                   
                   
               
               
                   
                   
                   
                 HRRQGVCELWGNLDPTVAARWHGTLPVQRLRALSQNERTEPAPH 
                   
                   
               
               
                   
               
               
                 8 
                 pNOP2004 
                 249 
                 HRRSRQPRPALLRKLGQGHGAEVPSDPPREPGVPPASASWIPTLAGRRQSPG 
                 GATA3 
                 0.09 
               
               
                   
                 alt splice a 
                   
                 QPPHRLPLESQPLLQDVHPPRLPGAPLRLPPGLVLLLVGGPRQPAPLHLPAHP 
                   
                   
               
               
                   
                   
                   
                 AEGRLPGPIAVHPRLGRLGPAGRERVPQVPGAPARQHEAGVVPLPWQHDRP 
                   
                   
               
               
                   
                   
                   
                 GWSLLVDPPPHHHLPALRARVQLRTLPPQQPAGRLPHRLRMQVQAQGPVQ 
                   
                   
               
               
                   
                   
                   
                 HRQGVCELWGNLDPTVAARWHGTLPVQRLRALSQNERTEPAPH 
                   
                   
               
               
                   
               
               
                 9 
                 pNOP104436 
                 59 
                 PPWVEPPRRPTTPSPPTRPTCPSTAPDSSPPAACWAAPPPASDASPGPRPGP 
                 GATA3 
                 &lt;.01 
               
               
                   
                 aft splice b 
                   
                 AQKAGSV 
                   
                   
               
               
                   
               
               
                 10 
                 pNOP107768 
                 58 
                 PPWVEPPRRPTTPSPPTRPTCPSTAPDSSPPAACWAAPPPASDASPGPRPGP 
                 GATA3 
                 &lt;.01 
               
               
                   
                 alt splice b 
                   
                 AQAGSV 
                   
                   
               
               
                   
               
               
                 11 
                 pNOP133723 
                 51 
                 HGGDGGPAALGEPPPPRRAQRAAPGHAPPGPQPLLHGRGAVPAAGGGGCA 
                 GATA3 
                 &lt;.01 
               
               
                   
                   
                   
                 F 
                   
                   
               
               
                   
               
               
                 12 
                 pNOP315778 
                 28 
                 TVGQPRPHCGGEMARDTTCATPAGSITK 
                 GATA3 
                 &lt;.01 
               
               
                   
               
               
                 13 
                 pNOP5779 
                 182 
                 ATTTPPCSTGSTRTRTTRASATPTWTRRSTRCRRRWMCFLTSTVKATTSRPTT 
                 GATA3 
                 &lt;.01 
               
               
                   
                   
                   
                 ETRSGPRCRGTLRPTTGARCAARLCFMDPYPGWTAAKPWAATTPPPPGISAP 
                   
                   
               
               
                   
                   
                   
                 SPRRPSTTAPRGPSPSTPRPRPPPCRGATPARTSSPSRPPRRRTSPRTHRCPPQ 
                   
                   
               
               
                   
                   
                   
                 ARPARPGRTRKSASSTRCPCPTA 
                   
                   
               
               
                   
               
               
                 14 
                 pNOP621902 
                 12 
                 ATSRPSATPATC 
                 GATA3 
                 &lt;.01 
               
               
                   
               
               
                 15 
                 pNOP668144 
                 11 
                 LTGGLPQEQLV 
                 GATA3 
                 &lt;.01 
               
               
                   
               
               
                 16 
                 pNOP716595 
                 10 
                 SWSRPTPVAA 
                 GATA3 
                 &lt;.01 
               
               
                   
               
               
                 17 
                 pNOP36301 
                 97 
                 TGGPSSPSSHWKTPVVIYWDGTALRCVFVPVLGETGAQRKRISARKGSLTTSC 
                 TP53 
                 1.07 
               
               
                   
                 alt splice c 
                   
                 PQGALSEHCPTTPAPLPSQRRNHWMENISPFRSVGVSASRCSES 
                   
                   
               
               
                   
               
               
                 18 
                 pNOP49591 
                 85 
                 SSQNARGCSPRGPCTSSSYTGGPCTSPLLAPVIFCPFPENLPGQLRFPSGLLAF 
                 TP53 
                 1.07 
               
               
                   
                   
                   
                 WDSQVCDLHVLPCPQQDVLPTGQDLPCAAVG 
                   
                   
               
               
                   
               
               
                 19 
                 pNOP31232 
                 103 
                 TGGPSSPSSHWKTPVVIYWDGTALRCVFVPVLGETGAQRKRISARKGSLTTSC 
                 TP53 
                 1.01 
               
               
                   
                 alt splice c 
                   
                 PQGALSEHCPTTPAPLPSQRRNHWMENISPFRTRPAFKKKIVKESMKMVL 
                   
                   
               
               
                   
               
               
                 20 
                 pNOP38141 
                 95 
                 TGGPSSPSSHWKTPVVIYWDGTALRCVFVPVLGETGAQRKRISARKGSLTTSC 
                 TP53 
                 1.01 
               
               
                   
                 alt splice c 
                   
                 PQGALSEHCPTTPAPLPSQRRNHWMENISPFRCYLTYDGVTS 
                   
                   
               
               
                   
               
               
                 21 
                 pNOP59073 
                 78 
                 CCPRTILNNGSLKTQVQMKLPECQRLLPPWPLHQQLLHRRPLHQPPPGPCHL 
                 TP53 
                 0.78 
               
               
                   
                   
                   
                 LSLPRKPTRAATVSVWASCILGQPSL 
                   
                   
               
               
                   
               
               
                 22 
                 pNOP70126 
                 72 
                 GAAPTMSAAQIAMVWPLLSILSEWKEICVWSIWMTETLFDIVWWCPMSRLR 
                 TP53 
                 0.63 
               
               
                   
                   
                   
                 LALTVPPSTTTTCVTVPAWAA 
                   
                   
               
               
                   
               
               
                 23 
                 pNOP158914 
                 46 
                 LARTPLPSTRCFANWPRPALCSCGLIPHPRPAPASAPWPSTSSHST 
                 TP53 
                 0.43 
               
               
                   
               
               
                 24 
                 pNOP272502 
                 31 
                 FHTPARHPRPRHGHLQAVTAHDGGCEALPPP 
                 TP53 
                 0.20 
               
               
                   
               
               
                 25 
                 pNOP252394 
                 33 
                 GACLCLSWERPAHRGRESPQERGASPRAAPREH 
                 TP53 
                 0.14 
               
               
                   
               
               
                 26 
                 pNOP193414 
                 40 
                 ASTAQQHQLLSPAKEETTGWRIFHPSGPDQLSKRKLLKRA 
                 TP53 
                 0.12 
               
               
                   
                 alt splice d 
                   
                   
                   
                   
               
               
                   
               
               
                 27 
                 pNOP281999 
                 30 
                 ASTAQQHQLLSPAKEETTGWRIFHPSDPWA 
                 TP53 
                 0.09 
               
               
                   
                 alt splice d 
                   
                   
                   
                   
               
               
                   
               
               
                 28 
                 pNOP293143 
                 29 
                 ASTAQQHQLLSPAKEETTGWRIFHPSDAT 
                 TP53 
                 0.09 
               
               
                   
                 alt splice d 
                   
                   
                   
                   
               
               
                   
               
               
                 29 
                 pNOP385655 
                 23 
                 QFLHGRHEPEAHPHHHHTGRLQW 
                 TP53 
                 0.09 
               
               
                   
               
               
                 30 
                 pNOP433152 
                 20 
                 HGHLQAVTAHDGGCEALPPP 
                 TP53 
                 0.09 
               
               
                   
               
               
                 31 
                 pNOP405064 
                 22 
                 RWSGPSSASYPSGRKFACGVFG 
                 TP53 
                 0.06 
               
               
                   
               
               
                 32 
                 pNOP136003 
                 51 
                 SPKRVSLPPAIKNSCSRQKGLTQTDILHFLFPTDSLPPPSLPPLPFWVLGL 
                 TP53 
                 0.03 
               
               
                   
               
               
                 33 
                 pNOP316190 
                 28 
                 VRKHFQTYGNYFLKTTFCPPCRPKQWMI 
                 TP53 
                 0.03 
               
               
                   
               
               
                 34 
                 pNOP367554 
                 24 
                 MRPWNSRMPRLGRSQGGAGLTPAT 
                 TP53 
                 0.03 
               
               
                   
               
               
                 35 
                 pNOP539666 
                 15 
                 DVLPTGQDLPCAAVG 
                 TP53 
                 0.03 
               
               
                   
               
               
                 36 
                 pNOP602122 
                 13 
                 KQRSVPLAVPSNG 
                 TP53 
                 0.03 
               
               
                   
               
               
                 37 
                 pNOP224126 
                 36 
                 CFANWPRPALCSCGLIPHPRPAPASAPWPSTSSHST 
                 TP53 
                 &lt;.01 
               
               
                   
               
               
                 38 
                 pNOP243169 
                 34 
                 GLGTQGCPGWEGARGEQGSLQPPEVQKGSVYLPP 
                 TP53 
                 &lt;.01 
               
               
                   
               
               
                 39 
                 pNOP445026 
                 20 
                 TRRKLKILSVGVSASRCSES 
                 TP53 
                 &lt;.01 
               
               
                   
               
               
                 40 
                 pNOP483390 
                 18 
                 RRAPSESGNIFRPMETTS 
                 TP53 
                 &lt;.01 
               
               
                   
               
               
                 41 
                 pNOP59708 
                 78 
                 LRLTFSTSCSPLTASHPHLSLPCHFGFWVFEPLLAIGVRQKHPGLPFALSRGSTE 
                 TP53 
                 &lt;.01 
               
               
                   
                   
                   
                 QVGLHWCFVVGRRMGSRTYQLRF 
                   
                   
               
               
                   
               
               
                 42 
                 pNOP604680 
                 13 
                 LTMVLLPDKLVVS 
                 TP53 
                 &lt;.01 
               
               
                   
               
               
                 43 
                 pNOP619453 
                 13 
                 WRSRSQILASSPL 
                 TP53 
                 &lt;.01 
               
               
                   
               
               
                 44 
                 pNOP703537 
                 10 
                 LYHHPLQLHV 
                 TP53 
                 &lt;.01 
               
               
                   
               
               
                 45 
                 pNOP68931 
                 73 
                 PTDPFLGLRLGLHLQKVFHQSHAEYSGAPPPPPAPSGLRFWNPSRIAHISQLLS 
                 CDH1 
                 0.49 
               
               
                   
                   
                   
                 WPQKTEERLGYSSHQLPRK 
                   
                   
               
               
                   
               
               
                 46 
                 pNOP35899 
                 97 
                 IQWGTTTAPRPIRPPFLESKQNCSHFPTPLLASEDRRETGLFLPSAAQKMKKAH 
                 CDH1 
                 0.40 
               
               
                   
                   
                   
                 FLKTWFRSNPTKTKKARFSTASLAKELTHPLLVSLLLKEKQDG 
                   
                   
               
               
                   
               
               
                 47 
                 pNOP162561 
                 45 
                 CRPSSQYISLHSRTNTRGECQLDHSVQRPNPRIYHFEAKDGLRGG 
                 CDH1 
                 0.32 
               
               
                   
               
               
                 48 
                 pNOP163094 
                 45 
                 FCCSCCFFGGERWSKSPYCPQRMTPGTTFITMMKKEAEKRTRTLT 
                 CDH1 
                 0.23 
               
               
                   
               
               
                 49 
                 pNOP137662 
                 50 
                 EGTACRSRIANSCHSGDSWRNSCFANSDSAALAVSSEESGGQRALTAPRG 
                 CDH1 
                 0.20 
               
               
                   
               
               
                 50 
                 pNOP123893 
                 54 
                 WALGAAASRRCCCCCRSPLGSARSRSPATLALTPRATRSRCPGATWREAASW 
                 CDH1 
                 0.17 
               
               
                   
                   
                   
                 AE 
                   
                   
               
               
                   
               
               
                 51 
                 pNOP164097 
                 45 
                 KESGSVRGLWRGPGNHILHCPGARHIYGTENNISDLERHCQLAGD 
                 CDH1 
                 0.14 
               
               
                   
               
               
                 52 
                 pNOP196613 
                 40 
                 MKPPSLCLLKREWKCPRTLAWARKSHPTLPRSQTHLWNRK 
                 CDH1 
                 0.12 
               
               
                   
               
               
                 53 
                 pNOP205726 
                 39 
                 RSACVTVKGPLASVGRHSLSKQDCKFLPFWGFLEEFLLC 
                 CDH1 
                 0.12 
               
               
                   
               
               
                 54 
                 pNOP359415 
                 25 
                 WWTICRHHKSSEQRWHFENSKGLGF 
                 CDH1 
                 0.12 
               
               
                   
               
               
                 55 
                 pNOP441910 
                 20 
                 RRCTNPHECPPVSSPPCQSR 
                 CDH1 
                 0.12 
               
               
                   
               
               
                 56 
                 pNOP266995 
                 32 
                 QSLWIENALPHTLSSLTLCHPTGMQLRIQWRF 
                 CDH1 
                 0.09 
               
               
                   
               
               
                 57 
                 pNOP312388 
                 28 
                 QEHRSHQCGHHWAGPREFPYVYPGGSSC 
                 CDH1 
                 0.09 
               
               
                   
               
               
                 58 
                 pNOP365712 
                 24 
                 KPGSDQIQQRQRRQGFLQHHWPRS 
                 CDH1 
                 0.09 
               
               
                   
               
               
                 59 
                 pNOP98858 
                 61 
                 TPTMPPSLTPSSAKILSSLTKICSPLTGTQESSVWSPLGWTERVSLRIPWWFKLL 
                 CDH1 
                 0.09 
               
               
                   
                   
                   
                 TFKVRG 
                   
                   
               
               
                   
               
               
                 60 
                 pNOP254972 
                 33 
                 LQSKGLYGFITHRSISWSTPGTPPTESFPPKSR 
                 CDH1 
                 0.06 
               
               
                   
               
               
                 61 
                 pNOP260719 
                 32 
                 AREEQHVHSPNHSYRQWFSSCYWNRDTSADPV 
                 CDH1 
                 0.06 
               
               
                   
               
               
                 62 
                 pNOP297360 
                 29 
                 KRLILTPQPRLMILCSCLTMKEAVPKLLV 
                 CDH1 
                 0.06 
               
               
                   
               
               
                 63 
                 pNOP311770 
                 28 
                 PIRMTTSPNSPRRSLRGLSWKVLFQEPL 
                 CDH1 
                 0.06 
               
               
                   
               
               
                 64 
                 pNOP335653 
                 26 
                 ILKIAPVDKGQPIFPSTPDSKWAQMV 
                 CDH1 
                 0.06 
               
               
                   
               
               
                 65 
                 pNOP499804 
                 17 
                 LMLMPPIPQRGRLYTPY 
                 CDH1 
                 0.06 
               
               
                   
               
               
                 66 
                 pNOP509630 
                 17 
                 TSLMQTFLPIHLPSQQN 
                 CDH1 
                 0.06 
               
               
                   
               
               
                 67 
                 pNOP523454 
                 16 
                 LQTMVLQLLLEQGHFC 
                 CDH1 
                 0.06 
               
               
                   
               
               
                 68 
                 pNOP243761 
                 34 
                 HIGFGETLPTGWRLIRTLVPFPLGLSWTGRILST 
                 CDH1 
                 0.03 
               
               
                   
               
               
                 69 
                 pNOP247500 
                 34 
                 RLHRSTKDSLFFPRHPIQSGHRWCDYSQKASTVS 
                 CDH1 
                 0.03 
               
               
                   
               
               
                 70 
                 pNOP290560 
                 30 
                 SQSLTPTIILRSSIPPRAWILRPSSSTFYT 
                 CDH1 
                 0.03 
               
               
                   
                 alt splice e 
                   
                   
                   
                   
               
               
                   
               
               
                 71 
                 pNOP302320 
                 29 
                 SQSLTPTIILRSSIPPRTRVRCLRTRLTS 
                 CDH1 
                 0.03 
               
               
                   
                 alt splice e 
                   
                   
                   
                   
               
               
                   
               
               
                 72 
                 pNOP371119 
                 24 
                 RRELHVHGAPAPPGERPRPGQSEF 
                 CDH1 
                 0.03 
               
               
                   
               
               
                 73 
                 pNOP383738 
                 23 
                 MTTPPYQNLELYSSVRGIQSLRS 
                 CDH1 
                 0.03 
               
               
                   
               
               
                 74 
                 pNOP387003 
                 23 
                 RMWYLLRSLSPPPQPPSPWMCWM 
                 CDH1 
                 0.03 
               
               
                   
               
               
                 75 
                 pNOP442322 
                 20 
                 RTHCHIHSLLSRCVIQRECS 
                 CDH1 
                 0.03 
               
               
                   
               
               
                 76 
                 pNOP472687 
                 18 
                 GLSHHLHSHRHRGCAGCE 
                 CDH1 
                 0.03 
               
               
                   
               
               
                 77 
                 pNOP490664 
                 17 
                 CEHLQCRHRLHHPQPRS 
                 CDH1 
                 0.03 
               
               
                   
               
               
                 78 
                 pNOP498423 
                 17 
                 KQQRAWILRPSSSTFYT 
                 CDH1 
                 0.03 
               
               
                   
               
               
                 79 
                 pNOP538388 
                 15 
                 CPQYPSVGGCIHHIE 
                 CDH1 
                 0.03 
               
               
                   
               
               
                 80 
                 pNOP633787 
                 12 
                 LEPAAQGPGRSA 
                 CDH1 
                 0.03 
               
               
                   
               
               
                 81 
                 pNOP656416 
                 11 
                 EESKASGHKHH 
                 CDH1 
                 0.03 
               
               
                   
               
               
                 82 
                 pNOP202693 
                 39 
                 HVRRRRGRLGDSREAGPRPMCWEMQKSRCWWFFSSLPLR 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 83 
                 pNOP263306 
                 32 
                 HGPLEPQPLGAAAAAAGLLLALPGAGALPPWL 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 84 
                 pNOP379760 
                 23 
                 GVCHGRCSSRNLCDGGHSHRRGR 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 85 
                 pNOP408019 
                 22 
                 VSPGIFPALPIPMKLEILLMKI 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 86 
                 pNOP414918 
                 21 
                 HTGRVPTGPFSTTTQPKNLSF 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 87 
                 pNOP462763 
                 19 
                 RWVTTKSISSSWITRIKTK 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 88 
                 pNOP467296 
                 19 
                 YGTENNISDLERHCQLAGD 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 89 
                 pNOP494963 
                 17 
                 GQAAVHSTRSSDECGTF 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 90 
                 pNOP556436 
                 15 
                 SELPELLRVRQRPGL 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 91 
                 pNOP585380 
                 14 
                 SSDLQSHHVQGSGA 
                 CDH1 
                 &lt;.01 
               
               
                   
                 alt splice f 
                   
                   
                   
                   
               
               
                   
               
               
                 92 
                 pNOP597964 
                 13 
                 GSNGDFDHGNRSE 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 93 
                 pNOP598198 
                 13 
                 GVKHNSNSCDHSH 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 94 
                 pNOP602266 
                 13 
                 KRNRMAEGDRASG 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 95 
                 pNOP603708 
                 13 
                 LLERMGQSLQEAG 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 96 
                 pNOP605229 
                 13 
                 MMMVDNLSSPQIQ 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 97 
                 pNOP609656 
                 13 
                 QRPHTRTSNYILL 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 98 
                 pNOP616731 
                 13 
                 TPQSQTKTRTMTT 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 99 
                 pNOP621664 
                 12 
                 ASCTGAWTLGLK 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 100 
                 pNOP646263 
                 12 
                 SSDLQSHHGLGF 
                 CDH1 
                 &lt;.01 
               
               
                   
                 alt splice f 
                   
                   
                   
                   
               
               
                   
               
               
                 101 
                 pNOP669584 
                 11 
                 NGDFDHGNRSE 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 102 
                 pNOP676234 
                 11 
                 RPSDHLRGQRV 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 103 
                 pNOP684840 
                 11 
                 WRSQPQTRTMM 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 104 
                 pNOP709338 
                 10 
                 QQARIHPGGL 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 105 
                 pNOP712539 
                 10 
                 RRRRRRGPGL 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 106 
                 pNOP714207 
                 10 
                 SGHWCHFHSG 
                 CDH1 
                 &lt;.01 
               
               
                   
               
               
                 107 
                 pNOP85108 
                 66 
                 TNGAIASRSWLTCTEAARTTRGLERGGPQTHVLGNAEITLLVVFQLPSLEMSF 
                 CDH1 
                 &lt;.01 
               
               
                   
                   
                   
                 WGKKRDWLVMQLV 
                   
                   
               
               
                   
               
               
                 108 
                 pNOP80315 
                 68 
                 VSRRGPGKCGFTSFQRPTQSFSWQLPIRSHSEIRISRSKEKKSFPSAFSEWQNH 
                 MAP3K1 
                 0.37 
               
               
                   
                   
                   
                 TTPKSPFTRWLLTI 
                   
                   
               
               
                   
               
               
                 109 
                 pNOP197571 
                 40 
                 QHWSETKNCRFWSCSQVGIKRNWCRRVSGTITGDNCIYGT 
                 MAP3K1 
                 0.23 
               
               
                   
               
               
                 110 
                 pNOP31404 
                 102 
                 CFQPCQPULLPHLYQLALQQMSLSIDFRDSFPAEYLLHLLKHSASFLYNSTETVL 
                 MAP3K1 
                 0.23 
               
               
                   
                   
                   
                 KTKTQINFPQSLLSQDPCPPVTYTGQSHLDLPQVIQVNREIPQKIA 
                   
                   
               
               
                   
               
               
                 111 
                 pNOP226356 
                 36 
                 IDCCWNFLLNFILILSVLMFHKLSLLKSGIRSCCPS 
                 MAP3K1 
                 0.14 
               
               
                   
               
               
                 112 
                 pNOP120630 
                 54 
                 CQWGPAVGKWGEHWKFWGQQWKCIPEWGSHQWVFPDQYLRRCGGGM 
                 MAP3K1 
                 0.12 
               
               
                   
                   
                   
                 LQRSVNGLC 
                   
                   
               
               
                   
               
               
                 113 
                 pNOP201764 
                 39 
                 FLSCRLKMEKISSLFNRIHQRLYQDIPKQNNRIEKTLNG 
                 MAP3K1 
                 0.12 
               
               
                   
               
               
                 114 
                 pNOP360358 
                 24 
                 ARRSSRSTKRRDKNDEPSESSKHH 
                 MAP3K1 
                 0.12 
               
               
                   
               
               
                 115 
                 pNOP122927 
                 54 
                 RILRLRVCSRNITVGVAQGSKLHLVTPSRSLFHACQILIHCHHLVLLRLVQKTA 
                 MAP3K1 
                 0.09 
               
               
                   
               
               
                 116 
                 pNOP139478 
                 50 
                 PCLQSVRCCFKNIESHAGIYSLPQFSGKNQTSETSPASCRHHPSQMCRCQ 
                 MAP3K1 
                 0.09 
               
               
                   
               
               
                 117 
                 pNOP194698 
                 40 
                 GGNCRSHPVGRRRHFGWSTGQLLAGICSQQLSGNHREQFP 
                 MAP3K1 
                 0.09 
               
               
                   
               
               
                 118 
                 pNOP224982 
                 36 
                 ELQILELQPGWHQKELVQESFRDNYWGQLHLWHLRY 
                 MAP3K1 
                 0.09 
               
               
                   
               
               
                 119 
                 pNOP336754 
                 26 
                 KWRRYHHYSTGYTRDSTRTYQSKTTV 
                 MAP3K1 
                 0.09 
               
               
                   
               
               
                 120 
                 pNOP367569 
                 24 
                 MSIQSSTPVLRTFLKHLCLQVIQQ 
                 MAP3K1 
                 0.09 
               
               
                   
               
               
                 121 
                 pNOP78143 
                 69 
                 YVPFVDLSGDLMISTATSCQVLWIPLLPSELHSSKPYSSSLWLDHEGIKRAILTLLI 
                 MAP3K1 
                 0.09 
               
               
                   
                   
                   
                 MELSKSLLLTKI 
                   
                   
               
               
                   
               
               
                 122 
                 pNOP116733 
                 55 
                 CNYCSIDPFTFVSWFTRCGSSLFRTSTSGQTSIKRATEASSLSYYMVANYADQL 
                 MAP3K1 
                 0.06 
               
               
                   
                   
                   
                 Q 
                   
                   
               
               
                   
               
               
                 123 
                 pNOP121052 
                 54 
                 FPLNGWQTFQKDLLEFCKNGYYSTPCVFKTVRNAECFQFHSLHQDASPFDGY 
                 MAP3K1 
                 0.06 
               
               
                   
                   
                   
                 CR 
                   
                   
               
               
                   
               
               
                 124 
                 pNOP187727 
                 41 
                 GEKEFPQCLFRVAESHHPEEPLHQMASHHIALRKQTAVLTK 
                 MAP3K1 
                 0.06 
               
               
                   
               
               
                 125 
                 pNOP194503 
                 40 
                 FLQPRVVKSCGFPFFPQSCTAANRTAAAFGWITKESREQF 
                 MAP3K1 
                 0.06 
               
               
                   
               
               
                 126 
                 pNOP212034 
                 38 
                 PHKSAVHINTVGTVQRPSRRVGSWQRNTKSWIHWYWWC 
                 MAP3K1 
                 0.06 
               
               
                   
               
               
                 127 
                 pNOP256729 
                 33 
                 QLSLQERSRQVRLHQLPKADAVLLLATPHQVAQ 
                 MAP3K1 
                 0.06 
               
               
                   
               
               
                 128 
                 pNOP264595 
                 32 
                 LEETAQTINTGCLLGLRTAAVHVEHSVFICYL 
                 MAP3K1 
                 0.06 
               
               
                   
               
               
                 129 
                 pNOP316970 
                 28 
                 YLRLLVQLLLHRSLHICLLVYEMWLFVV 
                 MAP3K1 
                 0.06 
               
               
                   
               
               
                 130 
                 pNOP64417 
                 75 
                 ERWPSGVFPMMSVGPCCWQMGRALEILGAAVEAARVGEPPVGLPRPVSQE 
                 MAP3K1 
                 0.06 
               
               
                   
                   
                   
                 MWWRHAAAFCQWSVLTLSTKCTLLL 
                   
                   
               
               
                   
               
               
                 131 
                 pNOP223009 
                 37 
                 VVEKKIMSAQAVSVVVRWRIKKLSKGCTRWMIVQRNE 
                 MAP3K1 
                 0.03 
               
               
                   
               
               
                 132 
                 pNOP239116 
                 35 
                 SNVQMPIAAQVSCPYQHCWNCAKAKQESWQLAEKY 
                 MAP3K1 
                 0.03 
               
               
                   
               
               
                 133 
                 pNOP244756 
                 34 
                 KTLWMVNRTASCRHLFPTTIWKPQRTVPLSAQSI 
                 MAP3K1 
                 0.03 
               
               
                   
               
               
                 134 
                 pNOP253661 
                 33 
                 IIQTSLGCWEPRVRRAITISSLNGWQGDRWLIC 
                 MAP3K1 
                 0.03 
               
               
                   
               
               
                 135 
                 pNOP271334 
                 31 
                 CMECWLCYYRNGLCKTTMECRKTLQSSCFDI 
                 MAP3K1 
                 0.03 
               
               
                   
               
               
                 136 
                 pNOP291483 
                 30 
                 TVVPNVMTALAVAAIVVMLLYPVTRQCSPQ 
                 MAP3K1 
                 0.03 
               
               
                   
               
               
                 137 
                 pNOP298479 
                 29 
                 MAERSTDRPWSIFFLLSGSRCGNWNFNGC 
                 MAP3K1 
                 0.03 
               
               
                   
               
               
                 138 
                 pNOP382700 
                 23 
                 LLSQKLLSCLLKRLKMMIPTKMM 
                 MAP3K1 
                 0.03 
               
               
                   
               
               
                 139 
                 pNOP397851 
                 22 
                 IVFLETKLNQTIGKNFLAAFVL 
                 MAP3K1 
                 0.03 
               
               
                   
               
               
                 140 
                 pNOP401956 
                 22 
                 PLLCSPLIIPTQWLANFPEGST 
                 MAP3K1 
                 0.03 
               
               
                   
               
               
                 141 
                 pNOP468088 
                 18 
                 ALEHFLLVIRLKMWELEL 
                 MAP3K1 
                 0.03 
               
               
                   
               
               
                 142 
                 pNOP535310 
                 16 
                 WLLQMRWKLPKPSSWA 
                 MAP3K1 
                 0.03 
               
               
                   
               
               
                 143 
                 pNOP594196 
                 13 
                 EAAVPLNLCFAVH 
                 MAP3K1 
                 0.03 
               
               
                   
               
               
                 144 
                 pNOP605820 
                 13 
                 NFNLRTDLHQESY 
                 MAP3K1 
                 0.03 
               
               
                   
               
               
                 145 
                 pNOP611013 
                 13 
                 RKESNFRDFSSQL 
                 MAP3K1 
                 0.03 
               
               
                   
               
               
                 146 
                 pNOP670032 
                 11 
                 NLQTQCYGEKL 
                 MAP3K1 
                 0.03 
               
               
                   
               
               
                 147 
                 pNOP692020 
                 10 
                 ETGQHFSRTF 
                 MAP3K1 
                 0.03 
               
               
                   
               
               
                 148 
                 pNOP94080 
                 62 
                 FNNNNNNNNRATKANGSNKRQTPQSVFELLSFISSFPINVSSLVNPFFFYPICT 
                 MAP3K1 
                 0.03 
               
               
                   
                   
                   
                 SWHCNRCL 
                   
                   
               
               
                   
               
               
                 149 
                 pNOP119437 
                 55 
                 SIQSFVLHGSQLCRSTTVETGCSTREKKLVGNHIDILLAMMPLNSYERGQWGT 
                 MAP3K1 
                 &lt;.01 
               
               
                   
                   
                   
                 LT 
                   
                   
               
               
                   
               
               
                 150 
                 pNOP133585 
                 51 
                 GRRRRRSPQGEQRARGCRGTAAGGGQRGPRAGGLAAAAAAQSAECGAGP 
                 MAP3K1 
                 &lt;.01 
               
               
                   
                   
                   
                 AA 
                   
                   
               
               
                   
               
               
                 151 
                 pNOP140186 
                 50 
                 RMKRNRCVLFACWACLMKKVLQCVKTAAGTSCTTTACQFGQKSVEEIENL 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 152 
                 pNOP158520 
                 46 
                 IPGVYWASELQLCTWNILYSSAICDAPGVSTRTFRPNVMEKNFKEF 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 153 
                 pNOP282791 
                 30 
                 DLLVQQQQQQQQQSNQSQWFKQKADPTVSV 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 154 
                 pNOP286430 
                 30 
                 LLTTLNSYSVAFRISMKTKSFTEMSKVPIC 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 155 
                 pNOP303263 
                 29 
                 TSGIHSLQNTFCISSNTAQVFSTIPQKLS 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 156 
                 pNOP314934 
                 28 
                 SSRGTNDQGETEGNLYASLEARMVGKEK 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 157 
                 pNOP341052 
                 26 
                 RRRSFSNCHGNVSVSGCPPHSSSAAG 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 158 
                 pNOP358464 
                 25 
                 VHSPFRENWKRIMCYKIECQFRGHF 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 159 
                 pNOP386384 
                 23 
                 RDSVHPSRGEMQIRCQYRAQLQY 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 160 
                 pNOP387248 
                 23 
                 RQPVCQPGSTNGWKGEIGEGLWW 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 161 
                 pNOP400051 
                 22 
                 LSHGFRCLEWNSLAAYFLETGM 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 162 
                 pNOP400858 
                 22 
                 NGGGGGESRLVVGIPGRQGYEP 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 163 
                 pNOP446688 
                 20 
                 WRIKKLSKGCTRWMIVQRNE 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 164 
                 pNOP455806 
                 19 
                 KWLVQNHHGMQKNTPIILL 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 165 
                 pNOP45906 
                 87 
                 AAALPCRLTAGLLDFPVAGARGRSGEWDRLPACGGAAAPRSREPRRRPPYRV 
                 MAP3K1 
                 &lt;.01 
               
               
                   
                   
                   
                 GGGAGQRRLESRSGRARGEAGARRRAVSCSGPRRS 
                   
                   
               
               
                   
               
               
                 166 
                 pNOP462198 
                 19 
                 RRLQEQAAPPLHVNLGRRV 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 167 
                 pNOP471366 
                 18 
                 EVNSMEGAVMYGVLAVLL 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 168 
                 pNOP473845 
                 18 
                 HHPEVCFTHVKFSYIVII 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 169 
                 pNOP496746 
                 17 
                 IIIKSAKRRWKLKKKKL 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 170 
                 pNOP503554 
                 17 
                 PYSLWNSANPSCLQRFS 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 171 
                 pNOP512752 
                 16 
                 AMDSGVWNGTRWLLIF 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 172 
                 pNOP534454 
                 16 
                 VLQEWLLQYPMCFQNC 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 173 
                 pNOP535898 
                 16 
                 YELLQGGGLEWWRKRS 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 174 
                 pNOP544644 
                 15 
                 IKQLARTSWPPLSYR 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 175 
                 pNOP546490 
                 15 
                 KPNHSQRCQRCQFAN 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 176 
                 pNOP553311 
                 15 
                 QSDAGQTVLTAADRA 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 177 
                 pNOP560491 
                 15 
                 VPVQRTFLRDWPAFQ 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 178 
                 pNOP572874 
                 14 
                 KLECERDGPQASFP 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 179 
                 pNOP5767 
                 183 
                 WRRRRGIAPRRRDSRAPGLRALRQAAAEEPSRRAARPRLPRDCCGRRAAGAA 
                 MAP3K1 
                 &lt;.01 
               
               
                   
                   
                   
                 SGRTGGGGSCAKCGVWSWTSCLSSRSSLPPHRRPPRLPRRRSPRTQRGVGPA 
                   
                   
               
               
                   
                   
                   
                 SSLWRCRRPTEPRAAAAPTLPSRWRRRTAAPRVPQRPSPGRSGRPPPSRLLQ 
                   
                   
               
               
                   
                   
                   
                 RPPPVVRWRIKKLSKGCTRWMIVQRNE 
                   
                   
               
               
                   
               
               
                 180 
                 pNOP580223 
                 14 
                 QLPWQCQRLRMPSP 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 181 
                 pNOP586366 
                 14 
                 TFPSLYSVKTLALQ 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 182 
                 pNOP587861 
                 14 
                 VFPVPLTSPGCVAV 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 183 
                 pNOP601588 
                 13 
                 KLDPLVLVVLIMS 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 184 
                 pNOP610732 
                 13 
                 RGTDVSYLLVGHA 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 185 
                 pNOP634633 
                 12 
                 LMSETHLLSKKK 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 186 
                 pNOP635798 
                 12 
                 MDGRGIGGSFAE 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 187 
                 pNOP652895 
                 11 
                 ARACGGKTNPS 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 188 
                 pNOP657154 
                 11 
                 EPCWYILLATV 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 189 
                 pNOP660722 
                 11 
                 GTKRSTVWKEL 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 190 
                 pNOP666179 
                 11 
                 LCLKLYSWKPN 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 191 
                 pNOP666458 
                 11 
                 LFPDWRRQPRQ 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 192 
                 pNOP670307 
                 11 
                 NQSQLKEMDLK 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 193 
                 pNOP675489 
                 11 
                 RKLEKDYVLQN 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 194 
                 pNOP688134 
                 10 
                 CGPDCTYCSR 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 195 
                 pNOP698339 
                 10 
                 IWSLQRISSY 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 196 
                 pNOP711852 
                 10 
                 RNSQRVAQDG 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 197 
                 pNOP717177 
                 10 
                 TGDLCQKHIF 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 198 
                 pNOP717868 
                 10 
                 TPLLYLIIPN 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 199 
                 pNOP718723 
                 10 
                 TVTPWPFVSP 
                 MAP3K1 
                 &lt;.01 
               
               
                   
               
               
                 200 
                 pNOP685 
                 338 
                 PSASESDAGYKADPTIPTVVLRSVLPIPGIHCLSFCAPSNAHFTWTCQRHDNPL 
                 RUNX1 
                 0.46 
               
               
                   
                 aft splice g 
                   
                 CRTFQSTLNGTRPDSVQRPAPVPRAALHLRPPHALSRRLHLLPDAGHLGHRHR 
                   
                   
               
               
                   
                   
                   
                 HVGHGLGHALPHLPAAALPRLVASAGRPVPSQLALLPPVLRRLGRLLPVLHGG 
                   
                   
               
               
                   
                   
                   
                 RRALAAAHPAALHQRLHRLRAAQPQPPEPERRGGGRGQPQQLPHQHGALR 
                   
                   
               
               
                   
                   
                   
                 APGGGRVEALLRRQAWPGWAPRAAAFASGRAGLLFATSPPGSRALGPATVL 
                   
                   
               
               
                   
                   
                   
                 GPRAPDGQDLAVGQARAASCAQKPTPPPSAGAPALAEVSEATHLEGVRRPQ 
                   
                   
               
               
                   
                   
                   
                 HPGDALESKQEDSRRETVNASDLAML 
                   
                   
               
               
                   
               
               
                 201 
                 pNOP28179 
                 107 
                 RRPRCQHEPPLHAAFHRAEPRQDERGVAAGRPGRRRCPGRQAEERRPQHG 
                 RUNX1 
                 0.37 
               
               
                   
                   
                   
                 GGAGRPPGRAGAHRQPQLPLLRAAYALALQQDPAHRFQGGGPRGCSRWHS 
                   
                   
               
               
                   
                   
                   
                 GHCDGWQ 
                   
                   
               
               
                   
               
               
                 202 
                 pNOP135011 
                 51 
                 PQVCRSKWKREKLHSDHHCLHKPTASRHLPQSHQNHSGWAPRTSKTSAETR 
                 RUNX1 
                 0.20 
               
               
                   
               
               
                 203 
                 pNOP352298 
                 25 
                 MIRPSPGACPFPSGSVNWSSCGAQP 
                 RUNX1 
                 0.14 
               
               
                   
               
               
                 204 
                 pNOP115907 
                 56 
                 TTPLPLTLSLRVRCRIQGRSNHPHRGPTISPTNTWDPLPLLLCTQQRPFHLDVP 
                 RUNX1 
                 0.12 
               
               
                   
                 aft splice h 
                   
                 AA 
                   
                   
               
               
                   
               
               
                 205 
                 pNOP142572 
                 49 
                 GAATAAWWRCWPTTRASWCAPTAPTSSAPCCLRTGAATRPCPSLSRWWP 
                 RUNX1 
                 0.12 
               
               
                   
                   
                   
                 QRSATRASSPRCPPSPTPACTIQAPSPTPRRRSPRASASACRPWARPRATTPTC 
                   
                   
               
               
                   
                   
                   
                 RRPTPARRKRREARSKPARPPTTCTTAPRPAPTSSPWWAASARRRASCRPAPT 
                   
                   
               
               
                   
                   
                   
                 PPPAPRCSTPASRTRATWWRPRAATATPPPTWRPPRAWRRPCGGPTEAPGL 
                   
                   
               
               
                   
                   
                   
                 ARLGPAGRRLRLRARGPPVRDKPAGIPGPGPGHRPGAEGARRPGSRCRSGPR 
                   
                   
               
               
                   
                   
                   
                 SLLRPEAHAAAVCWRPGPRGGVRGDAPRGCPPAPAPRGRAGKQTGRFPEG 
                   
                   
               
               
                   
               
               
                 206 
                 pNOP1630 
                 265 
                 NCECF 
                 RUNX1 
                 0.12 
               
               
                   
               
               
                 207 
                 pNOP246619 
                 34 
                 PSLSSCITHRKSPPTTEPSKSQWMGPENLEDIGRN 
                 RUNX1 
                 0.09 
               
               
                   
                 alt splice j 
                   
                   
                   
                   
               
               
                   
               
               
                 208 
                 pNOP269003 
                 32 
                 TGAAAAHSHEGQPTPPSPHAQPSCLPEPLHCL 
                 RUNX1 
                 0.06 
               
               
                   
               
               
                 209 
                 pNOP59911 
                 78 
                 PSASESDAGGRHSTLEMLRQKSVLLSIPLPSQDRAIFSISSSEETQKKDDSPQNA 
                 RUNX1 
                 0.03 
               
               
                   
                 alt splice g 
                   
                 RYEEGRMWVCNSSVSLLPLCKPS 
                   
                   
               
               
                   
               
               
                 210 
                 pNOP18884 
                 124 
                 NARSTAGTGAGGSGRPHAKPGCQHEPPLHAAFHRAEPRQDERGVAAGRPG 
                 RUNX1 
                 &lt;.01 
               
               
                   
                   
                   
                 RRRCPGROAEERRPQHGGGAGRPPGRAGAHRQPQLPLLRAAYALALQQDP 
                   
                   
               
               
                   
                   
                   
                 AHRFQGGGPRGCSRWHSGHCDGWQ 
                   
                   
               
               
                   
               
               
                 211 
                 pNOP206880 
                 39 
                 TTPLPLTLSLRVRCRRKTQHPGDVKAEVSSSVHPSPQPG 
                 RUNX1 
                 &lt;.01 
               
               
                   
                 alt splice h 
                   
                   
                   
                   
               
               
                   
               
               
                 212 
                 pNOP23436 
                 115 
                 VISFVPTVLHERCQHEPPLHAAFHRAEPRQDERGVAAGRPGRRRCPGRQAEE 
                 RUNX1 
                 &lt;.01 
               
               
                   
                 alt splice i 
                   
                 RRPQHGGGAGRPPGRAGAHRQPQLPLLRAAYALALQQDPAHRFQGGGPRG 
                   
                   
               
               
                   
                   
                   
                 CSRWHSGHCDGWQ 
                   
                   
               
               
                   
               
               
                 213 
                 pNOP241712 
                 34  
                 CPQHRGDRRRGKRPPAREAGMPARAAASRRLPPR 
                 RUNX1 
                 &lt;.01 
               
               
                   
               
               
                 214 
                 pNOP26418 
                 109 
                 DAYPRRCQHEPPLHAAFHRAEPRQDERGVAAGRPGRRRCPGRQAEERRPQH 
                 RUNX1 
                 &lt;.01 
               
               
                   
                   
                   
                 GGGAGRPPGRAGAHRQPQLPLLRAAYALALQQDPAHRFQGGGPRGCSRWH 
                   
                   
               
               
                   
                   
                   
                 SGHCDGWQ 
                   
                   
               
               
                   
               
               
                 215 
                 pNOP387672 
                 23 
                 RTRLQDLMTSGLSVEVEEGKASL 
                 RUNX1 
                 &lt;.01 
               
               
                   
               
               
                 216 
                 pNOP415291 
                 21 
                 ILLETSTMPARAAASRRLPPR 
                 RUNX1 
                 &lt;.01 
               
               
                   
               
               
                 217 
                 pNOP445668 
                 20 
                 VISFVPTVLHERMHTWNESF 
                 RUNX1 
                 &lt;.01 
               
               
                   
                 alt splice i 
                   
                   
                   
                   
               
               
                   
               
               
                 218 
                 pNOP458 
                 374 
                 PQVCRSKWKREKLHSDHHCLHKPTASRHLPQSHQNHSGWAPRTSKYKADPTI 
                 RUNX1 
                 &lt;.01 
               
               
                   
                   
                   
                 PTVVLRSVLPIPGIHCLSFCAPSNAHFTWTCQRHDNPLCRTFQSTLNGTRPDSV 
                   
                   
               
               
                   
                   
                   
                 QRPAPVPRAALHLRPPHALSRRLHLLPDAGHLGHRHRHVGHGLGHALPHLPA 
                   
                   
               
               
                   
                   
                   
                 AALPRLVASAGRPVPSQLALLPPVLRRLGRLLPVLHGGRRALAAAHPAALHQR 
                   
                   
               
               
                   
                   
                   
                 LHRLRAAQPQPPEPERRGGGRGQPQQLPHQHGALRAPGGGRVEALLRRQA 
                   
                   
               
               
                   
                   
                   
                 WPGWAPRAAAFASGRAGLLFATSPPGSRALGPATVLGPRAPDGQDLAVGQ 
                   
                   
               
               
                   
                   
                   
                 ARAASCAQKPTPPPSAGAPALAEVSEATHLEGVRRPQHPGDALESKQEDSRR 
                   
                   
               
               
                   
                   
                   
                 ETVNASDLAML 
                   
                   
               
               
                   
               
               
                 219 
                 pNOP468446 
                 18 
                 ARRCRWAPRTPALPWPAS 
                 RUNX1 
                 &lt;.01 
               
               
                   
               
               
                 220 
                 pNOP48701 
                 85 
                 DERGVAAGRPGRRRCPGRQAEERRPQHGGGAGRPPGRAGAHRQPQLPLLR 
                 RUNX1 
                 &lt;.01 
               
               
                   
                   
                   
                 AAYALALQQDPAHRFQGGGPRGCSRWHSGHCDGWQ 
                   
                   
               
               
                   
               
               
                 221 
                 pNOP511171 
                 17 
                 WLQTAYLSHFLRTHSAS 
                 RUNX1 
                 &lt;.01 
               
               
                   
               
               
                 222 
                 pNOP518241 
                 16 
                 GSAHTTQPPRPTLVPP 
                 RUNX1 
                 &lt;.01 
               
               
                   
               
               
                 223 
                 pNOP527804 
                 16 
                 QPSLQNFPVDSQRHPT 
                 RUNX1 
                 &lt;.01 
               
               
                   
               
               
                 224 
                 pNOP536626 
                 15 
                 AEKCYRSHEEPGCKI 
                 RUNX1 
                 &lt;.01 
               
               
                   
               
               
                 225 
                 pNOP540378 
                 15 
                 EMPARAAASRRLPPR 
                 RUNX1 
                 &lt;.01 
               
               
                   
               
               
                 226 
                 pNOP576438 
                 14 
                 MPARAAASRRLPPR 
                 RUNX1 
                 &lt;.01 
               
               
                   
               
               
                 227 
                 pNOP583929 
                 14 
                 SDQARELVLFRAAQ 
                 RUNX1 
                 &lt;.01 
               
               
                   
               
               
                 228 
                 pNOP684423 
                 11 
                 WLAMMKTTRLS 
                 RUNX1 
                 &lt;.01 
               
               
                   
               
               
                 229 
                 pNOP694394 
                 10 
                 GMFQMALW5L 
                 RUNX1 
                 &lt;.01 
               
               
                   
               
               
                 230 
                 pNOP74999 
                 70 
                 P5LSSQTHRKSPPTTEPSKSQWMGPENLEIQGR5NHPHRGPTISPTNTWDPL 
                 RUNX1 
                 &lt;.01 
               
               
                   
                 alt splice j 
                   
                 PLLLCTQQRPFHLDVPAA 
                   
                   
               
               
                   
               
               
                 231 
                   
                 71 
                 PLQHGHRHGLEPCSMLTGPPARVPAVPFDLHFCRSSIMKPKRDGYMFLKAES 
                 GATA3 
                   
               
               
                   
                   
                   
                 KIMFATLQRSSLWCLCSNH 
                   
                   
               
               
                   
               
               
                 232 
                   
                 37 
                 RDVLCELSDHHNHTLEEECQWGPCLQCLWALLQASQY 
                 GATA3 
                   
               
               
                   
               
               
                 233 
                   
                 51 
                 TRHPACPLDHTTPPAWSPPWVRALLDAHRAPSESPCSPFRLAFLQEQYHEA 
                 GATA3 
                   
               
               
                   
               
               
                 234 
                   
                 40 
                 GRPVRTVRPPQPHSGGGMPMGTLSAMPVGSTTSFTILTDP 
                 GATA3 
               
               
                   
               
            
           
         
       
     
     In a preferred embodiment the disclosure provides one or more frameshift-mutation peptides (also referred to herein as ‘neoantigens’) comprising an amino acid sequence selected from the groups: 
     (i) Sequences 1-16 or 231-234, an amino acid sequence having 90% identity to Sequences 1-16 or 231-234, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 1-16 or 231-234; 
     (ii) Sequences 17-44, an amino acid sequence having 90% identity to Sequences 17-44, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 17-44; 
     (iii) Sequences 45-107, an amino acid sequence having 90% identity to Sequences 45-107, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 45-107; 
     (iv) Sequences 108-199, an amino acid sequence having 90% identity to Sequences 108-199, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 108-199; and 
     (v) Sequences 200-230, an amino acid sequence having 90% identity to Sequences 200-230, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 200-230. 
     As will be clear to a skilled person, the preferred amino acid sequences may also be provided as a collection of tiled sequences, wherein such a collection comprises two or more peptides that have an overlapping sequence. Such ‘tiled’ peptides have the advantage that several peptides can be easily synthetically produced, while still covering a large portion of the NOP. In an exemplary embodiment, a collection comprising at least 3, 4, 5, 6, 10, or more tiled peptides each having between 10-50, preferably 12-45, more preferably 15-35 amino acids, is provided. As described further herein, such tiled peptides are preferably directed to the C-terminus of a pNOP. As will be clear to a skilled person, a collection of tiled peptides comprising an amino acid sequence of Sequence X, indicates that when aligning the tiled peptides and removing the overlapping sequences, the resulting tiled peptides provide the amino acid sequence of Sequence X, albeit present on separate peptides. As is also clear to a skilled person, a collection of tiled peptides comprising a fragment of 10 consecutive amino acids of Sequence X, indicates that when aligning the tiled peptides and removing the overlapping sequences, the resulting tiled peptides provide the amino acid sequence of the fragment, albeit present on separate peptides. When providing tiled peptides, the fragment preferably comprises at least 20 consecutive amino acids of a sequence as disclosed herein. 
     Specific NOP sequences cover a large percentage of breast cancer patients. Preferred NOP sequences, or subsequences of NOP sequence, are those that target the largest percentage of breast cancer patients. Preferred sequences are, preferably in this order of preference, Sequence 1 (4.6% of breast cancer patients) and Sequence 2 (2.8% of breast cancer patients), Sequence 17 (1.1% of breast cancer patients), Sequence 18 (1.1% of breast cancer patients), Sequence 3 (0.9% of breast cancer patients), Sequence 19 (1% of breast cancer patients), Sequence 20 (1% of breast cancer patients), Sequence 21 (0.8% of breast cancer patients), Sequence 22 (0.6% of breast cancer patients), Sequence 45 (0.5% of breast cancer patients), Sequence 200 (0.5% of breast cancer patients), Sequences 4, 46, 201, 108, 23 (each covering 0.4% of breast cancer patients) and Sequences 5-16, 24-44, 47-17, 109-199 and 202-230 (covering less than 0.4% of breast cancer patients). 
     As discussed further herein, neoantigens also include the nucleic acid molecules (such as DNA and RNA) encoding said amino acid sequences. The preferred sequences listed above are also the preferred sequences for the embodiments described further herein. 
     Preferably, the neoantigens and vaccines disclosed herein induce an immune response, or rather the neoantigens are immunogenic. Preferably, the neoantigens bind to an antibody or a T-cell receptor. In preferred embodiments, the neoantigens comprise an MHCI or MHCII ligand. 
     The major histocompatibility complex (MHC) is a set of cell surface molecules encoded by a large gene family in vertebrates. In humans, MHC is also referred to as human leukocyte antigen (HLA). An MHC molecule displays an antigen and presents it to the immune system of the vertebrate. Antigens (also referred to herein as ‘MHC ligands’) bind MHC molecules via a binding motif specific for the MHC molecule. Such binding motifs have been characterized and can be identified in proteins. See for a review Meydan et al. 2013 BMC Bioinformatics 14:S13. 
     MHC-class I molecules typically present the antigen to CD8 positive T-cells whereas MHC-class II molecules present the antigen to CD4 positive T-cells. The terms “cellular immune response” and “cellular response” or similar terms refer to an immune response directed to cells characterized by presentation of an antigen with class I or class II MHC involving T cells or T-lymphocytes which act as either “helpers” or “killers”. The helper T cells (also termed CD4+ T cells) play a central role by regulating the immune response and the killer cells (also termed cytotoxic T cells, cytolytic T cells, CD8+ T cells or CTLs) kill diseased cells such as cancer cells, preventing the production of more diseased cells. 
     In preferred embodiments, the present disclosure involves the stimulation of an anti-tumor CTL response against tumor cells expressing one or more tumor-expressed antigens (i.e., NOPs) and preferably presenting such tumor-expressed antigens with class I MHC. 
     In some embodiments, an entire NOP (e.g., Sequence 1) may be provided as the neoantigen (i.e., peptide). The length of the NOPs identified herein vary from around 10 to around 374 amino acids. Preferred NOPs are at least 20 amino acids in length, more preferably at least 30 amino acids, and most preferably at least 50 amino acids in length. While not wishing to be bound by theory, it is believed that neoantigens longer than 10 amino acids can be processed into shorter peptides, e.g., by antigen presenting cells, which then bind to MHC molecules. 
     In some embodiments, fragments of a NOP can also be presented as the neoantigen. The fragments comprise at least 8 consecutive amino acids of the NOP, preferably at least 10 consecutive amino acids, and more preferably at least 20 consecutive amino acids, and most preferably at least 30 amino acids. In some embodiments, the fragments can be about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, or about 120 amino acids or greater. Preferably, the fragment is between 8-50, between 8-30, or between 10-20 amino acids. As will be understood by the skilled person, fragments greater than about 10 amino acids can be processed to shorter peptides, e.g., by antigen presenting cells. A preferred fragment of sequence 1 is sequence 231; a preferred fragment of sequence 2 is sequence 232 a preferred fragment of sequence 3 is sequence 233; and a preferred fragment of sequence 5 is sequence 234. 
     The specific mutations resulting in the generation of a neo open reading frame may differ between individuals resulting in differing NOP lengths. However, as depicted in, e.g.,  FIG. 2 , such individuals share common NOP sequences, in particular at the C-terminus of an NOP. While suitable fragments for use as neoantigens may be located at any position along the length of an NOP, fragments located near the C-terminus are preferred as they are expected to benefit a larger number of patients. Preferably, fragments of a NOP correspond to the C-terminal (3′) portion of the NOP, preferably the C-terminal 10 consecutive amino acids, more preferably the C-terminal 20 consecutive amino acids, more preferably the C-terminal 30 consecutive amino acids, more preferably the C-terminal 40 consecutive amino acids, more preferably the C-terminal 50 consecutive amino acids, more preferably the C-terminal 60 consecutive amino acids, more preferably the C-terminal 70 consecutive amino acids, more preferably the C-terminal 80 consecutive amino acids, more preferably the C-terminal 90 consecutive amino acids, and most preferably the C-terminal 100 or more consecutive amino acids. In some embodiments a subsequence of the preferred C-terminal portion of the NOP may be highly preferred for reasons of manufacturability, solubility and MHC binding strength. 
     Suitable fragments for use as neoantigens can be readily determined. The NOPs disclosed herein may be analysed by known means in the art in order to identify potential MHC binding peptides (i.e., MHC ligands). Suitable methods are described herein in the examples and include in silico prediction methods (e.g., ANNPRED, BIMAS, EPIMHC, HLABIND, IEDB, KISS, MULTIPRED, NetMHC, PEPVAC, POPI, PREDEP, RANKPEP, SVMHC, SVRMHC, and SYFFPEITHI, see Lundegaard 2010 130:309-318 for a review). MHC binding predictions depend on HLA genotypes, furthermore it is well known in the art that different MHC binding prediction programs predict different MHC affinities for a given epitope. While not wishing to be limited by such predictions, at least 60% of NOP sequences as defined herein, contain one or more predicted high affinity MHC class I binding epitope of 10 amino acids, based on allele HLA-A0201 and using NetMHC4.0. 
     A skilled person will appreciate that natural variations may occur in the genome resulting in variations in the sequence of an NOP. Accordingly, a neoantigen of the disclosure may comprise minor sequence variations, including, e.g., conservative amino acid substitutions. Conservative substitutions are well known in the art and refer to the substitution of one or more amino acids by similar amino acids. For example, a conservative substitution can be the substitution of an amino acid for another amino acid within the same general class (e.g., an acidic amino acid, a basic amino acid, or a neutral amino acid). A skilled person can readily determine whether such variants retain their immunogenicity, e.g., by determining their ability to bind MHC molecules. 
     Preferably, a neoantigen has at least 90% sequence identity to the NOPs disclosed herein. Preferably, the neoantigen has at least 95% or 98% sequence identity. The term “% sequence identity” is defined herein as the percentage of nucleotides in a nucleic acid sequence, or amino acids in an amino acid sequence, that are identical with the nucleotides, resp. amino acids, in a nucleic acid or amino acid sequence of interest, after aligning the sequences and optionally introducing gaps, if necessary, to achieve the maximum percent sequence identity. The skilled person understands that consecutive amino acid residues in one amino acid sequence are compared to consecutive amino acid residues in another amino acid sequence. Methods and computer programs for alignments are well known in the art. Sequence identity is calculated over substantially the whole length, preferably the whole (full) length, of a sequence of interest. 
     The disclosure also provides at least two frameshift-mutation derived peptides (i.e., neoantigens), also referred to herein as a ‘collection’ of peptides. Preferably the collection comprises at least 3, at least 4, at least 5, at least 10, at least 15, or at least 20, or at least 50 neoantigens. In some embodiments, the collections comprise less than 20, preferably less than 15 neoantigens. Preferably, the collections comprise the top 20, more preferably the top 15 most frequently occurring neoantigens in cancer patients. The neoantigens are selected from:
     (i) Sequences 1-16, an amino acid sequence having 90% identity to Sequences 1-16 or 231-234, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 1-16 or 231-234;   (ii) Sequences 17-44, an amino acid sequence having 90% identity to Sequences 17-44, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 17-44;   (iii) Sequences 45-107, an amino acid sequence having 90% identity to Sequences 45-107, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 45-107;   (iv) Sequences 108-199, an amino acid sequence having 90% identity to Sequences 108-199, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 108-199; and   (v) Sequences 200-230, an amino acid sequence having 90% identity to Sequences 200-230, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 200-230.   

     Preferably, the collection comprises at least two frameshift-mutation derived peptides corresponding to the same gene. Preferably, a collection is provided comprising: 
     (i) at least two frameshift-mutation derived peptides, wherein each peptide, or collection of tiled peptides, comprises a different amino acid sequence selected from Sequences 1-16, an amino acid sequence having 90% identity to Sequences 1-16 or 231-234, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 1-16 or 231-234; 
     (ii) at least two frameshift-mutation derived peptides, wherein each peptide, or collection of tiled peptides, comprises a different amino acid sequence selected from Sequences 17-44, an amino acid sequence having 90% identity to Sequences 17-44, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 17-44; 
     (iii) at least two frameshift-mutation derived peptides, wherein each peptide, or collection of tiled peptides, comprises a different amino acid sequence selected from Sequences 45-107, an amino acid sequence having 90% identity to Sequences 45-107, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 45-107; 
     (iv) at least two frameshift-mutation derived peptides, wherein each peptide, or collection of tiled peptides, comprises a different amino acid sequence selected from Sequences 108-199, an amino acid sequence having 90% identity to Sequences 108-199, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 108-199; or 
     (v) at least two frameshift-mutation derived peptides, wherein each peptide, or collection of tiled peptides, comprises a different amino acid sequence selected from Sequences 200-230, an amino acid sequence having 90% identity to Sequences 200-230, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 200-230. 
     In some embodiments, the collection comprises two or more neoantigens corresponding to the same NOP. For example, the collection may comprise two (or more) fragments of Sequence 1 or the collection may comprise a peptide having Sequence 1 and a peptide having 95% identity to Sequence 1. 
     Preferably, the collection comprises two or more neoantigens corresponding to different NOPs. In some embodiments, the collection comprises two or more neoantigens corresponding to different NOPs of the same gene. For example the peptide may comprise the amino acid sequence of Sequence 1 (or a fragment or collection of tiled fragments thereof) and the amino acid sequence of Sequence 2 (or a fragment or collection of tiled fragments thereof). 
     Preferably, the collection comprises Sequences 1-4, preferably 1-6 (or a fragment or collection of tiled fragments thereof). 
     Preferably, the collection comprises Sequences 17-20, preferably 17-24, more preferably 17-30 (or a fragment or collection of tiled fragments thereof). 
     Preferably, the collection comprises Sequences 45-49, preferably 45-53, more preferably 45-55 (or a fragment or collection of tiled fragments thereof). 
     Preferably, the collection comprises Sequences 108-112, preferably 108-117 (or a fragment or collection of tiled fragments thereof). 
     Preferably, the collection comprises Sequences 200-206, preferably 200-212 (or a fragment or collection of tiled fragments thereof). 
     In some embodiments, the collection comprises two or more neoantigens corresponding to different NOPs of different genes. For example the collection may comprise a peptide having the amino acid sequence of Sequence 1 (or a fragment or collection of tiled fragments thereof) and a peptide having the amino acid sequence of Sequence 17 (or a fragment or collection of tiled fragments thereof). Preferably, the collection comprises at least one neoantigen from group (i) and at least one neoantigen from group (ii); at least one neoantigen from group (i) and at least one neoantigen from group (iii); at least one neoantigen from group (i) and at least one neoantigen from group (iv); at least one neoantigen from group (i) and at least one neoantigen from group (v); at least one neoantigen from group (ii) and at least one neoantigen from group (iii); at least one neoantigen from group (ii) and at least one neoantigen from group (iv); at least one neoantigen from group (ii) and at least one neoantigen from group (v); at least one neoantigen from group (iii) and at least one neoantigen from group (iv); at least one neoantigen from group (iii) and at least one neoantigen from group (v); or at least one neoantigen from group (iv) and at least one neoantigen from group (v). Preferably, the collection comprises at least one neoantigen from group (i), at least one neoantigen from group (ii), and at least one neoantigen from group (iii). Preferably, the collection comprises at least one neoantigen from each of groups (i) to (iv). Preferably, the collection comprises at least one neoantigen from each of groups (i) to (v). 
     In a preferred embodiment, the collections disclosed herein include Sequence 1, Sequence 2, and Sequence 17 (or a variant or fragment or collection of tiled fragments thereof as disclosed herein). In some embodiments, the collection further includes Sequence 18, Sequence 19, Sequence 20, and Sequence 3 (or a variant or fragment or collection of tiled fragments thereof as disclosed herein). In some embodiments, the collection further includes, Sequence 21, Sequence 22, Sequence 45, Sequence 200 and Sequence 23 (or a variant or fragment or collection of tiled fragments thereof as disclosed herein). In some embodiments, the collection even further includes Sequences 46, 4, 108, 201 and 47 (or a variant or fragment or collection of tiled fragments thereof as disclosed herein). In some embodiments, the collection even further includes Sequences 5, 48, 109, 110, 24, 49, 202 (or a variant or fragment or collection of tiled fragments thereof as disclosed herein); preferably, the collection further includes Sequences 50, 25, 51, 111, 203, 6, 26, 52, 53, 54, 55, 112, 113, 114, 204, 205, 206 (or a variant or fragment or collection of tiled fragments thereof as disclosed herein). In some embodiments, the collections includes all Sequences 1-234 (or a variant or fragment or collection of tiled fragments thereof as disclosed herein). 
     Such collections comprising multiple neoantigens have the advantage that a single collection (e.g, when used as a vaccine) can benefit a larger group of patients having different frameshift mutations. This makes it feasible to construct and/or test the vaccine in advance and have the vaccine available for off-the-shelf use. This also greatly reduces the time from screening a tumor from a patient to administering a potential vaccine for said tumor to the patient, as it eliminates the time of production, testing and approval. In addition, a single collection consisting of multiple neoantigens corresponding to different genes will limit possible resistance mechanisms of the tumor, e.g. by losing one or more of the targeted neoantigens. 
     In some embodiments, the neoantigens (i.e., peptides) are directly linked. Preferably, the neoantigens are linked by peptide bonds, or rather, the neoantigens are present in a single polypeptide. Accordingly, the disclosure provides polypeptides comprising at least two peptides (i.e., neoantigens) as disclosed herein. In some embodiments, the polypeptide comprises 3, 4, 5, 6, 7, 8, 9, 10 or more peptides as disclosed herein (i.e., neoantigens). Such polypeptides are also referred to herein as ‘polyNOPs’. A collection of peptides can have one or more peptides and one or more polypeptides comprising the respective neoantigens. 
     In an exemplary embodiment, a polypeptide of the disclosure may comprise 10 different neoantigens, each neoantigen having between 10-400 amino acids. Thus, the polypeptide of the disclosure may comprise between 100-4000 amino acids, or more. As is clear to a skilled person, the final length of the polypeptide is determined by the number of neoantigens selected and their respective lengths. A collection may comprise two or more polypeptides comprising the neoantigens which can be used to reduce the size of each of the polypeptides. 
     In some embodiments, the amino acid sequences of the neoantigens are located directly adjacent to each other in the polypeptide. For example, a nucleic acid molecule may be provided that encodes multiple neoantigens in the same reading frame. In some embodiments, a linker amino acid sequence may be present. Preferably a linker has a length of 1, 2, 3, 4 or 5, or more amino acids. The use of linker may be beneficial, for example for introducing, among others, signal peptides or cleavage sites. In some embodiments at least one, preferably all of the linker amino acid sequences have the amino acid sequence VDD. 
     As will be appreciated by the skilled person, the peptides and polypeptides disclosed herein may contain additional amino acids, for example at the N- or C-terminus. Such additional amino acids include, e.g., purification or affinity tags or hydrophilic amino acids in order to decrease the hydrophobicity of the peptide. In some embodiments, the neoantigens may comprise amino acids corresponding to the adjacent, wild-type amino acid sequences of the relevant gene, i.e., amino acid sequences located 5′ to the frame shift mutation that results in the neo open reading frame. Preferably, each neoantigen comprises no more than 20, more preferably no more than 10, and most preferably no more than 5 of such wild-type amino acid sequences. 
     In preferred embodiments, the peptides and polypeptides disclosed herein have a sequence depicted as follows: 
       A-B-C-(D-E) n , wherein 
     A, C, and E are independently 0-100 amino acids 
     B and D are amino acid sequences as disclosed herein and selected from sequences 1-234, or an amino acid sequence having 90% identity to Sequences 1-234, or a fragment thereof comprising at least 10 consecutive amino acids of Sequences 1-234, 
     n is an integer from 0 to 500. 
     Preferably, B and D are different amino acid sequences. Preferably, n is an integer from 0-200. Preferably A, C, and E are independently 0-50 amino acids, more preferably independently 0-20 amino acids. 
     The peptides and polypeptides disclosed herein can be produced by any method known to a skilled person. In some embodiments, the peptides and polypeptide are chemically synthesized. The peptides and polypeptide can also be produced using molecular genetic techniques, such as by inserting a nucleic acid into an expression vector, introducing the expression vector into a host cell, and expressing the peptide. Preferably, such peptides and polypeptide are isolated, or rather, substantially isolated from other polypeptides, cellular components, or impurities. The peptide and polypeptide can be isolated from other (poly)peptides as a result of solid phase protein synthesis, for example. Alternatively, the peptides and polypeptide can be substantially isolated from other proteins after cell lysis from recombinant production (e.g., using HPLC). 
     The disclosure further provides nucleic acid molecules encoding the peptides and polypeptide disclosed herein. Based on the genetic code, a skilled person can determine the nucleic acid sequences which encode the (poly)peptides disclosed herein. Based on the degeneracy of the genetic code, sixty-four codons may be used to encode twenty amino acids and translation termination signal. 
     In a preferred embodiment, the nucleic acid molecules are codon optimized. As is known to a skilled person, codon usage bias in different organisms can effect gene expression level. Various computational tools are available to the skilled person in order to optimize codon usage depending on which organism the desired nucleic acid will be expressed. Preferably, the nucleic acid molecules are optimized for expression in mammalian cells, preferably in human cells. Table 2 lists for each acid amino acid (and the stop codon) the most frequently used codon as encountered in the human exome. 
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 most frequently used codon for 
               
               
                 each amino acid and most 
               
               
                  frequently usedstop codon. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 A 
                 GCC 
               
               
                   
                   
               
               
                   
                 C 
                 TGC 
               
               
                   
                   
               
               
                   
                 D 
                 GAC 
               
               
                   
                   
               
               
                   
                 E 
                 GAG 
               
               
                   
                   
               
               
                   
                 F 
                 TTC 
               
               
                   
                   
               
               
                   
                 G 
                 GGC 
               
               
                   
                   
               
               
                   
                 H 
                 CAC 
               
               
                   
                   
               
               
                   
                 I 
                 ATC 
               
               
                   
                   
               
               
                   
                 K 
                 AAG 
               
               
                   
                   
               
               
                   
                 L 
                 CTG 
               
               
                   
                   
               
               
                   
                 M 
                 ATG 
               
               
                   
                   
               
               
                   
                 N 
                 AAC 
               
               
                   
                   
               
               
                   
                 P 
                 CCC 
               
               
                   
                   
               
               
                   
                 Q 
                 CAG 
               
               
                   
                   
               
               
                   
                 R 
                 CGG 
               
               
                   
                   
               
               
                   
                 S 
                 AGC 
               
               
                   
                   
               
               
                   
                 T 
                 ACC 
               
               
                   
                   
               
               
                   
                 V 
                 GTG 
               
               
                   
                   
               
               
                   
                 W 
                 TGG 
               
               
                   
                   
               
               
                   
                 Y 
                 TAC 
               
               
                   
                   
               
               
                   
                 Stop 
                 TGA 
               
               
                   
                   
               
            
           
         
       
     
     In some embodiments, at least 50%, 60%, 70%, 80%, 90%, or 100% of the amino acids are encoded by a codon corresponding to a codon presented in Table 2. 
     In some embodiments, the nucleic acid molecule encodes for a linker amino acid sequence in the peptide. Preferably, the nucleic acid sequence encoding the linker comprises at least one codon triplet that codes for a stop codon when a frameshift occurs. Preferably, said codon triplet is chosen from the group consisting of: ATA, CTA, GTA, TTA, ATO, CTG, GTG, TTG, AAA, AAC, AAG, AAT, AGA, AGC, AGG, AGT, GAA, GAC, GAG, and GAT. These codons do not code for a stop codon, but could create a stop codon in case of a frame shift, such as when read in the +1, +2, +4, +, 5, etc. reading frame. For example, two amino acid encoding sequences are linked by a linker amino acid encoding sequence as follows (linker amino acid encoding sequence in bold): 
     
       
         
           
               
               
            
               
                   
                 CTATACAGGCGA ATG AGATTATG 
               
            
           
         
       
     
     Resulting in the following amino acid sequence (amino acid linker sequence in bold): LYRRMRL 
     In case of a +1 frame shift, the following sequence is encoded: 
     
       
         
           
               
               
            
               
                   
                 YTGE[stop]DY 
               
            
           
         
       
     
     This embodiment has the advantage that if a frame shift occurs in the nucleotide sequence encoding the peptide, the nucleic acid sequence encoding the linker will terminate translation, thereby preventing expression of (part of) the native protein sequence for the gene related to peptide sequence encoded by the nucleotide sequence. 
     In some preferred embodiments, the linker amino acid sequences are encoded by the nucleotide sequence (TAGATGAC. This linker has the advantage that it contains two out of frame stop codons (TAG and TGA), one in the +1 and one in the −1 reading frame. The amino acid sequence encoded by this nucleotide sequence is VDD. The added advantage of using a nucleotide sequence encoding for this linker amino acid sequence is that any frame shift will result in a stop codon. 
     The disclosure also provides binding molecules and a collection of binding molecules that bind the neoantigens disclosed herein and or a neoantigen/MHC complex. In some embodiments the binding molecule is an antibody, a T-cell receptor, or an antigen binding fragment thereof. In some embodiments the binding molecule is a chimeric antigen receptor comprising i) a T cell activation molecule; ii) a transmembrane region; and iii) an antigen recognition moiety; wherein said antigen recognition moieties bind the neoantigens disclosed herein and or a neoantigen/MHC complex. 
     The term “antibody” as used herein refers to an immunoglobulin molecule that is typically composed of two identical pairs of polypeptide chains, each pair of chains consisting of one “heavy” chain with one “light” chain. The human light chains are classified as kappa and lambda. The heavy chains comprise different classes namely: mu, delta, gamma, alpha or epsilon. These classes define the isotype of the antibody, such as IgM, IgD, IgG IgA and IgE, respectively. These classes are important for the function of the antibody and help to regulate the immune response. Both the heavy chain and the light chain comprise a variable domain and a constant region. Each heavy chain variable region (VH) and light chain variable region (VL) comprises complementary determining regions (CDR) interspersed by framework regions (FR). The variable region has in total four FRs and three CDRs. These are arranged from the amino- to the carboxyl-terminus as follows: FR1. CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the light and heavy chain together form the antibody binding site and define the specificity for the epitope. 
     The term “antibody” encompasses murine, humanized, deimmunized, human, and chimeric antibodies, and an antibody that is a multimeric form of antibodies, such as dimers, trimers, or higher-order multimers of monomeric antibodies. The term antibody also encompasses monospecific, bispecific or multi-specific antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity. 
     Preferably, an antibody or antigen binding fragment thereof as disclosed herein is a humanized antibody or antigen binding fragment thereof. The term “humanized antibody” refers to an antibody that contains some or all of the CDRs from a non-human animal antibody while the framework and constant regions of the antibody contain amino acid residues derived from human antibody sequences. Humanized antibodies are typically produced by grafting CDRs from a mouse antibody into human framework sequences followed by back substitution of certain human framework residues for the corresponding mouse residues from the source antibody. The term “deimmunized antibody” also refers to an antibody of non-human origin in which, typically in one or more variable regions, one or more epitopes have been removed, that have a high propensity of constituting a human T-cell and/or B-cell epitope, for purposes of reducing immunogenicity. The amino acid sequence of the epitope can be removed in full or in part. However, typically the amino acid sequence is altered by substituting one or more of the amino acids constituting the epitope for one or more other amino acids, thereby changing the amino acid sequence into a sequence that does not constitute a human T-cell and/or B-cell epitope. The amino acids are substituted by amino acids that are present at the corresponding position(s) in a corresponding human variable heavy or variable light chain as the case may be. 
     In some embodiments, an antibody or antigen binding fragment thereof as disclosed herein is a human antibody or antigen binding fragment thereof. The term “human antibody” refers to an antibody consisting of amino acid sequences of human immunoglobulin sequences only. Human antibodies may be prepared in a variety of ways known in the art. 
     As used herein, antigen-binding fragments include Fab, F(ab′), F(ab′)2, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, and other antigen recognizing immunoglobulin fragments. 
     In some embodiments, the antibody or antigen binding fragment thereof is an isolated antibody or antigen binding fragment thereof. The term “isolated”, as used herein, refer to material which is substantially or essentially free from components which normally accompany it in nature. 
     In some embodiments, the antibody or antigen binding fragment thereof is linked or attached to a non-antibody moiety. In preferred embodiments, the non-antibody moiety is a cytotoxic moiety such as auristatins, maytanasines, calicheasmicins, duocarymycins, α-amanitin, doxorubicin, and centanamycin. Other suitable cytotoxins and methods for preparing such antibody drug conjugates are known in the art; see, e.g., WO2013085925A1 and WO2016133927A1. 
     Antibodies which bind a particular epitope can be generated by methods known in the art. For example, polyclonal antibodies can be made by the conventional method of immunizing a mammal (e.g., rabbits, mice, rats, sheep, goats). Polyclonal antibodies are then contained in the sera of the immunized animals and can be isolated using standard procedures (e.g., affinity chromatography, immunoprecipitation, size exclusion chromatography, and ion exchange chromatography). Monoclonal antibodies can be made by the conventional method of immunization of a mammal, followed by isolation of plasma B cells producing the monoclonal antibodies of interest and fusion with a myeloma cell (see, e.g., Mishell, B. B., et al., Selected Methods In Cellular Immunology, (W.H. Freeman, ed.) San Francisco (1980)). Peptides corresponding to the neoantigens disclosed herein may be used for immunization in order to produce antibodies which recognize a particular epitope. Screening for recognition of the epitope can be performed using standard immunoassay methods including ELISA techniques, radioimmunoassays, immunofluorescence, immunohistochemistry, and Western blotting. See, Short Protocols in Molecular Biology, Chapter 11, Green Publishing Associates and John Wiley &amp; Sons, Edited by Ausubel, F. M et al., 1992. In vitro methods of antibody selection, such as antibody phage display, may also be used to generate antibodies recognizing the neoantigens disclosed herein (see, e.g., Schirrmann et al. Molecules 2011 16:412-426). 
     T-cell receptors (TCRs) are expressed on the surface of T-cells and consist of an α chain and a β chain. TCRs recognize antigens bound to MHC molecules expressed on the surface of antigen-presenting cells. The T-cell receptor (TCR) is a heterodimeric protein, in the majority of cases (95%) consisting of a variable alpha (α) and beta (ß) chain, and is expressed on the plasma membrane of T-cells. The TCR is subdivided in three domains: an extracellular domain, a transmembrane domain and a short intracellular domain. The extracellular domain of both α and ß chains have an immunoglobulin-like structure, containing a variable and a constant region. The variable region recognizes processed peptides, among which neoantigens, presented by major histocompatibility complex (MHC) molecules, and is highly variable. The intracellular domain of the TCR is very short, and needs to interact with CD3 to allow for signal propagation upon ligation of the extracellular domain. 
     With the focus of cancer treatment shifted towards more targeted therapies, among which immunotherapy, the potential of therapeutic application of tumor-directed T-cells is increasingly explored. One such application is adoptive T-cell therapy (ATCT) using genetically modified T-cells that carry chimeric antigen receptors (CARs) recognizing a particular epitope (Ref Gomes-Silva 2018). The extracellular domain of the CAR is commonly formed by the antigen-specific subunit of (scFv) of a monoclonal antibody that recognizes a tumor-antigen (Ref Abate-Daga 2016). This enables the CAR T-cell to recognize epitopes independent of MHC-molecules, thus widely applicable, as their functionality is not restricted to individuals expressing the specific MHC-molecule recognized by the TCR. Methods for engineering TCRs that bind a particular epitope are known to a skilled person. See, for example, US20100009863A1, which describes methods of modifying one or more structural loop regions. The intracellular domain of the CAR can be a TCR intracellular domain or a modified peptide to enable induction of a signaling cascade without the need for interaction with accessory proteins. This is accomplished by inclusion of the CD3ζ-signaling domain, often in combination with one or more co-stimulatory domains, such as CD28 and 4-1BB, which further enhance CAR T-cell functioning and persistence (Ref Abate-Daga 2016). 
     The engineering of the extracellular domain towards an scFv limits CAR T-cell to the recognition of molecules that are expressed on the cell-surface. Peptides derived from proteins that are expressed intracellularly can be recognized upon their presentation on the plasma membrane by MHC molecules, of which human form is called human leukocyte antigen (HLA). The HLA-haplotype generally differs among individuals, but some HLA types, like HLA-A*02:01, are globally common. Engineering of CAR T-cell extracellular domains recognizing tumor-derived peptides or neoantigens presented by a commonly shared HLA molecule enables recognition of tumor antigens that remain intracellular. Indeed CAR T-cells expressing a CAR with a TCR-like extracellular domain have been shown to be able to recognize tumor-derived antigens in the context of HLA-A*02:01 (Refs Zhang 2014, Ma 2016, Liu 2017). 
     In some embodiments, the binding molecules are monospecific, or rather they bind one of the neoantigens disclosed herein. In some embodiments, the binding molecules are bispecific, e.g., bispecific antibodies and bispecific chimeric antigen receptors. 
     In some embodiments, the disclosure provides a first antigen binding domain that binds a first neoantigen described herein and a second antigen binding domain that binds a second neoantigen described herein. The first and second antigen binding domains may be part of a single molecule, e.g., as a bispecific antibody or bispecific chimeric antigen receptor or they may be provided on separate molecules, e.g., as a collection of antibodies, T-cell receptors, or chimeric antigen receptors. In some embodiments, 3, 4, 5 or more antigen binding domains are provided each binding a different neoantigen disclosed herein. As used herein, an antigen binding domain includes the variable (antigen binding) domain of a T-cell receptor and the variable domain of an antibody (e.g., comprising a light chain variable region and a heavy chain variable region). 
     The disclosure further provides nucleic acid molecules encoding the antibodies, TCRs, and CARs disclosed herein. In a preferred embodiment, the nucleic acid molecules are codon optimized as disclosed herein. 
     The disclosure further provides vectors comprising the nucleic acids molecules disclosed herein. A “vector” is a recombinant nucleic acid construct, such as plasmid, phase genome, virus genome, cosmid, or artificial chromosome, to which another nucleic acid segment may be attached. The term “vector” includes both viral and non-viral means for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. The disclosure contemplates both DNA and RNA vectors. The disclosure further includes self-replicating RNA with (virus-derived) replicons, including but not limited to mRNA molecules derived from mRNA molecules from alphavirus genomes, such as the Sindbis, Semliki Forest and Venezuelan equine encephalitis viruses. 
     Vectors, including plasmid vectors, eukaryotic viral vectors and expression vectors are known to the skilled person. Vectors may be used to express a recombinant gene construct in eukaryotic cells depending on the preference and judgment of the skilled practitioner (see, for example, Sambrook et al., Chapter 16). For example, many viral vectors are known in the art including, for example, retroviruses, adeno-associated viruses, and adenoviruses. Other viruses useful for introduction of a gene into a cell include, but a not limited to, arenavirus, herpes virus, mumps virus, poliovirus, Sindbis virus, and vaccinia virus, such as, canary pox virus. The methods for producing replication-deficient viral particles and for manipulating the viral genomes are well known. In some embodiments, the vaccine comprises an attenuated or inactivated viral vector comprising a nucleic acid disclosed herein. 
     Preferred vectors are expression vectors. It is within the purview of a skilled person to prepare suitable expression vectors for expressing the inhibitors disclosed hereon. An “expression vector” is generally a DNA element, often of circular structure, having the ability to replicate autonomously in a desired host cell, or to integrate into a host cell genome and also possessing certain well-known features which, for example, permit expression of a coding DNA inserted into the vector sequence at the proper site and in proper orientation. Such features can include, but are not limited to, one or more promoter sequences to direct transcription initiation of the coding DNA and other DNA elements such as enhancers, polyadenylation sites and the like, all as well known in the art. Suitable regulatory sequences including enhancers, promoters, translation initiation signals, and polyadenylation signals may be included. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector. The expression vectors may also contain a selectable marker gene which facilitates the selection of host cells transformed or transfected. Examples of selectable marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, ß-galactosidase, chloramphenicol acetyltransferase, and firefly luciferase. 
     The expression vector can also be an RNA element that contains the sequences required to initiate translation in the desired reading frame, and possibly additional elements that are known to stabilize or contribute to replicate the RNA molecules after administration. Therefore when used herein the term DNA when referring to an isolated nucleic acid encoding the peptide according to the invention should be interpreted as referring to DNA from which the peptide can be transcribed or RNA molecules from which the peptide can be translated. 
     Also provided for is a host cell comprising an nucleic acid molecule or a vector as disclosed herein. The nucleic acid molecule may be introduced into a cell (prokaryotic or eukaryotic) by standard methods. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art recognized techniques to introduce a DNA into a host cell. Such methods include, for example, transfection, including, but not limited to, liposome-polybrene, DEAE dextran-mediated transfection, electroporation, calcium phosphate precipitation, microinjection, or velocity driven microprojectiles (“biolistics”). Such techniques are well known by one skilled in the art. See, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2 ed. Cold Spring Harbor Lab Press, Plainview, N.Y.). Alternatively, one could use a system that delivers the DNA construct in a gene delivery vehicle. The gene delivery vehicle may be viral or chemical. Various viral gene delivery vehicles can be used with the present invention. In general, viral vectors are composed of viral particles derived from naturally occurring viruses. The naturally occurring virus has been genetically modified to be replication defective and does not generate additional infectious viruses, or it may be a virus that is known to be attenuated and does not have unacceptable side effects. 
     Preferably, the host cell is a mammalian cell, such as MRCS cells (human cell line derived from lung tissue), HuH7 cells (human liver cell line), CHO-cells (Chinese Hamster Ovary), COS-cells (derived from monkey kidney (African green monkey), Vero-cells (kidney epithelial cells extracted from African green monkey), Hela-cells (human cell line), BHK-cells (baby hamster kidney cells, HEK-cells (Human Embryonic Kidney), NSO-cells (Murine myeloma cell line), C127-cells (nontumorigenic mouse cell line), PerC6®-cells (human cell line, Crucell), and Madin-Darby Canine Kidney (MDCK) cells. In some embodiments, the disclosure comprises an in vitro cell culture of mammalian cells expressing the neoantigens disclosed herein. Such cultures are useful, for example, in the production of cell-based vaccines, such as viral vectors expressing the neoantigens disclosed herein. 
     In some embodiments the host cells express the antibodies, TCRs, or CARs as disclosed herein. As will be clear to a skilled person, individual polypeptide chains (e.g., immunoglobulin heavy and light chains) may be provided on the same or different nucleic acid molecules and expressed by the same or different vectors. For example, in some embodiments, a host cell is transfected with a nucleic acid encoding an α-TCR polypeptide chain and a nucleic acid encoding a β-polypeptide chain. 
     In preferred embodiments, the disclosure provides T-cells expressing a TCR or CAR as disclosed herein. T cells may be obtained from, e.g., peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, spleen tissue, and tumors. Preferably, the T-cells are obtained from the individual to be treated (autologous T-cells). T-cells may also be obtained from healthy donors (allogenic T-cells). Isolated T-cells are expanded in vitro using established methods, such as stimulation with cytokines (IL-2). Methods for obtaining and expanding T-cells for adoptive therapy are well known in the art and are also described, e.g., in EP2872533A1. 
     The disclosure also provides vaccines comprising one or more neoantigens as disclosed herein. In particular, the vaccine comprises one or more (poly)peptides, antibodies or antigen binding fragments thereof, TCRs, CARS, nucleic acid molecules, vectors, or cells (or cell cultures) as disclosed herein. 
     The vaccine may be prepared so that the selection, number and/or amount of neoantigens (e.g., peptides or nucleic acids encoding said peptides) present in the composition is patient-specific. Selection of one or more neoantigens may be based on sequencing information from the tumor of the patient. For any frame shift mutation found, a corresponding NOP is selected. Preferably, the vaccine comprises more than one neoantigen corresponding to the NOP selected. In case multiple frame shift mutations (multiple NOPs) are found, multiple neoantigens corresponding to each NOP may be selected for the vaccine. 
     The selection may also be dependent on the specific type of cancer, the status of the disease, earlier treatment regimens, the immune status of the patient, and, HLA-haplotype of the patient. Furthermore, the vaccine can contain individualized components, according to personal needs of the particular patient. 
     As is clear to a skilled person, if multiple neoantigens are used, they may be provided in a single vaccine composition or in several different vaccines to make up a vaccine collection. The disclosure thus provides vaccine collections comprising a collection of tiled peptides, collection of peptides as disclosed herein, as well as nucleic acid molecules, vectors, or host cells as disclosed herein. As is clear to a skilled person, such vaccine collections may be administered to an individual simultaneously or consecutively (e.g., on the same day) or they may be administered several days or weeks apart. 
     Various known methods may be used to administer the vaccines to an individual in need thereof. For instance, one or more neoantigens can be provided as a nucleic acid molecule directly, as “naked DNA”. Neoantigens can also be expressed by attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of a virus as a vector to express nucleotide sequences that encode the neoantigen. Upon introduction into the individual, the recombinant virus expresses the neoantigen peptide, and thereby elicits a host CTL response. Vaccination using viral vectors is well-known to a skilled person and vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin) as described in Stover et al. (Nature 351:456-460 (1991)). 
     Preferably, the vaccine comprises a pharmaceutically acceptable excipient and/or an adjuvant. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like. Suitable adjuvants are well-known in the art and include, aluminum (or a salt thereof, e.g., aluminium phosphate and aluminium hydroxide), monophosphoryl lipid A, squalene (e.g., MF59), and cytosine phosphoguanine (CpG), montanide, liposomes (e.g. CAF adjuvants, cationic adjuvant formulations and variations thereof), lipoprotein conjugates (e.g. Amplivant), Resiquimod, Iscomatrix, hiltonol, poly-ICLC (polyriboinosinic-polyribocytidylic acid-polylysine carboxymethylcellulose). A skilled person is able to determine the appropriate adjuvant, if necessary, and an immune-effective amount thereof. As used herein, an immune-effective amount of adjuvant refers to the amount needed to increase the vaccine&#39;s immunogenicity in order to achieve the desired effect. 
     The disclosure also provides the use of the neoantigens disclosed herein for the treatment of disease, in particular for the treatment of breast cancer in an individual. In some embodiments, the breast cancer is ductal carcinoma or lobular carcinoma. Preferably, the cancer is breast invasive carcinoma (BRCA). It is within the purview of a skilled person to diagnose an individual with as having breast cancer. 
     As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, or inhibiting the progress of a disease, or reversing, alleviating, delaying the onset of, or inhibiting one or more symptoms thereof. Treatment includes, e.g., slowing the growth of a tumor, reducing the size of a tumor, and/or slowing or preventing tumor metastasis. 
     The term ‘individual’ includes mammals, both humans and non-humans and includes but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines. Preferably, the human is a mammal. 
     As used herein, administration or administering in the context of treatment or therapy of a subject is preferably in a “therapeutically effective amount”, this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. 
     The optimum amount of each neoantigen to be included in the vaccine composition and the optimum dosing regimen can be determined by one skilled in the art without undue experimentation. The composition may be prepared for injection of the peptide, nucleic acid molecule encoding the peptide, or any other carrier comprising such (such as a virus or liposomes). For example, doses of between 1 and 500 mg 50 μg and 1.5 mg, preferably 125 μg to 500 μg, of peptide or DNA may be given and will depend from the respective peptide or DNA. Other methods of administration are known to the skilled person. Preferably, the vaccines may be administered parenterally, e.g., intravenously, subcutaneously, intradermally, intramuscularly, or otherwise. 
     In preferred embodiments, the vaccines disclosed herein may be provided as a neoadjuvant therapy, e.g., prior to the removal of tumors or prior to treatment, e.g., with radiation or chemotherapy. Neoadjuvant therapy is intended to reduce the size of the tumor before more radical treatment is used. For that reason being able to provide the vaccine off-the-shelf or in a short period of time is very important. 
     In preferred embodiments, the vaccines disclosed herein may be provided shortly after the surgical removal of tumors. This can be followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. 
     Also disclosed herein, the vaccine is capable of initiating a specific T-cell response. It is within the purview of a skilled person to measure such T-cell responses either in vivo or in vitro, e.g. by analyzing IFN-γ production or tumor killing by T-cells. In therapeutic applications, vaccines are administered to a patient in an amount sufficient to elicit an effective CTL response to the tumor antigen and to cure or at least partially arrest symptoms and/or complications. 
     In preferred embodiments, the vaccines disclosed herein may be provided in combination with other therapeutic agents. The therapeutic agent is for example, a chemotherapeutic agent, radiation, or immunotherapy, including but not limited to checkpoint inhibitors, such as nivolumab, ipilimumab, pembrolizumab, or the like. Any suitable therapeutic treatment for a particular, cancer may be administered. 
     The term “chemotherapeutic agent” refers to a compound that inhibits or prevents the viability and/or function of cells, and/or causes destruction of cells (cell death), and/or exerts anti-tumor/anti-proliferative effects. The term also includes agents that cause a cytostatic effect only and not a mere cytotoxic effect. Examples of chemotherapeutic agents include, but are not limited to bleomycin, capecitabine, carboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin, etoposide, interferon alpha, irinotecan, lansoprazole, levamisole, methotrexate, metoclopramide, mitomycin, omeprazole, ondansetron, paclitaxel, pilocarpine, rituxitnab, tamoxifen, taxol, trastuzumab, vinblastine, and vinorelbine tartrate. 
     Preferably, the other therapeutic agent is an anti-immunosuppressive/immunostimulatory agent, such as anti-CTLA antibody or anti-PD-1 or anti-PD-L1. Blockade of CTLA-4 or PD-L1 by antibodies can enhance the immune response to cancerous cells. In particular, CTLA-4 blockade has been shown effective when following a vaccination protocol. 
     As is understood by a skilled person the vaccine and other therapeutic agents may be provided simultaneously, separately, or sequentially. In some embodiments, the vaccine may be provided several days or several weeks prior to or following treatment with one or more other therapeutic agents. The combination therapy may result in an additive or synergistic therapeutic effect. 
     As disclosed herein, the present disclosure provides vaccines which can be prepared as off-the-shelf vaccines. As used herein “off-the-shelf” means a vaccine as disclosed herein that is available and ready for administration to a patient. For example, when a certain frame shift mutation is identified in a patient, the term “off-the-shelf” would refer to a vaccine according to the disclosure that is ready for use in the treatment of the patient, meaning that, if the vaccine is peptide based, the corresponding polyNOP peptide may, for example already be expressed and for example stored with the required excipients and stored appropriately, for example at −20° C. or −80° C. Preferably the term “off-the-shelf” also means that the vaccine has been tested, for example for safety or toxicity. More preferably the term also means that the vaccine has also been approved for use in the treatment or prevention in a patient. Accordingly, the disclosure also provides a storage facility for storing the vaccines disclosed herein. Depending on the final formulation, the vaccines may be stored frozen or at room temperature, e.g., as dried preparations. Preferably, the storage facility stores at least 20 or at least 50 different vaccines, each recognizing a neoantigen disclosed herein. 
     The present disclosure also contemplates methods which include determining the presence of NOPs in a tumor sample. In a preferred embodiment, a tumor of a patient can be screened for the presence of frame shift mutations and an NOP can be identified that results from such a frame shift mutation. Based on the NOP(s) identified in the tumor, a vaccine comprising the relevant NOP(s) can be provided to immunize the patient, so the immune system of the patient will target the tumor cells expressing the neoantigen. An exemplary workflow for providing a neoantigen as disclosed herein is as follows. When a patient is diagnosed with a cancer, a biopsy may be taken from the tumor or a sample set is taken of the tumor after resection. The genome, exome and/or transcriptome is sequenced by any method known to a skilled person. The outcome is compared, for example using a web interface or software, to the library of NOPs disclosed herein. A patient whose tumor expresses one of the NOPs disclosed herein is thus a candidate for a vaccine comprising the NOP (or a fragment thereof). 
     Accordingly, the disclosure provides a method for determining a therapeutic treatment for an individual afflicted with cancer, said method comprising determining the presence of a frame shift mutation which results in the expression of an NOP selected from sequences 1-234. Identification of the expression of an NOP indicates that said individual should be treated with a vaccine corresponding to the identified NOP. For example, if it is determined that tumor cells from an individual express Sequence 1, then a vaccine comprising Sequence 1 or a fragment thereof is indicated as a treatment for said individual. 
     Accordingly, the disclosure provides a method for determining a therapeutic treatment for an individual afflicted with cancer, said method comprising
     a. performing complete, targeted or partial genome, exome, ORFeome, or transcriptome sequencing of at least one tumor sample obtained from the individual to obtain a set of sequences of the subject-specific tumor genome, exome, ORFeome, or transcriptome;   b. comparing at least one sequence or portion thereof from the set of sequences with one or more sequences selected from:   (i) Sequences 1-16;   (ii) Sequences 17-44;   (iii) Sequences 45-107;   (iv) Sequences 108-199; and   (v) Sequences 200-230;   c. identifying a match between the at least one sequence or portion thereof from the set of sequences and a sequence from groups (i) to (v) when the sequences have a string in common representative of at least 8 amino acids to identify a neoantigen encoded by a frameshift mutation;   

     wherein a match indicates that said individual is to be treated with the vaccine as disclosed herein. 
     As used herein the term “sequence” can refer to a peptide sequence, DNA sequence or RNA sequence. The term “sequence” will be understood by the skilled person to mean either or any of these, and will be clear in the context provided. For example, when comparing sequences to identify a match, the comparison may be between DNA sequences, RNA sequences or peptide sequences, but also between DNA sequences and peptide sequences. In the latter case the skilled person is capable of first converting such DNA sequence or such peptide sequence into, respectively, a peptide sequence and a DNA sequence in order to make the comparison and to identify the match. As is clear to a skilled person, when sequences are obtained from the genome or exome, the DNA sequences are preferably converted to the predicted peptide sequences. In this way, neo open reading frame peptides are identified. 
     As used herein the term “exome” is a subset of the genome that codes for proteins. An exome can be the collective exons of a genome, or also refer to a subset of the exons in a genome, for example all exons of known cancer genes. 
     As used herein the term “transcriptome” is the set of all RNA molecules is a cell or population of cells. In a preferred embodiment the transcriptome refers to all mRNA. 
     In some preferred embodiments the genome is sequenced. In some preferred embodiments the exome is sequenced. In some preferred embodiments the transcriptome is sequenced. In some preferred embodiments a panel of genes is sequenced, for example TP53, GATA3, CDH1, RUNX1, MAP3K1. In some preferred embodiments a single gene is sequenced. Preferably the transcriptome is sequenced, in particular the mRNA present in a sample from a tumor of the patient. The transcriptome is representative of genes and neo open reading frame peptides as defined herein being expressed in the tumor in the patient. 
     As used herein the term “sample” can include a single cell or multiple cells or fragments of cells or an aliquot of body fluid, taken from an individual, by means including venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage sample, scraping, surgical incision, or intervention or other means known in the art. The DNA and/or RNA for sequencing is preferably obtained by taking a sample from a tumor of the patient. The skilled person knowns how to obtain samples from a tumor of a patient and depending on the nature, for example location or size, of the tumor. Preferably the tumor is a breast tumor. Preferably the sample is obtained from the patient by biopsy or resection. The sample is obtained in such manner that is allows for sequencing of the genetic material obtained therein. In order to prevent a less accurate identification of at least one antigen, preferably the sequence of the tumor sample obtained from the patient is compared to the sequence of other non-tumor tissue of the patient, usually blood, obtained by known techniques (e.g. venipuncture). 
     Identification of frame shift mutations can be done by sequencing of RNA or DNA using methods known to the skilled person. Sequencing of the genome, exome, ORFeome, or transcriptome may be complete, targeted or partial. In some embodiments the sequencing is complete (whole sequencing). In some embodiments the sequencing is targeted. With targeted sequencing is meant that purposively certain region or portion of the genome, exome, ORFeome or transcriptome are sequenced. For example targeted sequencing may be directed to only sequencing for sequences in the set of sequences obtained from the cancer patient that would provide for a match with one or more of the sequences in the sequence listing, for example by using specific primers. In some embodiment only portion of the genome, exome, ORFeome or transcriptome is sequenced. The skilled person is well-aware of methods that allow for whole, targeted or partial sequencing of the genome, exome, ORFeome or transcriptome of a tumor sample of a patient. For example any suitable sequencing-by-synthesis platform can be used including the Genome Sequencers from Illumina/Solexa, the Ion Torrent system from Applied BioSystems, and the RSII or Sequel systems from Pacific Biosciences. Alternatively Nanopore sequencing may be used, such as the MinION, GridION or PromethION platform offered by Oxford Nanopore Technologies. The method of sequencing the genome, exome, ORFeome or transcriptome is not in particular limited within the context of the present invention. 
     Sequence comparison can be performed by any suitable means available to the skilled person. Indeed the skilled person is well equipped with methods to perform such comparison, for example using software tools like BLAST and the like, or specific software to align short or long sequence reads, accurate or noisy sequence reads to a reference genome, e.g. the human reference genome GRCh37 or GRCh38. A match is identified when a sequence identified in the patients material and a sequence as disclosed herein have a string, i.e. a peptide sequence (or RNA or DNA sequence encoding such peptide (sequence) in case the comparison is on the level of RNA or DNA) in common representative of at least 8, preferably at least 10 adjacent amino acids. Furthermore, sequence reads derived from a patients cancer genome (or transcriptome) can partially match the genomic DNA sequences encoding the amino acid sequences as disclosed herein, for example if such sequence reads are derived from exon/intron boundaries or exon/exon junctions, or if part of the sequence aligns upstream (to the 5′ end of the gene) of the position of a frameshift mutation. Analysis of sequence reads and identification of frameshift mutations will occur through standard methods in the field. For sequence alignment, aligners specific for short or long reads can be used, e.g. BWA (Li and Durbin, Bioinformatics. 2009 Jul. 15; 25(14):1754-60) or Minimap2 (Li, Bioinformatics. 2018 Sep. 15; 34(18):3094-3100). Subsequently, frameshift mutations can be derived from the read alignments and their comparison to a reference genome sequence (e.g. the human reference genome GRCh37) using variant calling tools, for example Genome Analysis ToolKit (GATK), and the like (McKenna et al. Genome Res. 2010 September; 20(9):1297-303). 
     A match between an individual patient&#39;s tumor sample genome or transcriptome sequence and one or more NOPs disclosed herein indicates that said tumor expresses said NOP and that said patient would likely benefit from treatment with a vaccine comprising said NOP (or a fragment thereof). More specifically, a match occurs if a frameshift mutation is identified in said patient&#39;s tumor genome sequence and said frameshift leads to a novel reading frame (+1 or −1 with respect to the native reading from of a gene). In such instance, the predicted out-of-frame peptide derived from the frameshift mutation matches any of the sequences 1-230 as disclosed herein. In some embodiments, said patient is administered said NOP (e.g., by administering the peptides, nucleic acid molecules, vectors, host cells or vaccines as disclosed herein). 
     In some embodiments, the methods further comprise sequencing the genome, exome, ORFeome, or transcriptome (or a part thereof) from a normal, non-tumor sample from said individual and determining whether there is a match with one or more NOPs identified in the tumor sample. Although the neoantigens disclosed herein appear to be specific to tumors, such methods may be employed to confirm that the neoantigen is tumor specific and not, e.g., a germline mutation. 
     The disclosure further provides the use of the neoantigens and vaccines disclosed herein in prophylactic methods from preventing or delaying the onset of breast cancer. Approximately 1 in 10 women will develop breast cancer and the neo open reading frames disclosed herein occur in approximately 20% of breast cancer patients. Prophylactic vaccination based on frameshift resulting peptides disclosed herein would thus provide possible protection to approximately 2% of the general population. The vaccine may be specifically used in a prophylactic setting for women that have an increased risk of developing breast cancer. For example, prophylactic vaccination based on the peptides disclosed herein is expected to provide possible protection to around 29% of women having a germline predisposition mutation in BRCA1 and/or BRCA2, and who will obtain breast cancer as a result of their predisposing mutation. In some embodiments, the prophylactic methods are useful for individuals who are genetically related to individuals afflicted with breast cancer. In some embodiments, the prophylactic methods are useful for the general population. 
     In some embodiments, the individual is at risk of developing cancer. It is understood to a skilled person that being at risk of developing cancer indicates that the individual has a higher risk of developing cancer than the general population; or rather the individual has an increased risk over the average of developing cancer. Such risk factors are known to a skilled person and include being a woman; being 50 years or older; previously being afflicted with breast cancer, atypical hyperplasia or lobular carcinoma in situ; previously having radiation therapy to the chests or breast, having received hormone replacement therapy, having a family history of breast cancer, and having a mutation in a gene that predisposes an individual to breast cancer. 
     In some embodiments, said individual has a germline mutation in a gene that increases the chance that the individual will develop breast cancer, preferably the mutation is in the PALB2, CHEK2, BRCA1, and/or BRCA2 gene. Predisposing mutations in the PALB2, CHEK2, BRCA1, and BRCA2 genes are known to a skilled person and such mutations can be identified in individuals. Preferably, the prophylactic methods disclosed herein comprise determining the presence of a predisposing mutation in one or more of the PALB2, CHEK2, BRCA1, and BRCA2 genes and prophylactically administering the vaccine disclosed herein to an individual having said predisposing mutation in one or more of the PALB2, CHEK2, BRCA1, and BRCA2 genes, or other genes with known predisposing mutations as described in Frey et al. Plast Reconstr Surg Glob Open 2017; 5e1564. 
     As used herein, “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb “to consist” may be replaced by “to consist essentially of” meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. 
     The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. 
     The word “approximately” or “about” when used in association with a numerical value (approximately 10, about 10) preferably means that the value may be the given value of 10 more or less 1% of the value. 
     All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. 
     For the purpose of clarity and a concise description features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  Frame shift initiated translation in the TCGA (n=10,186) cohort is of sufficient size for immune presentation. A. Peptide length distribution of frame shift mutation initiated translation up to the first encountered stop codon. Dark shades are unique peptide sequences derived from frameshift mutations, light shade indicates the total sum (unique peptides derived from frameshifts multiplied by number of patients containing that frameshift). B. Gene distribution of peptides with length 10 or longer and encountered in up to 10 patients. 
         FIG. 2  Neo open reading frame peptides (TCGA cohort) converge on common peptide sequences. Graphical representation in an isoform of TP53, where amino acids are colored distinctly. A. somatic single nucleotide variants, B. positions of frame shift mutations on the −1 and the +1 frame. C. amino acid sequence of TP53. D. Peptide (10aa) library (n=1,000) selection. Peptides belonging to −1 or +1 frame are separated vertically E,F pNOPs for the different frames followed by all encountered frame shift mutations (rows), translated to a stop codon (lines) colored by amino acid. 
         FIG. 3  A recurrent peptide selection procedure can generate a ‘fixed’ library to corer up to 50% of the TCGA cohort. Graph depicts the number of unique patients from the TCGA cohort (10,186 patients) accommodated by a growing library of 10-mer peptides, picked in descending order of the number patients with that sequence in their NOPs. A peptide is only added if it adds a new patient from the TCGA cohort. The dark blue line shows that an increasing number of 10-mer peptides covers an increasing number of patients from the TCGA cohort (up to 50% if using 3000 unique 10-mer peptides). Light shaded blue line depicts the number of patients containing the peptide that was included (right Y-axis). The best peptide covers 89 additional patients from the TCGA cohort (left side of the blue line), the worst peptide includes only 1 additional patient (right side of the blue line). 
         FIG. 4  For some cancers up to 70% of patients contain a recurrent NOP. TCGA cohort ratio of patients separated by tumor type that could be ‘helped’ using optimally selected peptides for genes encountered most often within a cancer. Coloring represents the ratio, using 1, 2 . . . 10 genes, or using all encountered genes (lightest shade) 
         FIG. 5  Examples of NOPs. Selection of genes containing NOPs of 10 or more amino acids. 
         FIG. 6  Frame shift presence in mRNA from 58 CCLE colorectal cancer cell lines. 
       a. Cumulative counting of RNAseq allele frequency (Samtools mpileup (XO:1/a11)) at the genomic position of DNA detected frame shift mutations. 
       b. IGV examples of frame shift mutations in the BAM files of CCLE cell lines. 
         FIG. 7  Example of normal isoforms, using shifted frame. 
       Genome model of CDKN2A with the different isoforms are shown on the minus strand of the genome. Zoom of the middle exon depicts the 2 reading frames that are encountered in the different isoforms. 
         FIG. 8  Gene prevalence us Cancer type. 
       Percentage of frameshift mutations (resulting in peptides of 10 aa or longer), assessed by the type of cancer in the TOGA cohort. Genes where 50% or more of the frameshifts occur within a single tumor type are indicated in bold. Cancer type abbreviations are as follows: 
       LAML Acute Myeloid Leukemia 
       ACC Adrenocortical carcinoma 
       BLCA Bladder Urothelial Carcinoma 
       LGG Brain Lower Grade Glioma 
       BRCA Breast invasive carcinoma 
       CESC Cervical squamous cell carcinoma and endocervical adenocarcinoma 
       CHOL Cholangiocarcinoma 
       LCML Chronic Myelogenous Leukemia 
       COAD Colon adenocarcinoma 
       CNTL Controls 
       ESCA Esophageal carcinoma 
       GBM Glioblastoma multiforme 
       HNSC Head and Neck squamous cell carcinoma 
       KICH Kidney Chromophobe 
       KIRC Kidney renal clear cell carcinoma 
       KIRP Kidney renal papillary cell carcinoma 
       LIHC Liver hepatocellular carcinoma 
       LUAD Lung adenocarcinoma 
       LUSC, Lung squamous cell carcinoma 
       DLBC Lymphoid Neoplasm Diffuse Large B-cell Lymphoma 
       MESO Mesothelioma 
       MISC Miscellaneous 
       OV Ovarian serous cystadenocarcinoma 
       PAAD Pancreatic adenocarcinoma 
       PCPG Pheochromocytoma and Paraganglioma 
       PRAD Prostate adenocarcinoma 
       READ Rectum adenocarcinoma 
       SARC Sarcoma 
       SKCM Skin Cutaneous Melanoma 
       STAD Stomach adenocarcinoma 
       TGCT Testicular Germ Cell Tumors 
       THYM Thymoma 
       THCA Thyroid carcinoma 
       UCS Uterine Carcinosarcoma 
       UCEC Uterine Corpus Endometrial Carcinoma 
       UVM Uveal Melanoma 
         FIG. 9  NOPs in the MSK-IMPACT study 
       Frame shift analysis in the targeted sequencing panel of the MSK-IMPACT study, covering up to 410 genes in more 10,129 patients (with at least 1 somatic mutation). a. FS peptide length distribution, b. Gene count of patients containing NOPs of 10 or more amino acids. c. Ratio of patients separated by tumor type that possess a neo epitope using optimally selected peptides for genes encountered most often within a cancer. Coloring represents the ratio, using 1, 2 . . . 10 genes, or using all encountered genes (lightest shade) d. Examples of NOPs for 4 genes. 
         FIG. 10  GATA3 out-of-frame peptide sequences based on frameshift mutations in breast cancer patients. Data are obtained from TCGA (986 patients), Hartwig Medical Foundation (568 patients; Priestley et al, 2018, biorxiv.org), and Memorial Sloan Kettering (1918; Razavi et al, 2018, Cancer Cell). Stacks of recurrent out-of-frame peptide subsequences are indicated with red boxes. 
         FIG. 11  Number of mutations in cancer susceptibility genes observed in patients with breast cancer from the cancer sequencing resource described in Priestley et al. 2019 at https://doi.org/10.1101/415133. 
         FIG. 12  Number of breast cancer patients with frameshift mutations per gene leading to neo-peptides&gt;=10 amino acids, as based on the cancer genome sequencing resource described in Razavi et al. 2018 Cancer Cell 34:427-438. 
         FIG. 13  Neo-peptide sequences predicted based on somatic frameshifts in GATA3 in breast cancer patients in the cancer genome sequencing resource described in Priestley et al. 2019 at https://doi.org/10.1101/415133. Two blocks of overlapping sequences can be observed, all derived from the −1 shifted frame of GATA3. For the +1 shifted frame of GATA3 smaller blocks of overlapping sequences are observed in this data resource (not shown). 
         FIG. 14  Number of breast cancer patients with germline predisposition mutation, that carry a cancer specific (i.e. somatic) frameshift mutation leading to a neo-peptide (&gt;=10 amino acids) in the indicated genes (x-axis). 
     
    
    
     EXAMPLES 
     We have analyzed 10,186 cancer genomes from 33 tumor types of the 40 TCGA (The Cancer Genome Atlas 22 ) and focused on the 143,444 frame shift mutations represented in this cohort. Translation of these mutations after re-annotation to a RefSeq annotation, starting in the protein reading frame, can lead to 70,439 unique peptides that are 10 or more amino acids in length (a cut off we have set at a size sufficient to shape a distinct epitope in the context of MHC ( FIG. 1 a   ). The list of genes most commonly represented in the cohort and containing such frame shift mutations is headed nearly exclusively by tumor driver genes, such as NF1, RB, BRCA2 ( FIG. 1 b   ) whose whole or partial loss of function apparently contributes to tumorigenesis. Note that a priori frame shift mutations are expected to result in loss of gene function more than a random SNV, and more independent of the precise position. NOPs initiated from a frameshift mutation and of a significant size are prevalent in tumors, and are enriched in cancer driver genes. Alignment of the translated NOP products onto the protein sequence reveals that a wide array of different frame shift mutations translate in a common downstream stretch of neo open reading frame peptides (‘NOPs’), as dictated by the −1 and +1 alternative reading frames. While we initially screened for NOPs of ten or more amino acids, their open reading frame in the out-of-frame genome often extends far beyond that search window. As a result we see ( FIG. 2 ) that hundreds of different frame shift mutations all at different sites in the gene nevertheless converge on only a handful of NOPs. Similar patterns are found in other common driver genes ( FIG. 5 ).  FIG. 2  illustrates that the precise location of a frame shift does not seem to matter much; the more or less straight slope of the series of mutations found in these 10,186 tumors indicates that it is not relevant for the biological effect (presumably reduction/loss of gene function) where the precise frame shift is, as long as translation stalls in the gene before the downstream remainder of the protein is expressed. As can also be seen in  FIG. 2 , all frame shift mutations alter the reading frame to one of the two alternative frames. Therefore, for potential immunogenicity the relevant information is the sequence of the alternative ORFs and more precisely, the encoded peptide sequence between 2 stop codons. We term these peptides ‘proto Neo Open Reading Frame peptides’ or pNOPs, and generated a full list of all thus defined out of frame protein encoding regions in the human genome, of 10 amino acids or longer. We refer to the total sum of all Neo-ORFs as the Neo-ORFeome. The Neo-ORFeome contains all the peptide potential that the human genome can generate after simple frame-shift induced mutations. The size of the Neo-ORFeome is 46.6 Mb. To investigate whether or not Nonsense Mediated Decay would wipe out frame shift mRNAs, we turned to a public repository containing read coverage for a large collection of cell lines (CCLE). We processed the data in a similar fashion as for the TCGA, identified the locations of frame shifts and subsequently found that, in line with the previous literature 23-25 , at least a large proportion of expressed genes also contained the frame shift mutation within the expressed mRNAs ( FIG. 6 ). On the mRNA level, NOPs can be detected in RNAseq data. We next investigated how the number of patients relates to the number of NOPs. We sorted 10-mer peptides from NOPs by the number of new patients that contain the queried peptide. Assessed per tumor type, frame shift mutations in genes with very low to absent mRNA expression were removed to avoid overestimation. Of note NOP sequences are sometimes also encountered in the normal ORFeome, presumably as result of naturally occuring isoforms (e,g,  FIG. 7 ). Also these peptides were excluded. We can create a library of possible ‘vaccines’ that is optimally geared towards covering the TCGA cohort, a cohort large enough that, also looking at the data presented here, it is representative of future patients ( FIG. 10 ). Using this strategy 30% of all patients can be covered with a fixed collection of only 1,244 peptides of length 10 ( FIG. 3 ). Since tumors will regularly have more than 1 frame shift mutation, one can use a ‘cocktail’ of different NOPs to optimally attack a tumor. Indeed, given a library of 1,244 peptides, 27% of the covered TCGA patients contain 2 or more ‘vaccine’ candidates. In conclusion, using a limited pool with optimal patient inclusion of vaccines, a large proportion of patients is covered. Strikingly, using only 6 genes (TP53, ARID1A, KMT2D, GATA3, APC, PTEN), already 10% of the complete TOGA cohort is covered. Separating this by the various tumor types, we find that for some cancers (like Pheochromocytoma and Paraganglioma (PCPG) or Thyroid carcinoma (THCA)) the hit rate is low, while for others up to 39% can be covered even with only 10 genes (Colon adenocarcinoma (COAD) using 60 peptides, Uterine Corpus Endometrial Carcinoma (UCEC) using 90 peptides),  FIG. 4 . At saturation (using all peptides encountered more than once) 50% of TCGA is covered and more than 70% can be achieved for specific cancer types (COAD, UCEC, Lung squamous cell carcinoma (LUSC) 72%, 73%, 73% respectively). As could be expected, these roughly follow the mutational load in the respective cancer types. In addition some frame shifted genes are highly enriched in specific tumor types (e.g. VHL, GATA3.  FIG. 8 ). We conclude that at saturating peptide coverage, using only very limited set of genes, a large cohort of patients can be provided with off the shelf vaccines. To validate the presence of NOPs, we used the targeted sequencing data on 10,129 patients from the MSK-IMPACT cohort 26. For the 341-410 genes assessed in this cohort, we obtained strikingly similar results in terms of genes frequently affected by frame shifts and the NOPs that they create ( FIG. 9 ). Even within this limited set of genes, 86% of the library peptides (in genes targeted by MSK-IMPACT) were encountered in the patient set. Since some cancers, like glioblastoma or pancreatic cancer, show survival expectancies after diagnosis measured in months rather than years (e.g. see 27), it is of importance to move as much of the work load and time line to the moment before diagnosis. Since the time of whole exome sequencing after biopsy is currently technically days, and since the scan of a resulting sequence against a public database describing these NOPs takes seconds, and the shipment of a peptide of choice days, a vaccination can be done theoretically within days and practically within a few weeks after biopsy. This makes it attractive to generate a stored and quality controlled peptide vaccine library based on the data presented here, possibly with replicates stored on several locations in the world. The synthesis in advance will—by economics of scale—reduce costs, allow for proper regulatory oversight, and can be quality certified, in addition to saving the patient time and thus provide chances. The present invention will likely not replace other therapies, but be an additional option in the treatment repertoire. The advantages of scale also apply to other means of vaccination against these common neoantigens, by RNA- or DNA-based approaches (e.g. 28), or recombinant bacteria (e.g. 29). The present invention also provides neoantigen directed application of the CAR-T therapy (For recent review see 30, and references therein), where the T-cells are directed not against a cell-type specific antigens (such as CD19 or CD20), but against a tumor specific neoantigen as provided herein. E.g. once one functional T-cell against any of the common p53 NOPs ( FIG. 2 ) is identified, the recognition domains can be engineered into T-cells for any future patient with such a NOP, and the constructs could similarly be deposited in an off-the-shelf library. In the present invention, we have identified that various frame shift mutations can result in a source for common neo open reading frame peptides, suitable as pre-synthesized vaccines. This may be combined with immune response stimulating measures such as but not limited checkpoint inhibition to help instruct our own immune system to defeat cancer. 
     It is estimated that around 20% of breast cancers can arise as a result of germline mutations in breast cancer predisposition genes, amongst which BRCA1, BRCA2, PALB2 and CHEK2 (ref: https://www.nebi.nlm.nih.gov/pmc/articles/PMC5732672/). Individuals which are known to carry such mutations (particularly BRCA1 and BRCA2 mutations) have a very high risk (˜70%) of developing breast cancer. Somewhat lower expectancies have been reported for PALB2 (33%) and CHEK2 (22-44%). 
     Thus, there is an opportunity for prophylactic vaccination to reduce the risk of breast cancer in individuals with predisposition mutations in these genes. A prophylactic vaccine would be of highest efficacy if it vaccinates against (i) strongly immunogenic antigens, and (ii) antigens that are expected to be present in a large proportion of breast tumors observed in patients with predisposition mutations in any of the BRCA1, BRCA2, PALB2 and CHEK2 genes. 
     We exploited a recent data source from the Hartwig Medical Foundation (HMF) (Priestley et al. 2019 at https://doi.org/10.1101/415133) for the presence of targetable neoantigens in breast tumors in patients with germline mutations a range of (breast) cancer predisposition genes. 
     Amongst 568 individuals with breast tumors, we observed that 112 (19.7%) of the patients have a mutation in one or more possible predisposition genes ( FIG. 11 ). As can be seen in  FIG. 11 , germline predisposing mutations in CHEK2 and BRCA2 are most frequently observed (22 and 13, respectively) in the HMF breast cancer cohort. 
     Next, we explored the idea of using neo-open reading frame peptides, resulting from somatic frameshift mutations, as an attractive source of neoantigens in human cancers. Therefore, we calculated the number of breast cancer patients in the HMF data resource with frameshift mutations leading to possible out of frame neo-peptides ( FIG. 12 ). Neo-peptides larger than or equal to 10 amino acids are most frequently found in TP53 (40/568; 7%), GATA3 (30/568; 5.2%) and CDH1 (22/568; 3.9%) in the total group of breast cancer patients. 
     Analyzing the sequences of the neo-peptides resulting from the frameshifts of the HMF data resource confirmed again that many of these sequences converge on a specific set of out-of-frame sequences. E.g. for GATA3, the C-terminal portion of the gene frequently contains frameshifts, leading to overlapping neo-peptides (See  FIG. 13 ). 
     To explore the possibility for prophylactic vaccination of individuals at risk of breast cancer based on germline predisposing mutations, we determined the out-of-frame peptide sequences in 5 genes that are most often hit by frameshifts that result in neo-peptides (&gt;=10 amino acids), namely: GATA3, TP53, RUNX1, MAP3K1, CDH1. 
     Out of 112 breast cancer patients with presumed germline predisposition mutations, we found 21 (19%) patients that have tumors with a frameshift mutation in one the genes GATA3, TP53, RUNX1, MAP3K1, CDH1 leading to the formation of a neoantigen ( FIG. 14 ). 
     When limiting the list to breast cancer patients with BRCA1, BRCA2, CHEK2 or PALB2 germline mutations. A total of 41 different patients have a predisposing mutation in any of these four genes (one patient has two different predisposing mutations). Out of these 41 patients, 8 (20%) have a neo-peptide in either TP53 (3), CDH1 (1), RUNX1 (1), GATA3 (2), MAP3K1 (1). 
     When limited to patients with a BRCA1 and/or BRCA2 predisposing germline mutation (17), we observed 5 (29%) that developed breast cancer with a neo-peptide in TP53 (3), GATA3 (1) and RUNX1 (1). Surprisingly, 10 of the 17 breast cancer patients with a BRCA1 and/or BRCA2 predisposing germline mutation have a neo-peptide resulting from a frameshift mutation in one or more genes (if not limiting to TP53, GATA3, RUNX1, CDH1, MAP3K1). The top genes recurrently hit by somatic frameshifts in breast tumors of BRCA1/BRCA2 germline mutation carriers are: TP53 (3), CDKN1B (2) and SPEN (2). 
     We conclude that a considerable fraction of breast cancer patients with predisposing germline mutations, may benefit from vaccination against frameshift-induced neopeptides. With a peptide vaccine covering only 5 genes (TP53, MAP3K1, GATA3, CDH1, RUNX1), for 29% of patients with BRCA1/BRCA2 germline mutations, cancer development can possibly be prevented. 
     Methods: 
     TCGA frameshift mutations—Frame shift mutations were retrieved from Varscan and mutect files per tumor type via https://portal.gdc.cancer.gov/. Frame shift mutations contained within these files were extracted using custom perl scripts and used for the further processing steps using HG38 as reference genome build. 
     CCLE frameshift mutations—For the CCLE cell line cohort, somatic mutations were retrieved from http://www.broadinstitute.org/ccle/data/browseData?conversationPropagation=begin (CCLE_hybrid_capture1650_hg19_NoCommonSNPs_NoNeutralVariants_CDS_2012.02.20.maf). Frame shift mutations were extracted using custom perl scripts using hg19 as reference genome. 
     Refseq annotation—To have full control over the sequences used within our analyses, we downloaded the reference sequences from the NCBI website (2018-02-27) and extracted mRNA and coding sequences from the gbff files using custom perl scripts. Subsequently, mRNA and every exon defined within the mRNA sequences were aligned to the genome (hg19 and hg38) using the BLAT suite. The best mapping locations from the psl files were subsequently used to place every mRNA on the genome, using the separate exons to perform fine placement of the exonic borders. Using this procedure we also keep track of the offsets to enable placement of the amino acid sequences onto the genome. 
     Mapping genome coordinate onto Refseq—To assess the effect of every mentioned frame shift mutation within the cohorts (CCLE or TCGA), we used the genome coordinates of the frameshifts to obtain the exact protein position on our reference sequence database, which were aligned to the genome builds. This step was performed using custom perl scripts taking into account the codon offsets and strand orientation, necessary for the translation step described below. 
     Translation of FS peptides—Using the reference sequence annotation and the positions on the genome where a frame shift mutation was identified, the frame shift mutations were used to translate peptides until a stop codon was encountered. The NOP sequences were recorded and used in downstream analyses as described in the text. 
     Verification of FS mRNA expression in the CCLE colorectal cancer cell lines—For a set of 59 colorectal cancer cell lines, the HG19 mapped bam files were downloaded from https://portal.gdc.cancer.gov/. Furthermore, the locations of FS mutations were retrieved from CCLE_hybrid_capture1650_hg19_NoCommonSNPs_NoNeutralVariants_CDS_2012.02.20.maf (http://www.broadinstitute.org/ccle/data/browseData?conversationPropagation=begin), by selection only frameshift entries. Entries were processed similarly to the TCGA data, but this time based on a HG19 reference genome. To get a rough indication that a particular location in the genome indeed contains an indel in the RNAseq data, we first extracted the count at the location of a frameshift by making use of the pileup function in samtools. Next we used the special tag XO:1 to isolate reads that contain an indel in it. On those bam files we again used the pileup function to count the number of reads containing an indel (assuming that the indel would primarily be found at the frameshift instructed location). Comparison of those 2 values can then be interpreted as a percentage of indel at that particular location. To reduce spurious results, at least 10 reads needed to be detected at the FS location in the original bam file. 
     Defining peptide library—To define peptide libraries that are maximized on performance (covering as many patients with the least amount of peptides) we followed the following procedure. From the complete TCGA cohort, FS translated peptides of size 10 or more (up to the encountering of a stop codon) were cut to produce any possible 10-mer. Then in descending order of patients containing a 10-mer, a library was constructed. A new peptide was added only if an additional patient in the cohort was included. peptides were only considered if they were seen 2 or more times in the TCGA cohort, if they were not filtered for low expression (see Filtering for low expression section), and if the peptide was not encountered in the orfeome (see Filtering for peptide presence orfeome). In addition, since we expect frame shift mutations to occur randomly and be composed of a large array of events (insertions and deletions of any non triplet combination), frame shift mutations being encountered in more than 10 patients were omitted to avoid focusing on potential artefacts. Manual inspection indicated that these were cases with e.g. long stretches of Cs, where sequencing errors are common. 
     Filtering for low expression—Frameshift mutations within genes that are not expressed are not likely to result in the expression of a peptide. To take this into account we calculated the average expression of all genes per TCGA entity and arbitrarily defined a cutoff of 2 log 2 units as a minimal expression. Any frameshift mutation where the average expression within that particular entity was below the cutoff was excluded from the library. This strategy was followed, since mRNA gene expression data was not available for every TCGA sample that was represented in the sequencing data set. Expression data (RNASEQ v2) was pooled and downloaded from the R2 platform (http://r2.amc.nl). In current sequencing of new tumors with the goal of neoantigen identification such mRNA expression studies are routine and allow routine verification of presence of mutant alleles in the mRNA pool. 
     Filtering for peptide presence orfeome—Since for a small percentage of genes, different isoforms can actually make use of the shifted reading frame, or by chance a 10-mer could be present in any other gene, we verified the absence of any picked peptide from peptides that can be defined in any entry of the reference sequence collection, once converted to a collection of tiled 10-mers. 
     Generation of cohort coverage by all peptides per gene To generate overviews of the proportion of patients harboring exhaustive FS peptides starting from the most mentioned gene, we first pooled all peptides of size 10 by gene and recorded the largest group of patients per tumor entity. Subsequently we picked peptides identified in the largest set of patients and kept on adding a new peptide in descending order, but only when at least 1 new patient was added. Once all patients containing a peptide in the first gene was covered, we progressed to the next gene and repeated the procedure until no patient with FS mutations leading to a peptide of size 10 was left. 
     proto-NOP (pNOP) and Neo-ORFeome proto—NOPs are those peptide products that result from the translation of the gene products when the reading frame is shifted by −1 or +1 base (so out of frame). Collectively, these pNOPs form the Neo-Orfeome. As such we generated a pNOP reference base of any peptide with length of 10 or more amino acids, from the RefSeq collection of sequences. Two notes: the minimal length of 10 amino acids is a choice; if one were to set the minimal window at 8 amino acids the total numbers go up a bit, e.g. the 30% patient covery of the library goes up. On a second note: we limited our definition to ORFs that can become in frame after a single insertion deletion on that location; this includes obviously also longer insertion or deletion stretches than +1 or −1. The definition has not taken account more complex events that get an out-of-frame ORF in frame, such as mutations creating or deleting splice sites, or a combination of two frame shifts at different sites that result in bypass of a natural stop codon; these events may and will occur, but counting those in will make the definition of the Neo-ORFeome less well defined. For the magnitude of the numbers these rare events do not matter much. 
     Visualizing flops—Visualization of the nops was performed using custom perl scripts, which were assembled such that they can accept all the necessary input data structures such as protein sequence, frameshifted protein sequences, somatic mutation data, library definitions, and the peptide products from frameshift translations. 
     Detection of frameshift resulting neopeptides in breast cancer patients with cancer predisposition mutations—Somatic and germline mutation data were downloaded from the supplementary files attached to the manuscript posted here: https://www.biorxiv.org/content/biorxiv/early/2019/01/16/415133.full.pdf. Frameshift mutations were selected from the somatic mutation files and out-of-frame peptides were predicted using custom Perl and Python scripts, based on the human reference genome GRCh37. Out-of-frame peptides were selected based on their length (&gt;=10 amino acids) and mapped against out of frame peptide sequences for each possible alternative transcript for genes present in the human genome, based on Ensembl annotation (ensembl.org). 
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