Selection of neoepitopes as disease-specific targets for therapies with enhanced efficacy

Abstract

The present invention relates to methods for determining whether neoepitopes that are expressed only within or on the surface of diseased cells are suitable disease-specific targets, such that the diseased cells are less likely to be able to evade immune surveillance, and the use of neoepitopes in providing an immune response against diseased cells that express the neoepitopes. [Solution] The present invention relates to a method for determining the suitability of a neoepitope resulting from a disease-specific mutation in an allele (mutated allele) in a gene as a disease-specific target, the method comprising a step of determining the copy number of the mutated allele encoding the neoepitope in a diseased cell or a population of diseased cells.

Description

Technical Field

[0001] The present invention relates to a method for determining the suitability of disease-specific neoepitopes as disease-specific targets, and to the use of such identified suitable neoepitopes in immunotherapy specifically targeted to diseased tissues of a patient, such as tumor tissue, expressing one or more of the identified suitable neoepitopes.

Background Art

[0002] Cancer is a major cause of mortality, accounting for one in four deaths worldwide. Cancer treatment has traditionally been based on the law of averages, which works best for the majority of patients. However, due to the molecular heterogeneity in cancer, in many cases, less than 25% of treated individuals benefit from approved therapies. Personalized medicine based on individually tailored treatments for patients is considered a potential solution to the low efficacy and high cost for technological innovation in drug discovery.

[0003] Personalized cancer immunotherapy is emerging as a promising breakthrough in cancer treatment, with the potential to revolutionize the standard of care for the millions of cancer patients diagnosed worldwide each year. An integrated aspect of personalized cancer immunotherapy is that the immune system is enabled to target genetic abnormalities (mutations) specific to the patient's cancer. Such disease-specific mutations can encode neoepitopes, which are disease-specific targets. The most prevalent genetic abnormality that the cancer genome undergoes and can be used as a disease-specific target for personalized immunotherapy is non-synonymous single nucleotide variation (SNV). Therefore, the accurate and thorough identification of a patient's SNVs in the coding region of the genome is a decisive step in the process of producing personalized cancer immunotherapy.

[0004] However, as described herein, knowing the identity of disease-specific mutations is only part of the picture. Rather, a complete genetic profiling of the mutations requires knowledge of the exact number of copies of the genes containing the mutations in the diseased cells, e.g., tumor cells (including both wild-type and mutated alleles), the number of copies of the mutated alleles in the tumor cells (referred to herein as the mutational status), and the degree of subclonality of the mutations in a sample of the diseased cells, such as a tumor sample. Indeed, copy number diversity that occurs in diseased cells is an important component of genetic diversity in diseased cells across most disease phenotypes. Furthermore, the degree of copy number diversity, the identity of the genes affected by copy number diversity, and the exact genetic makeup of the copy number diversity are specific to each individual and can vary widely between individuals. Generally, see Shlien and Malkin, 2009, Genome Med. 1:62; Yang et al., 2013, Cell 153:919-929. Accurate knowledge of such genetic features can be decisive in the selection of mutations that, when targeted, serve as immunity against tumors and thus have the potential to confer overall tumor control.

Prior Art Documents

Patent Documents

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[0006] [Non-Patent Document 1] Shlien and Malkin, 2009, Genome Med. 1: 62 [Non-Patent Document 2] Yang et al., 2013, Cell 153: 919 - 929 [Non-Patent Document 3] Tamborero et al., 2013, Comprehensive identification of mutational cancer driver genes across 12 tumor types, Scientific Reports 3: 2650 [Non-Patent Document 4] Youn et al., 2011, Identifying cancer driver genes in tumor genome sequencing studies, Bioinformatics 27(2): 175 - 181 [Non-Patent Document 5] Sakoparnig et al., 2015, Identification of constrained cancer driver genes based on mutation timing, PLoS Comput. Biol. 11(1): e1004027 [Non-Patent Document 6] Forbes et al., 2008, Current protocols in human genetics 10 - 11 [Non-Patent Document 7] Liao et al., 2008, Proc. Nat. Acad. Sci. USA 105: 6987 - 6992 [Non-Patent Document 8] Georgi et al., 2013, PLoS Genetics 9 (5): e1003484

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[0007] To maximize the effectiveness of personalized cancer immunotherapy and confer sustained tumor control on the majority of treated patients, the treatment method needs to circumvent, in some way, the ability of tumors to evade immune surveillance, for example, by silencing the expression of mutated targets, for example, by deleting genes. If the mutation is not expressed, for example, deleted from the genome, the immunotherapy cannot target the mutation, so if this problem is not addressed, the immunotherapy risks recurrence. The selection of suitable neoepitopes to enhance tumor control will benefit any personalized immunotherapy approach targeting neoepitopes, regardless of how it is carried out. Thus, there is a need in the art for a method of selecting neoepitopes resulting from disease - specific mutations in genes that confer enhanced tumor control. Means for Solving the Problems

[0008] The present invention provides a method for overcoming the deficiencies of the art by determining the suitability of neoepitopes resulting from disease - specific mutations in genes as disease - specific targets that will confer enhanced tumor control in the case of cancer, where the diseased tissue cannot easily evade immune surveillance. Once suitable neoepitopes are identified, such suitable epitopes can be used as disease - specific targets to induce a specific immune response in patients with the disease. For example, the disease can be cancer and potentially primary tumors, and tumor metastases expressing suitable neoepitopes can be targeted for more effective treatment.

[0009] The present invention relates to a method for determining the suitability of a neoepitope resulting from a disease-specific mutation in an allele (mutated allele) in a gene as a disease-specific target, the method comprising determining the copy number of the mutated allele encoding the neoepitope in an affected cell or population of affected cells. As used herein, for example, when the copy number of the mutated allele is 4, the copy number can also be referred to as the zygosity such that the mutated allele has a zygosity of 4. As used herein, an allele is a site in the genome having a specific nucleotide identity, which identity can be the same in both the maternal and paternal copies of the genome (homozygous genotype), or the identity can be different in the maternal and paternal copies of the genome (heterozygous genotype). The mutated allele has an identity different from that of the corresponding normal genome, for example, the genome from non-affected cells of the same individual, preferably non-affected cells of the same tissue type as the affected cells (matched genome), due to the disease-specific mutation. A neoepitope suitable as a disease-specific target (suitable neoepitope), as used herein, is a neoepitope whose expression is less likely to be downregulated or silenced (e.g., by deletion) by the affected tissue when targeted by the immune system, such that the likelihood of avoiding a response, preferably an immunological response, generated against the neoepitope by, for example, vaccination against the neoepitope or administration of immune cells capable of targeting (binding to) the neoepitope, is reduced in the affected tissue. In one embodiment, the copy number of the mutated allele can be the same as the copy number of the gene containing the mutated allele, and thus the present invention also relates to a method for determining the suitability of a neoepitope resulting from a disease-specific mutation in a gene as a disease-specific target, the method comprising determining the copy number of the gene having the disease-specific mutation in an affected cell or population of affected cells.

[0010] In one embodiment, the higher the copy number of a mutated allele or gene having a disease-specific mutation, the higher the suitability of the neoepitope as a disease-specific target. Thus, the high copy number of a mutated allele or gene having a disease-specific mutation indicates the suitability of the neoepitope as a disease-specific target. In one embodiment, if the copy number of a mutated allele or gene having a disease-specific mutation in diseased cells exceeds 2, this indicates the suitability of the neoepitope as a disease-specific target. In one embodiment, if the copy number of a gene having a disease-specific mutation exceeds 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or exceeds 100, this indicates the suitability of the neoepitope as a disease-specific target.

[0011] If not all copies of a gene having at least one copy of a mutated allele have a mutation, it is preferred that many copies of the gene have the mutated allele, i.e., it is preferred that a higher rather than a lower fraction of the copies of the gene have the mutated allele (higher rather than lower fractional zygosity). Thus, in certain embodiments, the mutated allele is found at a high fraction (fractional zygosity) in the copies of the gene having at least one copy of the mutated allele, where fractional zygosity is the ratio of the number of copies of the mutated allele (the zygosity state of the mutated allele) to the total number of copies of the nucleotide site to which the mutated allele maps, particularly relative to a reference genome or corresponding wild-type genome or matched genome, i.e., the wild-type genome from the same individual. The higher the fractional zygosity of the copies of the mutated allele, the higher the suitability as a disease-specific target for neoepitopes. Preferably, the fractional zygosity can exceed 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, and most preferably, the fractional zygosity is 1, i.e., all copies of the gene in the diseased cells have the mutated allele. When the fractional zygosity is 1, there is no wild-type copy of the gene such that the diseased cells cannot revert to the expression of the corresponding wild-type epitope. As used herein, a fraction of 1 is the case where the hypothesis that the genetic arrangement of the mutated allele / gene, e.g., copy number, zygosity state is the same, is not disproven by the data, i.e., is statistically consistent.

[0012] It is known that diseased tissues such as tumors can have heterogeneous genetic compositions and gene expressions. Therefore, not all diseased cells in a diseased tissue have the same copy number of genes and / or genes with at least one copy of an allele having a mutation (total copy number of genes), and / or copies of genes having a mutated allele, and this may be even more so in the case of disease-specific mutations themselves. Thus, for example, if it is found that the copy number of a mutated allele, and / or the zygosity, and / or the total number of copies of the nucleotide site to which the mutated allele is mapped is the same or similar in a high proportion of diseased cells rather than a low proportion of diseased cells in the diseased tissue, this is preferable (high rather than low clonal proportion). For example, the higher the proportion of diseased cells having the same or similar copy number of a mutated allele, and / or the zygosity, and / or the total number of copies of the nucleotide site to which the mutated allele is mapped, the higher the suitability of the neoepitope as a disease-specific target. For example, the clonal proportion can be at least 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or at least 0.9. In a preferred embodiment, all diseased cells in the diseased tissue have the same or similar copy number, i.e., the clonal proportion is 1, i.e., statistically consistent. As used herein, the same or similar copy number includes the same copy number, or a copy number within 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or less of the copy number, with or without error correction, of the copy number or absolute copy number.

[0013] Preferably, the clonal fraction of a mutation can be obtained by the fraction of diseased cells having the same or a similar genetic configuration of the mutation, and the genetic configuration of the mutation includes the total number of copies of the nucleotide site to which the mutation is mapped and the number of copies of the mutated allele. A feature is said to be fixed in a population of diseased cells if it is present in all diseased cells to an extent that cannot be statistically disproven by the available data. Preferably, a clonal fraction of 1 means that the genetic configuration of the mutation is fixed in the population of diseased cells. Preferably, if the mutation is fixed in the population of diseased cells and the copy number variation (CNV) affecting the site encoding the mutation is fixed in the population of diseased cells, the genetic configuration of the mutation is fixed in the population of diseased cells. Preferably, if the total number of copies of the nucleotide site to which the mutation is mapped is 2 and a mutation present in the balanced region of the diseased (tumor) genome is determined to be fixed in the population of diseased cells, the genetic configuration of the mutation is fixed.

[0014] Genes in which disease-specific mutations are found can potentially be present in any gene in the genome. A preferred type of gene in which mutations that result in suitable neoepitopes are found is a gene whose expression leads to the transformation of cells into a cancerous phenotype, or the lack of whose expression leads to cancerous cells that lose that cancerous phenotype, i.e., a gene whose expression contributes to tumor progression. Such genes are known as driver genes. Examples of driver genes for many types of tumors are well known. For example, a list of 291 high-confidence cancer driver genes that act on 3,205 tumors derived from 12 different cancer types is disclosed in Tamborero et al., 2013, Comprehensive identification of mutational cancer driver genes across 12 tumor types, Scientific Reports 3:2650. Additional driver genes have been identified using the methods disclosed in Youn et al., 2011, Identifying cancer driver genes in tumor genome sequencing studies, Bioinformatics 27(2):175-181, Sakoparnig et al., 2015, Identification of constrained cancer driver genes based on mutation timing, PLoS Comput. Biol. 11(1):e1004027, and Forbes et al., 2008, Current protocols in human genetics 10-11. Disease-specific mutations in driver genes may or may not contribute to the cancerous phenotype. Preferably, all copies of the driver gene found in the diseased cells have disease-specific mutations. Also preferably, all cells in the diseased tissue are diseased cells in which all copies of the driver gene have disease-specific mutations.

[0015] Another preferred type of gene is an essential gene. In certain embodiments, an essential gene is a gene that, when silenced or its expression is reduced (e.g., by deletion), results in at least growth arrest or reduced fitness of a cell, preferably an affected cell. Such genes are herein named essential genes. In one embodiment, an essential gene is a gene in which at least a 10% reduction in growth or fitness is seen in an affected cell in which the gene is silenced or its expression is reduced, compared to a cell in which the gene is not silenced and its expression is not reduced. In one embodiment, the reduction in growth or fitness is at least 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or at least 95%, and most preferably, silencing or reduced expression of the essential gene results in lethality of the affected cell. Preferably, all copies of the essential gene found in the affected cell have a disease-specific mutation.

[0016] Essential genes are well-known in the art. For example, a list of essential genes in humans (e.g., in human cell lines or inferred from other organisms) is disclosed in Liao et al., 2008, Proc. Nat. Acad. Sci. USA 105: 6987-6992 and Georgi et al., 2013, PLoS Genetics 9 (5):e1003484, along with corresponding orthologs in other eukaryotes such as mouse (Liao et al., 2007, Trends Genet. 23:378-381), fruit fly (Spradling et al., 1999, Genetics 153:135-177), C. elegans (Kamath et al., 2003, Nature 421:231-237), zebrafish (Amsterdam et al., 2004, Proc. Natl. Acad. Sci. USA 101:12792-12797), Arabidopsis thaliana (Tzafrir et al., 2004, Plant Physiol. 135:1206-1220), yeast (Kim et al., 2010, Nat. Biotechnol. 28:617-623), etc. A list of essential genes derived from human cancer cell lines is disclosed in Wang et al., 2015, Science 350:1096-1101, and the list of essential genes can be found in the database of essential genes, DEG5.0 (Zhang et al., 2009, Nucleic Acids Res. 37:D455-D458).

[0017] Furthermore, a list of essential genes whose deletion / silencing significantly reduces the fitness of a cohort of cell lines can be empirically generated from multiple healthy tissues and / or cancer cell lines, which can be derived from donors or patients. Deletion / silencing of genes can be performed experimentally using various molecular biology techniques such as CRISPR technology, RNA interference, etc., and cell survival or fitness is determined by the presence or absence of expression of the putative essential genes. The list of essential genes can also be determined experimentally from cells or cell lines, or the list of essential genes can be obtained by bioinformatics approaches. The cells or cell lines can be diseased cells or cell lines (tumor cells or cell lines) or non-diseased (healthy / normal) cells or cell lines and can be obtained from donors or patients with a disease. Preferably, the non-diseased cells or cell lines are derived from the same tissue type as the diseased cells, and more preferably, from the same patient. In embodiments where the disease is cancer, the cells or cell lines can be obtained from the primary tumor or, if present, any metastasis. Furthermore, the list of essential genes can be essentially the same as the minimal set of genes expressed in a wide variety of tissues in the body. For example, essential genes are genes expressed in a wide variety of different tissues and are expressed at an RPKM (minimum reads per kilobase of transcript per million mapped reads) threshold greater than 0, preferably greater than 0.1, 0.5, 1, 2, 3, 4, 5, 10, 20, 25. Such a list of essential genes can be obtained by analyzing RNA expression data (e.g., RNAseq) from a panel of cell samples obtained from at least 5, 6, 7, 8, 9, 10, 15, 20, 25 or more different tissues. Furthermore, if patient-derived tumor cell lines are available, genes whose entire copies contain mutations encoding neoepitopes can be deleted one by one, and the growth rate of each modified cell line can be measured.Such measurement / analysis can be carried out by high-throughput methods known in the art, which allow screening of at least one gene at a time, preferably many genes at a time, in order to evaluate its effect on the fitness of diseased or non-diseased cells. Such methods also allow detection of synthetically lethal or sub-lethal combinations of genes, as described below. Briefly, a library of cell lines, each lacking one gene, can be used to examine the deletion of one or more candidate genes so as to be able to determine the effect of the deletion of the gene in the cells.

[0018] The present invention further relates to a method for determining the suitability of a neoepitope resulting from a disease-specific mutation in a gene as a disease-specific target, the method comprising determining the copy number of the gene in an affected cell or population of affected cells, i.e., determining the copy number of a gene at least one copy of which has a disease-specific mutation. When a gene has a high copy number in affected cells such as tumor cells, for example, by focal amplification, the probability that the gene can be a driver gene is very high. Therefore, a high copy number of a gene indicates the suitability of the neoepitope as a disease-specific target, and the higher the copy number of the gene, the higher the suitability of the neoepitope as a disease-specific target. For example, the high copy number can be 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or more than 100 copies. The high copy number may be at least 50% greater than the copy number of the gene in the corresponding non-affected cells. The high copy number may be such that the copy number of a gene at least one copy of which has a disease-specific mutation is at least 2×, 3×, 5×, 10×, 15×, 20×, 25×, 30×, 40×, 50×, 60×, 70×, 80×, 90× or at least 100× greater than the copy number of the gene in the corresponding non-affected cells. Due to copy number variations that can also exist in the normal genome, the copy number of a gene in the normal genome is not necessarily 2. Furthermore, focal amplification is known to be observed more frequently in certain diseases such as glioblastoma where the epidermal growth factor receptor gene is frequently focally amplified, and thus this embodiment is well suited for use in these diseases.

[0019] Furthermore, it is preferable that the gene copy number is the same or similar in a high proportion of diseased cells rather than a low proportion of diseased cells, such that the higher the proportion of diseased cells having the same or similar copy number, the higher the suitability of the neoepitope as a disease-specific target. In a preferred embodiment, all diseased cells in the diseased tissue have the same or similar copy number of a gene with at least one copy having a disease-specific mutation, i.e., the clonal proportion is 1. As used herein, the same or similar copy number includes the same copy number, or a copy number within 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or less of the copy number, with or without error correction, of the copy number or absolute copy number.

[0020] Furthermore, a gene having a high copy number with at least one copy having a disease-specific mutation that results in a neoepitope is preferably a gene whose expression results in the transformation of cells into a cancerous phenotype, or whose lack of expression results in cancerous cells losing their cancerous phenotype, i.e., it can be a driver gene such as a driver gene known in the art, or an essential gene, for example, a gene whose silencing or reduced expression results in at least growth arrest or reduced fitness of diseased cells.

[0021] The present invention also relates to a method for determining the suitability of neoepitopes resulting from disease-specific mutations in genes as disease-specific targets, the method comprising determining, in an affected cell or population of affected cells, whether the gene having the disease-specific mutation is an essential gene. In one embodiment, an essential gene is a gene that, when silenced or its expression is reduced (e.g., by deletion of the gene), results in at least growth arrest or reduced fitness of the affected cells. In this embodiment, when the gene is an essential gene and all copies of the essential gene have the disease-specific mutation (1 zygosity), it indicates the suitability of the neoepitope as a preferred disease-specific target. In certain embodiments, an essential gene is expressed in a wide variety of different tissues and is expressed at an RPKM (reads per kilobase of transcript per million mapped reads) threshold greater than 0, preferably greater than 0.1, 0.5, 1, 2, 3, 4, 5, 10, 20, 25. Preferably, all copies of the essential gene contain the mutation. Further, the higher the proportion of affected cells containing a copy of the essential gene whose all copies have the disease-specific mutation, the higher the suitability of the neoepitope as a disease-specific target, so it is preferred that a high proportion of affected cells contain a copy of the essential gene whose all copies have the disease-specific mutation (high rather than low clonal proportion). In a more preferred embodiment, all affected cells in the affected tissue have an essential gene whose all copies have the disease-specific mutation, i.e., the clonal proportion is 1.

[0022] It is known that when certain specific genes are individually silenced or their expression is individually reduced, there may be only a very slight effect, if any, on the fitness or growth ability of diseased cells. However, it has been observed that when two such genes are both silenced or their respective expressions are reduced, it can lead to a much stronger growth impairment that is lethal. Such genetic combinations are referred to as synthetic lethal or synthetically sick / defective. For considerations regarding synthetic lethal and synthetically sick genes, and methods for identifying such genes, see Nijman, 2011, Synthetic lethality: General principles, utility and detection using genetic screens in human cells, FEBS Lett. 585: 1 - 6. Since both genes are required for cell survival, it is unlikely that the cell will silence or reduce the expression of both genes. Thus, a suitable combination of neoepitopes as disease - specific targets can result from disease - specific mutations in at least two genes that are synthetically lethal or synthetically sick together. Considering this, the present invention further relates to a method for determining the suitability of a combination of at least two neoepitopes resulting from disease - specific mutations in at least two genes as a combination of disease - specific targets, the method comprising the step of determining whether a combination of at least two genes each having a disease - specific mutation is a synthetic lethal or synthetically sick gene. If a combination of at least two genes results in a synthetic lethal or synthetically sick phenotype, this indicates that the resulting neoepitopes are a suitable combination of disease - specific targets. In a preferred embodiment, synthetically sick results in at least a more pronounced effect on cell growth / fitness than would be expected from the additive effect of individual gene deletions / expression reductions. The larger the number of neoepitopes, the greater the number of combinations that can be synthetically sick or lethal, so this approach is preferred when there are a large number of suitable neoepitopes.For example, 10 mutations correspond to 45 possible combinations, 100 mutations correspond to 4,950 combinations, and 1,000 mutations correspond to approximately 500,000 combinations. In certain embodiments, at least two genes each have a higher, rather than lower, zygosity rate, preferably a zygosity rate of 1, and / or each have a higher, rather than lower, clonal fraction, preferably a clonal fraction of 1, and both are in diseased cells and diseased cells of diseased tissue. As used herein, each neoepitope found in a suitable combination of neoepitopes is considered to be a neoepitope suitable for the purposes of the present invention.

[0023] As referred to herein, the copy number of a gene in either diseased or non-diseased cells can be a relative copy number, but is preferably an absolute copy number, and more preferably a ploidy, e.g., the ploidy of the genome of diseased cells, i.e., the absolute copy number normalized to the copy number of the genome. Even more preferably, the relative, absolute, and normalized copy numbers are error-corrected.

[0024] For example, when estimating the absolute copy number or its zygosity state of a mutated allele or gene, the estimate can be inaccurate, and it is desirable to correct the absolute copy number to account for sources of error. In embodiments where next-generation sequencing is used to obtain sequence information from genomes and exomes, sources of error can include: bias in the estimated purity of a sample of diseased tissue such as a tumor sample, bias in the estimated parameters required to derive purity and / or absolute copy number, probabilistic errors due to the finite coverage of the sequenced sample, limited detection ability due to low purity, low clonal fraction, and so on.

[0025] The absolute copy number is determined using balanced heterozygous segments containing heterozygous SNPs, such as those disclosed in the international PCT patent application titled "Tumor Modeling Based on Primary Balanced Heterozygous Segments" filed on the same date as this specification and incorporated herein by reference in its entirety. In certain embodiments, errors in the absolute copy number of the segment can propagate to other estimated parameters, such as the absolute copy number of the mutated allele, e.g., SNV encoding a neoepitope, the zygosity of the mutated allele, the clonal fraction, etc. Since such downstream estimated parameters have clinical significance for determining the suitability of neoepitopes as disease-specific targets for the patients described herein, it is desirable to correct the error in the absolute copy number to obtain the most accurate value of the absolute copy number. Furthermore, mutations encoding neoepitopes can be prioritized for their suitability for inclusion in vaccines administered to patients using the criteria described herein. Also, particularly when the gene containing the mutation is an essential gene, a zygosity of 1 is qualitatively superior to a zygosity less than 1, so it is beneficial to have the most accurate estimated values of the absolute copy number and any parameters derived therefrom.

[0026] In preferred embodiments, the absolute copy number of the mutated allele, e.g., SNV, and / or the zygosity of the SNV can be error-corrected. In certain embodiments, the absolute copy number of the SNV is first error-corrected, and then the zygosity of the SNV is corrected to reflect the error-corrected absolute copy number of the SNV. Once the absolute copy number of the SNV and / or the zygosity of the SNV has been error-corrected, the estimated value of the clonal fraction can also be corrected to reflect the error-corrected absolute copy number, including a determination as to whether the clonal fraction is statistically consistent with a value of 1.

[0027] The absolute copy number of the SNV is preferably obtained by the absolute copy number of the segment to which the SNV is mapped. The absolute copy number of all segments in a diseased, e.g., tumor genome, can be error-corrected, which includes the absolute copy number of the SNV. Once the absolute copy number of all segments in the genome has been error-corrected, an error-corrected ploidy can be calculated based on the error-corrected absolute copy number of the segments in the diseased, e.g., tumor genome.

[0028] In a specific embodiment, when the absolute copy number of the SNV is error-corrected such that the new absolute copy number is different from the original absolute copy number, this can be interpreted as a sign that the estimated absolute copy number of the SNV is unreliable.

[0029] An example of error-correction of the absolute copy number of a segment is parity error-correction, which includes correcting the odd absolute copy number of a segment to the nearest higher even absolute copy number or the nearest lower even absolute copy number when the segment is in a balanced region. A balanced region is a region of a diseased, e.g., tumor genome, where the maternal and paternal alleles within this region have undergone equal (balanced) amplification or neither the maternal nor the paternal allele has undergone any amplification at all.

[0030] The decision to error-correct the odd absolute copy number of a segment to the nearest higher even absolute copy number or the nearest lower even absolute copy number can depend on a comparison to disease read data and normal read data mapped to the segment, and to prediction boundaries that define the absolute copy number of the segment. When the normal read data is read data related to a sequenced normal sample, the disease read data is read data related to a sequenced diseased sample. In particular, when the disease is cancer, the tumor read data is read data related to a sequenced tumor sample.

[0031] For example, in a first type of parity error-correction, CN mutIf the absolute copy number in the diseased genome of the segment to which the mutation is mapped is CN mut The absolute copy number of the segment predicted to have a value of is r > ρ th Then it can be corrected to CN mut +1, and if r < ρ th Then it can be corrected to CN mut -1. r is the ratio of the diseased segment read data count to the normal (the ratio of the number of tumor read data, which is the diseased read data mapped to the segment, to the number of normal read data mapped to the segment), and ρ th Is the prediction decision boundary, and its value also depends on the purity of the diseased tissue sample, for example, the tumor sample.

[0032] The allele-specific copy number of a segment is the number of copies in the diseased genome of either the maternal or paternal allele of the segment. If the segment contains a heterozygous SNP (heterozygous segment), the heterozygous SNP can be used to determine the allele-specific copy number of the segment. Heterozygous segments can be assigned to preferred nodes, and the nodes can be defined as a unique combination of the absolute copy number of the heterozygous segment and the allele-specific copy number of the heterozygous segment. Even nodes are a subset of nodes where the absolute copy number of the segment is even. If a heterozygous segment contains more than one heterozygous SNP, as long as the allele frequencies of each heterozygous SNP are calculated in a consistent manner for either the allele with a greater or lesser number of copies in the diseased genome, a group of two or more heterozygous SNPs can be represented by a single member of the group, or the allele frequencies of all members of the group can be averaged, or the median can be adopted.

[0033] The first type of parity error correction for a heterozygous segment may involve finding the even node most likely to correspond to the heterozygous segment, based on, for example, a maximum likelihood framework that considers measured disease (e.g., tumor) and normal read data mapped to the segment.

[0034] In the second type of parity error correction, the upstream and downstream neighboring segments that do not require parity error correction are preferably identified within 10 Mb, 5 Mb, or 1 Mb of the segment containing the SNV. If the absolute copy numbers of both the upstream and downstream neighboring segments are the same, the absolute copy number of the segment containing the SNV is changed to the absolute copy number of the neighboring segment. Generally, the second type of parity error correction is preferred over the first type, except when it cannot be performed because suitable adjacent segments cannot be identified, in which case the first type of parity error correction can be provided.

[0035] Instead of or in addition to parity error correction, other forms of error correction for the absolute copy number of a segment can also be introduced. For example, a method that considers the absolute copy number of the segment closest to a gene containing a mutation in the diseased genome, preferably when the change in the absolute copy number is 3, 2 or less, preferably 1, and also preferably when most (50%, 60%, 70%, 80%, 90%, 100%) of the adjacent segments have an absolute copy number equivalent to the mode, the absolute copy number of the segment containing the SNV is changed to the mode of the absolute copy numbers of the adjacent segments.

[0036] Different error correction schemes for the absolute copy number can be combined. In a preferred embodiment, the first parity error correction is applied as the first layer of error correction, and additional error correction methods can be applied on top of it.

[0037] As used herein, a segment can be a predetermined genomic region, e.g., a predetermined one based on a reference genome. A segment can span, for example, a gene defined in a reference genome to which read data is aligned. A segment can be a fragment of a gene, an exon, a union of exons, or a union of exons related to a given gene. A segment can be another set of predetermined regions (with or without introns) in the reference genome, or another set of predetermined regions in the reference genome based on the normal genome. In a specific embodiment, a segment can be a region of the reference genome having a given copy number and / or a given allele-specific copy number in an affected, e.g., tumor genome, or a fragment of a gene having a given copy number and / or an allele-specific copy number in an affected, e.g., tumor genome. A segment can be defined to include or exclude introns.

[0038] Multiple copies of a segment in a given genome (e.g., a normal genome or a tumor genome) can be defined as the frequency at which the nucleotide sequence of the segment occurs in the genome, ignoring diversity due to SNPs and / or SNVs and / or other cancer-related changes, such as mutations, insertions, deletions, and / or other cancer-related genetic variants, etc. Preferably, different copies of a segment in a given genome have the same length or approximately the same length.

[0039] The number of copies of a segment in a genome can mean the number of physical copies of the segment in a cell containing the genome. The absolute copy number of a segment in a normal genome can be defined as the number of physical copies of a given segment in a healthy cell. The absolute copy number of a segment in an affected, e.g., tumor genome can be defined as the number of physical copies of a given segment in an affected, e.g., tumor cell. The number of copies of a segment in a genome can be referred to as the absolute copy number of the segment in the genome. The copy number can mean the absolute copy number.

[0040] In a specific embodiment, when only a portion of the segment is amplified or deleted in the genome, such a partial copy of the segment may or may not be counted as a copy of the segment. In a preferred embodiment, copies of segments less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or 5% of the segment length may be ignored.

[0041] The reference genome is used for mapping read data and providing a coordinate system for a normal genome and an affected, e.g., tumor genome, and the coordinate system can include providing a chromosome number, a nucleotide position on the chromosome, and the directionality of the read data, and the position on the chromosome is indicated by a line.

[0042] The reference genome can be based on the genome of one or more members of the same species as the subject providing the sample of the affected tissue, or can be based on the normal genome of the subject.

[0043] A sample of affected tissue, such as a tumor sample, can also include contamination from a normal genome, particularly the normal genome of the same patient from whom the sample was taken, and / or, in the case of intratumoral heterogeneity, more than one tumor genome. Purity, tumor sample purity, tumor purity, and sample purity are all to be interpreted as equivalent terms and preferably mean the proportion of tumor cells present in the tumor sample. Normal contamination preferably means the proportion of normal cells present in the tumor sample and can be obtained by one minus the purity.

[0044] Normalization against the ploidy of cells controls for the presence of copies of genes due to genomic duplication events. In certain embodiments, the absolute copy number can be normalized against the ploidy of the genome, which is the average of the absolute copy numbers of all segments in a given genome in a given cell weighted by the length of each segment. In certain embodiments, the absolute copy number can be normalized against the ploidy of the chromosome containing the mutant gene of interest (including the mutation), which is the average of the absolute copy numbers of all segments on a given chromosome in a given cell weighted by the length of each segment on the chromosome. In certain embodiments, the absolute copy number can be normalized against the ploidy of the adjacent region of the chromosome containing the mutant gene of interest, which is the average of each segment in a given region in a given cell weighted by the length of each segment in the region. The adjacent region can be within a predetermined distance of the gene having the disease-specific mutation, for example, within 100 megabases (Mb), 75 Mb, 50 Mb, 25 Mb, 10 Mb, 5 Mb, 4 Mb, 3 Mb, 2 Mb, or 1 Mb of the gene having the disease-specific mutation. The copy number of a segment can be routinely calculated by methods known in the art, both experimentally and computationally. For example, European Patent No. 2 198 292 (B1) and European Patent No. 2 002 016 (B1) disclose methods for determining the relative copy number and copy number frequency of nucleic acid sequences. Further, European Patent Application Publication No. 2 835 752 (A) and International Patent Application Publication Numbers WO 2014 / 014497 and WO 2014 / 138153 also disclose methods for determining copy number variation. See also Machado et al., 2013, Copy Number Variation of Fc Gamma Receptor Genes in HIV-Infected and HIV-Tuberculosis Co-Infected Individuals in Sub-Saharan Africa, PLoS, 8(11):e78165. Other methods include the use of FACS, FISH, or other fluorescence-based methods, spectral karyotyping (SKY), and digital PCR.The segment may be a gene.

[0045] A disease-specific mutation can preferably be any mutation that results in the expression of a neoepitope on the surface of the diseased cells. In particular, the mutation can be an indel or a gene fusion event, or a single nucleotide polymorphism (point mutation). Preferably, the disease-specific mutation is a non-synonymous mutation, preferably a non-synonymous mutation of a protein expressed in a tumor or cancer cell. Any method known in the art for determining a disease-specific mutation can be used, and in particular, using next-generation sequencing data to determine any changes between the genome / exome of the diseased cells compared to the genome / exome of the corresponding non-diseased wild-type cells is preferred. For example, Carter et al., 2012, Absolute quantification of somatic DNA alterations in human cancer, Nature Biotechnology 30:413-421; Cibulskis et al., 2013, Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples, Nature Biotechnology 31:213-219; and Li and Li, 2014, A general framework for analyzing tumor subclonality using SNP array and DNA sequencing data, Genome Biology 15:473-495 not only disclose methods for identifying disease-specific mutations, but also methods for determining the fractional subclonality and allelic fraction of gene copy number, allelic ratio and allelic state.Another way to determine the copy number, such as the absolute copy number, relates to the use of segments of the genome, each segment containing at least one heterozygous single nucleotide polymorphism (SNP), the segment being balanced (equal numbers of each version of the heterozygous SNP), sharing a common number of copies (the primary copy number), which is preferably the absolute copy number most frequently observed for all balanced segments of the genome, as disclosed in the international PCT patent application titled "Tumor Modeling Based on Primary Balanced Heterozygous Segments" filed on the same date as this specification, the entire disclosure of which is incorporated herein by reference. Further, in addition to determining the absolute copy number, the present application can also determine the zygosity, allelic fraction, and subclonality of a gene containing a mutated allele or at least one copy of a mutated allele. Further, the methodology therein also performs error correction for the absolute copy number, which improves the accuracy of the absolute copy number and zygosity as well as the parameters derived therefrom, such as subclonality, ploidy, etc.

[0046] Generally, the total number of copies of the nucleotide site to which a mutated allele maps can mean the absolute copy number of the mutation, which can mean the absolute copy number of the SNV, particularly when the mutation is an SNV. Generally, the absolute copy number of a mutation can preferably be obtained by the absolute copy number of the segment to which the mutation maps (the absolute copy number being in a diseased state, e.g., in a tumor genome).

[0047] Generally, the copy number of a mutated allele encoding a neoantigen can mean the absolute copy number of the mutated allele of the mutation, which can mean the absolute copy number of the alternative allele of the SNV (the zygosity of the SNV), particularly when the mutation is an SNV, the alternative allele of the SNV being the mutated allele.

[0048] Preferably, when the mutation is not an SNV, the absolute copy number of the mutated allele of the mutation can be estimated in a manner similar to the method applied to SNVs.

[0049] Generally, the copy number of a gene can mean the absolute copy number of a segment, and the segment can be a gene or can include a gene. The copy number of a gene can mean the absolute copy number of the gene.

[0050] Preferably, the disease can be any disease for which an immune response against diseased cells / tissues, such as virus-infected cells, is desired. Preferably, the disease is cancer.

[0051] The method of the present invention can further include another step of determining the utility / validity of a suitable neoepitope identified by the method of the present invention as a disease-specific target for use in a method for eliciting an immune response against a suitable neoepitope, such as the inclusion of a suitable neoepitope in a cancer vaccine. Thus, the another step can involve one or more of the following: determining the antigenicity and / or immunogenicity of a suitable neoepitope; assessing whether a suitable neoepitope is expressed on the surface of diseased cells; the ability of a peptide containing a suitable neoepitope presented as an MHC-presented epitope; determining the efficacy of expression of a suitable neoepitope from the encoding nucleic acid; determining whether a putative suitable neoepitope can stimulate T cells, such as T cells of a patient with the desired specificity, particularly when present in the context of its native sequence, for example, when flanked by an amino acid sequence that also flanks the neoepitope in a protein of natural origin and when expressed in antigen-presenting cells.

[0052] Once a neoepitope has been determined to be suitable / appropriate for use as a target, taking into account its antigenicity / immunogenicity, ability to be expressed, ability to be presented as an MHC-presented epitope, etc., the identified suitable neoepitopes can be ranked, i.e., prioritized, with respect to their potential to not be downregulated or deleted from diseased cells, such that the diseased tissue has a low likelihood of being able to avoid targeting of the neoepitope. For example, one prioritization could start with the "best" neoepitopes, which are neoepitopes encoded by essential genes, where all copies of the essential gene have a mutation encoding the neoepitope, followed by a pair of synthetic lethal or essential genes, where each copy of each gene has a mutation encoding the neoepitope, followed by neoepitopes encoded by known driver genes with a very high absolute copy number, where all copies of the gene have a mutation, followed by neoepitopes encoded by genes with a very high absolute copy number and a high ploidy state that are not known to be driver genes, followed by neoepitopes encoded by genes with a high copy number (ploidy state), etc.

[0053] In certain embodiments, neoepitopes encoded by essential genes with a ploidy of 1 are preferred over other neoepitopes not encoded by essential genes. In certain embodiments, between two essential genes encoding a neoepitope with a ploidy of 1, the neoepitope encoded by the gene with a higher absolute copy number is preferred. In certain embodiments, between two essential genes encoding a neoepitope, the neoepitope encoded by the gene whose deletion results in a lower fitness is preferred. In certain embodiments, between genes encoding neoepitopes with a ploidy of 1 for all genes, the neoepitope encoded by the gene with a higher absolute copy number is preferred. In certain embodiments, when the ploidy is less than 1, neoepitopes encoded by genes with a high ploidy state are preferred over those with a high ploidy rate, and when the ploidy states are the same or similar, genes with a higher absolute copy number are preferred over those with a high ploidy rate (a mutated allele of 10 copies / 20 total nucleotide sites is superior to 3 / 4 due to a higher ploidy state; 10 / 100 is superior to 10 / 20, possibly because the former can be a driver gene; 9 / 100 is superior to 10 / 20, possibly because the ploidy states are similar and the former can be a driver gene). In certain embodiments, neoepitopes encoded by driver genes whose disease-specific mutations cause the transformation of cells into a cancerous phenotype are preferred over those whose mutations have no role in the transformation of cells into a cancerous phenotype. Further, it is preferred that the neoepitope has a higher rather than a lower clonal fraction.

[0054] A further embodiment of the invention relates to the use of a method for determining the suitability of neoepitopes as disease-specific targets for the manufacture of medicaments, such as vaccines, for example, personalized cancer vaccines and the like. The vaccine can be derived from one or more suitable neoepitopes, or a combination of suitable neoepitopes identified by the method of the invention. In a preferred embodiment, the vaccine comprises a peptide or polypeptide comprising one or more suitable neoepitopes, or a combination of suitable neoepitopes identified by the method of the invention, or a nucleic acid encoding said peptide or polypeptide.

[0055] In particular, it is possible to provide a recombinant vaccine which, when administered to a patient, preferably results in a collection of MHC-presented epitopes, at least one of the collection being a suitable neoepitope, or at least two of the collection being a suitable combination of neoepitopes identified by the method of the invention, such as 2 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, preferably up to 60, up to 55, up to 50, up to 45, up to 40, up to 35 or up to 30 MHC-presented epitopes. Presentation of these epitopes by the patient's cells, in particular antigen-presenting cells, preferably results in T cells that target the patient's tumor, preferably the primary tumor and tumor metastases, which express the antigen from which the MHC-presented epitope is derived and present the same epitope on the surface of the tumor cells.

[0056] The method of the invention is also useful in the manufacture of recombinant immune cells that express an antigen receptor targeted to a suitable neoepitope, or to one neoepitope in a suitable combination of neoepitopes. Preferably, the immune cells are T cells and the antigen receptor is a T cell receptor.

[0057] The present invention also relates to a method for generating recombinant immune cells targeted to a suitable neoepitope, or to one epitope in a combination of suitable neoepitopes, comprising the step of transfecting immune cells with a recombinant antigen receptor targeted to a suitable neoepitope identified by the method of the present invention for determining the suitability of a neoepitope as a disease-specific target, or to one epitope in a combination of suitable epitopes, as well as to recombinant immune cells produced by such method.

[0058] The present invention also provides a method for targeting a cell population or tissue expressing one or more neoepitopes. For example, an antibody against one or more of the neoepitopes can be used to target cells or tissues expressing one or more neoepitopes identified by the methods described herein. In one embodiment, the present invention is a method for eliciting an immune response against a target cell population or target tissue expressing one or more neoepitopes in a mammal, comprising: (a) administering to the mammal one or more immune cells expressing one or more antigen receptors targeted to one or more neoepitopes; (b) administering a nucleic acid encoding one or more of the neoepitopes; or (c) administering a peptide or polypeptide comprising one or more of the neoepitopes, wherein the neoepitope is identified according to the method of the present invention for determining the suitability of a neoepitope as a disease-specific target. In one embodiment, the method for eliciting an immune response against a target cell population or target tissue expressing one or more neoepitopes in a mammal comprises: (i) determining the copy number of a mutated allele in a gene encoding a neoepitope in diseased cells or a diseased cell population (disease-specific mutation); and (ii) (a) administering immune cells expressing an antigen receptor targeted to a neoepitope resulting from the disease-specific mutation; (b) administering a nucleic acid encoding a neoepitope resulting from the disease-specific mutation; or (c) administering a peptide or polypeptide comprising a neoepitope resulting from the disease-specific mutation.

[0059] In one embodiment, a method for eliciting an immune response against a target cell population or target tissue expressing one or more neoepitopes in a mammal comprises: (i) determining the copy number of a gene in an affected cell or affected cell population, wherein at least one copy of the gene has a disease-specific mutation that gives rise to a neoepitope; and (ii) (a) administering immune cells expressing an antigen receptor targeted to a neoepitope resulting from the disease-specific mutation; (b) administering a nucleic acid encoding a neoepitope resulting from the disease-specific mutation; or (c) administering a peptide or polypeptide comprising a neoepitope resulting from the disease-specific mutation. In one embodiment, a method for eliciting an immune response against a target cell population or target tissue expressing one or more neoepitopes in a mammal comprises: (i) determining whether a gene having a disease-specific mutation that gives rise to a neoepitope is an essential gene in an affected cell or affected cell population; and (ii) (a) administering immune cells expressing an antigen receptor targeted to a neoepitope resulting from the disease-specific mutation; (b) administering a nucleic acid encoding a neoepitope resulting from the disease-specific mutation; or (c) administering a peptide or polypeptide comprising a neoepitope resulting from the disease-specific mutation. Preferably, all copies of the essential gene have the disease-specific mutation, i.e., the zygosity is 1.

[0060] In one embodiment, a method for eliciting an immune response against a target cell population or target tissue expressing one or more neoepitopes in a mammal comprises: (i) determining, in an affected cell or affected cell population, whether a combination of at least two genes, each having a disease-specific mutation that gives rise to a neoepitope, is a synthetic lethal or synthetic sick gene; and (ii) (a) administering one or more immune cells expressing one or more antigen receptors targeted to one or more neoepitopes resulting from the disease-specific mutations of the at least two genes; (b) administering a nucleic acid encoding one or more neoepitopes resulting from the disease-specific mutations of the at least two genes; or (c) administering a peptide or polypeptide comprising one or more neoepitopes resulting from the disease-specific mutations of the at least two genes. Preferably, all neoepitopes resulting from the disease-specific mutations of the at least two genes are targeted by the administered immune cells, encoded by the administered nucleic acid, or contained within the administered peptide or polypeptide.

[0061] Furthermore, a disease, disorder, or condition can elicit an immune response in a mammal having a disease, disorder, or condition associated with the expression of neoepitopes resulting from disease-specific mutations so as to be treated or prevented. Preferably, the disease, disorder, or condition is cancer.

[0062] Preferably, the immune cells are T cells, the antigen receptors are T cell receptors, and the immune response is a T cell-mediated immune response. More preferably, the immune response is an anti-tumor immune response, and the target cell population or target tissue expressing one or more suitable neoepitopes is tumor cells or tumor tissue.

[0063] Other features and advantages of the present invention will be apparent from the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

[0064] The present invention will be described in detail below, but it should be understood that since the methods, protocols, and reagents can vary, the present invention is not limited to the specific methods, protocols, and reagents described herein. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0065] The elements of the present invention are described below. These elements are described using specific embodiments, but it should be understood that additional embodiments can be created by combining them in any manner and in any number. The various preferred embodiments described should not be construed as limiting the present invention to only the explicitly described embodiments. This description is to be understood as supporting and encompassing embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Further, unless otherwise indicated by the context, any permutation and combination of all the described elements in this application should be considered to be disclosed by the description of this application. For example, in a preferred embodiment, the neoepitope has a high junction state rather than a high junction rate, and in a preferred embodiment, the neoepitope results from a mutation in an essential gene. In a preferred embodiment, a suitable neoepitope has a high junction rate and results from a mutation in an essential gene. In a more preferred embodiment, the junction rate is equal to 1.

[0066] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", edited by H. G. W. Leuenberger, B. Nagel, and H. Kolbl, (1995) Helvetica Chimica Acta, CH-4010, Basel, Switzerland.

[0067] Unless otherwise specified, conventional methods of biochemistry, cell biology, immunology, and recombinant DNA techniques described in the literature of this field (for example, see Molecular Cloning: A Laboratory Manual, 2nd edition, edited by J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989) are used in the practice of the present invention.

[0068] Unless the context requires otherwise, throughout this specification and the following claims, the word "comprise", and variations such as "comprises" and "comprising", imply the inclusion of the stated members, integers or steps or groups of members, integers or steps, but do not exclude any other member, integer or step or group of members, integers or steps, provided that in some embodiments such other member, integer or step or group of members, integers or steps may be excluded, that is, it is to be understood that the subject consists of the inclusion of the stated members, integers or steps or groups of members, integers or steps. The terms "a", "an", "the" and similar references used in connection with the description of the present invention (especially in connection with the claims) are to be construed as covering both the singular and the plural unless otherwise specified herein or clearly contradicted by the context. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value within the range. Unless otherwise specified herein, each individual value is incorporated herein as if it were individually recited herein.

[0069] All of the methods described in this specification can be performed in any suitable order, unless otherwise specified herein or clearly contradicted by the context. For example, determining whether a neoepitope is a suitable disease-specific target by determining the copy number of the coding gene can be determined before, after, or simultaneously with determining whether the gene is a driver gene or an essential gene, or before, after, or simultaneously with determining whether the neoepitope is expressed on the surface of a cell or induces a good immune response suitable for use in a vaccine.

[0070] Any and all examples provided herein, or the use of exemplary language (e.g., "such as"), are intended only to better illustrate the invention and do not present a limitation to the scope of the invention as claimed elsewhere. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0071] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), regardless of whether above or below, is hereby incorporated by reference in its entirety. Nothing in this specification should be construed as an admission that the invention has no right to antedate such disclosure based on prior invention.

[0072] The present invention contemplates a method of treating diseases including immunotherapy and radiotherapy in certain cancers by targeting a neoepitope (a "suitable neoepitope") having a feature of being expressed only inside or on the surface of diseased cells and being less likely to be silenced by the diseased cells so that the diseased cells are less likely to evade immune surveillance by the targeted neoepitope. The immunotherapy can be brought about by methods of active and / or passive immunotherapy. For example, in one embodiment, an antibody or other molecule conjugated to a toxic agent capable of specifically targeting the neoepitope and killing cells expressing the neoepitope is used according to the present invention to target and kill such cells.

[0073] The present invention particularly targets the identification of such suitable neoepitopes as disease-specific targets in immunotherapy. Once a suitable neoepitope is identified, it can be used in a vaccine to induce and / or activate appropriate effector cells such as T cells that recognize the identified suitable neoepitope, particularly when presented in the context of MHC, via an appropriate antigen receptor such as a T cell receptor or an artificial T cell receptor, thereby inducing an immune response against the neoepitope, which results in the death of diseased cells expressing the suitable neoepitope. Alternatively or additionally, immune cells that recognize the identified suitable neoepitope can be administered via an appropriate antigen receptor, which will also result in the death of cells expressing the suitable neoepitope.

[0074] The immunotherapy approaches according to the present invention include immunization with a peptide or polypeptide containing a suitable neoepitope, ii) a nucleic acid encoding a peptide or polypeptide containing a suitable neoepitope, iii) a recombinant cell encoding a peptide or polypeptide containing a suitable neoepitope, iv) a recombinant virus encoding a peptide or polypeptide containing a neoepitope, and v) antigen-presenting cells pulsed with a peptide or polypeptide containing a neoepitope or transfected with a nucleic acid encoding a peptide or polypeptide. Other immunotherapy approaches according to the present invention include vi) a T cell receptor that recognizes a neoepitope, and vii) transfer of effector cells (such as T cells) encoding a receptor that recognizes a neoepitope, particularly when presented in the context of MHC.

[0075] The term "disease-specific mutation" relates, in the context of the present invention, to somatic mutations that are present in the nucleic acid of diseased cells but not in the nucleic acid of the corresponding normal, non-diseased cells. Since the disease can be cancer, the terms "tumor-specific mutation" or "cancer-specific mutation" relate to somatic mutations that are present in the nucleic acid of a tumor or cancer cell but not in the nucleic acid of the corresponding normal, i.e., non-tumorigenic or non-cancerous cells. The terms "tumor-specific mutation" and "tumor mutation" as well as the terms "cancer-specific mutation" and "cancer mutation" are used interchangeably herein.

[0076] As used herein, a single nucleotide polymorphism (SNP) is a site in the normal genome where at least one of two alleles (maternal or paternal) has an identity different from that in the normal genome or, for example, different from a reference genome.

[0077] As used herein, a heterozygous single nucleotide polymorphism (heterozygous SNP) is defined as a site in the normal genome where the two alleles (maternal and paternal alleles) have different identities.

[0078] As used herein, the term "zygosity rate" refers to the ratio of the copy number of a gene having a disease-specific mutation, taking into account the total copy number of the gene, regardless of whether the gene has a mutation. For example, if there are a total of 20 copies of a gene and 10 of the copies have a disease-specific mutation, the zygosity rate is 0.5. If all copies of the gene have a disease-specific mutation, the zygosity rate is 1. The zygosity rate can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or at least 0.95. In the context of the present invention, a higher zygosity rate is preferred over a lower zygosity rate. In certain embodiments, the zygosity rate of a mutated allele encoding an epitope, preferably a neoepitope, is, for example, the ratio of the copy number of the mutated allele to the total number of copies of the nucleotide site to which the mutated allele maps in a reference genome.

[0079] As used herein, the term "clonal fraction" refers to the ratio of the number of diseased cells containing the same disease-specific mutation in the same gene, as well as its genetic characteristics such as copy number and zygosity rate, taking into account the total number of diseased cells, regardless of whether the diseased cells have the same mutation in the same gene. This term can also be applied to tumor tissue in that the clonal fraction is the ratio of diseased cells in a tumor tissue containing the same disease-specific mutation and genetic characteristics such as copy number in the same gene, taking into account the total number of cells in the tumor tissue. For example, in a sample obtained from a tumor in which only half of the total number of tumor cells have the same mutation in the same gene, the clonal fraction is 0.5. The clonal fraction can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or at least 0.95. In the context of the present invention, a higher clonal fraction is preferred over a lower clonal fraction. A clonal fraction of 1, where all diseased cells have the same disease-specific mutation in the same gene, is most preferred. "Clonal fraction", "fractional clonality" and "fractional subclonality" are used interchangeably herein.

[0080] The term "local amplification" refers to the amplification or copy number increase of a portion of the genome, for example, the amplification of one or more genes located together on the same chromosome, which results in a copy number greater than two, preferably greater than 5, 10, 15, 20, 25, 50, 75, 100, with respect to that portion of the genome when no deletion event has occurred for the same portion of the genome. Thus, for the purposes of the present invention, a gene that is locally amplified in diseased cells can be a gene that has an increased copy number compared to the wild-type copy number of two or, in the case of genes on the X and Y chromosomes in males, one wild-type copy number. Local amplification is distinct from whole-genome duplication and / or amplification events.

[0081] The term "immune response" refers to an integrated bodily response to an antigen, preferably a cellular immune response or a cellular and humoral immune response. The immune response can be protective / preventive and / or therapeutic.

[0082] "Induction of an immune response" can mean that there was no immune response to a specific antigen prior to induction, but it can also mean that there was a certain level of immune response to a specific antigen prior to induction and that the immune response was enhanced after induction. Thus, "inducing an immune response" also includes "enhancing an immune response". Preferably, after inducing an immune response in a subject, the subject is protected from developing a disease such as a cancer disease or the medical condition is remitted by inducing the immune response. For example, an immune response against a tumor-expressed antigen can be induced in a patient suffering from a cancer disease or a subject at risk of developing a cancer disease. In this case, inducing an immune response can mean that the medical condition of the subject is remitted, that the subject does not develop metastases, or that a subject at risk of developing a cancer disease does not develop a cancer disease.

[0083] The terms "cellular immune response", "cellular response", "cellular response to an antigen", or similar terms are meant to include a cellular response to cells characterized by the presentation of an antigen having class I or class II MHC. The cellular response relates to cells called T cells or T lymphocytes that act as either "helper" or "killer". Helper T cells (also called CD4 + T cells) play a central role by regulating the immune response, and killer cells (cytotoxic T cells, cytolytic T cells, also called CD8 + T cells or CTLs) kill diseased cells such as cancer cells and prevent the production of further diseased cells. Preferably, the anti-tumor CTL response is stimulated against tumor cells that express one or more tumor-expressed antigens and preferably present tumor-expressed antigens having class I MHC.

[0084] The "antigen" according to the present invention covers any substance that is the target of and / or induces an immune response, such as a specific reaction with an antibody or a T lymphocyte (T cell), preferably a peptide or a protein. Preferably, the antigen contains at least one epitope such as a T cell epitope. Preferably, this antigen is a molecule that optionally induces an immune reaction that is specific for the antigen (including cells expressing the antigen) after processing, preferably in the context of the invention. The antigen or its T cell epitope is presented by a cell, preferably an antigen-presenting cell, including diseased cells, particularly cancer cells, in the context of MHC molecules, resulting in an immune response against the antigen (including cells expressing the antigen).

[0085] Preferably, an antigen is, in the context of the present invention, a molecule which, optionally after processing, preferably induces an immune reaction specific for the antigen. According to the present invention, any suitable antigen which is a candidate for an immune reaction can be used, and the immune reaction can be both a humoral and a cellular immune reaction. In the context of the present invention, an antigen is preferably presented by a cell, preferably an antigen-presenting cell, in the context of MHC molecules, which results in an immune reaction against the antigen. The antigen is preferably a product corresponding to or derived from an antigen of natural origin. Such antigens of natural origin can include or be derived from allergens, viruses, bacteria, fungi, parasites and other infectious pathogens and pathogens, or the antigen can be a tumor antigen. According to the present invention, the antigen can correspond to a product of natural origin, for example a viral protein or a part thereof. In a preferred embodiment, the antigen is a surface polypeptide, i.e. a polypeptide which is naturally displayed on the surface of a cell, pathogen, bacterium, virus, fungus, parasite, allergen or tumor. The antigen can induce an immune response against a cell, pathogen, bacterium, virus, fungus, parasite, allergen or tumor.

[0086] The term "disease-related antigen" or "disease-specific antigen" is used in its broadest sense to refer to any antigen related to or specific for a disease. Such an antigen is a molecule containing epitopes that will stimulate the host's immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Thus, disease-related antigens can be used for therapeutic purposes. Disease-related antigens are preferably related to an infection by a microorganism, typically related to a microbial antigen, or to cancer, typically related to a tumor.

[0087] The term "pathogen" refers to a biological material that can cause disease in an organism, preferably a vertebrate organism. Pathogens include viruses, together with microorganisms such as bacteria, unicellular eukaryotes (protozoa), fungi, etc.

[0088] In the context of the present invention, the terms "tumor antigen" or "tumor-associated antigen" relate to proteins that are specifically expressed under normal conditions in a limited number of tissues and / or organs, or at specific developmental stages. For example, a tumor antigen can be specifically expressed under normal conditions in gastric tissue, preferably gastric mucosa, genital organs such as the testis, trophoblast tissue such as the placenta, or germ line cells, and is expressed or abnormally expressed in one or more tumors or cancer tissues. In this context, "a limited number" preferably means three or less, more preferably two or less. In the context of the present invention, tumor antigens include, for example, differentiation antigens, preferably cell type-specific differentiation antigens, i.e., proteins that are specifically expressed in a specific cell type at a specific differentiation stage under normal conditions, cancer / testis antigens, i.e., proteins that are specifically expressed in the testis and optionally in the placenta under normal conditions, and germ line-specific antigens. In the context of the present invention, a tumor antigen is preferably associated with the cell surface of cancer cells and is preferably not expressed or very rarely expressed in normal tissues. Preferably, a tumor antigen or an abnormal expression of a tumor antigen identifies cancer cells. In the context of the present invention, a tumor antigen expressed by cancer cells in a subject, for example, a patient suffering from a cancer disease, is preferably a self-protein in said subject. In a preferred embodiment, a tumor antigen, in the context of the present invention, is specifically expressed under normal conditions in a tissue or organ that is non-essential, i.e., a tissue or organ that does not cause the death of the subject if damaged by the immune system, or in a body organ or structure that is not accessible or only slightly accessible by the immune system. Preferably, the amino acid sequence of a tumor antigen is identical between the tumor antigen expressed in normal tissues and the tumor antigen expressed in cancer tissues.

[0089] According to the present invention, the terms "tumor antigen", "tumor-expressed antigen", "cancer antigen" and "antigen expressed in cancer" are equivalents and are used interchangeably herein.

[0090] The terms "epitope", "antigenic peptide", "antigenic epitope", "immunogenic peptide", and "MHC-binding peptide" are used interchangeably herein and refer to an antigenic determinant in a molecule such as an antigen, i.e., a part or fragment of an immunologically active compound that is recognized by the immune system, for example, particularly when presented in the context of an MHC molecule, recognized by T cells. An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein and preferably has a length of 5 to 100, preferably 5 to 50, more preferably 8 to 30, and most preferably 10 to 25 amino acids. For example, an epitope can preferably have a length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. According to the present invention, an epitope can be a "MHC-binding peptide" or "antigenic peptide" because it can bind to an MHC molecule such as an MHC molecule on the surface of a cell. The terms "major histocompatibility complex" and the abbreviation "MHC" relate to a complex of genes present in all vertebrates and include MHC class I and MHC class II molecules. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting cells or diseased cells in an immune response, and MHC proteins or molecules bind to peptides and present them for recognition by the T cell receptor. Proteins encoded by MHC are expressed on the surface of cells and display both self-antigens (peptide fragments derived from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms) to T cells. Such a preferred immunogenic moiety binds to an MHC class I or class II molecule. As used herein, an immunogenic moiety is said to "bind to" an MHC class I or class II molecule if binding can be detected using any assay known in the art. The term "MHC-binding peptide" relates to a peptide that binds to an MHC class I and / or MHC class II molecule. In the case of a class I MHC / peptide complex, the binding peptide typically has a length of 8 to 10 amino acids, although longer or shorter peptides may also be effective.In the case of a class II MHC / peptide complex, the binding peptide is typically 10 to 25 amino acids in length, particularly 13 to 18 amino acids in length, although longer and shorter peptides may also be effective.

[0091] As used herein, the term "neoepitope" refers to an epitope that is not present in a reference such as normal non-cancerous or germline cells, but is found in diseased cells such as cancer cells. This includes, in particular, situations where a corresponding epitope is found in normal non-cancerous cells or germline cells, but a neoepitope is generated by a change in the sequence of the epitope due to one or more mutations in the cancer cells. Furthermore, a neoepitope may be specific not only to the diseased cells, but also to a patient having the disease. Since the neoepitopes and suitable neoepitopes identified by the methods of the present invention are a subset of epitopes, the disclosures herein regarding epitopes as immunological targets generally apply equally to neoepitopes and suitable neoepitopes.

[0092] In a particularly preferred embodiment of the present invention, the epitope or neoepitope is a T cell epitope. As used herein, the term "T cell epitope" refers to a peptide that binds to an MHC molecule in a conformation recognized by a T cell receptor. Typically, a T cell epitope is presented on the surface of an antigen-presenting cell.

[0093] As used herein, the term "prediction of immunogenic amino acid modifications" refers to a prediction as to whether a peptide containing such amino acid modifications will be immunogenic in vaccination and thus useful as an epitope, particularly a T cell epitope.

[0094] According to the present invention, a T cell epitope can be present in a vaccine as part of a larger entity such as a vaccine sequence and / or polypeptide containing two or more T cell epitopes. The presented peptide or T cell epitope is produced after suitable processing.

[0095] The T cell epitope can be modified at one or more residues that are not essential for TCR recognition or binding to MHC. Such modified T cell epitopes can be considered immunologically equivalent.

[0096] Preferably, when the T cell epitope is presented by MHC and recognized by a T cell receptor, in the presence of an appropriate co-stimulatory signal, it can induce clonal expansion of T cells carrying a T cell receptor that specifically recognizes the peptide / MHC complex.

[0097] Preferably, the T cell epitope comprises an amino acid sequence that substantially corresponds to the amino acid sequence of a fragment of the antigen. Preferably, the said fragment of the antigen is an MHC class I and / or class II presenting peptide.

[0098] The T cell epitope according to the present invention preferably relates to a part or fragment of an antigen that can stimulate an immune response, preferably a cellular response, against an antigen, or a cell characterized by the expression of an antigen, particularly a disease cell, especially a cancer cell, etc., preferably by antigen presentation. Preferably, the T cell epitope can stimulate a cellular response against cells characterized by the presentation of an antigen having class I MHC, and preferably can stimulate antigen-responsive cytotoxic T lymphocytes (CTL).

[0099] In some embodiments, the antigen is a self-antigen, particularly a tumor antigen. Tumor antigens and their determinants are known to those skilled in the art.

[0100] The term "immunogenicity" preferably relates to the relative effectiveness for inducing an immune response associated with a therapeutic treatment, such as a treatment for cancer. As used herein, the term "immunogenic (ity)" relates to the property of having immunogenicity. For example, the term "immunogenic modification", when used in the context of a peptide, polypeptide or protein, relates to the effectiveness of the peptide, polypeptide or protein for inducing an immune response resulting from and / or directed against the modification. Preferably, an unmodified peptide, polypeptide or protein does not induce an immune response, induces a different immune response, or induces a different level, preferably a lower level, of immune response.

[0101] According to the present invention, the term "immunogenicity" or "immunogenic" preferably relates to the relative effectiveness for inducing a biologically relevant immune response, particularly an immune response useful for vaccination. Thus, in a preferred embodiment, an amino acid modification or a modified peptide is immunogenic if it induces an immune response against the target modification in a subject, and this immune response can be beneficial for therapeutic or prophylactic purposes.

[0102] "Antigen processing" or "processing" refers to the degradation of a polypeptide or antigen into processing products of fragments of the polypeptide or antigen (e.g., degradation of a polypeptide into peptides), and the association (e.g., by binding) of one or more of these fragments with MHC molecules for presentation to specific T cells by a cell, preferably an antigen-presenting cell.

[0103] An "antigen-presenting cell" (APC) is a cell that presents peptide fragments of a protein antigen associated with MHC molecules on its cell surface. Some APCs can activate antigen-specific T cells.

[0104] Professional antigen-presenting cells are very efficient at internalizing antigens either by phagocytosis or receptor-mediated endocytosis and then displaying fragments of the antigens bound to class II MHC molecules on their membranes. T cells recognize and interact with the antigen-class II MHC molecule complexes on the membrane of the antigen-presenting cells. Subsequently, additional co-stimulatory signals are generated by the antigen-presenting cells, leading to the activation of the T cells. The expression of co-stimulatory molecules is a defining feature of professional antigen-presenting cells.

[0105] The main types of professional antigen-presenting cells are dendritic cells, macrophages, B-cells, and certain activated epithelial cells, which have the broadest range of antigen presentation and are probably the most important antigen-presenting cells.

[0106] Dendritic cells (DCs) are a population of leukocytes that present antigens captured in peripheral tissues to T cells via both the MHC class II and I antigen presentation pathways. It is well known that dendritic cells are powerful inducers of immune responses and that the activation of these cells is an important step in the induction of anti-tumor immunity. Dendritic cells can be conveniently classified into "immature" and "mature" cells, which can be used as a simple way to distinguish between two well-characterized phenotypes. However, this nomenclature should not be interpreted as excluding all possible intermediate stages of differentiation. Immature dendritic cells are characterized as antigen-presenting cells with a high capacity for antigen uptake and processing, which correlates with the high expression of Fcγ receptors and mannose receptors. The mature phenotype is typically characterized by lower expression of these markers but high expression of cell surface molecules that govern T cell activation, such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11), and co-stimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB).

[0107] The maturation of dendritic cells refers to the state of dendritic cell activation in which such antigen-presenting dendritic cells bring about the primary stimulation of T cells, while presentation by immature dendritic cells leads to tolerance. The maturation of dendritic cells is mainly caused by biomolecules having microbial features detected by innate receptors (such as bacterial DNA, viral RNA, endotoxin, etc.), pro-inflammatory cytokines (TNF, IL-1, IFN), ligation of CD40 on the surface of dendritic cells by CD40L, and substances released from cells experiencing stress-induced cell death. Dendritic cells can be induced by culturing bone marrow cells in vitro with cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha.

[0108] Non-professional antigen-presenting cells do not constitutively express MHC class II proteins required for interaction with naive T cells. These are expressed only upon stimulation of non-professional antigen-presenting cells by specific cytokines such as IFNγ.

[0109] An "antigen-presenting cell" can be loaded with MHC class I-presented peptides by transducing a cell with a nucleic acid encoding a peptide or polypeptide containing the peptide to be presented, preferably RNA, for example, a nucleic acid encoding an antigen.

[0110] In some embodiments, a pharmaceutical composition or vaccine of the invention comprising a gene delivery vehicle targeting dendritic or other antigen-presenting cells can be administered to a patient to effect transfection occurring in vivo. For example, in vivo transfection of dendritic cells can generally be performed using any method known in the art, such as those described in WO 97 / 24447 or the gene gun technique described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.

[0111] The term "antigen-presenting cell" also includes target cells.

[0112] The term "target cell" shall mean a cell that is the target of an immune response such as a cellular immune response. Target cells include cells that present an antigen or antigen epitope, i.e., a peptide fragment derived from an antigen, and include all undesirable cells such as cancer cells. In a preferred embodiment, the target cell is a cell that expresses the antigen described herein and preferably presents the antigen using class I MHC.

[0113] The term "portion" refers to a fraction. With respect to a specific structure such as an amino acid sequence or a protein, the term, its "portion" may designate a continuous or discontinuous fraction of the structure. Preferably, a portion of an amino acid sequence comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, preferably at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, most preferably at least 90% of the amino acids of the amino acid sequence. Preferably, when the portion is a discontinuous fraction, the discontinuous fraction is composed of 2, 3, 4, 5, 6, 7, 8, or more parts of the structure, and each part is a continuous element of the structure. For example, a discontinuous fraction of an amino acid sequence may be composed of 2, 3, 4, 5, 6, 7, 8, or more, preferably 4 or fewer parts of the amino acid sequence, and each part preferably comprises at least 5 consecutive amino acids, at least 10 consecutive amino acids, preferably at least 20 consecutive amino acids, preferably at least 30 consecutive amino acids of the amino acid sequence.

[0114] The terms "portion" and "fragment" are used interchangeably herein and refer to contiguous elements. For example, a part of a structure such as an amino acid sequence or a protein refers to contiguous elements of the structure. A portion, part or fragment of a structure preferably includes one or more functional properties of the structure. For example, a portion, part or fragment of an epitope, peptide or protein is preferably immunologically equivalent to the epitope, peptide or protein from which it is derived. In the context of the present invention, a "part" of a structure such as an amino acid sequence preferably comprises at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99% of the whole structure or amino acid sequence and preferably consists of it.

[0115] In the context of the present invention, the term "immunoreactive cell" relates to a cell that exerts an effector function during an immune response. An "immunoreactive cell" can preferably bind to a cell characterized by the presentation of an antigen, or an antigen peptide derived from an antigen, and mediate an immune response. For example, such cells secrete cytokines and / or chemokines, secrete antibodies, recognize cancer cells and optionally eliminate such cells. For example, immunoreactive cells include T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present invention, an "immunoreactive cell" is a T cell, preferably CD4 + and / or CD8 + T cell.

[0116] Preferably, an "immunoreactive cell" recognizes an antigen or an antigen peptide derived from the antigen with a certain degree of specificity, particularly when presented on the surface of an antigen-presenting cell or a diseased cell such as a cancer cell in the context of MHC molecules. Preferably, the recognition enables a cell that recognizes the antigen or the antigen peptide derived from the antigen to become responsive or reactive. When the cell is a helper T cell (CD4 + T cell) that possesses a receptor for recognizing an antigen or an antigen peptide derived from the antigen in the context of MHC class II molecules, such responsiveness or reactivity may include the release of cytokines and / or the activation of CD8 + lymphocytes (CTLs) and / or B cells. When the cell is a CTL, such responsiveness or reactivity may include the elimination of cells presented in the context of MHC class I molecules, i.e., cells characterized by antigen presentation using class I MHC, for example, by apoptosis or perforin-mediated cytolysis. CTL responsiveness may include a sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of antigen-expressing target cells. Also, CTL responsiveness can be determined using an artificial reporter that accurately indicates CTL responsiveness. A CTL that recognizes an antigen or an antigen peptide derived from the antigen and is responsive or reactive is also referred to herein as an "antigen-responsive CTL". When the cell is a B cell, such responsiveness may include the release of immunoglobulins.

[0117] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include cytotoxic T cells (CTLs, CD8+ T cells) including helper T cells (CD4+ T cells).

[0118] T cells belong to a group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells, by the presence of a special receptor called the T cell receptor (TCR) on their cell surface. The thymus is the main organ responsible for the maturation of T cells. Several different subsets of T cells have been discovered, each with distinct functions.

[0119] T helper cells assist other white blood cells in immune processes, including, among other functions, the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 protein on their surface. Helper T cells are activated when peptide antigens are presented by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). After activation, they rapidly divide and secrete small proteins called cytokines that regulate or assist the active immune response.

[0120] Cytotoxic T cells destroy virus-infected cells and tumor cells and are also associated with transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which is present on the surface of almost all cells in the body.

[0121] In most T cells, the T cell receptor (TCR) exists as a complex of several proteins. The actual T cell receptor is composed of two separate peptide chains produced from the independent T cell receptor alpha and beta (TCRα and TCRβ) genes and is called the α- and β-TCR chains. Gamma delta T cells (γδ T cells) represent a small subset of T cells that possess a distinctly different T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR consists of one γ-chain and one δ-chain. This group of T cells is much rarer than αβ T cells (2% of all T cells).

[0122] According to the present invention, the term "antigen receptor" includes receptors of natural origin such as T cell receptors and engineered receptors that confer any specificity, such as the specificity of a monoclonal antibody, in immune effector cells such as T cells. In this way, a large number of antigen-specific T cells can be generated for adoptive cell transfer. Thus, an antigen receptor according to the present invention can be present in a T cell, for example, instead of or in addition to the T cell receptor of the T cell itself. Such T cells do not necessarily require antigen processing and presentation for the recognition of target cells, but rather, preferably by specificity, can recognize any antigen present on the target cells. Preferably, the antigen receptor is expressed on the surface of the cell. For the purposes of the present invention, T cells containing an antigen receptor are included by the term "T cell" as used herein. In particular, according to the present invention, the term "antigen receptor" includes an artificial receptor comprising a single molecule or a complex of molecules that can recognize, i.e., bind to (e.g., by binding of an antigen-binding site or antigen-binding domain to an antigen expressed on the surface of a target cell), a target structure (e.g., an antigen) in a target cell such as a cancer cell, and confer specificity to immune effector cells such as T cells expressing the antigen receptor on the cell surface. Preferably, the recognition of the target structure by the antigen receptor results in the activation of the immune effector cell expressing the antigen receptor. The antigen receptor can comprise one or more protein units, and the protein units can comprise one or more domains described herein. According to the present invention, "antigen receptor" may be a "chimeric antigen receptor (CAR)", a "chimeric T cell receptor", or an "artificial T cell receptor".

[0123] An antigen can be recognized by an antigen receptor via any antigen recognition domain (also simply referred to herein as "domain") that can form an antigen-binding site, such as via the antigen-binding portions of antibodies and T cell receptors, which can exist on the same or different peptide chains. In one embodiment, the two domains forming the antigen-binding site are derived from immunoglobulins. In one embodiment, the two domains forming the antigen-binding site are derived from T cell receptors. Single-chain variable fragments (scFv) derived from monoclonal antibodies, as well as antibody variable domains, particularly TCR alpha and beta single chains, etc., are particularly preferred. Virtually anything that binds to a given target with high affinity can be used as an antigen recognition domain.

[0124] The first signal in T cell activation is provided by the binding of the T cell receptor to a short peptide presented by the major histocompatibility antigen complex (MHC) in another cell. This ensures that only T cells with a TCR specific for the peptide are activated. The partner cell is usually a professional antigen-presenting cell (APC), usually a dendritic cell in the case of a naive response, although B cells and macrophages can also be important APCs. Peptides presented to CD8+ T cells by MHC class I molecules are typically 8 - 10 amino acids in length; peptides presented to CD4+ T cells by MHC class II molecules are typically longer because the ends of the binding cleft of MHC class II molecules are open.

[0125] According to the present invention, a molecule has significant affinity for a target in a standard assay and can bind to the pre-determined target when binding to the pre-determined target. "Affinity" or "binding affinity" is often measured by the equilibrium dissociation constant (K D ). A molecule that does not have significant affinity for a target in a standard assay and does not bind significantly to the target cannot (substantially) bind to the target.

[0126] Cytotoxic T lymphocytes can be produced in vivo by causing an antigen or antigen peptide to be taken up in vivo by antigen-presenting cells. The antigen or antigen peptide can be represented as a protein, as DNA (e.g., within a vector), or as RNA. While the antigen can be processed to yield the peptide partner of the MHC molecule, its fragments can be presented without further processing, particularly in the latter situation when they can bind to the MHC molecule. Generally, administration to a patient by intradermal injection is possible. However, intranodal injection into lymph nodes can also be carried out (Maloy et al., 2001, Proc Natl Acad Sci USA 98:3299-303). The resulting cells present the complex of interest and are recognized by autologous cytotoxic T lymphocytes, which then proliferate.

[0127] Specific activation of CD4+ or CD8+ T cells can be detected in various ways. Methods for detecting specific T cell activation include detecting the proliferation of T cells, the production of cytokines (e.g., lymphokines), or the development of cytolytic activity. For CD4+ T cells, a preferred method for detecting specific T cell activation is detection of T cell proliferation. For CD8+ T cells, a preferred method for detecting specific T cell activation is detection of the development of cytolytic activity.

[0128] The term "cells characterized by antigen presentation" or "cells presenting an antigen" or similar expressions means, in the context of MHC molecules, particularly MHC class I molecules, cells such as diseased cells, e.g., cancer cells, or antigen-presenting cells that present the antigen they express or fragments derived from said antigen, e.g., by processing of the antigen. Similarly, the term "disease characterized by antigen presentation" refers to a disease that includes cells characterized by antigen presentation, particularly using class I MHC. Antigen presentation by cells can be achieved by transfecting the cells with a nucleic acid such as RNA encoding the antigen.

[0129] The term "fragment of the antigen being presented" or similar expressions means, for example, a fragment that can be presented by MHC class I or class II, preferably MHC class I, when added directly to an antigen-presenting cell. In one embodiment, the fragment is a fragment that is naturally presented by a cell expressing the antigen.

[0130] The term "immunologically equivalent" means that immunologically equivalent molecules, such as immunologically equivalent amino acid sequences, exhibit the same or essentially the same immunological properties and / or exert the same or essentially the same immunological effects with respect to, for example, the type of immunological effect such as induction of humoral and / or cellular immune responses, the intensity and / or duration of the induced immune response, or the specificity of the induced immune response. In the context of the present invention, the term "immunologically equivalent" is preferably used with respect to the immunological effects or properties of the peptide used for immunization. For example, when an amino acid sequence induces an immune response having a specificity that reacts with a reference amino acid sequence when exposed to the immune system of a subject, the amino acid sequence is immunologically equivalent to the reference amino acid sequence.

[0131] The term "immune effector function" in the context of the present invention includes any function mediated by components of the immune system that results in, for example, inhibition of tumor growth and / or inhibition of tumorigenesis, including killing of tumor cells or inhibition of intravasation and metastasis of tumors. Preferably, the immune effector function in the context of the present invention is an effector function mediated by T cells. Such functions, in the case of helper T cells (CD4 + T cells), include recognition of an antigen or an antigen-derived antigen peptide by a T cell receptor in the context of MHC class II molecules, release of cytokines, and / or CD8 +Activation of lymphocytes (CTL) and / or B cells, in the case of CTL, recognition of an antigen or an antigen-derived antigen peptide by a T cell receptor in the context of an MHC class I molecule, elimination of cells presented in the context of an MHC class I molecule, i.e., cells characterized by antigen presentation using class I MHC, for example, by apoptosis or perforin-mediated cytolysis, production of cytokines such as IFN-γ and TNF-α, and specific cytolytic killing of antigen-expressing target cells.

[0132] The terms "major histocompatibility antigen complex" and the abbreviation "MHC" relate to a complex of genes that occur in all vertebrates and include MHC class I and MHC class II molecules. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting cells or diseased cells in an immune response, and MHC proteins or molecules bind to peptides and present them for recognition by a T cell receptor. Proteins encoded by MHC are expressed on the surface of cells and display both self-antigens (peptide fragments derived from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms) to T cells.

[0133] The MHC region is divided into three subgroups: class I, class II, and class III. MHC class I proteins contain an α chain and β2-microglobulin (not part of the MHC encoded by chromosome 15). These present antigen fragments to cytotoxic T cells. In most immune system cells, particularly antigen-presenting cells, MHC class II proteins contain α and β chains, which present antigen fragments to T helper cells. The MHC class III region encodes other immune components such as complement components, some of which encode cytokines.

[0134] MHC is both polygenic (there are several MHC class I and MHC class II genes) and polymorphic (there are multiple alleles of each gene).

[0135] As used herein, the term "haplotype" refers to the HLA alleles found on one chromosome and the proteins encoded thereby. A haplotype can also refer to an allele present at any one locus within the MHC. Each class of the MHC is represented by several loci: for example, class I includes HLA-A (human leukocyte antigen-A), HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-H, HLA-J, HLA-K, HLA-L, HLA-P and HLA-V, and class II includes HLA-DRA, HLA-DRB1-9, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1, HLA-DMA, HLA-DMB, HLA-DOA and HLA-DOB. The terms "HLA allele" and "MHC allele" are used interchangeably herein.

[0136] The MHC exhibits extreme polymorphism. Within the human population, at each locus, there are a large number of haplotypes containing distinct alleles. Different polymorphic MHC alleles of both class I and class II have different peptide specificities in that each allele encodes a protein that binds to peptides presenting a particular sequence pattern.

[0137] In the context of the present invention, the MHC molecule is preferably an HLA molecule.

[0138] In the context of the present invention, the term "MHC-binding peptide" includes MHC class I and / or class II-binding peptides, or peptides that can be processed to produce MHC class I and / or class II-binding peptides. In the case of class I MHC / peptide complexes, the binding peptides are typically 8 - 12, preferably 8 - 10 amino acids in length, although longer or shorter peptides may also be effective. In the case of class II MHC / peptide complexes, the binding peptides are typically 9 - 30, preferably 10 - 25, particularly 13 - 18 amino acids in length, although longer and shorter peptides may also be effective.

[0139] "Antigenic peptide" preferably relates to a part or fragment of an antigen that can stimulate an immune response, preferably a cellular response, against an antigen, or an affected cell, particularly a cancer cell, etc., characterized by the expression of the antigen, preferably the presentation of the antigen. Preferably, the antigenic peptide can stimulate a cellular response against cells characterized by the presentation of an antigen having class I MHC, and preferably can stimulate antigen-responsive cytotoxic T lymphocytes (CTL). Preferably, the antigenic peptide is an MHC class I and / or class II presentation peptide, or can be processed to produce an MHC class I and / or class II presentation peptide. Preferably, the antigenic peptide contains an amino acid sequence substantially corresponding to the amino acid sequence of a fragment of the antigen. Preferably, the said fragment of the antigen is an MHC class I and / or class II presentation peptide. Preferably, the antigenic peptide contains an amino acid sequence substantially corresponding to the amino acid sequence of such a fragment and is processed to produce such a fragment, i.e., an MHC class I and / or class II presentation peptide derived from the antigen.

[0140] When a peptide needs to be presented directly, i.e., without processing, particularly without cleavage, it has a length suitable for binding to MHC molecules, particularly class I MHC molecules, preferably a length of 7 to 20 amino acids, more preferably a length of 7 to 12 amino acids, more preferably a length of 8 to 11 amino acids, particularly a length of 9 or 10 amino acids.

[0141] When the peptide is part of an additional array, for example, of a vaccine array or a larger entity containing a polypeptide and needs to be presented after processing, in particular after cleavage, the peptide produced by processing has a length suitable for binding to MHC molecules, in particular class I MHC molecules, preferably a length of 7 to 20 amino acids, more preferably a length of 7 to 12 amino acids, more preferably a length of 8 to 11 amino acids, in particular a length of 9 or 10 amino acids. Preferably, the sequence of the peptide that needs to be presented after processing is derived from the amino acid sequence of the antigen, i.e., its sequence substantially corresponds to a fragment of the antigen and is preferably identical to this. Thus, the MHC-binding peptide contains a sequence that substantially corresponds to a fragment of the antigen and is preferably identical to this.

[0142] A peptide having an amino acid sequence that substantially corresponds to the sequence of a peptide presented by class I MHC may have one or more residues that are not essential for TCR recognition of the peptide presented by class I MHC or for peptide binding to MHC that differ. Such substantially corresponding peptides can also stimulate antigen-responsive CTLs and can be considered immunologically equivalent. A peptide having an amino acid sequence different from that of a peptide presenting a residue that does not affect TCR recognition but improves the stability of binding to MHC can improve the immunogenicity of the antigen peptide and can be referred to herein as an "optimized peptide". Using existing knowledge regarding which of these residues are likely to affect binding to either MHC or TCR, a rational approach can be used for the design of substantially corresponding peptides. The resulting functional peptide is considered as an antigen peptide.

[0143] When presented by MHC, an antigen peptide should be recognizable by a T cell receptor. Preferably, when recognized by a T cell receptor, the antigen peptide can induce clonal expansion of T cells carrying a T cell receptor that specifically recognizes the antigen peptide in the presence of appropriate co-stimulatory signals. Preferably, the antigen peptide can, particularly when presented in the context of an MHC molecule, stimulate an immune response, preferably a cellular response, against the cell from which it is derived or characterized by the expression of the antigen, preferably characterized by the presentation of the antigen. Preferably, the antigen peptide can stimulate a cellular response against cells characterized by the presentation of an antigen having class I MHC, preferably can stimulate antigen-responsive CTLs. Such cells are preferably target cells.

[0144] The term "genome" relates to the total amount of genetic information in the chromosomes of an organism or cell.

[0145] The term "exome" refers to a part of an organism's genome formed by exons, which are the coding portions of expressed genes. The exome provides the genetic blueprint used in the synthesis of proteins and other functional gene products. This is the most functionally relevant part of the genome and thus most likely to contribute to the phenotype of the organism. The exome of the human genome is estimated to constitute 1.5% of the entire genome (Ng et al., 2008, PLoS Gen., 4(8): pp. 1-15).

[0146] The term "transcriptome" relates to the set of all RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNAs, produced in a single cell or a population of cells. In the context of the present invention, the transcriptome or RNA sequence means the set of all RNA molecules produced in a single cell, a population of cells, preferably a population of cancer cells, or all cells of a given individual at a given time point.

[0147] "Nucleic acid" preferably refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), more preferably RNA, and most preferably in vitro transcribed RNA (IVT RNA) or synthetic RNA. Nucleic acids include genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. Nucleic acids can exist as single-stranded or double-stranded linear or covalently closed circular molecules. Nucleic acids can be isolated. The term "isolated nucleic acid" means that the nucleic acid has been (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified by separation, for example by cleavage and gel electrophoresis, or (iv) synthesized, for example by chemical synthesis. Nucleic acids can be used in the form of RNA, which can be prepared, in particular, by in vitro transcription from a DNA template, for introduction into cells, i.e., transfection. RNA can be further modified by sequence stabilization, capping, and polyadenylation before administration.

[0148] The term "genetic material" refers to an isolated nucleic acid of either DNA or RNA, a section of a double helix, a section of a chromosome, or the entire genome of an organism or cell, particularly its exome or transcriptome.

[0149] The term "mutation" refers to a change or difference (nucleotide substitution, addition or deletion) in the nucleic acid sequence in an affected genome compared to a reference, preferably a matched normal genome. "Somatic mutations" can occur in any of the body's cells other than germ cells (sperm and eggs) and thus are not passed on to offspring. These changes can (but not necessarily) cause cancer or other diseases. Preferably, the mutation is a non-synonymous mutation. The term "non-synonymous mutation" refers to a mutation, preferably a nucleotide substitution, that results in an amino acid change such as an amino acid substitution in the translation product, which preferably results in the formation of neoepitopes.

[0150] The term "single nucleotide variant / diversity (SNV)" refers to a difference in the nucleic acid sequence at a specific site (allele) when comparing the genome derived from diseased cells such as tumor cells and preferably the genome of matched (corresponding) normal, non-diseased cells or a reference genome. As used herein, the term mutation preferably encompasses SNVs.

[0151] Copy number variation (CNV) events in the diseased (tumor) genome are somatic copy number variation events that occur only in diseased cells and are defined as changes in the number of copies of the maternal and / or paternal alleles of a region of the diseased (tumor) genome relative to the matched normal genome, and this alteration preferably affects a genomic region spanning approximately 1 kb or more.

[0152] The term "mutation" includes point mutations, indels, fusions, chromothripsis, and RNA editing.

[0153] The term "indel" describes a special class of mutations defined as mutations that result in co-existing insertions and deletions and a net increase or decrease in nucleotides. In the coding regions of the genome, these result in frameshift mutations unless the length of the indel is a multiple of 3. Indels can be contrasted with point mutations. Whereas indels insert and delete nucleotides from a sequence, point mutations are a form of substitution that replaces one of the nucleotides.

[0154] Fusion can result in hybrid genes formed from two previously separate genes. This can occur as a result of translocation, interstitial deletion, or chromosomal inversion. Often, fusion genes are oncogenes. Oncogenic fusion genes can result in new or different gene products with functions from the two fusion partners. Alternatively, a proto-oncogene is fused to a strong promoter, and thus, the oncogenic function begins to function by upregulation caused by the strong promoter of the upstream fusion partner. Also, oncogenic fusion transcripts can be caused by trans-splicing or read-through events.

[0155] The term "chromothripsis" refers to a genetic phenomenon in which a specific region of the genome is shattered by a single disruptive event and then stitched back together.

[0156] The term "RNA editing" or "RNA edit" refers to a molecular process in which the information content in an RNA molecule is altered by chemical changes in the base composition. RNA editing includes nucleoside modifications such as cytidine (C) to uridine (U) and adenosine (A) to inosine (I), deamination, as well as non-template nucleotide addition and insertion. RNA editing of mRNA effectively changes the amino acid sequence of the encoded protein so that it differs from that predicted by the genomic DNA sequence.

[0157] The term "cancer mutation signature" refers to a set of mutations present in cancer cells when compared to non-cancerous reference cells.

[0158] In the context of the present invention, "reference" can be used to correlate and compare results obtained from tumor specimens. Typically, "reference" can be obtained based on one or more normal specimens, particularly specimens not affected by cancer diseases, obtained from either the patient or one or more different individuals, preferably healthy individuals, particularly individuals of the same species. "Reference" can be determined empirically by testing a sufficiently large number of normal specimens.

[0159] The term "reference genome" refers to a genome that provides a coordinate system for a normal genome and an affected genome. The reference genome is used for mapping read data and providing a coordinate system for a normal genome and a tumor genome, and the coordinate system enables the provision of chromosome numbers, nucleotide positions in the chromosome, and the directionality of read data. The reference genome can be based on the genome of one or more members from the same species as the subject providing the affected sample, or can be based on the normal genome (matched genome) of the subject.

[0160] Any suitable sequencing method can be used in the context of the present invention to identify disease-specific mutations, with next-generation sequencing (NGS) technology being preferred, optionally in combination with SNP arrays to obtain absolute copy number information. In order to increase the speed of the sequencing step of the method, in the future, third-generation sequencing methods may replace NGS technology. For the purpose of clarity, the term "next-generation sequencing" or "NGS" in the context of the present invention refers to all new high-throughput sequencing technologies, as opposed to the "conventional" sequencing method known as Sanger chemistry, which reads nucleic acid templates randomly in parallel along the entire genome by cutting the entire genome into small pieces. Such NGS technologies (also known as ultra-parallel sequencing technologies) can deliver nucleic acid sequence information of the entire genome, exome, transcriptome (all transcribed sequences of the genome), or methylome (all methylated sequences of the genome) in a very short period of time, for example, within 1 to 2 weeks, preferably within 1 to 7 days, or most preferably within less than 24 hours, and in principle enable single-cell sequencing techniques. A plurality of NGS platforms, which are commercially available or described in detail in the literature, for example, Zhang et al., 2011, The impact of next-generation sequencing on genomics. J. Genet Genomics 38(3): 95-109, or Voelkerding et al., 2009, Next generation sequencing: From basic research to diagnostics. Clinical chemistry 55: 641-658, can be used in the context of the present invention. Non-limiting examples of such NGS technologies / platforms are as follows. 1) For example, first, sequencing by a synthesis technique known as pyrosequencing implemented in the GS-FLX 454 Genome Sequencer (trademark) of Roche affiliate 454 Life Sciences (Branford, Connecticut), as described in Ronaghi et al., 1998, A sequencing method based on real-time pyrophosphate, Science 281: 363-365. This technique uses emulsion PCR, in which single-stranded DNA-binding beads are encapsulated by vigorously vortexing an aqueous micelle containing PCR reactants surrounded by oil for emulsion PCR amplification. During the pyrosequencing step, the light released from phosphate molecules during nucleotide incorporation as polymerase synthesizes the DNA strand is recorded. 2) Sequencing by a synthesis approach developed by Solexa (now part of Illumina Inc., San Diego, California), which is based on reversible dye-terminators and implemented, for example, in the Illumina / Solexa Genome Analyzer (trademark) and the Illumina HiSeq 2000 Genome Analyzer (trademark). In this technique, all four nucleotides are added simultaneously, along with DNA polymerase, to oligonucleotide-primed cluster fragments in a flow cell channel. The cluster strands having all four fluorescently labeled nucleotides are extended for sequencing by bridge amplification. 3) For example, sequencing by ligation implemented on the SOLid™ platform of Applied Biosystems (now Life Technologies Corporation, Carlsbad, California). In this technique, a pool of all possible oligonucleotides of a fixed length is labeled according to the sequenced position. The oligonucleotides are annealed and ligated, and the preferential ligation of the matching sequences by DNA ligase results in a signal that gives the information of the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR. The resulting beads, each containing only copies of the same DNA molecule, are placed on a slide glass. As a second example, the Polonator™ G.007 platform of Dover Systems (Salem, New Hampshire) also uses emulsion PCR based on randomly arrayed beads to amplify DNA fragments for parallel sequencing, thereby using sequencing by ligation. 4) For example, single molecule sequencing technologies implemented on the PacBio RS system of Pacific Biosciences (Menlo Park, California) or the HeliScope™ platform of Helicos Biosciences (Cambridge, Massachusetts). A distinct feature of this technology is its ability to sequence a single DNA or RNA molecule without amplification, defined as single molecule real-time (SMRT) DNA sequencing. For example, HeliScope uses a high-sensitivity fluorescence detection system to directly detect each nucleotide as it is being synthesized. Similar techniques based on fluorescence resonance energy transfer (FRET) have been developed by Visigen Biotechnology (Houston, Texas). Other fluorescence-based single molecule techniques are from U.S. Genomics (GeneEngine™) and Genovoxx (AnyGene™). 5) Nanotechnologies for single molecule sequencing that use various nanostructures placed on a chip, for example, to monitor the movement of polymerase molecules on a single strand during replication. Non-limiting examples of techniques based on nanotechnology are the GridON™ platform of Oxford Nanopore Technologies (Oxford, UK), the hybridization-assisted nanopore sequencing (HANS™) platform developed by Nabsys (Providence, Rhode Island), and a ligase-based DNA sequencing platform with a trademark using DNA nanoball (DNB) technology called combinatorial probe anchor ligation (cPAL™). 6) Electron microscopy-based techniques for single molecule sequencing, such as those developed by LightSpeed Genomics (Sunnyvale, California) and Halcyon Molecular (Redwood City, California). 7) Ion semiconductor sequencing based on the detection of hydrogen ions released during DNA polymerization. For example, Ion Torrent Systems (San Francisco, California) uses a high-density array of microscale measurement wells to perform this biochemical process in a massively parallel fashion. Each well has a different DNA template. There is an ion-sensitive layer under the well and an ion sensor with a trademark under that.

[0161] In one embodiment, as disclosed in the international PCT patent application titled "Highly Accurate Mutation Detection, In Particular for Personalized Therapeutics" filed on the same date as this specification and incorporated herein by reference in its entirety, whether a disease-specific mutation has occurred can be determined by a method related to the determination that a site in the normal genome is consistent with the homozygous genotype reflected by the normal allele and three noise alleles and the ideal noise distribution. When the corresponding site in the tumor genome is not consistent with the homozygous genotype and the ideal noise distribution, it is determined that a mutation has occurred. When the read data is mapped to each of the noise alleles with a probability of one-third of the error rate per base, the read data is consistent with the ideal noise distribution.

[0162] Preferably, the DNA and RNA preparations serve as starting materials for NGS. Such nucleic acids can be readily obtained from samples such as biological materials, for example, from fresh, flash-frozen or formalin-fixed paraffin-embedded tumor tissue (FFPE), or from freshly isolated cells, or from CTCs present in the peripheral blood of a patient. Normal non-mutated genomic DNA or RNA can be extracted from normal somatic tissues, but germline cells are preferred in the context of the present invention. Germline DNA or RNA is extracted from peripheral blood mononuclear cells (PBMCs) in patients suffering from non-hematological malignancies. Nucleic acids extracted from FFPE tissue or freshly isolated single cells are highly fragmented, but they are suitable for NGS applications.

[0163] Several targeted NGS methods for exome sequencing are described in the literature (for reviews, see, e.g., Teer and Mullikin, 2010, Human Mol Genet 19(2): pp. R145-51), and all of these can be used in conjunction with the present invention. Many of these methods (described, e.g., as genome capture, genome partitioning, genome enrichment, etc.) use hybridization techniques, including array-based (e.g., Hodges et al., 2007, Nat. Genet. 39: 1522-1527) and solution-based (e.g., Choi et al., 2009, Proc. Natl. Acad. Sci USA 106: 19096-19101) hybridization approaches. Commercially available kits for the preparation of DNA samples and subsequent capture of exomes are also available; for example, Illumina Inc. (San Diego, Calif.) offers the TruSeq™ DNA Sample Preparation Kit and the TruSeq™ Exome Enrichment Kit.

[0164] For example, when comparing the sequence of a tumor sample to the sequence of a reference sample such as the sequence of a germline sample, in order to reduce the number of false positive discoveries in the detection of cancer-specific somatic mutations or sequence differences, it is preferable to determine the sequence during replication of one or both of these sample types. Thus, it is preferable to determine the sequence of a reference sample such as the sequence of a germline sample two, three, or more times. Alternatively or in addition thereto, determine the sequence of the tumor sample two, three, or more times. Also, the sequence of a reference sample such as the sequence of a germline sample and / or the sequence of the tumor sample can also be determined multiple times by determining the sequence in genomic DNA at least once and determining the sequence in RNA of the reference sample and / or the tumor sample at least once. For example, by determining the mutations between replicates of a reference sample such as a germline sample, the false discovery rate (FDR) of expected somatic mutations can be estimated as a statistical quantity. Technical replicates of one sample should yield the same result, and all mutations detected during this "comparison to the identical" are false positives. In particular, technical replicates of the reference sample can be used as a reference for estimating the number of false positives in order to determine the false discovery rate of somatic mutation detection in the tumor sample relative to the reference sample. Furthermore, various quality-related metrics (e.g., coverage or SNP quality) can be combined into a single quality score using machine learning techniques. Optionally, for a given somatic mutation, all other mutations having a quality score above can be counted, thereby enabling the ranking of all mutations in the dataset.

[0165] In the context of the present invention, the term "RNA" relates to a molecule containing at least one ribonucleotide residue and preferably consisting entirely or substantially of ribonucleotide residues. "Ribonucleotide" relates to a nucleotide having a hydroxyl group at the 2'-position of the β-D-ribofuranosyl group. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially or completely purified RNA, essentially pure RNA, synthetic RNA, and recombinantly produced RNA such as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations can include, for example, the addition of non-nucleotide substances to the ends or within the RNA, such as at one or more nucleotides of the RNA. Also, the nucleotides in the RNA molecule can include non-standard nucleotides such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally occurring RNA.

[0166] The term "RNA" includes and preferably relates to "mRNA". The term "mRNA" means "messenger RNA" and relates to a "transcript" produced by using a DNA template and encoding a peptide or polypeptide. Typically, mRNA includes a 5'-UTR, a protein-coding region, and a 3'-UTR. mRNA has a limited half-life both in cells and in vitro. In the context of the present invention, mRNA can be produced by in vitro transcription from a DNA template. In vitro transcription methods are known to those skilled in the art. For example, various in vitro transcription kits are commercially available.

[0167] The stability and translation efficiency of the RNA can be modified as needed. For example, the RNA can be stabilized and its translation increased by one or more modifications having an RNA stabilizing effect and / or increasing the translation efficiency. Such modifications are described, for example, in PCT / EP2006 / 009448, which is incorporated herein by reference. To increase the expression of the RNA used in embodiments of the present invention, within the coding region, i.e., the sequence encoding the expressed peptide or protein, the GC content can be increased and the mRNA stability increased, and codon optimization performed, thus enhancing translation in the cell, preferably without altering the sequence of the expressed peptide or protein, by modifying.

[0168] The term "modification" in the context of the RNA used in the present invention includes any modification of the RNA that is not naturally present in said RNA.

[0169] In one embodiment of the present invention, the RNA used in accordance with the present invention does not have an uncapped 5'-triphosphate. Removal of such an uncapped 5'-triphosphate can be achieved by treating the RNA with a phosphatase.

[0170] The RNA according to the present invention may have modified ribonucleotides in order to increase its stability and / or decrease its cytotoxicity. For example, in one embodiment, within the RNA used in accordance with the present invention, cytidine is partially or completely, preferably completely, replaced with 5-methylcytidine. Alternatively or in addition, in one embodiment, within the RNA used in accordance with the present invention, uridine is partially or completely, preferably completely, replaced with pseudouridine.

[0171] In one embodiment, the term "capping" relates to providing a 5'-cap or a 5'-cap analog to RNA. The term "5'-cap" refers to the cap structure found on the 5' end of an mRNA molecule, and generally consists of a guanosine nucleotide linked to the mRNA via an unusual 5'-to-5' triphosphate bond. In one embodiment, this guanosine is methylated at the 7 position. The term "conventional 5'-cap" refers to a naturally occurring RNA 5'-cap, preferably a 7-methylguanosine cap (m 7 G). In the context of the present invention, the term "5'-cap" includes 5'-cap analogs that are similar to the RNA cap structure and are preferably modified to have the ability to stabilize RNA and / or enhance RNA translation when attached thereto in vivo and / or intracellularly.

[0172] Providing a 5'-cap or a 5'-cap analog to RNA may be achieved by in vitro transcription of a DNA template in the presence of said 5'-cap or 5'cap analog, wherein the 5'-cap is co-transcriptionally incorporated into the RNA strand being produced, or the RNA may be produced, for example, by in vitro transcription, and the 5'-cap may be attached to the RNA post-transcriptionally using a capping enzyme, such as the capping enzyme of vaccinia virus.

[0173] The RNA may contain further modifications. For example, further modifications of the RNA used in the present invention may include elongation or cleavage of a naturally occurring poly(A) tail, or introduction of a UTR not associated with the coding region of said RNA, for example, replacement or insertion of an existing 3'-UTR with one or more, preferably 2 copies of a 3'-UTR derived from a globin gene, such as alpha2-globin, alpha1-globin, beta-globin, preferably beta-globin, more preferably human beta-globin, etc., which may be a change in the 5'- or 3'-untranslated region (UTR).

[0174] RNA having an unmasked poly-A sequence is translated more efficiently than RNA having a masked poly-A sequence. The terms "poly(A) tail" or "poly-A sequence" typically refer to a sequence of adenyl (A) residues located on the 3'-end of an RNA molecule, and "unmasked poly-A sequence" means that the poly-A sequence at the 3'-end of the RNA molecule ends with an A of the poly-A sequence and is not followed by nucleotides other than A located 3' of, i.e., downstream of, the 3'-end of the poly-A sequence. Further, a poly-A sequence about 120 base pairs in length results in optimal transcriptional stability and translational efficiency of the RNA.

[0175] Thus, to increase the stability and / or expression of the RNA used according to the present invention, it can be modified to be present with a poly-A sequence preferably having a length of 10 to 500, more preferably 30 to 300, even more preferably 65 to 200, particularly 100 to 150 adenosine residues. In a particularly preferred embodiment, the poly-A sequence has a length of about 120 adenosine residues. To further increase the stability and / or expression of the RNA used according to the present invention, the poly-A sequence can be unmasked.

[0176] Furthermore, the incorporation of a 3'-untranslated region (UTR) into the 3'-untranslated region of an RNA molecule can result in enhanced translational efficiency. A synergistic effect can be achieved by incorporating two or more such 3'-untranslated regions. The 3'-untranslated regions can be self or heterologous with respect to the RNA into which they are introduced. In one particular embodiment, the 3'-untranslated region is derived from the human β-globin gene.

[0177] The combination of the above-described modifications, i.e., the incorporation of a poly-A sequence, the unmasking of the poly-A sequence, and the incorporation of one or more 3'-untranslated regions, has a synergistic effect on increasing the stability and translational efficiency of the RNA.

[0178] The term "stability" of RNA is related to the "half-life" of RNA. The "half-life" refers to the period required to eliminate half of the activity, amount, or number of molecules. In the context of the present invention, the half-life of RNA is an indicator of the stability of said RNA. The half-life of RNA can affect the "duration of expression" of RNA. An RNA having a long half-life can be predicted to be expressed for a long period of time.

[0179] Of course, when it is desirable to reduce the stability and / or translation efficiency of RNA, it is possible to modify the RNA so as to interfere with the functions of the above-described elements that increase the stability and / or translation efficiency of RNA.

[0180] The term "expression" is used in its most general sense and includes, for example, the production of RNA and / or peptides or polypeptides by transcription and / or translation. With respect to RNA, the term "expression" or "translation" particularly relates to the production of peptides or polypeptides. Further, this also includes partial expression of nucleic acids. Furthermore, expression can be transient or stable.

[0181] The term expression also includes "abnormal expression" or "dysregulated expression". "Abnormal expression" or "dysregulated expression" means that the expression is altered, preferably increased, compared to the state of a subject not suffering from a disease associated with abnormal expression or dysregulated expression of a reference, for example, a specific protein, such as a tumor antigen. An increase in expression means an increase of at least 10%, particularly at least 20%, at least 50% or at least 100%, or more. In one embodiment, the expression is found only in diseased tissue and the expression in healthy tissue is suppressed.

[0182] The term "specifically expressed" means that a protein is expressed essentially only in a specific tissue or organ. For example, a tumor antigen that is specifically expressed in the gastric mucosa means that the protein is mainly expressed in the gastric mucosa and is not expressed in other tissues or is not expressed to a significant extent in other tissue or organ types. Thus, a protein that is exclusively expressed in cells of the gastric mucosa and is expressed to a significantly lesser extent in any other tissue such as the testis is specifically expressed in cells of the gastric mucosa. In some embodiments, the tumor antigen may also be specifically expressed, under normal conditions, in a plurality of tissue types or organs, for example two or three tissue types or organs, but preferably in three or fewer different tissue or organ types. In this case, the tumor antigen is specifically expressed in these organs. For example, if a tumor antigen is preferably expressed to approximately the same extent in the lung and the stomach under normal conditions, the tumor antigen is specifically expressed in the lung and the stomach.

[0183] In the context of the present invention, the term "transcription" relates to the process by which the genetic code of a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into a protein. According to the present invention, the term "transcription" includes "in vitro transcription", which relates to the process by which RNA, particularly mRNA, is synthesized in vitro in a cell-free system, preferably using an appropriate cell extract. Preferably, a cloning vector is applied to the production of the transcript. These cloning vectors are generally named transcription vectors and are encompassed by the term "vector". The RNA used in the present invention is preferably in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter of any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into an appropriate vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0184] The term "translation" relates to the process in the ribosome of a cell in which a strand of messenger RNA directs the assembly of an amino acid sequence to produce a peptide or polypeptide.

[0185] An expression control sequence or regulatory sequence that can be functionally linked to a nucleic acid in the context of the present invention can be homologous or heterologous with respect to the nucleic acid. A coding sequence and a regulatory sequence are "functionally" linked together when they are covalently linked together such that the transcription or translation of the coding sequence is under the control or influence of the regulatory sequence. When using the functional linkage of a regulatory sequence and a coding sequence to translate the coding sequence into a functional protein, induction of the regulatory sequence results in transcription of the coding sequence without causing a shift in the reading frame of the coding sequence or preventing the coding sequence from being translated into the desired protein or peptide.

[0186] In the context of the present invention, the term "expression control sequence" or "regulatory sequence" includes promoters, ribosome binding sequences and other control elements that control the transcription of a nucleic acid or the translation of the induced RNA. In certain embodiments, the regulatory sequence can be controlled. The exact structure of the regulatory sequence can vary depending on the species or cell type, but generally includes 5'-untranscribed and 5'- and 3'-untranslated sequences involved in the initiation of transcription or translation such as TATA boxes, capping sequences, CAAT sequences, etc. In particular, the 5'-untranscribed regulatory sequence includes a promoter region containing the promoter sequence for transcriptional control of the functionally linked gene. Also, the regulatory sequence can include enhancer sequences or upstream activation sequences.

[0187] Preferably, the RNA to be expressed in the cell is introduced into the cell. In one embodiment of the method according to the present invention, the RNA introduced into the cell is obtained by in vitro transcription of an appropriate DNA template.

[0188] Terms such as "RNA capable of being expressed" and "encoding RNA" are used interchangeably herein. For a specific peptide or polypeptide, RNA means that it can be expressed to produce the peptide or polypeptide when present in a suitable environment, preferably inside a cell. Preferably, the RNA can interact with the translation machinery of the cell to provide the peptide or polypeptide that it can be expressed into.

[0189] Terms such as "transfer", "introduce" or "transfect" are used interchangeably herein and relate to introducing nucleic acids, particularly exogenous or heterologous nucleic acids, particularly RNA, into a cell. According to the present invention, a cell can form part of an organ, tissue and / or organism. According to the present invention, administration of the nucleic acid can be achieved either as naked nucleic acid or in combination with an administration reagent. Preferably, the administration of the nucleic acid is in the form of naked nucleic acid. Preferably, the RNA is administered in combination with a stabilizing substance such as an RNase inhibitor. The present invention also contemplates repeatedly introducing the nucleic acid into the cell to enable long-term sustained expression.

[0190] The cell can be transfected using any carrier that can associate with the RNA, for example by forming a complex with the RNA or forming vesicles that encapsulate or encapsulate the RNA, resulting in increased RNA stability compared to naked RNA. Useful carriers include, for example, cationic lipids, liposomes, particularly cationic liposomes, and lipid-containing carriers such as micelles, as well as nanoparticles. Cationic lipids can form complexes with negatively charged nucleic acids. Any cationic lipid can be used.

[0191] Preferably, introduction of RNA encoding a peptide or polypeptide into cells, particularly cells present in vivo, results in expression of the peptide or polypeptide in the cells. In certain embodiments, it is preferred to target the nucleic acid to specific cells. In such embodiments, the carrier (e.g., retrovirus or liposome) applied to administer the nucleic acid to the cells displays a targeting molecule. For example, a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be incorporated into or conjugated to the nucleic acid carrier. When the nucleic acid is administered by liposome, a protein that binds to a surface membrane protein associated with endocytosis can be incorporated into the liposome formulation to enable targeting and / or uptake. Such proteins include capsid proteins or fragments thereof specific for a particular cell type, an antibody against an internalized protein, a protein that targets an intracellular location, and the like.

[0192] The terms "cell" or "host cell" preferably refer to untreated cells, i.e., cells having an untreated membrane that has not released its normal intracellular components such as enzymes, organelles, or genetic material. The untreated cells are preferably living cells, i.e., living cells that can perform any normal metabolic function. Preferably, the terms relate to any cell that can be transformed or transfected with exogenous nucleic acid. The term "cell" includes prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., dendritic cells, B cells, CHO cells, COS cells, K562 cells, HEK293 cells, HELA cells, yeast cells, and insect cells). The exogenous nucleic acid can be found within the cell (i) freely dispersed by itself, (ii) incorporated within a recombinant vector, or (iii) incorporated within the host cell genome or mitochondrial DNA. Mammalian cells such as cells from humans, mice, hamsters, pigs, goats, and primates are particularly preferred. The cells can be derived from a number of tissue types and include primary cells and cell lines. Specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells. In further embodiments, the cells are antigen-presenting cells, particularly dendritic cells, monocytes, or macrophages.

[0193] Cells containing nucleic acids preferably express peptides or polypeptides encoded by the nucleic acids.

[0194] The term "clonal expansion" refers to the process by which a particular entity increases. In the context of the present invention, this term is preferably used in relation to an immunological response in which lymphocytes are stimulated by an antigen, proliferate, and the specific lymphocytes that recognize the antigen are amplified. Preferably, clonal expansion results in lymphocyte differentiation.

[0195] Terms such as "reduce" or "inhibit" preferably relate to the ability to cause a decrease in the overall level of at least 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more. The term "inhibit" or similar phrases includes complete or essentially complete inhibition, i.e., a decrease to zero or essentially zero.

[0196] Terms such as "increase", "enhance", "promote", or "extend" preferably relate to an increase, enhancement, promotion, or extension of at least about 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 80%, preferably at least 100%, preferably at least 200%, and particularly at least 300%. Also, these terms can relate to an increase, enhancement, promotion, or extension from zero or an unmeasurable or undetectable level to a level higher than zero or a measurable or detectable level.

[0197] According to the present invention, the term "peptide" refers to a substance composed of two or more, preferably three or more, preferably four or more, preferably six or more, preferably eight or more, preferably ten or more, preferably thirteen or more, preferably sixteen or more, preferably twenty-one or more, and preferably up to 8, 10, 20, 30, 40 or 50, and particularly up to 100 amino acids covalently linked by peptide bonds. The terms "polypeptide" or "protein" refer to large peptides, preferably peptides having more than 100 amino acid residues. Generally, the terms "peptide", "polypeptide" and "protein" are synonyms and are used interchangeably herein. According to the present invention, the terms "modification" or "sequence change" with respect to a peptide, polypeptide or protein relate to a sequence change in a peptide, polypeptide or protein as compared to a parental sequence, such as the sequence of a wild-type peptide, polypeptide or protein. This term includes amino acid insertion mutants, amino acid addition mutants, amino acid deletion mutants and amino acid substitution mutants, preferably amino acid substitution mutants. All of these sequence changes according to the present invention can potentially generate new epitopes.

[0198] An amino acid insertion mutant includes the insertion of one or two or more amino acids into a specific amino acid sequence.

[0199] An amino acid addition mutant includes the amino and / or carboxy-terminal fusion of one or more amino acids such as 1, 2, 3, 4 or 5, or more amino acids.

[0200] An amino acid deletion mutant is characterized by the removal of one or more amino acids from the sequence, for example, the removal of 1, 2, 3, 4 or 5 or more amino acids.

[0201] An amino acid substitution mutant is characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place.

[0202] According to the present invention, the modified or modified peptides used in the assays in the methods of the present invention can be derived from proteins containing the modification.

[0203] According to the present invention, the term "derived from" means that a particular entity, particularly a particular peptide sequence, is present in the object from which it is derived. In the case of an amino acid sequence, particularly a particular sequence region, "derived from" particularly means that the relevant amino acid sequence is derived from the amino acid sequence in which it is present.

[0204] Using the agents, compositions, and methods described herein, a subject having a disease, such as a disease characterized by the presence of diseased cells that express an antigen and present antigenic peptides, can be treated. Particularly preferred diseases are cancer diseases. The agents, compositions, and methods described herein can also be used for immunization or vaccination to prevent the diseases described herein.

[0205] One such agent is a vaccine, such as a cancer vaccine, designed based on a suitable neoepitope that resists immune evasion identified by the method of the present invention.

[0206] According to the present invention, the term "vaccine" relates to a pharmaceutical preparation (pharmaceutical composition) or product that induces an immune response, particularly a cellular immune response, that recognizes and attacks diseased cells such as pathogens or cancer cells upon administration. Vaccines can be used for the prevention or treatment of diseases. The term "personalized cancer vaccine" or "individualized cancer vaccine" relates to a particular cancer patient and means that the cancer vaccine is adapted to the needs or special circumstances of the individual cancer patient.

[0207] The cancer vaccine provided according to the present invention provides one or more T cell epitopes for the stimulation, priming and / or expansion of T cells specific to the patient's tumor when administered to the patient (patent). The T cells are preferably directed against cells expressing the antigen from which the T cell epitope is derived. Thus, the vaccines described herein can preferably induce or promote a cellular response, preferably cytotoxic T cell activity, against cancer diseases characterized by the presentation of one or more tumor-associated neoantigens by class I MHC. Since the vaccines provided herein target cancer-specific mutations, this is specific to the patient's tumor.

[0208] In the context of the present invention, a vaccine relates to a vaccine that, when administered to a patient, preferably provides one or more T cell epitopes (neoepitopes, suitable neoepitopes, combinations of suitable neoepitopes identified herein), such as two or more, five or more, ten or more, fifteen or more, twenty or more, twenty-five or more, thirty or more, preferably up to sixty, up to fifty-five, up to fifty, up to forty-five, up to forty, up to thirty-five or up to thirty T cell epitopes, which incorporate amino acid modifications or modified peptides predicted to be suitable epitopes. Presentation of these epitopes by the patient's cells, particularly antigen-presenting cells, preferably results in the targeting of the epitope by T cells when bound to MHC, and thus the patient's tumor, preferably the primary tumor and tumor metastases, expressing the antigen from which the T cell epitope presented by MHC is derived and presenting the same epitope on the surface of the tumor cells.

[0209] The method of the invention can further comprise another step of determining the usefulness of a modified peptide comprising an identified amino acid modification for cancer vaccination or a suitable neoepitope identified herein. Thus, the further step can comprise one or more of the following: (i) an evaluation of whether the modification is located within an epitope presented by a known or predicted MHC, (ii) in vitro and / or in silico testing of whether the modification is located within an epitope presented by an MHC, e.g., testing whether the modification is part of a peptide sequence that is processed and / or presented as an epitope presented by an MHC, and (iii) an in vitro test of whether an envisioned modified epitope can stimulate T cells, such as T cells of a patient having a desired specificity, particularly when present in its native sequence configuration, e.g., when adjacent to an amino acid sequence that also flanks said epitope in a naturally occurring protein and when expressed in an antigen-presenting cell. Such flanking sequences can each comprise 3 or more, 5 or more, 10 or more, 15 or more, 20 or more, preferably up to 50, up to 45, up to 40, up to 35 or up to 30 amino acids and can be adjacent to the epitope sequence at the N-terminus and / or C-terminus.

[0210] The modified peptides determined according to the invention can be ranked for their usefulness as epitopes for cancer vaccination. Thus, in one aspect, the method of the invention comprises an analytical process, manual or computer-based, in which the identified modified peptides are analyzed and selected for their usefulness in each vaccine provided. In a preferred embodiment, the analytical process is a computer algorithm-based process. Preferably, the analytical process comprises one or more of the following steps, preferably, the analytical method comprises the step of determining and / or ranking epitopes according to a prediction of their ability to be immunogenic.

[0211] Epitopes identified according to the present invention and provided in a vaccine are preferably present in the form of a polyepitope polypeptide or a nucleic acid encoding said polypeptide, in particular RNA, etc., a polypeptide comprising said epitope (neoepitope, suitable neoepitope, neoepitope found in a combination of suitable neoepitopes identified herein). Further, the epitope may be present in a polypeptide in the form of a vaccine sequence, i.e., in the context of its native sequence, for example, adjacent to the amino acid sequence that similarly flanks said epitope in a protein of natural origin. Such flanking sequences may each contain 5 or more, 10 or more, 15 or more, 20 or more amino acids, preferably up to 50, up to 45, up to 40, up to 35 or up to 30 amino acids, and may be adjacent to the epitope sequence at the N-terminus and / or C-terminus. Thus, the vaccine sequence may contain 20 or more, 25 or more, 30 or more, 35 or more, 40 or more amino acids, preferably up to 50, up to 45, up to 40, up to 35 or up to 30 amino acids. In one embodiment, the epitope and / or vaccine sequence are arranged head-to-tail in a polypeptide.

[0212] In one embodiment, the epitopes / neoepitopes and / or vaccine sequences identified herein are separated by linkers, particularly neutral linkers. As used in the context of the present invention, the term "linker" refers to a peptide added between two peptide domains, such as an epitope or a vaccine sequence, to connect said peptide domains. There are no particular restrictions regarding the linker sequence. However, the linker sequence preferably reduces steric hindrance between the two peptide domains, is well translated, and aids or permits processing of the epitope. Furthermore, the linker should have no or only few immunogenic sequence elements. The linker should preferably not create non-native epitopes, such as those resulting from the junction stitching between neighboring epitopes, which can give rise to unwanted immune reactions. Thus, the multi-epitope vaccine should preferably contain a linker sequence that can reduce the number of unwanted MHC-binding junction epitopes. Hoyt et al. (EMBO J. 25(8), pp. 1720-1729, 2006) and Zhang et al. (J. Biol. Chem., 279(10), pp. 8635-8641, 2004) have shown that glycine-rich sequences impair proteasomal processing, and thus the use of glycine-rich linker sequences serves to minimize the number of peptides contained in linkers that are processable by the proteasome. Furthermore, glycine has been observed to inhibit strong binding at MHC-binding groove positions (Abastado et al., 1993, J. Immunol. 151(7): pp. 3569-3575). Schlessinger et al., 2005, Proteins, 61(1): pp. 115-126 discovered that the amino acids glycine and serine included in the amino acid sequence result in a more flexible protein that is more efficiently translated and processed by the proteasome, allowing for better access to the encoded epitope. The linker may contain 3 or more, 6 or more, 9 or more, 10 or more, 15 or more, 20 or more amino acids, preferably up to 50, up to 45, up to 40, up to 35 or up to 30 amino acids. Preferably, the linker is rich in glycine and / or serine amino acids.Preferably, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the amino acids of the linker are glycine and / or serine. In a preferred embodiment, the linker is substantially composed of the amino acids glycine and serine. In one embodiment, the linker has the amino acid sequence (GGS). a (GSS) b (GGG) c (SSG) d (GSG) e and wherein a, b, c, d, and e are independently numbers selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and a + b + c + d + e is different from 0, preferably 2 or more, 3 or more, 4 or more, or 5 or more. In one embodiment, the linker comprises a sequence described herein, including the linker sequences described in the examples, such as the sequence GGSGGGGSG.

[0213] In a particularly preferred embodiment, a polypeptide incorporating one or more suitable neoepitopes identified by the methods herein, such as a polyepitope polypeptide, is expressed in a patient's cells, such as antigen-presenting cells, to produce the polypeptide, and is preferably administered to the patient in the form of a nucleic acid capable of producing the polypeptide, such as RNA that has been in vitro transcribed or synthetic RNA. Further, the present invention contemplates administering one or more multi-epitope polypeptides included by the term "polyepitope polypeptide" for the purposes of the present invention, preferably in the form of a nucleic acid capable of expressing the one or more polypeptides in a patient's cells, such as antigen-presenting cells, preferably RNA that has been transcribed in vitro or synthetic RNA. When administering multiple multi-epitope polypeptides, the suitable neoepitopes provided by different multi-epitope polypeptides may be different or partially overlapping. Once present in a patient's cells, such as antigen-presenting cells, the polypeptides according to the present invention are processed to produce suitable neoepitopes identified in accordance with the present invention. Administration of the vaccine provided according to the present invention can preferably provide epitopes presented by MHC class II that can induce a CD4+ helper T cell response against cells expressing the antigen from which the epitopes presented by MHC are derived. Alternatively or additionally, administration of the vaccine provided according to the present invention can provide neoepitopes presented by MHC class I that can induce a CD8+ T cell response against cells expressing the antigen from which the neoepitopes presented by MHC are derived. Further, administration of the vaccine provided according to the present invention can provide one or more neoepitopes (including known neoepitopes and suitable neoepitopes identified in accordance with the present invention), as well as one or more epitopes that do not contain cancer-specific somatic mutations but are expressed by cancer cells and preferably induce an immune response against cancer cells, preferably a cancer-specific immune response. In one embodiment, administration of the vaccine provided according to the present invention is capable of inducing a CD4+ helper T cell response against cells expressing the antigen from which the epitopes presented by MHC class II are derived and / or the epitopes presented by MHC, and does not contain cancer-specific somatic mutations, MH Provided are epitopes that can induce a CD8+ T cell response against cells expressing an antigen that is an epitope presented by class I MHC and / or derived from an epitope presented by MHC. In one embodiment, an epitope that does not contain a cancer-specific somatic mutation is derived from a tumor antigen. In one embodiment, a neoepitope and an epitope that do not contain a cancer-specific somatic mutation have a synergistic effect in the treatment of cancer. Preferably, the vaccine provided according to the present invention is useful for polyepitope stimulation of cytotoxic and / or helper T cell responses.

[0214] The vaccine provided according to the present invention can be a recombinant vaccine.

[0215] Another type of agent is an immune cell such as a T cell expressing a T cell receptor, or a T cell recombinantly expressing a T cell receptor, or expressing an artificial or chimeric T cell receptor (CAR), and this receptor is targeted to an antigen, for example, a suitable neoepitope identified by the method of the present invention as a suitable disease-specific target, and preferably, such a neoepitope is expressed on the surface of a cell in complex with an MHC molecule. Preferably, when an immune cell recognizes an antigen by receptor-antigen binding, the immune cell (immunoreactive cell) is stimulated, primed and / or increased, or exerts the effector function of the above-mentioned immunoreactive cell.

[0216] The term "antigen-specific T cell" or similar terms relate to a T cell that recognizes an antigen complexed within an MHC class I molecule, for example, a suitable neoepitope, and preferably exerts the effector function of the above-mentioned T cell after binding to the antigen. T cells and other lymphoid cells are considered antigen-specific when they kill target cells expressing the antigen. T cell specificity can be evaluated using any of various standard techniques within, for example, a chromium release assay or a proliferation assay. Alternatively, the synthesis of lymphokines (such as interferon-γ) can be measured.

[0217] T cell receptors and other antigen receptors are described above. The term "CAR" (or "chimeric antigen receptor") refers to an artificial receptor that recognizes, i.e., binds to, a target structure (e.g., an antigen) in a target cell such as a cancer cell, and can confer specificity to an immune effector cell such as a T cell expressing the CAR on the cell surface (e.g., by binding of an antigen-binding domain to an antigen expressed on the surface of the target cell). Preferably, recognition of the target structure by the CAR results in activation of the immune effector cell expressing the CAR. A CAR can comprise one or more protein units, and the protein units can comprise one or more domains described herein. The term "CAR" does not include a T cell receptor.

[0218] In one embodiment, a single-chain variable fragment (scFv) derived from a monoclonal antibody is fused to the CD3-zeta transmembrane and endodomain. Such a molecule results in zeta signal transduction in response to recognition of its antigen target in a target cell by the scFv and death of the target cell expressing the target antigen. Antigen recognition domains that can be used similarly include, among others, the T cell receptor (TCR) alpha and beta single chains. Virtually anything that binds to a given target with high affinity can be used as an antigen recognition domain.

[0219] After antigen recognition, the receptors cluster and signals are transmitted to the cell. In this regard, a "T cell signaling domain" is a domain that transmits an activation signal to a T cell after antigen binding, preferably an endodomain. The most commonly used endodomain component is CD3-zeta.

[0220] CAR-modified T cells can be engineered to target substantially any antigen expressed on diseased cells, such as tumor antigens, and thus adoptive cell transfer therapy with CAR-engineered T cells expressing chimeric antigen receptors is a promising mode of therapy. Preferably, the tumor antigen is a neoepitope resulting from a tumor-specific mutation identified by the method of the present invention as a suitable tumor-specific target. For example, a patient's T cells can be genetically engineered (genetically modified) to express a CAR specifically made against a tumor-specific neoepitope complexed with MHC molecules on the surface of the patient's tumor cells and then reinfused into the patient.

[0221] CAR can replace the function of the T cell receptor and, in particular, can confer reactivity such as cytolytic activity on cells such as T cells. However, in contrast to the binding of the T cell receptor to the antigen peptide-MHC complex described above, CAR can also bind to an antigen, particularly when expressed on the cell surface.

[0222] According to the present invention, a CAR can generally comprise three domains. The first domain is a binding domain that recognizes and binds an antigen. The second domain is a co-stimulatory domain. The co-stimulatory domain functions to enhance the proliferation and survival of cytotoxic lymphocytes after binding of the CAR to the targeting moiety. The identity of the co-stimulatory domain is limited only in that it has the ability to enhance cellular proliferation and survival after binding of the targeting moiety by the CAR. Suitable co-stimulatory domains include CD28, CD137 (4-1BB), members of the tumor necrosis factor (TNF) receptor family, CD134 (OX40), members of the TNFR superfamily of receptors, and CD278 (ICOS), CD28 superfamily co-stimulatory molecules expressed on activated T cells. The third domain is an activation signaling domain (or T cell signaling domain). The activation signaling domain functions to activate cytotoxic lymphocytes after binding of the CAR to the antigen. The identity of the activation signaling domain is limited only in that it has the ability to induce activation of selected cytotoxic lymphocytes after binding of the antigen by the CAR. Suitable activation signaling domains include the T cell CD3[zeta] chain and the Fc receptor[gamma].

[0223] The CAR can contain three domains together in the form of a fusion protein. Such a fusion protein will generally include a binding domain, one or more co-stimulatory domains, and an activation signaling domain, which are linked in the direction from the N-terminus to the C-terminus. However, the CAR is not limited to this arrangement, and other arrangements are allowed, which include a binding domain, an activation signaling domain, and one or more co-stimulatory domains. Since the binding domain must be able to freely bind to the antigen, it will be understood that the placement of the binding domain in the fusion protein will generally result in the display of the region outside the cell. In the same way, since the co-stimulatory and activation signaling domains function to induce the activity and proliferation of cytotoxic lymphocytes, the fusion protein will generally display these two domains inside the cell. The CAR can include additional elements such as a signal peptide to ensure proper export of the fusion protein to the cell surface, a transmembrane domain to ensure that the fusion protein is maintained as an integral membrane protein, and a hinge domain (or spacer region) that confers mobility to the binding domain and allows strong binding to the antigen.

[0224] Cells used in connection with CARs and other artificial antigen receptors are preferably T cells, particularly cytotoxic lymphocytes, and are preferably selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. After activation, each of these cytotoxic lymphocytes induces the destruction of target cells. For example, cytotoxic T cells induce the destruction of target cells by either or both of the following means. First, after activation, T cells release cytotoxins such as perforin, granzyme, and granulysin. Perforin and granulysin create pores in the target cells, and granzyme enters the cells and induces a caspase cascade in the cytoplasm, which induces apoptosis (programmed cell death) of the cells. Second, apoptosis can be induced via the Fas-Fas ligand interaction between the T cells and the target cells. The cytotoxic lymphocytes are preferably autologous cells, but heterologous or allogeneic cells may also be used.

[0225] The binding domain for an antigen that may be present within a CAR has the ability to bind (target) the antigen, i.e., the ability to bind (target) an epitope present on the antigen, and preferably the ability to bind (target) a neoepitope identified by the methods of the present invention as a suitable disease-specific target, where the neoepitope is presented in the context of MHC on the surface of the cell. Preferably, the binding domain for the antigen is specific for the antigen.

[0226] Another type of agent is immune cells such as lymphoid cells loaded with peptides containing suitable neoepitopes identified by the method of the present invention. In a preferred embodiment, the lymphoid cells are dendritic cells. In the context of the present invention, lymphoid cells, preferably isolated from the patient to be treated, are incubated with the antigen to be targeted, and then the incubated cells are administered to the patient, inducing an immune response against the cells expressing the antigen. Thus, peptides containing suitable epitopes can be incubated with dendritic cells, and the incubated cells can be administered to induce an immune response against cells expressing the suitable neoepitopes.

[0227] The term "recombinant", in the context of the present invention, means "produced by genetic manipulation". Preferably, a "recombinant entity" such as a recombinant polypeptide in the context of the present invention does not occur naturally and is preferably the result of a combination of entities such as amino acid or nucleic acid sequences that are not naturally combined. For example, a recombinant polypeptide may contain several amino acid sequences in the context of the present invention, such as a vaccine sequence derived from a neoepitope, or a fusion of different proteins or different parts of the same protein together by, for example, a peptide bond or a suitable linker.

[0228] As used herein, the term "naturally occurring" means that an object can be found in nature. For example, a peptide or nucleic acid that exists in a living organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by a human in a laboratory is naturally occurring.

[0229] According to the present invention, the term "disease" refers to any pathological condition, including cancer diseases, particularly the forms of cancer diseases described herein.

[0230] The term "normal" refers to a healthy state or a healthy subject or tissue, i.e., a state in a non-diseased state, and "healthy" preferably means non-cancerous.

[0231] "Disease containing cells expressing an antigen" means that the expression of the antigen is detected in the cells of the affected tissue or organ. The expression in the cells of the affected tissue or organ may be increased compared to the state of healthy tissue or organ. The increase means at least 10%, particularly at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more increase. In one embodiment, the expression is found only in the affected tissue and the expression in healthy tissue is suppressed. According to the present invention, diseases containing or related to cells expressing an antigen include cancer diseases.

[0232] Cancer (medical term is malignant neoplasm) is a class of diseases in which a group of cells shows uncontrolled growth (division beyond normal limits), invasion (invasion and destruction of adjacent tissues), and in some cases metastasis (spread to other locations in the body via lymph or blood). Due to these three malignant characteristics of cancer, they are distinguished from benign tumors that are self-limiting and do not infiltrate or metastasize. Most cancers form tumors, but some, such as leukemia, do not.

[0233] Malignant tumor is essentially synonymous with cancer. Malignant disease, malignant neoplasm, and malignant tumor are essentially synonymous with cancer.

[0234] According to the present invention, the terms "tumor" or "tumor disease" preferably refer to abnormal growth of cells (referred to as new cells, tumor-forming cells or tumor cells) that form a swelling or lesion. "Tumor cells" mean abnormal cells that grow by rapid uncontrolled cell proliferation and continue to grow after the stimulus that initiated the new growth has ended. Tumors show partial or complete lack of structural organization and functional coordination with normal tissues and usually form distinct tissue masses that can be either benign, pre-malignant or malignant.

[0235] A benign tumor is a tumor that lacks all three of the malignant characteristics of cancer. Thus, by definition, a benign tumor does not grow in an unlimited invasive pattern, does not invade surrounding tissues, and does not spread (metastasize) to non-adjacent tissues.

[0236] A neoplasm is an abnormal mass of tissue as a result of new formation. Neoplasia (from the Greek for new growth) is the abnormal proliferation of cells. The growth of the cells exceeds that of the normal tissue around it and is uncoordinated. The growth persists in the same excessive manner even after the stimulus has ended. This usually causes a mass or tumor. A neoplasm can be benign, pre-malignant or malignant.

[0237] "Tumor growth" or "tumor growth" in the context of the present invention relates to the tendency for the size of the tumor to increase and / or the tendency for tumor cells to proliferate.

[0238] For the purposes of the present invention, the terms "cancer" and "cancer disease" are used interchangeably with the terms "tumor" and "tumor disease".

[0239] Cancers are classified by the type of cells that resemble tumors and thus the type of tissue from which the tumor is presumed to originate. These are histological and positional respectively.

[0240] The term "cancer" according to the present invention includes leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer and lung cancer and their metastases. Examples thereof are lung tumors, breast tumors, prostate tumors, colon tumors, renal cell tumors, cervical tumors, or metastases of the above cancer types or tumors. Also, according to the present invention, the term cancer includes cancer metastases and cancer relapses.

[0241] "Metastasis" means the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a very complex process and depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter body cavities and blood vessels, and then infiltration of the target organ after being transported by the blood. Finally, the growth of a new tumor, i.e., a secondary or metastatic tumor, at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor because tumor cells or components may remain and develop the ability to metastasize. In one embodiment, the term "metastasis" according to the present invention relates to "distant metastasis" relating to metastases away from the primary tumor and the associated lymph node system.

[0242] The cells of a secondary or metastatic tumor resemble those of the original tumor. This means that, for example, when ovarian cancer metastasizes to the liver, the secondary tumor is composed of abnormal ovarian cells rather than abnormal liver cells. In this case, the tumor in the liver is called metastatic ovarian cancer rather than liver cancer.

[0243] The term "circulating tumor cell" or "CTC" refers to cells that detach from a primary tumor or tumor metastasis and circulate in the bloodstream. CTCs can constitute the seeds for the growth of further tumors (metastases) in various subsequent tissues. Circulating tumor cells are found at a frequency of approximately 1 to 10 CTCs per milliliter of whole blood in patients suffering from metastatic disease. Research methods for isolating CTCs have been developed. Several research methods for isolating CTCs in the art are described, for example, techniques that utilize the fact that epithelial cells generally express the cell adhesion protein EpCAM, which is not present in normal blood cells. Immunomagnetic bead-based capture involves treating a blood sample with an antibody against EpCAM conjugated to magnetic particles, followed by separating the tagged cells in a magnetic field. Subsequently, rare CTCs are discriminated from contaminating white blood cells by staining the isolated cells with antibodies against another epithelial marker, cytokeratin, and the common white blood cell marker CD45. This robust and semi-automated approach identifies CTCs with an average yield of approximately 1 CTC / mL and a purity of 0.1% (Allard et al., 2004, Clin Cancer Res 10: 6897-6904). A second method for isolating CTCs involves using a microfluidics-based CTC capture device that includes flowing whole blood through a chamber embedded with 80,000 micro-posts functionalized by coating with an antibody against EpCAM. Subsequently, the CTCs are stained with a secondary antibody against either cytokeratin or a tissue-specific marker such as PSA in prostate cancer or HER2 in breast cancer, and visualized by automatically scanning the micro-posts along three-dimensional coordinates in multiple planes. The CTC-chip can identify cytokerating-positive circulating tumor cells in patients with a median yield of 50 cells / ml and a purity range of 1-80% (Nagrath et al., 2007, Nature 450: 1235-1239). Another possibility for isolating CTCs is to use the CellSearch™ Circulating Tumor Cell (CTC) Test from Veridex, LLC (Raritan, NJ), which captures, identifies, and counts CTCs in a blood tube.The CellSearch (trademark) system is a method approved by the US Food and Drug Administration (FDA) for counting CTCs in whole blood and is based on a combination of immunomagnetic labeling and automated digital microscopy. Other methods for isolating CTCs are described in the literature, and all of them can be used in combination with the present invention.

[0244] Relapse or recurrence occurs when a person is affected again by a condition that previously affected them. For example, if a patient has a tumor disease, undergoes treatment for the disease successfully, and develops the disease again, the newly developed disease can be regarded as a relapse or recurrence. However, according to the present invention, a relapse or recurrence of a tumor disease may occur at the site of the original tumor, but it is not necessarily so. Thus, for example, if a patient has an ovarian tumor and the treatment received is successful, a relapse or recurrence can be the occurrence of an ovarian tumor or the occurrence of a tumor at a site different from the ovary. Also, a relapse or recurrence of a tumor includes the situation where the tumor occurs at a site different from the site of the original tumor and the situation where it occurs at the site of the original tumor. Preferably, the original tumor from which the patient received treatment is a primary tumor, and the tumor at a site different from the site of the original tumor is a secondary or metastatic tumor.

[0245] The term "immune response" relates to the reaction of the immune system to immunogenic organisms such as bacteria or viruses, cells or substances. The term "immune response" includes innate immune responses and adaptive immune responses. Preferably, the immune response is related to the activation of immune cells, the induction of cytokine biosynthesis, and / or antibody production.

[0246] The immune response induced by the composition of the present invention preferably includes the steps of activation of antigen-presenting cells such as dendritic cells and / or macrophages, presentation of an antigen or a fragment thereof by the antigen-presenting cells, and activation of cytotoxic T cells by this presentation.

[0247] The term "immune cell" refers to cells of the immune system involved in the defense of an individual's body. The term "immune cell" encompasses specific types of immune cells and their precursors, including leukocytes such as macrophages, monocytes (precursors of macrophages), neutrophils, eosinophils, and basophils, dendritic cells, mast cells, and lymphocytes such as B cells, T cells, and natural killer (NK) cells. Macrophages, monocytes (precursors of macrophages), neutrophils, dendritic cells, and mast cells are phagocytic cells.

[0248] The term "immunotherapy" relates to the treatment of a disease or condition by inducing, enhancing, or suppressing an immune response. Immunotherapies designed to induce or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress an immune response are classified as suppression immunotherapies. The term "immunotherapy" includes antigen immunization or antigen vaccination, or tumor immunization or tumor vaccination. The term "immunotherapy" also relates to the manipulation of an immune response such that an inappropriate immune response is modulated to a more appropriate response in the context of autoimmune diseases such as rheumatoid arthritis, allergy, diabetes, or multiple sclerosis.

[0249] The term "immunization" or "vaccination" represents the process of administering an antigen to an individual for the purpose of inducing an immune response, for example for therapeutic or prophylactic reasons.

[0250] "Treating" means administering to a subject a compound or composition described herein for the purpose of preventing or eliminating a disease, including reducing the size or number of tumors in the subject, halting or delaying a disease in the subject, inhibiting or delaying the onset of a new disease in the subject, reducing the frequency or severity of symptoms and / or recurrence in a subject currently or previously suffering from the disease, and / or prolonging, i.e., increasing, the lifespan of the subject. In particular, the term "treatment of a disease" includes curing the disease or its symptoms, shortening the duration, remission, preventing, delaying or inhibiting progression or worsening, or preventing or delaying recurrence.

[0251] "At risk" means a subject, i.e., a patient, identified as having a higher than normal likelihood of developing a disease, particularly cancer, compared to the general population. Further, a subject who had or currently has a disease, particularly cancer, is a subject with an increased risk of developing the disease because such a subject can continue to develop the disease. Also, a subject who currently has or had cancer also has an increased risk of cancer metastasis.

[0252] Prophylactic administration of immunotherapy, e.g., prophylactic administration of the compositions of the present invention, preferably protects the recipient from the onset of the disease. Therapeutic administration of immunotherapy, e.g., therapeutic administration of the compositions of the present invention, can result in inhibition of disease progression / growth. This preferably includes deceleration of disease progression / growth that results in elimination of the disease, particularly disruption of disease progression.

[0253] Immunotherapy can be performed using any of a variety of techniques in which the drugs provided herein function to remove disease cells from the patient. Such removal can occur as a result of enhancing or inducing an immune response specific to an antigen or cells expressing an antigen in the patient.

[0254] In certain embodiments, immunotherapy can be active immunotherapy, and the treatment depends on in vivo stimulation of the host's innate immune system using administration of immune response modifiers (such as polypeptides and nucleic acids provided herein) that react to disease cells.

[0255] The drugs and compositions provided herein can be used alone or in combination with conventional treatment regimens such as surgery, irradiation, chemotherapy, and / or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated).

[0256] The term "in vivo" relates to the situation within a subject.

[0257] The terms "subject", "individual", "organism" or "patient" relate to vertebrates, particularly mammals. For example, mammals in the context of the present invention include domesticated mammals such as humans, non-human primates, dogs, cats, sheep, cows, goats, pigs, horses, etc., experimental animals such as mice, rats, rabbits, guinea pigs, etc., and animals kept in enclosures such as zoo animals. These terms also relate to non-mammalian vertebrates such as birds (particularly domesticated birds such as chickens, ducks, geese, pigeons, etc.) and fish (particularly farmed fish such as salmon or catfish). Also, the term "animal" as used herein includes humans.

[0258] The term "autologous" is used to describe all things derived from the same subject. For example, "autologous transplantation" refers to the transplantation of tissue or an organ derived from the same subject. Such a procedure is advantageous for overcoming immunological barriers that would otherwise result in rejection.

[0259] The term "heterologous" is used to describe something consisting of a plurality of different elements. As an example, transplanting the bone marrow of one individual into another individual constitutes a heterologous transplantation. A heterologous gene is a gene derived from a source other than the subject.

[0260] As part of a composition for immunization or vaccination, preferably one or more of the agents described herein are administered together with one or more adjuvants for inducing or increasing an immune response. The term "adjuvant" relates to a compound that extends or enhances or accelerates an immune response. The compositions of the present invention preferably exert their effects without the addition of an adjuvant. Nevertheless, the compositions of the present application may contain any known adjuvant. Adjuvants include heterogeneous groups of compounds such as oil emulsions (e.g., Freund's adjuvant), inorganic compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), liposomes, and immunostimulating complexes. Examples of adjuvants are saponins such as monophosphoryl-lipid-A (MPL SmithKline Beecham), QS21 (SmithKline Beecham), DQS21 (SmithKline Beecham, WO96 / 33739), QS7, QS17, QS18, and QS-L1 (So et al., 1997, Mol. Cells 7: 178-186), incomplete Freund's adjuvant, complete Freund's adjuvant, vitamin E, montanid, alum, CpG oligonucleotides (Krieg et al., 1995, Nature 374: 546-549), and various water-in-oil emulsions prepared from biodegradable oils such as squalene and / or tocopherol.

[0261] Also, other substances that stimulate the patient's immune response may be administered. For example, cytokines can be used during vaccination due to their regulatory properties on lymphocytes. Such cytokines include, for example, interleukin-12 (IL-12) (see Hall, 1995, IL-12 at the crossroads, Science 268: 1432-1434), which has been shown to increase the protective effect of vaccines, GM-CSF, and IL-18.

[0262] There are several compounds that enhance the immune response and can thus be used in vaccination. The compounds include costimulatory molecules provided in the form of proteins or nucleic acids such as B7-1 and B7-2 (CD80 and CD86 respectively).

[0263] According to the present invention, a "tumor specimen" is a body sample containing tumor or cancer cells such as circulating tumor cells (CTCs), in particular, a sample such as a tissue sample and / or a cellular sample containing body fluid. According to the present invention, a "non-tumor specimen" is a body sample that does not contain tumor or cancer cells such as circulating tumor cells (CTCs), in particular, a sample such as a tissue sample and / or a cellular sample containing body fluid. Such body samples can be obtained in conventional manners such as by tissue biopsy including punch biopsy and by collecting blood, bronchial aspirate, sputum, urine, feces or other body fluids. According to the present invention, the term "sample" also includes processed samples such as fractions or isolates of biological samples, for example, isolates of nucleic acids or cells.

[0264] The therapeutic agents, vaccines and compositions described herein can be administered by any conventional route including by injection or infusion. Administration can be effected, for example, orally, intravenously, intraperitoneally, intramuscularly, subcutaneously or transdermally. In one embodiment, administration is effected intranodally such as by injection into a lymph node. Other forms of administration contemplate in vitro transfection of antigen-presenting cells such as dendritic cells with the nucleic acids described herein, followed by administration of the antigen-presenting cells.

[0265] The agents described herein are administered in an effective amount. "Effective amount" means an amount that achieves a desired response or desired effect, either alone or together with further dosages. In the case of treating a particular disease or a particular condition, the desired response preferably relates to inhibition of the course of the disease. This includes delaying the progression of the disease, in particular interrupting or reversing the progression of the disease. Also, the desired response in the treatment of a disease or condition may be delaying or preventing the onset of the disease or the condition.

[0266] The effective amount of the agents described herein depends on the condition being treated, the severity of the disease, the age, physiological state, size and weight of the patient, including individual parameters, the duration of the treatment, the type of concomitant therapy (if any), the particular route of administration and similar factors. Accordingly, the dosage of the agents described herein can vary depending on such parameters. If the response in the patient is inadequate at the initial dosage, higher dosages (or effectively higher dosages achieved by a different, more local route of administration) may be used.

[0267] The term "pharmaceutically acceptable" refers to the non-toxicity of materials that do not interact with the action of the active ingredient of a pharmaceutical composition.

[0268] The pharmaceutical compositions of the present invention can contain salts, buffers, preservatives, carriers and optionally other therapeutic agents. Preferably, the pharmaceutical compositions of the present invention include one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0269] The term "excipient" is intended to denote any substance in a pharmaceutical composition that is not an active ingredient, such as binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring agents or coloring agents.

[0270] The term "diluent" relates to agents for dilution and / or attenuation. Further, the term "diluent" includes any one or more of fluids, liquids or solid suspensions and / or mixed media.

[0271] The term "carrier" relates to one or more compatible solid or liquid fillers or diluents suitable for administration to humans. The term "carrier" relates to natural or synthetic organic or inorganic components that are combined with the active ingredient to facilitate the application of the active ingredient. Preferably, the carrier component is a sterile liquid such as water or oil, including peanut oil, soybean oil, sesame oil, sunflower oil, etc., mineral oil, those derived from animals or plants. Saline solutions and aqueous dextrose and glycerol solutions can also be used as aqueous carrier compounds.

[0272] Pharmaceutically acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (edited by A. R Gennaro, 1985). Examples of suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, carboxymethylcellulose sodium, low melting wax, cocoa butter and others. Examples of suitable diluents include ethanol, glycerol and water.

[0273] The pharmaceutical carrier, excipient or diluent can be selected with respect to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition of the present invention can contain, as or in addition to a carrier, excipient or diluent, any suitable binder, lubricant, suspending agent, coating agent and / or solubilizing agent. Examples of suitable binders include starch, gelatin, glucose, anhydrous lactose, free-flow lactose, beta-lactose, natural sugars such as corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and others. Preservatives, stabilizers, dyes and even flavorings can be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be used.

[0274] In one embodiment, the composition is an aqueous composition. The aqueous composition can optionally contain a solute, such as a salt. In one embodiment, the composition is in the form of a freeze-dried composition. The freeze-dried composition can be obtained by freeze-drying each aqueous composition.

[0275] The agents and compositions provided herein can be used alone or in combination with other treatment regimens such as surgery, radiation, chemotherapy and / or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated).

[0276] The present invention is described in detail by the figures and examples, which are used for illustrative purposes only and are not intended to be limiting. Thanks to the description and examples, further embodiments included in the present invention are also available to those skilled in the art.

Brief Description of the Drawings

[0277]

Fig. 1a

Fig. 1b

Fig. 2

Fig. 3

Example 1

[0278] Targeting of disease-specific mutations in genes with high copy numbers Genomic information regarding glioblastoma samples (Chin et al., 2008, Comprehensive genomic characterization defines human glioblastoma genes and core pathways, Nature 455: 1061-1068) was analyzed by searching for genes with high copy numbers and containing at least one copy of a gene with a disease-specific mutation. Quality analysis showed that there was high fidelity in the copy number assignment of 11,574 individual genes analyzed, and the genomic ploidy of the sample was determined to be 1.95. Figure 1a shows a graphical representation of local genes around the epidermal growth factor receptor (EGFR) on chromosome 7, which is a known driver gene and a target for treatment. It was shown that EGFR in this genome had 76 error-corrected absolute copy numbers, 13 of which contained disease-specific single nucleotide polymorphisms. Figure 1b provides a list of genes in this genomic sample with the highest absolute copy numbers. There are four additional genes with absolute copy numbers greater than 2. Indeed, EGFR amplification is a known genetic feature of primary glioblastoma (Benito et al., 2009, Neuropathology 30 (4): 392-400), and this gene has been considered as a target for treatment (Taylor, 2012, Curr Cancer Drug Targets. Mar; 12(3):197-209).

Example 2

[0279] Targeting Disease-Specific Mutations in the Homozygous State Genes having at least one copy of a disease-specific mutation were searched, and by focusing on the number of copies of the gene having the disease-specific mutation, the total number of gene copies, and whether the gene has a mutation or not, an exome obtained from a sample of tumor-derived melanoma cells in humans was analyzed. Figure 2 provides a list of genes selected by the zygosity state in which disease-specific mutations are found in multiple copies of the gene. For example, the disease-specific mutation in the OXGR1 gene has the highest zygosity state (4), and in particular, there are a total of 5 copies of the OXGR1 gene, 4 of which contain the disease-specific mutation, so it also has the highest zygosity rate of 4 / 5 or 0.8. The list provides 10 additional genes in which 3 out of a total of 4 copies of the gene have a mutation, indicating that the disease-specific mutations in these genes have a zygosity rate of 3 / 4 or 0.75. The remaining listed genes have a mutation with a zygosity rate of 2 / 3 or 0.66 because 2 out of a total of 3 copies have a mutation.

Example 3

[0280] Targeting of disease-specific mutations present in all copies of essential genes Exomes obtained from samples of tumor-derived melanoma cells in humans were analyzed by searching for genes in which all copies of the gene have the same disease-specific mutation and determining which of these genes are essential genes. These genes were determined to be essential by inferring their degree of essentiality in humans from the knowledge that they are essential in mice (Georgi et al., 2013, From mouse to human: evolutionary genomics analysis of human orthologs of essential genes, PLoS Genetics 9(5): e1003484; Liao et al., 2007, Mouse duplicate genes are as essential as singletons, Trends Genet. 23: 378-381). Figure 3 lists a number of genes in which all copies of the gene have the same disease-specific mutation. Furthermore, three highlighted genes were determined to be essential by inferring their degree of essentiality from mouse data.

Claims

1. Use of an individualized cancer vaccine in the manufacture of a medicament for use in a method for treating or preventing cancer in a patient, wherein the cancer of the patient is characterized by cancer cells comprising one or more cancer-specific neoepitopes, each neoepitope being due to a mutation in an allele of the patient's gene, the manufacture of the medicament comprising the following steps (1) A step of determining the copy number of one or more mutated alleles in an affected cell or population of affected cells, each mutated allele encoding a cancer-specific neoepitope, said determination comprising (a) determining that the copy number of each mutated allele encoding each neoepitope exceeds 2, and / or (b) determining that the copy number of the mutated allele encoding each neoepitope relative to the total copy number of the gene exceeds 0.5, a step comprising, and (2) (i) A recombinant peptide or recombinant polyepitope polypeptide, wherein the recombinant peptide comprises one cancer-specific neoepitope identified in said steps (a) and (b), or the recombinant polyepitope polypeptide comprises two or more cancer-specific neoepitopes identified in said steps (a) and (b), and the neoepitopes of the recombinant polyepitope polypeptide are fused together by peptide bonds or linkers, a recombinant peptide or recombinant polyepitope polypeptide, or (ii) RNA encoding said recombinant peptide comprising one cancer-specific neoepitope identified in said steps (a) and (b) or said recombinant polyepitope polypeptide comprising two or more cancer-specific neoepitopes identified in said steps (a) and (b) manufacturing a medicament comprising a vaccine comprising, a step comprising, Use.

2. The use according to claim 1, wherein the method comprises a step of determining that the copy number of the mutated allele encoding each neoepitope relative to the total copy number of the gene is 1.

3. The use according to claim 1, wherein the patient is characterized by a copy number of mutated alleles exceeding 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or exceeding 100.

4. The use according to any one of claims 1 to 3, wherein the recombinant polyepitope polypeptide comprises neoepitopes derived from different proteins or neoepitopes derived from different parts of the same protein fused together by peptide bonds or linkers.

5. The medicament containing the vaccine comprises RNA encoding the recombinant peptide containing 1 cancer-specific neoepitope identified in the steps (a) and (b) or the recombinant polyepitope polypeptide containing 2 or more cancer-specific neoepitopes identified in the steps (a) and (b), The RNA contains a 5'-cap or a 5'-cap analog, the use according to claim 1.

6. The medicament containing the vaccine comprises RNA encoding the recombinant peptide containing 1 cancer-specific neoepitope identified in the steps (a) and (b) or the recombinant polyepitope polypeptide containing 2 or more cancer-specific neoepitopes identified in the steps (a) and (b), The RNA includes a 7-methylguanosine cap (m 7 G), and is used according to any one of claims 1 to 5.

7. The medicament containing the vaccine comprises RNA encoding the recombinant peptide containing 1 cancer-specific neoepitope identified in the steps (a) and (b) or the recombinant polyepitope polypeptide containing 2 or more cancer-specific neoepitopes identified in the steps (a) and (b), In the RNA, cytidine is partially or completely replaced with 5-methylcytidine, or uridine is partially or completely replaced with pseudouridine, the use according to any one of claims 1 to 6.

8. The medicament containing the vaccine comprises RNA encoding the recombinant peptide containing 1 cancer-specific neoepitope identified in the steps (a) and (b) or the recombinant polyepitope polypeptide containing 2 or more cancer-specific neoepitopes identified in the steps (a) and (b), The RNA contains a 5'- or 3'-untranslated region (UTR), the use according to any one of claims 1 to 7.

9. The medicament containing the vaccine comprises RNA encoding the recombinant peptide containing 1 cancer-specific neoepitope identified in the steps (a) and (b) or the recombinant polyepitope polypeptide containing 2 or more cancer-specific neoepitopes identified in the steps (a) and (b), Use according to any one of claims 1 to 8, wherein the RNA comprises a poly(A) sequence.

10. Use according to claim 9, wherein the poly(A) sequence has a length of 100 to 150 adenosine residues.

11. The medicament containing the vaccine comprises RNA encoding the recombinant peptide containing the one cancer-specific neoepitope identified in the steps (a) and (b) or the recombinant polyepitope polypeptide containing two or more cancer-specific neoepitopes identified in the steps (a) and (b), Use according to any one of claims 1 to 10, wherein the RNA is bound to a carrier.

12. Use according to claim 11, wherein the carrier comprises a lipid-containing carrier.

13. Use according to claim 11, wherein the carrier comprises a cationic lipid, liposome, micelle or nanoparticle.

14. The cancer is selected from the group consisting of leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, gastric cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and pharynx (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer and lung cancer and metastases thereof. Use according to any one of claims 1 to 13.

15. Use according to any one of claims 1 to 14, wherein the one or more neoepitopes are T cell epitopes.

16. The suitability of the recombinant peptide or recombinant polyepitope polypeptide for the cancer vaccine is (i) evaluation of whether the mutation is located in an epitope presented by a known or predicted MHC, (ii) in vitro and / or in silico tests of whether the mutation is located in an epitope presented by the MHC, and (iii) whether the putative modified epitope, when adjacent to the amino acid sequence adjacent to the epitope in a naturally occurring protein and expressed in an antigen-presenting cell, can stimulate T cells of a patient with the desired specificity. Use according to any one of claims 1 to 15, determined by one or more of the in vitro tests.

17. A method for manufacturing a cancer vaccine, comprising Determining the copy number of one or more mutated alleles encoding one or more cancer-specific neoepitopes in a diseased cell or population of diseased cells, (a) the copy number of the mutated allele encoding the neoepitope being greater than 2, or (b) the copy number of the mutated allele encoding the neoepitope being greater than 0.5 relative to the total copy number of the gene, which indicates the suitability of the neoepitope as a cancer-specific target, and manufacturing a vaccine comprising a peptide or polypeptide comprising one or more neoepitopes or a nucleic acid encoding said peptide or polypeptide, comprising wherein the copy number is determined using a heterozygous segment comprising at least one heterozygous SNP, and the segment is a predetermined region based on a reference genome or a fluorescence-based method such as FACS, FISH, spectral karyotyping (SKY) or digital PCR.

18. The method according to claim 17, wherein the copy number of the mutated allele encoding each neoepitope relative to the total copy number of the gene is 1.

19. The method according to claim 18, wherein the heterozygous segment comprises an equal number of each version of the heterozygous SNP.

20. The method according to claim 17, wherein the heterozygous SNP is used for determining the allele-specific copy number of the segment.

21. The method according to claim 17, wherein the copy number is a relative or absolute copy number, preferably the copy number is an absolute copy number.

22. The method according to claim 21, wherein the absolute copy number is determined using next-generation sequencing (NGS) combined with a single nucleotide polymorphism (SNP) array.

23. The method according to claim 21, wherein the absolute copy number is an error-corrected absolute copy number.

24. The method according to claim 21, wherein the absolute copy number or the error-corrected absolute copy number is normalized relative to the ploidy, preferably the ploidy of the genome of the diseased cell, or a chromosome, or a portion of the chromosome where the mutated or mutated gene is located in the diseased cell.

25. A computer-based analysis processing method for analyzing and selecting one or more neoepitopes resulting from cancer-specific mutations (mutated alleles) in alleles of one or more genes for use in the provision of a vaccine, wherein the computer-based analysis processing method (a) identifying one or more neoepitopes determined to be suitable as cancer-specific targets by the method according to claim 17, and (b) ranking one or more neoepitopes according to their suitability as cancer-specific targets, The method comprising:

26. The method according to claim 25, wherein the copy number of the mutated allele encoding each neoepitope is 1 relative to the total copy number of the gene.

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