Individualized vaccines for cancer

Abstract

The invention relates to an individualized vaccine for cancer, and belongs to the field of tumor immunotherapy. In particular, the present invention provides individualized cancer vaccines specific for a tumor of a patient based on transcriptome analysis of RNA transcripts that are over-upregulated in one or more cancer cells of the patient in a tumor specimen of the patient. These individualized cancer vaccines, when administered to a patient, induce an immune response against a tumor-associated antigen expressed in a tumor of the patient through an RNA transcript that is over-upregulated in one or more cancer cells of the patient. Due to excessive upregulation of the RNA transcript, the individualized cancer vaccine is effective for vaccination and breaking of self-tolerance to tumor-associated antigens that are autoproteins in a subject.

Description

[0001] This application is a divisional application of the Chinese patent application with an application date of July 23, 2019, application number 201980048922.0, and invention name “Personalized vaccine for cancer”. Technical Field

[0002] The present invention belongs to the field of tumor immunotherapy. In particular, the present invention provides a personalized cancer vaccine specific to the patient's tumor based on the analysis of the transcriptome of RNA transcripts over-regulated in one or more cancer cells of the patient in the patient's tumor specimen. These personalized cancer vaccines, when administered to the patient, induce an immune response to tumor-associated antigens expressed in the patient's tumor by RNA transcripts over-regulated in one or more cancer cells of the patient. Due to the excessive upregulation of RNA transcripts, personalized cancer vaccines are effective for vaccination of the subject and breaking self-tolerance to tumor-associated antigens that are self-proteins in the subject. Background Art

[0003] Cancer is the leading cause of death, accounting for a quarter of all deaths. Traditionally, cancer treatments have been based on the law of averages - that is, what works best for the largest number of patients. However, due to molecular heterogeneity in cancer, typically less than 25% of treated individuals benefit from approved treatments. Personalized medicine, based on customized treatments for patients, is considered a potential solution to the inefficiencies and high costs of innovation in drug development.

[0004] Antigen-specific immunotherapy aims to enhance or induce specific immune responses in patients to control malignant diseases. The identification of an increasing number of pathogen-associated antigens and tumor-associated antigens leads to a wide collection of suitable targets for immunotherapy. Cells that specifically present immunogenic peptides (epitopes) derived from these antigens can be targeted by active or passive immunization strategies. Active immunization tends to induce and amplify antigen-specific T cells in patients, which can specifically recognize and kill diseased cells. In contrast, passive immunization relies on the adoptive transfer of T cells that are amplified in vitro and optionally genetically modified (adoptive T cell therapy; ACT).

[0005] Tumor vaccines aim to induce endogenous tumor-specific immune responses through active immunization. Different antigen formats can be used for tumor vaccination, including intact diseased cells, proteins, peptides, or immune vectors (e.g., RNA, DNA, or viral vectors), which can be applied directly in vivo or in vitro by pulsing DCs followed by transfer into patients.

[0006] The discovery of a variety of pathogen-associated antigens and tumor-associated antigens provides the basis for the concept of antigen-specific immunotherapy. Tumor-associated antigens (TAA) are uncommon proteins expressed on tumor cells due to genetic instability, which have no or limited expression in normal cells. These TAAs can lead to specific recognition of malignant cells by the immune system. In addition, cancer can be caused by the accumulation of genomic mutations and epigenetic changes, some of which can have a causal effect. In addition to tumor-associated antigens, human cancers also carry an average of 100 to 120 non-synonymous mutations, many of which can be targeted by vaccines. More than 95% of mutations in tumors are unique and patient-specific. The number of protein-changing somatic mutations that can produce tumor-specific T cell epitopes is 30 to 400. Mutations are considered to be ideal targets for cancer immunotherapy. As a new epitope that is strictly lacking expression in any healthy tissue, it is expected to be safe and can avoid central tolerance mechanisms. We have recently proposed a personalized immunotherapy approach targeting an individual's mutation profile (Castle, JC, et al., Cancer Res 72, 1081 (2012)).

[0007] Despite the increasing number of attractive target structures for immunotherapeutic approaches, the definition of suitable epitopes for immunotherapy remains a challenge. The induction of an immune response against self-proteins requires breaking immune self-tolerance. Due to immune tolerance, self-proteins or their peptides are only weakly immunogenic.

[0008] Therefore, there is a need to provide target structures for vaccination of cancer patients. These target structures should in particular overcome self-tolerance mechanisms when used in vaccine approaches. Summary of the invention

[0009] The inventors have found that many tumors are defined by characteristic patterns of RNA transcripts. In particular, specific RNA transcripts were found to be highly upregulated in specific tumors and individual patients. The present invention is based on the following discovery: tumors of individual patients have characteristic transcriptome patterns and over-regulated RNA transcripts, which are not expressed or expressed in significantly lower amounts in normal healthy cells and tissues of the same or different tissue types. The transcriptome pattern of the tumor makes the tumor unique at the RNA level and can be distinguished from non-cancerous tissues (including corresponding non-cancerous tissues and other non-cancerous tissues). Such a unique transcriptome pattern enables the production of vaccines that specifically target tumor cells and tumor tissues. The present disclosure relates to providing personalized (i.e., patient-specific) cancer vaccines based on tumor-associated antigens encoded by such over-regulated RNA transcripts. In particular, the personalized cancer vaccines provided herein include or encode at least one epitope derived from an amino acid sequence encoded by such over-regulated RNA transcripts. The present invention aims to immunotherapeutically target the expression products of RNA transcripts that are over-regulated in a patient's tumor cells but are not expressed or expressed in significantly lower amounts in the patient's non-tumorigenic cells. For example, Next Generation Sequencing (NGS) enables rapid and cost-effective identification of patient-specific transcriptome profiles in cancer cells.

[0010] Transcript species that are over-upregulated in cancer cells are identified and provided to a patient by administering a polypeptide comprising one or more epitopes derived from an amino acid sequence encoded by one or more over-upregulated transcript species or a nucleic acid (e.g., RNA) encoding the polypeptide, with the goal of inducing an immune response to the epitope after appropriate processing of the polypeptide and presentation of the epitope by an MHC molecule to display the epitope to the patient's immune system to stimulate, priming, and / or expand appropriate T cells against the patient's cancer cells, providing a new strategy that is specific to the patient's cancer. Utilizing the transcriptome profile present in circulating tumor cells (CTCs) enables the provision of vaccines that induce immune responses that potentially target primary tumors as well as tumor metastases. The present invention utilizes the characteristic transcriptome profile present in the cancer cells of an individual patient, rather than retrieving a common molecular signature (denominator) present in many patients for targeting.

[0011] In one aspect, the present invention relates to a method for producing a personalized cancer vaccine comprising the following steps:

[0012] (a) identifying one or more RNA transcripts present in a tumor specimen of a cancer patient, wherein each of the one or more RNA transcripts encodes an amino acid sequence, and wherein each of the one or more RNA transcripts is present in the tumor specimen at a copy number exceeding a predetermined expression threshold; and

[0013] (b) providing a vaccine characterized in that at least one epitope is derived from an amino acid sequence encoded by said one or more RNA transcripts.

[0014] In one embodiment, the step of identifying one or more RNA transcripts comprises determining the copy number of RNA transcripts present in a tumor specimen of a cancer patient, and comparing the copy number of each of the RNA transcripts to a respective predetermined expression threshold.

[0015] In one embodiment, the copy number of the RNA transcript in a tumor specimen is at least 10 times, preferably at least 1000 times, that in a non-tumor tissue.

[0016] In one embodiment, the copy number of the RNA transcript in the tumor specimen is at least 50, 100, 500, 1000, 2000, 3000, 4000 or 5000 transcripts per million (TPM), and the copy number of the RNA transcript in the non-tumor tissue is less than 10%, 5%, 1%, 0.1% or 0.01% of the copy number of the RNA transcript in the tumor specimen.

[0017] In one embodiment, the step of identifying one or more RNA transcripts comprises performing single cell sequencing on one or more cancer cells.

[0018] In one embodiment, the cancer cells are circulating tumor cells.

[0019] In one embodiment, the step of identifying one or more RNA transcripts comprises using next generation sequencing (NGS).

[0020] In one embodiment, the step of identifying one or more RNA transcripts comprises sequencing RNA from a tumor specimen and / or a DNA library obtained from RNA from a tumor specimen.

[0021] In one embodiment, the step of identifying one or more RNA transcripts is repeated at least in duplicate.

[0022] In one embodiment, the method comprises the further step of determining the usability of epitopes encoded by one or more RNA transcripts for cancer vaccination.

[0023] In one embodiment, the vaccine comprises a polypeptide comprising one or more epitopes encoded by one or more RNA transcripts, or a nucleic acid encoding the polypeptide.

[0024] In one embodiment, the polypeptide comprises up to 30 epitopes.

[0025] In one embodiment, the polypeptide further comprises one or more epitopes that are not encoded by the one or more RNA transcripts but are expressed by the cancer cell.

[0026] In one embodiment, the one or more epitopes not encoded by the one or more RNA transcripts are cancer-specific neo-epitopes.

[0027] In one embodiment, the epitope is in its natural sequence context so as to form a vaccine sequence.

[0028] In one embodiment, the vaccine sequence is about 30 amino acids in length.

[0029] In one embodiment, the epitopes and / or vaccine sequences are lined up head-to-tail.

[0030] In one embodiment, the epitope and / or vaccine sequence are separated by a linker.

[0031] In one embodiment, the epitope and / or vaccine sequence has the same amino acid sequence in a tumor specimen as in non-tumor tissue.

[0032] In one embodiment, the vaccine is an RNA vaccine.

[0033] In one embodiment, the vaccine is a prophylactic and / or therapeutic vaccine.

[0034] In another aspect, the present invention relates to a personalized cancer vaccine obtained by the method described herein.

[0035] In another aspect, the present invention relates to a personalized cancer vaccine comprising a recombinant polypeptide or a nucleic acid encoding the polypeptide, wherein the recombinant polypeptide comprises an epitope produced by the expression of an RNA transcript in a tumor specimen of a cancer patient, wherein the RNA transcript is present in the tumor specimen at a copy number exceeding a predetermined expression threshold.

[0036] In one embodiment, the copy number of RNA transcripts in a tumor specimen is at least 10-fold, preferably at least 1000-fold, higher than in a non-tumor tissue (eg, non-tumor tissue of the same or different tissue type as the tumor specimen).

[0037] In one embodiment, the copy number of the RNA transcript in the tumor specimen is at least 50, 100, 500, 1000, 2000, 3000, 4000 or 5000 transcripts per million (TPM), and the copy number of the RNA transcript in the non-tumor tissue is less than 10%, 5%, 1%, 0.1% or 0.01% of the copy number of the RNA transcript in the tumor specimen.

[0038] In one embodiment, the polypeptide further comprises an epitope that is not encoded by the RNA transcript but is expressed by the cancer cell.

[0039] In another aspect, the present invention relates to a method of treating a cancer patient comprising the steps of:

[0040] (a) providing a personalized cancer vaccine by the methods described herein; and

[0041] (b) administering the vaccine to a patient.

[0042] In another aspect, the invention relates to a method of treating a patient with cancer comprising administering to the patient a vaccine described herein.

[0043] In one embodiment, the vaccine is administered intravenously, dermally, muscularly, or subcutaneously.

[0044] In one aspect, the present invention relates to a vaccine comprising one or more polypeptides or nucleic acids (e.g., RNA) encoding the one or more polypeptides, each polypeptide comprising one or more epitopes derived from one or more RNA transcripts that are over-regulated in a tumor specimen of a cancer patient. Some preferred embodiments of such a vaccine are as described above in the context of the method of the present invention.

[0045] The vaccine provided according to the present invention may comprise a pharmaceutically acceptable carrier and may optionally comprise one or more adjuvants, stabilizers, etc. The vaccine may be in the form of a therapeutic or prophylactic vaccine.

[0046] Another aspect relates to a method for inducing an immune response in a patient, comprising administering to the patient a vaccine provided according to the present invention.

[0047] In other aspects, the present invention provides vaccines as described herein for use in the methods of treatment as described herein, particularly for the treatment or prevention of cancer.

[0048] Treatment of cancer described herein may be combined with surgical resection and / or radiation and / or traditional chemotherapy.

[0049] Other features and advantages of the present invention will become apparent from the following detailed description and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 , Figure 1 (Continued): Some examples of differentially expressed genes in the B16-F10 melanoma cell line

[0051] Expression values ​​in transcripts per million (TPM) of selected genes in melanoma cell line B16-F10 and murine tissues (subdivided into three categories: normal tissue (n=46), reproductive tissue (n=5) and embryonic tissue (n=14). Tissue expression is given as the median of all samples for each tissue. Circles indicate single tissue medians. The center line of the box plot indicates the median of all tissues, the box shows the first and third quartiles, and the whiskers are 2.5 times the quartile from the median.

[0052] Figure 2 :Immunogenicity testing of Tyrp1 RNA vaccine

[0053] C57BL / 6 mice (n=5 per group) were immunized with 40 μg of Tyrp1 antigen encoding RNA formulated as a lipoplex or 40 μg of an irrelevant RNA on days 0, 7 and 14 (see timeline). Five days after the last vaccination, splenocytes from mice were isolated and tested for recognition of two Tyrp1 peptides encoding MHC I epitopes (TAPDNLGYA) and MHC II epitopes (CRPGWRGAACNQKI) by IFNγ ELISpot, respectively. The average number of spots (dots) for triplicates of a single mouse and the average for all mice (bars) are shown on the left. A picture of the ELISpot plate is shown on the right. Each row represents one mouse.

[0054] Figure 3 :Immunogenicity testing of Dct RNA vaccine

[0055] C57BL / 6 mice (n=5 per group) were repeatedly vaccinated with 20 μg Dct-encoding RNA lipoplexes or NaCl (vaccination times are indicated by dashed lines). CD8 against Dct (SVYDFFVWL) was measured in the blood by flow cytometry with MHC tetramer (MBL international) staining on days 5, 12, 19, 26 and 33 after the first vaccination. + T cell response. Tetramers in all CD8+ lymphocytes are shown + CD8 + Frequency of T cells (mean ± standard error of the mean).

[0056] Figure 4:Immunogenicity testing of Pmel RNA vaccine

[0057] As shown in the timeline, C57BL / 6 mice (n=8 in each group) were repeatedly vaccinated with 20 μg Pmel encoding RNA lipid complexes. 27 days after the first vaccination, the splenocytes of mice were detected for Pmel peptide (EGSRNQDWL) or irrelevant peptide (VSV-NP, PGYVYQGL) by IFNγELISpot. The average spot number (point) of single mouse triplicate and the mean value (bar) of all mice are shown.

[0058] Figure 5 :Immunogenicity testing of Fkbp6 RNA vaccine

[0059] As shown in the timeline, C57BL / 6 mice (n=3 per group) were repeatedly vaccinated with 20 μg of Fkbp6 encoding RNA lipoplexes. Five days after the last vaccination, the recognition of BMDC electroporated with Fkbp6 RNA or irrelevant RNA (control) by IFNγ ELISpot was tested for splenocytes of mice. The average number of spots plus standard deviation of duplicate (control) or triplicate (Fkbp9) of pooled mouse splenocytes is shown.

[0060] Figure 6 : Tumor control after therapeutic Tyrp1 vaccination

[0061] Use 3×10 5 C57BL / 6 mice (n=11 to 12 per group) were iv vaccinated with 100 B16-F10 tumor cells. RNA lipid complex vaccination was initiated with Tyrp1 or irrelevant control RNA as indicated in the timeline. Lung tumor nodule counts (left), exemplary lungs (middle), and survival (right) for each mouse are shown.

[0062] Figure 7 : Tumor control after therapeutic Tyrp1 or Dct vaccination

[0063] As shown in the timeline, 3×10 5 Luciferase transgenic B16-F10 tumor cells (B16-F10-LUC) were injected iv into naive C57BL / 6 mice (n=12 per group) and treated. Median tumor growth determined by luciferase bioluminescence (left), lung tumor nodule counts per mouse (middle, mean ± standard error of the mean), and lung weight per mouse (right, mean ± standard error of the mean) are shown.

[0064] Fig. 8A , Fig. 8A (Continued) Figure 8B , Figure 8B (Continued) Figure 8C and Fig.8D :Some examples of differentially expressed genes in tumor models CT26, MC38, TRAMP-C2 and 4T1

[0065] Expression values ​​in transcripts per million (TPM) of selected genes in mouse tumor cell lines CT26 (A), MC38 (B), TRAMP-C2 (C) and 4T1 (D) and murine tissues (divided into three categories: normal tissue (n=46), reproductive tissue (n=5) and embryonic tissue (n=14). Tissue expression is given as the median of all samples for each tissue. Circles indicate single tissue medians. The center line of the box plot indicates the median of all tissues, the box shows the first and third quartiles, and the whiskers are 2.5 times the quartile from the median.

[0066] Fig. 9 : Immunogenicity testing of identified targets in the CT26 tumor cell line

[0067] As shown in the timeline, BALB / c mice (n=3 per group) were repeatedly vaccinated with 20 μg antigen-encoding RNA lipid complexes. Five days after the last vaccination, mouse splenocytes were tested for recognition of BMDC electroporated with antigen-encoding RNA or irrelevant RNA (control) by IFNγ ELISpot. The average number of spots plus standard deviation of triplicates of pooled mouse splenocytes is shown.

[0068] Fig.10 : Immunogenicity testing of identified targets in the MC38 tumor cell line

[0069] As shown in the timeline, C57BL / 6 mice (n=3 per group) were repeatedly vaccinated with 20 μg antigen-encoding RNA lipid complexes. Five days after the last vaccination, mouse splenocytes were tested for recognition of BMDC electroporated with antigen-encoding RNA or irrelevant RNA (control) by IFNγELISpot. The upper figure shows the average number of spots plus standard deviation of duplicate (control) or triplicate (Gm14819, Rhox2h, Gm15091, Ash21, Gm15097, Dppa4) of combined mouse splenocytes. The lower figure shows the average number of spots plus standard error of the average of triplicate splenocytes from a single mouse for targets Mageb2, Gm773, Prl2c2, Fmr1nb and Luzp4.

[0070] Fig.11 : Immunogenicity testing of identified targets in the TRAMP-C2 tumor cell line

[0071] As shown in the timeline, C57BL / 6 mice (n=3 per group) were repeatedly vaccinated with 20 μg antigen-encoding RNA lipid complexes. Five days after the last vaccination, mouse splenocytes were tested for recognition of BMDC electroporated with antigen-encoding RNA or irrelevant RNA (control) by IFNγ ELISpot. The average number of spots plus standard deviation of duplicate (control) or triplicate (antigen) of pooled mouse splenocytes is shown. DETAILED DESCRIPTION

[0072] Although the present disclosure is described in detail below, it should be understood that the present disclosure is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing some specific embodiments, and are not intended to limit the scope of the present disclosure, which will only be limited by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art are generally understood.

[0073] Preferably, the terms used herein are as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B.Nagel and H. The definition is as described in Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0074] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods in chemistry, biochemistry, cell biology, immunology and recombinant DNA techniques, which are described in the literature in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0075] Hereinafter, the elements of the present disclosure will be described. These elements are listed with some specific embodiments, however, it should be understood that they can be combined in any way and in any number to produce other embodiments. Some examples and embodiments of different descriptions should not be interpreted as limiting the present disclosure to only some embodiments clearly described. This specification should be understood to disclose and cover the embodiments that will clearly describe the embodiment and any number of disclosed elements combined. In addition, unless the context otherwise indicates, any arrangement and combination of all description elements should be considered to be disclosed by this specification.

[0076] The term "about" means approximately or close to, and in the context of a numerical value or range listed herein, means in one embodiment ±20%, ±10%, ±5%, or ±3% of the recited or claimed numerical value or range.

[0077] Unless otherwise noted herein or obviously contradictory with context, otherwise the nouns modified by quantifiers used in the context of describing the present disclosure (especially in the context of claims) should be interpreted as covering one / kind and / or more / kind. The recording of numerical ranges herein is only intended to be used as a shorthand method for referring to each individual value falling within the scope individually. Unless otherwise noted herein, each individual value is incorporated into this specification as it is recorded individually in this article. Unless otherwise noted herein or obviously contradictory with context in addition, all methods described herein can be carried out in any suitable order. The use of any and all examples or exemplary language (for example, "for example") provided herein is only intended to better illustrate the present disclosure, without limiting the scope of claims. The language in this specification should not be interpreted as indicating any unclaimed element necessary for the implementation of the present disclosure.

[0078] Unless explicitly stated otherwise, the term "includes / comprising" is used in the context of this document to indicate that other members may optionally be present in addition to the members of the list introduced by "includes / comprising". However, it is contemplated that as a specific embodiment of the present disclosure, the term "includes / comprising" encompasses the possibility that no other members are present, that is, for this purpose, the embodiment "includes / comprising" should be understood to have the meaning of "consisting of ..."

[0079] Several documents are cited throughout the text of this specification. Each document cited herein, whether above or below (including all patents, patent applications, scientific publications, manufacturer specifications, guidelines, etc.) is hereby incorporated by reference in its entirety. None of the content herein should be construed as admitting that the present disclosure is not entitled to predate such disclosure.

[0080] definition

[0081] Definitions applicable to all aspects of the present disclosure are provided below. Unless otherwise indicated, the following terms have the following meanings. Any undefined term has its art-recognized meaning.

[0082] According to the present disclosure, the term "peptide" includes oligopeptides and polypeptides, and refers to a substance comprising about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids connected to each other by peptide bonds. The term "protein" or "polypeptide" refers to large peptides, particularly peptides with at least about 151 amino acids, but the terms "peptide", "protein" and "polypeptide" are often used as synonyms in this article.

[0083] According to the present invention, "nucleic acid" is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0084] In the present disclosure, the term "RNA" refers to a nucleic acid molecule comprising ribonucleotide residues. In some preferred embodiments, RNA comprises all or most of the ribonucleotide residues. "Ribonucleotide" as used herein refers to a nucleotide having a hydroxyl group at the 2'-position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA), substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that is different from naturally occurring RNA by adding, deleting, replacing, and / or changing one or more nucleotides. Such a change may refer to adding a non-nucleotide substance to an internal RNA nucleotide or an RNA end. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered RNAs are considered to be analogs of naturally occurring RNA.

[0085] In certain embodiments of the present disclosure, RNA is a messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As recognized in the art, mRNA generally comprises a 5' untranslated region (5'-UTR), a peptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, mRNA is produced by in vitro transcription using a DNA template, wherein DNA refers to a nucleic acid comprising deoxyribonucleotides.

[0086] According to the present disclosure, the term "RNA encoding" means that, if present in a suitable environment, such as within a cell of a target tissue, the RNA can direct the assembly of amino acids during the translation process to produce a peptide or protein encoded by it. In one embodiment, the RNA is capable of interacting with the cellular translation machinery, thereby allowing translation of the peptide or protein. The cell can produce the encoded peptide or protein within the cell (e.g., in the cytoplasm and / or in the nucleus), can secrete the encoded peptide or protein, or can cause it to be produced on the surface.

[0087] For example, a "reference" as a predetermined expression threshold value can be used to correlate and compare the results obtained from the tumor specimen in the method of the present invention. Generally speaking, the "reference" can be obtained based on one or more normal tissues, especially tissues that are not affected by tumors or cancer diseases (i.e., non-tumor tissues), and the tissues are usually obtained from one or more individuals different from the patient from which the tumor specimen is derived, preferably healthy individuals, especially individuals of the same species. Non-tumor tissues generally include tissues different from the tissues from which the tumor specimen is derived, and may include tissues corresponding to the tissues from which the tumor specimen is derived.

[0088] As used herein, terms such as "reduce" or "inhibit" mean the ability to cause an overall decrease in levels, such as about 5% or greater, about 10% or greater, about 20% or greater, about 50% or greater, or about 75% or greater. The term "inhibit" or similar phrases include complete or substantially complete inhibition, i.e., a decrease to zero or substantially to zero.

[0089] In one embodiment, terms such as "increase" or "enhance" refer to an increase or enhancement of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, or at least about 100%.

[0090] In the context of the present disclosure, the term "recombinant" means "prepared by genetic engineering." In one embodiment, "recombinant material" in the context of the present disclosure does not occur naturally.

[0091] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including a virus) and can be isolated from a natural source and has not been intentionally modified by a person in a laboratory is naturally occurring. The term "found in nature" means "existing in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature but may be discovered and / or isolated from natural sources in the future.

[0092] The term "transcripts per million" or "TPM" is a normalization method for RNA-seq and should be understood as "for every 1,000,000 RNA molecules in the sample, x comes from this gene / transcript."

[0093] Over-regulated RNA transcripts

[0094] The present invention relates to a method for producing a personalized cancer vaccine, comprising the following steps:

[0095] (a) identifying one or more RNA transcripts present in a tumor specimen of a cancer patient, wherein each of the one or more RNA transcripts encodes an amino acid sequence, and wherein each of the one or more RNA transcripts is present in the tumor specimen at a copy number exceeding a predetermined expression threshold; and

[0096] (b) providing a vaccine characterized in that at least one epitope is derived from an amino acid sequence encoded by said one or more RNA transcripts.

[0097] The term "expression threshold value" as used herein relates to a quantitative limit / value associated with the expression level (i.e., copy number) of an RNA transcript, and will be considered for implementing the methods described herein. The expression threshold value of an RNA transcript is generally a characteristic of the RNA transcript. Generally speaking, expression levels below the expression threshold value are not considered to reflect excessive upregulation of RNA transcripts (e.g., in tumor or cancer tissue), and are particularly considered to reflect the (potential) expression of RNA transcripts in healthy tissues. Generally speaking, expression levels above the expression threshold value are considered to reflect excessive upregulation of RNA transcripts (e.g., in tumor or cancer tissue), and are particularly not considered to reflect the (potential) expression of RNA transcripts in healthy tissues. Therefore, the term "expression threshold value" refers to such an RNA transcript expression level, above which the RNA transcript is considered suitable for providing a vaccine characterized in that at least one epitope is derived from an amino acid sequence encoded by the RNA transcript. The expression threshold value is generally selected so that the expression level of the RNA transcript in healthy tissue does not exceed the threshold value. On the contrary, the expression threshold value is generally selected so that the expression of the RNA transcript in healthy tissue is far below the threshold value. The expression level of the RNA transcript in a diseased tissue, such as a cancerous tissue (e.g., in a tumor specimen) above the expression threshold value generally indicates that the RNA transcript is suitable for providing a vaccine characterized in that at least one epitope is derived from an amino acid sequence encoded by the RNA transcript. The expression threshold value is generally determined by obtaining information about the expression level of the RNA transcript in (healthy) tissue, preferably a tissue group of different tissue types, optionally tissues of different subjects (preferably subjects of the same species), and the threshold value is defined taking into account the information obtained about the expression level of the RNA transcript. Such a tissue group may include 10 or more, such as 100 or more different tissue types. Information about the expression level of the RNA transcript can be obtained, for example, from publicly available databases such as Sequence Read Archive (SRA). The expression threshold value is generally calculated to achieve a useful prediction about the effectiveness and / or safety of the RNA transcript suitable for providing a vaccine, wherein the vaccine is characterized in that at least one epitope is derived from an amino acid sequence encoded by the RNA transcript. Typically, the expression threshold is much higher than the highest expression level determined for RNA transcripts in healthy tissue and includes a safety margin; for example, a certain multiplication factor of the (e.g., highest or median) expression level determined for RNA transcripts in healthy tissue. Such a safety factor can be, for example, 10 times, 100 times, or 1000 times higher than the expression level in healthy tissue. Preferably, the expression threshold in the methods described herein can be used for many RNA transcripts, in particular coding transcripts, such as at least 100, at least 1000, or at least 10000 RNA transcripts.Generally speaking, such an expression threshold value is a predetermined value, wherein the term "predetermined" or "predefined" means a constant value that is independent of the determination of the expression level of the RNA transcript in the tumor specimen, i.e., it has not been determined when the expression level of the RNA transcript in the tumor specimen is determined, and this value is selected as the value that must be compared with the expression level of the RNA transcript determined in the tumor specimen. Thus, the expression threshold value is a quantitative value that is related to and provides information about the expression level of the RNA transcript in a subject, in particular in different tissues of the subject. In the methods described herein, it is not necessary to determine the expression level of the RNA transcript in a non-tumor specimen of the subject, and it is not necessary to compare the expression level of the RNA transcript determined in the tumor specimen of the subject with the expression level of the same RNA transcript in a non-tumor specimen of the same subject. Instead, the expression level of a plurality of RNA transcripts (e.g., at least 10, at least 100, at least 1000 or at least 10,000 RNA transcripts) is determined in a tumor specimen of a subject and compared to a predetermined expression threshold for each of the plurality of RNA transcripts (e.g., at least 100, at least 1000 or at least 10,000 RNA transcripts) (e.g., as described above). Thus, the methods of the invention may involve the use of a data set, such as a database comprising information about expression thresholds for a plurality of RNA transcripts, particularly coding transcripts, such as at least 100, at least 1000 or at least 10,000 RNA transcripts. The methods of the invention may involve comparing the expression level of each of the plurality of RNA transcripts in a tumor specimen of a subject with a data set comprising information about expression thresholds for the plurality of RNA transcripts to determine whether the compared expression level exceeds the respective expression threshold contained in the data set. Those RNA transcripts whose expression levels exceed the respective expression thresholds contained in the data set may be considered suitable or potentially suitable for providing a vaccine. In one embodiment, the method of the present invention can be carried out over time until a sufficient number of RNA transcripts are obtained that are considered suitable or potentially suitable for providing a vaccine. Once this point is reached, the method can be stopped.

[0098] The terms "overly upregulated RNA transcript", "RNA transcript present in a tumor specimen at a copy number exceeding a predetermined expression threshold" or similar terms refer to an RNA whose presence or expression in a tumor specimen is strongly increased compared to non-tumor tissue. In various embodiments, the presence or expression of the RNA is at least 10-fold, at least 100-fold, at least 10-fold, or at least 10-fold greater than that in non-tumor tissue. 3 times, at least 10 4 times, at least 10 5 Preferred according to the invention are those RNA transcripts which are not expressed or are expressed only in small amounts in the thymus and / or in key organs (eg heart, brain etc.).

[0099] In one embodiment, the method of the present invention comprises the following steps:

[0100] i) Providing tumor specimens from cancer patients;

[0101] ii) identification of RNA transcripts that are hyper-upregulated in tumor specimens;

[0102] iii) designing a polypeptide comprising an epitope of an amino acid sequence encoded by one or more of the over-regulated RNA transcripts identified in step (ii);

[0103] iv) providing the polypeptide designed in step (iii) or a nucleic acid encoding the polypeptide, preferably RNA; and

[0104] v) providing a vaccine comprising the polypeptide or nucleic acid provided in step (iv).

[0105] According to the present invention, a "tumor specimen" is a sample such as a body sample derived from a patient containing or suspected of containing a tumor or cancer cell (e.g., circulating tumor cell (CTC)), in particular a tissue sample comprising a body fluid such as blood, and / or a cell sample, and may be derived from a tumor tissue. In one embodiment, a tumor specimen relates to one or more isolated tumors or cancer cells (e.g., circulating tumor cells (CTC)) or a sample comprising one or more isolated tumors or cancer cells (e.g., circulating tumor cells (CTC)).

[0106] According to the present invention, "non-tumor tissue" is tissue that is not affected by a tumor or cancer and does not contain tumor or cancer cells (eg, circulating tumor cells (CTCs)).

[0107] Body samples can be obtained in conventional ways, for example by tissue biopsy, including punch biopsy, and by collecting blood, bronchial aspirates, 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, such as nucleic acid or cell isolates.

[0108] The present invention may involve identifying all over-upregulated RNA transcripts present in one or more cancer cells of a patient, or it may involve identifying only a portion of over-upregulated RNA transcripts present in one or more cancer cells of a patient. Generally speaking, the methods of the present invention provide for the identification of a number of over-upregulated RNA transcripts that provide a sufficient number of epitopes to be included in a vaccine.

[0109] In the context of the present invention, a transcriptome refers to a collection of all RNA molecules (including mRNA, rRNA, tRNA and other non-coding RNA) produced by a cell, a cell group (preferably a cancer cell group) or all cells of a given individual at a specific time point. Therefore, the method of the present invention may include identifying the transcriptome of one or more cancer cells, preferably the over-regulated RNA transcripts of the entire transcriptome. In one embodiment, the step of identifying the over-regulated RNA transcripts in the tumor specimen of a cancer patient includes identifying the over-regulated RNA transcript profile of the entire transcriptome.

[0110] The transcriptome differs from the exome in that it contains only those RNA molecules found in a particular cell or population of cells and may contain the amount or concentration of each RNA molecule in addition to its molecular identity. Unlike the genome, which is largely fixed to a given cell line (excluding mutations), the transcriptome can vary with external environmental conditions.

[0111] In one embodiment, the step of identifying overly upregulated RNA transcripts comprises single cell sequencing of one or more, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or even more cancer cells. Thus, the method of the present invention may comprise identifying the transcriptome signature of the one or more cancer cells. In one embodiment, the cancer cells are circulating tumor cells. Cancer cells (e.g., circulating tumor cells) may be isolated prior to single cell sequencing.

[0112] In one embodiment, the step of identifying over-regulated RNA transcripts comprises using next generation sequencing (NGS).

[0113] In one embodiment, the step of identifying over-regulated RNA transcripts comprises sequencing RNA from a tumor specimen.

[0114] In order to reveal over-regulated RNA transcripts, the information about the properties and levels of RNA transcripts obtained from the tumor specimen is compared with a predetermined expression threshold. Such an expression threshold is usually determined for RNA transcripts based on their expression levels in normal non-cancerous cells or tissues. In one embodiment, the corresponding information is obtained from a database. In one embodiment, the corresponding information includes more than one tissue type, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 50 or at least 100 tissue types of non-tumor tissues (and their respective RNA expression levels). In one embodiment, the corresponding information includes, for example, non-tumor tissues (and their respective RNA expression levels) of all available tissue types whose RNA expression levels can be obtained in the database. In one embodiment, the corresponding information includes non-tumor tissues (and their respective RNA expression levels) of one or more key organs (e.g., one or more, preferably all, of the lungs, heart, brain, CNS, kidneys, and liver). In one embodiment, over-regulation of RNA transcripts in tumor specimens relative to non-tumor tissues of one or more, preferably all such key organs is desired, and preferably the RNA transcripts are not expressed or not significantly expressed in non-tumor specimens of such key organs. In one embodiment, the corresponding information excludes non-tumor tissues (and their respective RNA expression levels) of one or more reproductive organs (e.g., one or more, preferably all, of the testes, ovaries, and placenta). In one embodiment, over-regulation of RNA transcripts in tumor specimens relative to non-tumor tissues of one or more, preferably all such reproductive organs is not desired.

[0115] Any suitable method can be used to determine the RNA transcripts that are over-regulated in a tumor specimen of a cancer patient. Generally speaking, such methods include determining a partial or complete transcriptome profile, i.e., the relative or absolute amount of one or more transcripts of a tumor tissue or tumor cell of a cancer patient. Such transcriptome profiles are then evaluated relative to the RNA species that are deregulated or over-regulated.

[0116] Transcriptomic studies (which include expression profiling, splice variant analysis, etc.) examine the expression levels of transcripts in a given cell population, usually focusing on mRNA but sometimes including others such as tRNA, sRNA.

[0117] Transcriptomics technology is a technology used to study the transcriptome of an organism (the sum of all its RNA transcripts). The information content of an organism is recorded in the DNA of its genome and expressed through transcription. Here, mRNA is used as a transient intermediary molecule in the information network, while non-coding RNA performs additional multiple functions. The transcriptome captures a time snapshot of all transcripts present in a cell. The first attempt to study the entire transcriptome began in the early 1990s, and technological advances since the late 1990s have made transcriptomics a broad discipline. There are two important contemporary technologies in this field: microarrays, which can quantify a set of predetermined sequences; and RNA-Seq, which uses high-throughput sequencing to capture all sequences. Transcription can also be studied at the single cell level by single-cell transcriptomics.

[0118] Microarray

[0119] Microarrays, which measure the abundance of a defined set of transcripts by their hybridization to an array of complementary probes, were first published in 1995. Microarray technology allows the simultaneous determination of thousands of transcripts, greatly reducing cost / gene and saving labor. Until the late 2000s, spotted oligonucleotide arrays and Affymetrix high-density arrays were both the preferred methods for transcriptional profiling. During this time, a series of microarrays were produced to cover known genes in model or economically important organisms. Advances in array design and manufacture have improved the specificity of probes and allowed more genes to be tested on a single array. Advances in fluorescence detection have increased the sensitivity and measurement accuracy of low-abundance transcripts.

[0120] RNA-Seq

[0121] RNA-Seq refers to the sequencing of transcript cDNA, where the abundance is derived from the number of counts of each transcript. Therefore, the technology is seriously affected by the development of high-throughput sequencing technology. Massively Parallel Signature Sequencing (MPSS) is based on an early example of generating 16 to 20 bp sequences through a complex hybridization series, and was used to verify the expression of 10,000 genes in Arabidopsis thaliana in 2004. The earliest RNA-Seq work was published in 2006, which used 454 technology to sequence 100,000 transcripts. This is sufficient coverage for quantitative relative transcript abundance. When the new Solexa / Illumina technology allowed the recording of 1 billion transcript sequences, RNA-Seq began to become increasingly popular after 2008. This yield now allows quantification and comparison of human transcriptomes.

[0122] Publicly available transcriptome databases

[0123] The function of most genes is unknown. Searching the transcriptome database can give researchers a list of all tissues in which a gene is expressed, providing clues about its possible function. For example, if the transcriptome database shows that the expression level of an unknown gene in cancer cells is significantly higher than in healthy cells, the unknown gene may play a role in cell growth. Transcriptome data provides technicians with relevant information to identify RNA transcripts that are over-regulated in cancer cells.

[0124] The National Human Genome Research Institute (NHGRI), as part of the National Institutes of Health (NIH), has participated in two projects that create transcriptome resources for researchers around the world: the Mammalian Gene Collection initiative and the Mouse Transcriptome Project. The Mammalian Gene Collection initiative has established a free public library of human, mouse and rat mRNA sequences, and can be used as a suitable database in the context of the present invention. The project is led by NHGRI and the National Cancer Institute (NCI), which is also a part of the NIH. Mouse and rat are important models for studying human biology. The Mouse Transcriptome Project is an initiative supported by the NIH, which has produced a free public database of gene transcripts of many mouse tissues, and can be used as a suitable database in the context of the present invention. These tissue-specific gene expression data mapped to the mouse genome can be obtained in a searchable format in the mouse reference transcriptome database. There are several other transcriptome resources, including those in the NIH project such as the Genotype Tissue Expression Project (GTEx) and the Encyclopedia of DNA Elements (ENCODE), all of which can be used as suitable databases in the context of the present invention. Therefore, GTEx is creating a catalog of human genes expressed in a variety of different tissues. ENCODE researchers aim to characterize and understand the working part of the genome including the transcriptome. Novartis and the European Molecular Biology Laboratory both have well-established gene expression databases, all of which can be used as suitable databases in the context of the present invention.

[0125] A number of organism-specific transcriptome databases have been constructed and annotated to aid in the identification of genes differentially expressed in different cell populations, all of which may be used as suitable databases in the context of the present invention.

[0126] Although older DNA microarray technology is still used, RNA-Seq is becoming the method of choice for measuring the transcriptome of an organism. According to the present invention, RNA-Seq is preferably used to identify overly upregulated RNA transcripts.

[0127] According to the present invention, any suitable sequencing method can be used, preferably next generation sequencing (NGS) technology. The third generation sequencing method can replace NGS technology in the future to accelerate the sequencing step of method. For illustration purposes, the term "next generation sequencing" or "NGS" means all new high-throughput sequencing technologies in the context of the present invention, which is contrasted with the "conventional" sequencing method called Sanger chemistry, by breaking the whole genome into small fragments to randomly read nucleic acid templates along the whole genome in parallel. Such NGS technology (also referred to as large-scale parallel sequencing technology) can be in a very short time, for example, in 1 to 2 weeks, preferably in 1 to 7 days or most preferably in less than 24 hours, the nucleic acid sequence information of the whole genome, exon group, transcript group (all transcription sequences of genome) or methylation group (all methylation sequences of genome) is delivered and in principle allows single cell sequencing method. In the context of the present invention, a number of NGS platforms that are commercially available or mentioned in the literature can be used, such as those described in detail in Zhang et al. 2011: The impact of next-generation sequencing ongenomics. J. Genet Genomics 38(3), 95-109; or Voelkerding et al. 2009: Nextgeneration sequencing: From basic research to diagnostics. Clinical chemistry 55, 641-658.

[0128] Non-limiting examples of such NGS technologies / platforms are:

[0129] 1) For example, the GS-FLX454 Genome Sequencer from Roche 454 Life Sciences (Branford, Connecticut) TMThe sequencing-by-synthesis technique called pyrophosphate sequencing, which was first described in Ronaghi et al. 1998: A sequencing method based on real-time pyrophosphate". Science 281(5375), 363-365. This technique uses emulsion PCR, in which single-stranded DNA-bound beads are encapsulated in aqueous micelles containing PCR reactants surrounded by oil by vigorous vortexing for emulsion PCR amplification. During the pyrophosphate sequencing process, as the polymerase synthesizes the DNA chain, the light emitted from the phosphate molecules during the incorporation of nucleotides is recorded;

[0130] 2) The sequencing-by-synthesis method developed by Solexa (now part of Illumina Inc., San Diego, California), which is based on reversible dye-terminators and is used, for example, in the Illumina / Solexa Genome Analyzer TM Neutralization in Illumina HiSeq 2000 Genome Analyzer TM In this technique, all four nucleotides are added simultaneously to the oligo-sensitized cluster fragments in the flow cell channel along with DNA polymerase. Bridge amplification extends the cluster chain with all four fluorescently labeled nucleotides for sequencing;

[0131] 3) For example, at Applied Biosystems (now Life Technologies Corporation, Carlsbad, California) SOLid TM The sequencing-by-ligation method is implemented in the platform. In this technology, a library of all possible oligonucleotides of fixed length is labeled according to the sequencing position. The oligonucleotides are annealed and ligated; the preferential ligation of matching sequences by DNA ligase generates a signal of the information of the nucleotide at that position. Prior to sequencing, the DNA is amplified by emulsion PCR. The resulting beads, each containing only copies of the same DNA molecule, are deposited on a slide. As a second example, the Polonator by Dover Systems (Salem, New Hampshire) TM The G.007 platform also employs a sequencing-by-ligation approach by using randomly arrayed bead-based emulsion PCR to amplify DNA fragments for parallel sequencing;

[0132] 4) For example, the PacBio RS system from Pacific Biosciences (Menlo Park, California) or the HeliScope from Helicos Biosciences (Cambridge, Massachusetts) TM Single-molecule sequencing technology implemented in the platform. The unique feature of this technology is its ability to sequence single DNA or RNA molecules without amplification, which is defined as single-molecule real-time (SMRT) DNA sequencing. For example, HeliScope uses a highly sensitive fluorescence detection system to detect each nucleotide directly as it is synthesized. Visigen Biotechnology (Houston, Texas) has developed a similar method based on fluorescence resonance energy transfer (FRET). Other fluorescence-based single-molecule technologies come from USGenomics (GeneEngine TM ) and Genovoxx (AnyGene TM );

[0133] 5) Nanotechnology for single-molecule sequencing, where a variety of nanostructures are used, for example, arranged on a chip to monitor the movement of polymerase molecules on a single strand during replication. Some non-limiting examples of nanotechnology-based methods are GridON from Oxford Nanopore Technologies (Oxford, UK). TM platform, hybridization-assisted nanopore sequencing (HANS) developed by Nabsys (Providence, Rhode Island) TM ) platform, and a combination probe-anchor ligation (cPAL TM )'s proprietary ligase-based DNA sequencing platform with DNA nanoball (DNB) technology;

[0134] 6) Electron microscopy-based techniques for single-molecule sequencing, such as those developed by LightSpeed ​​Genomics (Sunnyvale, California) and Halcyon Molecular (Redwood City, California);

[0135] 7) Ion semiconductor sequencing based on detecting hydrogen ions released during DNA polymerization. For example, the Ion Torrent System (San Francisco, California) uses a high-density array of micromachined holes to perform this biochemical process in a massively parallel manner. Each hole accommodates a different DNA template. Below the hole is an ion-sensitive layer and below that is a proprietary ion sensor.

[0136] RNA preparations can be used as starting materials for NGS. Such nucleic acids can be easily obtained from samples of, for example, biological materials, such as fresh, rapidly frozen or formalin-fixed paraffin-embedded tumor tissue (FFPE) or from freshly isolated cells or from CTCs present in the peripheral blood of patients.

[0137] According to the present invention, a high-throughput whole genome single cell genotyping method can be applied. Such a method may include the following steps:

[0138] 1. Obtain a tumor specimen from a given patient.

[0139] 2. Extract the transcriptome (RNA) from the tumor cells, convert it to cDNA and sequence it to determine the amount of transcripts expressed by the tumor cells.

[0140] 3. Identification of over-regulated RNA transcripts.

[0141] vaccine

[0142] The vaccines for cancer treatment described herein are characterized in that at least one epitope is derived from an amino acid sequence encoded by one or more over-regulated RNA transcripts. In one embodiment, the vaccine comprises a polypeptide containing one or more epitopes encoded by one or more over-regulated RNA transcripts. In one embodiment, the vaccine comprises a nucleic acid (particularly RNA) encoding a polypeptide containing one or more epitopes encoded by one or more over-regulated RNA transcripts.

[0143] Therefore, for vaccination, one or more epitopes of the over-regulated RNA transcript are provided to the patient in the form of a polypeptide comprising the one or more epitopes or a nucleic acid (particularly RNA) encoding the polypeptide. Such a polypeptide may be a single epitope or a multi-epitope. In addition, such a polypeptide may correspond to a tumor-associated antigen expressed by an over-regulated RNA transcript, or may be a recombinant polypeptide, such as a polypeptide comprising an epitope derived from one or more tumor-associated antigens expressed by an over-regulated RNA transcript. The nucleic acid may be translated in the patient's cells, particularly antigen presenting cells, to produce a polypeptide. After the cell appropriately processes the polypeptide, the epitope is presented by the MHC and displayed to the patient's immune system for stimulating appropriate T cells.

[0144] The vaccines provided according to the methods of the present invention relate to vaccines that, when administered to a patient, preferably provide a collection of MHC presented epitopes, such as 2 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, and 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, derived from amino acid sequences encoded by over-upregulated RNA transcripts. Presentation of these epitopes by the patient's cells, particularly antigen presenting cells, preferably results in T cells targeting the epitopes when bound to MHC, and thus targeting the patient's tumor, preferably primary tumor as well as tumor metastasis, expressing the antigen from which the MHC presented epitope is derived and presenting the same epitope on the surface of tumor cells.

[0145] For providing a vaccine, the method of the present invention may comprise optionally including in the vaccine a sufficient number of epitopes encoded by one or more cancer-specific over-regulated RNA transcripts (preferably in the form of encoding nucleic acids), or it may comprise a further step of determining the usability of over-regulated RNA transcripts and / or epitopes for cancer vaccination. Thus, further steps may include one or more of the following: (i) sorting or ranking the over-upregulated RNA transcripts, e.g. according to the level of over-upregulation; generally, the more over-upregulated RNA transcripts are, the more suitable they are for providing a vaccine; (ii) assessing whether the amino acid sequence encoded by the over-upregulated RNA transcripts comprises a known or predicted MHC-presenting epitope, (iii) testing in vitro and / or in silico whether the amino acid sequence encoded by the over-upregulated RNA transcripts comprises an MHC-presenting epitope, e.g. testing whether the amino acid sequence encoded by the over-upregulated RNA transcripts comprises a sequence that is processed into an MHC-presenting epitope and / or is presented as an MHC-presenting epitope, and (iv) testing in vitro whether the envisioned epitope is capable of stimulating T cells of a patient with the desired specificity, particularly when present in its natural sequence context, e.g. when flanked by amino acid sequences that also flank the epitope in naturally occurring proteins, and when expressed in antigen presenting cells. Such flanking sequences may each comprise 3 or more, 5 or more, 10 or more, 15 or more, 20 or more and preferably up to 50, up to 45, up to 40, up to 35 or up to 30 amino acids and may flank the epitope sequence at the N-terminus and / or C-terminus.

[0146] The step of identifying potentially immunogenic epitopes may comprise determining and / or ranking the epitopes according to their predicted MHC binding capacity, preferably MHC class I binding capacity.

[0147] The epitope set identified according to the present invention and provided by the vaccine of the present invention is preferably present in the form of a polypeptide (multi-epitope or multi-epitope polypeptide) comprising the epitope or a nucleic acid (particularly RNA) encoding the polypeptide. In certain embodiments of the present disclosure, the polypeptide comprises at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes or at least ten epitopes derived from the same or different tumor-associated antigens encoded by the over-regulated RNA transcript. These epitopes can be present in the polypeptide in the form of vaccine sequences, i.e., in its natural sequence environment, for example, flanked by amino acid sequences that also flank the epitopes in naturally occurring proteins. Such flanking sequences can each contain 3 or more, 5 or more, 10 or more, 15 or more, 20 or more and preferably up to 50, up to 45, up to 40, up to 35 or up to 30 amino acids, and can be flanked by epitope sequences at the N-terminus and / or C-terminus. Thus, the vaccine sequence may comprise 20 or more, 25 or more, 30 or more, 35 or more, 40 or more and 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 is arranged head to tail in the polypeptide.

[0148] In a particularly preferred embodiment, the multi-epitope or multi-epitope polypeptide according to the invention is administered to a patient in the form of a nucleic acid (preferably RNA, such as in vitro transcribed or synthetic RNA), which can be expressed in the patient's cells, such as antigen presenting cells, to produce the polypeptide.

[0149] In one embodiment, epitope and / or vaccine sequence are separated by a joint (particularly a neutral joint). According to the term "joint" of the present invention, it is related to a peptide added between two peptide domains (e.g., epitope or vaccine sequence) to connect the peptide domains. There are no particular restrictions on the joint sequence. However, preferably, the joint sequence reduces the steric hindrance between the two peptide domains, is well translated and supports or allows epitope processing. In addition, the joint should not have or only have very few immunogenic sequence elements. The joint should preferably not produce non-endogenous epitopes, such as those produced by the junction seam (junction suture) between adjacent epitopes, which may produce an unwanted immune response. Therefore, a multi-epitope vaccine should preferably include a joint sequence that can reduce the number of unwanted MHC-bound junction epitopes. Hoyt et al. (EMBO J. 25 (8), 1720-9, 2006) and Zhang et al. (J. Biol. Chem., 279 (10), 8635-41, 2004) have shown that glycine-rich sequences impair proteasomal processing, and therefore use of glycine-rich linker sequences minimizes the number of linker-containing peptides that can be processed by the proteasome. In addition, glycine was observed to inhibit strong binding in the MHC binding groove position (Abastado et al., J. Immunol. 151 (7), 3569-75, 1993). Schlessinger et al. (Proteins, 61 (1), 115-26, 2005) have found that inclusion of the amino acids glycine and serine in an amino acid sequence results in more efficiently translated and more flexible proteins that are processed by the proteasome, thereby enabling better access to the encoded epitope. Each of the linkers may comprise 3 or more, 6 or more, 9 or more, 10 or more, 15 or more, 20 or more and preferably at most 50, at most 45, at most 40, at most 35 or at most 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 consists essentially of the amino acids glycine and serine. In one embodiment, the linker comprises the amino acid sequence (GGS) a (GSS) b (GGG) c (SSG) d (GSG) e, wherein a, b, c, d and e are independently a number 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 wherein a+b+c+d+e is different from 0 and is preferably 2 or more, 3 or more, 4 or more or 5 or more. In one embodiment, the linker comprises the sequence GGSGGGGSG.

[0150] In another embodiment of the invention, the set of epitopes identified according to the invention and provided by the vaccine of the invention is preferably present in the form of a set of polypeptides comprising said epitopes on different polypeptides, wherein each of said polypeptides comprises one or more epitopes, which may also be overlapping or a set of nucleic acids (particularly RNA) encoding said polypeptides. In the case of administering more than one multi-epitope and / or multi-epitope polypeptides, the epitopes provided by different polypeptides may be different or partially overlapping.

[0151] Once present in the patient's cells, such as antigen presenting cells, the polypeptide according to the present invention is processed to produce the epitope identified according to the present invention. The use of the vaccine provided according to the present invention can provide class II MHC presented epitopes, which can induce CD4+ helper T cell responses for cells expressing the antigen from which the MHC presented epitopes are derived. As an alternative or supplement, the use of the vaccine provided according to the present invention can provide class I MHC presented epitopes, which can induce CD8+ T cell responses for cells expressing the antigen from which the MHC presented epitopes are derived. Preferably, the vaccine provided according to the present invention can be used for multi-epitope stimulation of cytotoxicity and / or helper T cell responses.

[0152] The polypeptides comprising one or more epitopes for vaccination described herein or nucleic acids encoding the polypeptides (particularly RNA) preferably cause stimulation, sensitization and / or amplification of T cells in the subject of administering the polypeptides or nucleic acids. The T cells stimulated, sensitized and / or amplified are preferably directed against target antigens, particularly target antigens expressed by cancer cells, tissues and / or organs, i.e., tumor-associated antigens expressed by over-regulated RNA transcripts. Therefore, the polypeptides comprising one or more epitopes may comprise tumor-associated antigens or fragments thereof (e.g., epitopes or vaccine sequences), or may comprise variants of tumor-associated antigens or fragments thereof. In one embodiment, such variants are immunologically equivalent to tumor-associated antigens or fragments. In the context of the present disclosure, the term "variants of tumor-associated antigens or fragments thereof" means sequences that cause stimulation, sensitization and / or amplification of T cells, and the T cells stimulated, sensitized and / or amplified target tumor-associated antigens, particularly when presented by diseased cells, tissues and / or organs. Thus, a polypeptide comprising one or more epitopes may correspond to or may comprise a tumor-associated antigen, may correspond to or may comprise a fragment of a tumor-associated antigen, or may correspond to or may comprise an amino acid sequence homologous to a tumor-associated antigen or a fragment thereof. If a polypeptide comprising one or more epitopes comprises a fragment of a tumor-associated antigen or an amino acid sequence homologous to a fragment of a tumor-associated antigen, the fragment or amino acid sequence may comprise an epitope (e.g., a T cell epitope of a tumor-associated antigen) or a sequence homologous to an epitope (e.g., a T cell epitope of a tumor-associated antigen). Thus, according to the present disclosure, a polypeptide comprising one or more epitopes may comprise an immunogenic fragment of a tumor-associated antigen encoded by an over-upregulated RNA transcript or an amino acid sequence homologous to an immunogenic fragment of a tumor-associated antigen encoded by an over-upregulated RNA transcript. An "immunogenic fragment of an antigen" according to the present disclosure preferably relates to a fragment of an antigen capable of stimulating, sensitizing and / or amplifying T cells when present in the presence of an MHC molecule. Preferably, a polypeptide comprising one or more epitopes (similar to a tumor associated antigen) can be presented by a cell such as an antigen presenting cell, thereby providing the relevant epitopes for binding by a T cell.

[0153] The term "immunologically equivalent" means, for example, immunologically equivalent molecules, such as immunologically equivalent amino acid sequences, that exhibit the same or substantially the same immunological properties and / or exert the same or substantially the same immunological effects, for example with respect to the type of immune effector. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effects or properties of an antigen or antigenic variant for immunization. For example, if an amino acid sequence induces an immune response with specificity that reacts with a reference amino acid sequence when exposed to the immune system of a subject (e.g., a T cell that binds to a reference amino acid sequence or a cell that expresses a reference amino acid sequence), then the amino acid sequence is immunologically equivalent to the reference amino acid sequence. Therefore, a molecule that is immunologically equivalent to an antigen exhibits the same or substantially the same properties and / or exerts the same or substantially the same effect as the antigen targeted by the T cell in terms of stimulation, sensitization and / or amplification of T cells.

[0154] In one embodiment, the vaccine described herein comprises one or more tumor-associated antigens encoded by one or more over-regulated RNA transcripts or one or more nucleic acids encoding one or more tumor-associated antigens, particularly one or more RNAs. In one embodiment, the vaccine described herein comprises two or more tumor-associated antigens encoded by over-regulated RNA transcripts or two or more nucleic acids encoding tumor-associated antigens, particularly two or more RNAs. In different embodiments, two or more include 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more. Two or more tumor-associated antigens or nucleic acids may be present in a mixture, or may be present in a vaccine separately from each other, and therefore may be administered separately from each other, for example, at different time points and / or administered to patients by different routes.

[0155] The term "priming" refers to the process in which a T cell first comes into contact with its specific antigen and results in differentiation into an effector T cell.

[0156] The term "clonal expansion" or "amplification" refers to a process in which a specific entity is multiplied. In the context of the present disclosure, the term is preferably used in the context of an immune response in which lymphocytes are stimulated by an antigen, proliferate, and amplify specific lymphocytes that recognize the antigen. Preferably, clonal expansion results in differentiation of lymphocytes.

[0157] The peptide or polypeptide encoded by the over-upregulated RNA transcript is also referred to herein as a "tumor-associated antigen" or "tumor antigen". In one embodiment, the peptide or polypeptide encoded by the over-upregulated RNA transcript can be a "standard" antigen, which has no difference in amino acid sequence between cancer tissue and healthy tissue. Alternatively or in addition, the peptide or polypeptide encoded by the over-upregulated RNA transcript can be a "new antigen", which is specific to an individual's tumor and has not been previously recognized by the immune system. The new antigen or new epitope can be caused by one or more cancer-specific mutations in the cancer cell genome that cause amino acid changes. However, for the purposes of the present disclosure, the over-upregulated RNA transcript encoding the standard antigen and the corresponding over-upregulated RNA transcript encoding the new antigen are considered to be the same transcript and both contribute to the amount or copy number of the over-upregulated RNA transcript.

[0158] Cancer mutations vary from individual to individual. Therefore, cancer mutations encoding new epitopes (neoepitopes) represent attractive targets in the development of vaccine compositions and immunotherapies. The efficacy of tumor immunotherapy depends on the selection of cancer-specific antigens and epitopes that can induce a potent immune response in the host.

[0159] The term "mutation" refers to a change or difference (nucleotide substitution, addition or deletion) in a nucleic acid sequence compared to a reference. "Somatic mutations" can occur in any cell of the body except germ cells (sperm and eggs) and are therefore not passed on to children. These changes can (but not always) cause cancer or other diseases. Preferably, the mutation is a non-synonymous mutation. The term "non-synonymous mutation" refers to a mutation that results in an amino acid change (e.g., an amino acid substitution) in a translation product, preferably a nucleotide substitution.

[0160] According to the present invention, the term "mutation" includes point mutation, indel, fusion, chromothripsis and RNA editing.

[0161] According to the present invention, the term "indel" describes a special class of mutations, which is defined as mutations that result in co-localized insertions and deletions and a net gain or loss of nucleotides. In the coding regions of the genome, unless the length of the indel is a multiple of 3, it produces a frameshift mutation. Indels can be contrasted with point mutations, where indels insert and delete nucleotides from a sequence, and point mutations are substitution forms that replace one nucleotide.

[0162] Fusion can produce a hybrid gene formed by two previously separated genes. It can occur due to translocation, interstitial deletion or chromosomal inversion. Generally speaking, fusion genes are oncogenes. Oncogenic fusion genes can lead to gene products with new functions or different functions from two fusion partners. Alternatively, the proto-oncogene is fused with a strong promoter, and the oncogenic function is thus set to play a role by the upregulation caused by the strong promoter of the upstream fusion partner. Oncogenic fusion transcripts can also be produced by trans-splicing or read-through events.

[0163] According to the present invention, the term "chromothripsis" refers to the genetic phenomenon whereby a specific region of the genome is broken apart and then pieced together by a single devastating event.

[0164] According to the present invention, the term "RNA editing" refers to a molecular process in which the information content in an RNA molecule is altered by chemical changes in base composition. RNA editing includes nucleoside modifications, such as deamination of cytidine (C) to uridine (U) and adenosine (A) to inosine (I), as well as non-templated nucleotide additions and insertions. RNA editing in mRNA effectively changes the amino acid sequence of the encoded protein so that it is different from that predicted by the genomic DNA sequence.

[0165] The term "antigen" refers to a substance comprising an epitope to which an immune response can be generated. In particular, the term "antigen" includes proteins and peptides. In one embodiment, the antigen is presented by cells of the immune system (e.g., antigen presenting cells such as dendritic cells or macrophages). In one embodiment, an antigen or its processed product, such as a T cell epitope, binds to an antibody through a T or B cell receptor or through an immunoglobulin molecule such as an antibody. Therefore, an antigen or its processed product can react specifically with an antibody or a T lymphocyte (T cell). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, and the epitope is derived from such an antigen.

[0166] The term "tumor-associated antigen" is used in its broadest sense to refer to any antigen associated with a tumor or cancer. Tumor-associated antigens are preferably encoded by over-regulated RNA transcripts and may be molecules that contain 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 tumor or cancer. Therefore, tumor-associated antigens or epitopes thereof may be used for therapeutic purposes.

[0167] The term "epitope" refers to a part or fragment of a molecule (e.g., an antigen) that is recognized by the immune system. For example, an epitope can be recognized by a T cell, a B cell, or an antibody. The epitope of an antigen can comprise a continuous or discontinuous portion of the antigen, and the length can be from about 5 to about 100, for example, from about 5 to about 50, more preferably from about 8 to about 30, most preferably from about 10 to about 25 amino acids, for example, the length of the epitope can be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In one embodiment, the length of the epitope is from about 10 to about 25 amino acids. The term "epitope" comprises a T cell epitope.

[0168] As used herein, the term "neo-epitope" refers to an epitope that is not present in a reference, such as a normal non-cancerous cell or germline cell, but is present in a cancer cell. This particularly includes situations where the corresponding epitope is found in a normal non-cancerous cell or germline cell, however, due to one or more mutations in the cancer cell, the sequence of the epitope is altered so as to generate a neo-epitope.

[0169] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of an MHC molecule.

[0170] The term "major histocompatibility complex" and the abbreviation "MHC" include class I MHC and class II MHC molecules, and relate to gene complexes present in all vertebrates. MHC proteins or molecules are important for signal transduction between lymphocytes and antigen presenting cells or diseased cells in immune responses, wherein MHC proteins or molecules bind peptide epitopes and present them to be recognized by T cell receptors on T cells. Proteins encoded by MHC are expressed on the cell surface, and both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) are displayed to T cells. In the case of class I MHC / peptide complexes, binding peptides are generally about 8 to about 10 amino acids long, although longer or shorter peptides may be effective. In the case of class II MHC / peptide complexes, binding peptides are generally about 10 to about 25 amino acids long, and particularly about 13 to about 18 amino acids long, although longer or shorter peptides may be effective.

[0171] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+T cells) and cytotoxic T cells (CTL, CD8+T cells) including cell lytic T cells. The term "antigen-specific T cell" or similar terms relate to T cells that recognize antigens targeted by T cells, particularly when presented on the surface of antigen presenting cells or diseased cells (e.g., cancer cells) in the case of MHC molecules, and preferably play the effector function of T cells. If T cells kill target cells expressing antigens, it is believed that T cells have specificity for antigens. Any of a variety of standard techniques can be used to evaluate T cell specificity, for example, in chromium release analysis or proliferation assays. Alternatively, the synthesis of lymphokines (e.g., interferon-γ) can be measured.

[0172] For amino acid sequences (peptides or proteins), "fragment" refers to a part of the amino acid sequence, i.e., a sequence representing an amino acid sequence shortened at the N-terminus and / or the C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains a start codon for initiating translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50 or at least 100 consecutive amino acids from the amino acid sequence.

[0173] For the purposes of this disclosure, "variants" of an amino acid sequence (peptide or protein) include amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. The term "variant" includes all splice variants, post-translationally modified variants, conformers, isomers and species homologs, particularly those naturally expressed by cells.

[0174] Amino acid insertion variants include the insertion of a single or two or more amino acids in a specific amino acid sequence. In the case of an amino acid sequence variant with insertion, one or more amino acid residues are inserted into a specific site in the amino acid sequence, although random insertion and suitable screening of the resulting product are also possible. Amino acid addition variants include one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids of amino and / or carboxyl terminal fusions. Amino acid deletion variants are characterized by removing one or more amino acids from the sequence, for example, removing 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion can be in any position of the protein. Amino acid deletion variants comprising a deletion at the N-terminal and / or C-terminal end of the protein are also referred to as N-terminal and / or C-terminal truncated variants. Amino acid substitution variants are characterized by removing at least one residue in the sequence and inserting another residue in its position. It is preferred to consider modifications in positions in non-conserved amino acid sequences between homologous proteins or peptides and / or consider replacing amino acids with other amino acids with similar properties. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., replacements of similar charged or uncharged amino acids. Conservative amino acid changes involve replacements of one of the amino acid families that are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid); basic amino acids (lysine, arginine, histidine); non-polar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes collectively classified as aromatic amino acids.

[0175] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence will be at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the full length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180 or about 200 amino acids, preferably continuous amino acids. In some preferred embodiments, the degree of similarity or identity is given for the total length of the reference amino acid sequence. Comparisons for determining sequence similarity, preferably sequence identity, can use tools known in the art, preferably using optimal sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, matrix: Blosum62, gap open (Gap Open) 10.0, gap extension (Gap Extend) 0.5 to complete.

[0176] "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences.

[0177] The term "percent identity" is intended to mean the percentage of identical amino acid residues between the two sequences to be compared obtained after optimal alignment, which percentage is purely statistical, and the differences between the two sequences are randomly distributed and randomly distributed over their entire length. The sequence comparison between two amino acid sequences is conventionally performed by comparing these sequences after optimal alignment thereof, the comparison being performed by segments or by a "comparison window" in order to identify and compare local areas of sequence similarity. Optimal alignment of sequences for comparison can be produced, in addition to manual work, by the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or by computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0178] The percent identity is calculated by determining the number of identical positions between the two sequences being compared, dividing that number by the number of positions being compared, and multiplying the result by 100 in order to obtain the percent identity between the two sequences.

[0179] According to the present disclosure, homologous amino acid sequences show at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and preferably at least 95%, at least 98% or at least 99% identity of the amino acid residues.

[0180] The amino acid sequence variants described herein can be easily prepared by a skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing peptides or proteins with substitutions, additions, insertions or deletions is described in detail, for example, in Sambrook et al. (1989). In addition, the peptides and amino acid variants described herein can be easily prepared by means of known peptide synthesis techniques, for example, such as by solid phase synthesis and similar methods.

[0181] In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant that exhibits one or more functional properties that are identical or similar to the functional properties of the amino acid sequence from which the fragment or variant is derived (i.e., it is functionally equivalent).

[0182] An amino acid sequence (peptide or protein) "derived from" a specified amino acid sequence (peptide or protein) refers to the source of the first amino acid sequence. Preferably, the amino acid sequence derived from a specific amino acid sequence has an amino acid sequence that is identical, substantially identical, or homologous to the specific sequence or a fragment thereof. The amino acid sequence derived from a specific amino acid sequence may be a variant of the specific sequence or a fragment thereof. For example, one of ordinary skill in the art will appreciate that epitopes suitable for use herein may be altered so that their sequence is different from the naturally occurring or native sequence from which they are derived, while retaining the desired activity of the native sequence.

[0183] RNA encoding a polypeptide comprising one or more epitopes described herein can be used to deliver epitopes derived from tumor-associated antigens encoded by over-regulated RNA transcripts to a patient. Dendritic cells (DCs) present in the spleen represent antigen presenting cells of particular interest for RNA expression of epitopes.

[0184] In one embodiment, the RNA is an in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning nucleic acids, particularly cDNA, and introducing them into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0185] In one embodiment, the RNA may have modified ribonucleotides. Some examples of modified ribonucleotides include, but are not limited to, 5-methylcytidine, pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ) or 5-methyl-uridine (m 5 U).

[0186] In some embodiments, the RNA according to the present disclosure comprises a 5'-cap. In one embodiment, the RNA of the present disclosure does not have an uncapped 5'-triphosphate. In one embodiment, the RNA may be modified by a 5'-cap analog. The term "5'-cap" refers to a structure found at the 5'-end of an mRNA molecule, and is typically composed of a guanosine nucleotide linked to the mRNA via a 5' to 5' triphosphate linkage. In one embodiment, the guanosine is methylated at position 7. Providing RNA with a 5'-cap or a 5'-cap analog can be achieved by in vitro transcription, wherein the 5'-cap is co-transcribed into the RNA chain, or can be linked to the RNA post-transcriptionally using a capping enzyme.

[0187] In some embodiments, the RNA according to the present disclosure comprises a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to a corresponding region in an RNA molecule (e.g., an mRNA molecule). An untranslated region (untranslated region, UTR) may be present at the 5' (upstream) (5'-UTR) and / or the 3' (downstream) (3'-UTR) of an open reading frame. The 5'-UTR, if present, is located at the 5' end, upstream of the start codon of the protein coding region. The 5'-UTR is located downstream of the 5'-cap (if present), for example, directly adjacent to the 5' cap. The 3'-UTR, if present, is located at the 3' end, downstream of the stop codon of the protein coding region, but the term "3'-UTR" preferably does not include a poly (A) tail. Therefore, the 3'-UTR is located upstream of the poly (A) sequence (if present), for example, directly adjacent to the poly (A) sequence.

[0188] In some embodiments, the RNA according to the present disclosure comprises a 3'-poly (A) sequence. The term "poly (A) sequence" refers to a sequence of adenylic acid (A) residues that are typically located at the 3' end of an RNA molecule. According to the present disclosure, in one embodiment, the poly (A) sequence comprises at least about 20, at least about 40, at least about 80, or at least about 100, and at most about 500, at most about 400, at most about 300, at most about 200, or at most about 150 A nucleotides, and in particular about 120 A nucleotides.

[0189] In the context of the present disclosure, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into a peptide or protein.

[0190] With respect to RNA, the term "expression" or "translation" refers to the process in the ribosomes of a cell that directs the assembly of an amino acid sequence through its strand of mRNA to produce a peptide or protein.

[0191] RNA-containing particles

[0192] The RNA to be administered may be present in particles comprising RNA and one or more components that associate with the RNA to form RNA particles. The RNA particles may comprise RNA in complex and / or encapsulated form. The particles described herein are preferably not viral particles (particularly infectious viral particles), i.e. they are not able to virally infect cells. The RNA-containing particles may be in the form of, for example, protein particles or lipid-containing particles. The term "particle-forming component" or "particle-forming substance" comprises a suitable protein or lipid. The term "particle-forming component" or "particle-forming substance" refers to any component that associates with RNA to form RNA particles.

[0193] In the context of the present disclosure, the term "particle" relates to a structured entity formed by a molecule or a molecular complex. In one embodiment, the term "particle" relates to a micrometer or nanometer sized structure, such as a compact micrometer or nanometer sized structure.

[0194] In the context of the present disclosure, the term "RNA particle" refers to a particle comprising RNA. Electrostatic interactions between positively charged molecules (e.g., polymers and lipids) and negatively charged RNA lead to complexation and spontaneous formation of RNA particles. In one embodiment, the RNA particle is a nanoparticle.

[0195] As used in this disclosure, in one embodiment, "nanoparticle" refers to particles having an average diameter suitable for intravenous administration.

[0196] Lipids, polymers or amphiphiles are typical components of RNA particle formulations.

[0197] Protein particles and lipid-containing particles suitable for delivering RNA in particle form have been described previously (e.g., Kaczmarek, JCet al., 2017, Genome Medicine 9, 60). For non-viral RNA delivery vehicles, nanoparticle encapsulation of RNA physically protects RNA from degradation and, depending on the specific chemical properties, can facilitate cellular uptake and endosomal escape. In view of its high chemical flexibility, polymers are commonly used materials for nanoparticle-based delivery. Generally speaking, cationic polymers are used to electrostatically condense negatively charged RNA into nanoparticles. These positively charged groups are generally composed of amines protonated at physiological pH (pKa ~ 7.4), which is believed to cause ion imbalance and thus endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have been applied to RNA delivery. In addition, some researchers have synthesized polymers specifically for nucleic acid delivery. In particular, poly (β-amino esters) have been widely used in nucleic acid delivery due to their ease of synthesis and biodegradability.

[0198] As used herein, "polymer" is given in its usual sense, i.e., a molecular structure comprising one or more repeating units (monomers) connected by covalent bonds. The repeating units may all be identical, or in some cases, more than one type of repeating unit may be present in the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer, such as a protein. In some cases, additional moieties may also be present in the polymer, such as targeting moieties such as those described herein.

[0199] If there are more than one type of repeating unit in a polymer, the polymer is referred to as a "copolymer". It should be understood that in any embodiment using a polymer, the polymer used may be a copolymer in some cases. The repeating units forming the copolymer may be arranged in any manner. For example, the repeating units may be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e., it comprises one or more regions (e.g., a first block) each comprising a first repeating unit, and one or more regions (e.g., a second block) each comprising a second repeating unit, etc. Block copolymers may have two (diblock copolymers), three (triblock copolymers), or more different blocks.

[0200] In certain embodiments, polymer is biocompatible. Biocompatible polymer is a polymer that does not cause significant cell death usually at moderate concentrations. In certain embodiments, biocompatible polymer is biodegradable, that is, the polymer can be chemically and / or biologically degraded in a physiological environment (e.g., in the human body). In certain embodiments, polymer can be protamine or polyethyleneimine.

[0201] RNA can be delivered to the spleen by so-called lipoplex formulations, in which RNA is combined with liposomes comprising cationic lipids and optionally additional or helper lipids to form injectable nanoparticle formulations. Liposomes can be obtained by injecting a lipid solution in ethanol into water or a suitable aqueous phase. RNA lipoplex particles can be prepared by mixing liposomes with RNA. Spleen targeting RNA lipoplex particles is described in WO 2013 / 143683, which is incorporated herein by reference. It has been found that RNA lipoplex particles with a net negative charge can be used to preferentially target spleen tissue or spleen cells, such as antigen presenting cells, particularly dendritic cells. Therefore, after administration of RNA lipoplex particles, RNA accumulation and / or RNA expression occur in the spleen. Therefore, the RNA lipoplex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, after administration of RNA lipoplex particles, there is no or substantially no RNA accumulation and / or RNA expression in the lungs and / or liver. In one embodiment, RNA accumulation and / or RNA expression occurs in antigen presenting cells (e.g., professional antigen presenting cells) in the spleen following administration of the RNA lipid complex particles. Thus, the RNA lipid complex particles of the present disclosure can be used to express RNA in such antigen presenting cells. In one embodiment, the antigen presenting cells are dendritic cells and / or macrophages.

[0202] In the context of the present disclosure, the term "RNA lipid complex particle" relates to a particle comprising a lipid (particularly a cationic lipid) and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA lead to complexation and spontaneous formation of RNA lipid complex particles. Positively charged liposomes can typically be synthesized using a cationic lipid (e.g., DOTMA) and an additional lipid (e.g., DOPE). In one embodiment, the RNA lipid complex particle is a nanoparticle.

[0203] "Cationic lipid" as used herein refers to a lipid with a net positive charge. Cationic lipids bind to negatively charged RNA through electrostatic interactions with lipid matrices. Generally speaking, cationic lipids have a lipophilic portion, such as a sterol, an acyl group, or a diacyl chain, and the head group of the lipid generally carries a positive charge. Some examples of cationic lipids include, but are not limited to: 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dimethyldioctadecyl ammonium (DDAB), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-diacyloxy-3-dimethylammonium propane, 1,2-dialkoxy-3-dimethylammonium propane, dioctadecyl dimethyl ammonium chloride (DODAC), 2,3-di-tetradecyloxy-propyl-(2-hydroxyethyl)-dimethylammonium (2,3- di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium, DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-trifluoroacetic acid propylammonium (DOSPA). Preferred are DOTMA, DOTAP, DODAC and DOSPA. In some specific embodiments, the cationic lipid is DOTMA and / or DOTAP.

[0204] Additional lipids may be incorporated to adjust the overall positive-negative charge ratio and the physical stability of the RNA lipid complex particles. In certain embodiments, the additional lipids are neutral lipids. "Neutral lipids" as used herein refer to lipids with a net charge of zero. Some examples of neutral lipids include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glyceryl-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glyceryl-3-phosphocholine (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, and cerebrosides. In some specific embodiments, additional lipids are DOPE, cholesterol, and / or DOPC.

[0205] In certain embodiments, the RNA lipid complex particle comprises both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0206] In some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In some specific embodiments, the molar ratio can be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of at least one cationic lipid to at least one additional lipid is about 2:1.

[0207] In one embodiment, the RNA lipid complex particles described herein have an average diameter of about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 to about 700 nm, about 400 to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In some specific embodiments, the average diameter of the RNA lipid complex particles is about 200nm, about 225nm, about 250nm, about 275nm, about 300nm, about 325nm, about 350nm, about 375nm, about 400nm, about 425nm, about 450nm, about 475nm, about 500nm, about 525nm, about 550nm, about 575nm, about 600nm, about 625nm, about 650nm, about 700nm, about 725nm, about 750nm, about 775nm, about 800nm, about 825nm, about 850nm, about 875nm, about 900nm, about 925nm, about 950nm, about 975nm, or about 1000nm. In one embodiment, the average diameter of the RNA lipid complex particles is about 250nm to about 700nm. In another embodiment, the average diameter of the RNA lipid complex particles is about 300 nm to about 500 nm. In an exemplary embodiment, the average diameter of the RNA lipid complex particles is about 400 nm.

[0208] The term "mean diameter" refers to the average hydrodynamic diameter of the particles as measured by dynamic light scattering (DLS) and data analysis using a so-called cumulant algorithm, the result of which provides a so-called Z having a length dimension. 平均值 and dimensionless polydispersity index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, the "average diameter", "diameter" or "size" of the particles is related to the Z 平均值 The value of is used synonymously.

[0209] The term "polydispersity index" is used herein as a measure of the size distribution of an ensemble of particles (eg nanoparticles).The polydispersity index is calculated by so-called cumulant analysis based on dynamic light scattering measurements.

[0210] The term "extrusion" refers to the production of particles with a fixed cross-sectional profile. In particular, it refers to the miniaturization of particles, thereby forcing the particles to pass through a filter with defined pores.

[0211] The charge of the RNA lipid particles of the present disclosure is the sum of the charge present in at least one cationic lipid and the charge present in the RNA. The charge ratio is the ratio of the positive charge present in at least one cationic lipid to the negative charge present in the RNA. The charge ratio of the positive charge present in at least one cationic lipid to the negative charge present in the RNA is calculated by the following equation: charge ratio=[(cationic lipid concentration (mol))*(total number of positive charges in the cationic lipid)] / [(RNA concentration (mol))*(total number of negative charges in the RNA)].

[0212] The spleen-targeted RNA lipid complex particles described herein preferably have a net negative charge at physiological pH, for example, a charge ratio of positive charge to negative charge of about 1.9:2 to about 1:2. In some specific embodiments, the charge ratio of positive charge to negative charge in the RNA lipid complex particles at physiological pH is about 1.9:2.0, about 1.8:2.0, about 1.7:2.0, about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0.

[0213] As used herein, "physiological pH" refers to a pH of about 7.5.

[0214] Pharmaceutical composition

[0215] The agents described herein can be used as or in the preparation of a pharmaceutical composition or medicament, in particular a vaccine for therapeutic or prophylactic treatment.

[0216] The term "pharmaceutical composition" relates to a formulation comprising a therapeutically effective agent, preferably a therapeutically effective agent together with a pharmaceutically acceptable carrier, diluent and / or excipient. The pharmaceutical composition can be used to treat, prevent or alleviate the severity of a disease or condition by administering the pharmaceutical composition to a subject. Pharmaceutical compositions are also referred to in the art as pharmaceutical formulations. In the context of the present disclosure, a pharmaceutical composition may comprise RNA, RNA particles and / or other agents as described herein.

[0217] According to the present invention, the term "vaccine" relates to a pharmaceutical preparation (pharmaceutical composition) or product that induces an immune response, in particular a cellular immune response, after administration, which recognizes and attacks pathogens or diseased cells such as cancer cells. Vaccines can be used to prevent or treat diseases. The term "personalized cancer vaccine" relates to a specific cancer patient and means that the cancer vaccine is adapted to the needs or special circumstances of an individual cancer patient.

[0218] The pharmaceutical compositions of the present disclosure preferably comprise one or more adjuvants or may be administered with one or more adjuvants. The term "adjuvant" relates to a compound that prolongs, enhances or accelerates an immune response. Adjuvants include heterogeneous groups of compounds such as oil emulsions (e.g., Freund's adjuvant), mineral compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immunostimulatory complexes. Some examples of adjuvants include, but are not limited to: LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, chemokines. Chemokines can be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, LT-a. Other known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils, such as ISA 51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys.

[0219] The pharmaceutical compositions according to the present disclosure are generally administered in a "pharmaceutically effective amount" and a "pharmaceutically acceptable formulation."

[0220] The term "pharmaceutically acceptable" refers to the non-toxicity of a substance that does not interact with the effects of the active ingredients of the pharmaceutical composition.

[0221] The term "pharmaceutically effective amount" refers to an amount that achieves a desired response or desired effect alone or in combination with another dose. In the case of treating a specific disease, the desired response preferably involves inhibition of the disease process. This includes slowing the progression of the disease, and in particular interrupting or reversing the progression of the disease. The desired response in the treatment of a disease may also be to delay the occurrence of the disease or the condition or to prevent its occurrence. The effective amount of the composition described herein will depend on: the condition to be treated, the severity of the disease, the individual parameters of the patient including age, physiological condition, stature and weight, duration of treatment, type of concomitant treatment (if present), specific route of administration and similar factors. Therefore, the dosage of the composition described herein may depend on a variety of such parameters. In the case of an insufficient response of the patient with the initial dose, a higher dose may be used (or an effectively higher dose may be achieved by a different, more localized route of administration).

[0222] The pharmaceutical compositions of the present disclosure may comprise salts, buffers, preservatives, and optionally other therapeutic agents.In one embodiment, the pharmaceutical compositions of the present disclosure comprise one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0223] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0224] The term "excipient" as used herein refers to a substance that may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Some examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0225] The term "diluent" refers to a diluting agent and / or a thinning agent. In addition, the term "diluent" includes any one or more of a fluid, a liquid or solid suspension and / or a mixed medium. Some examples of suitable diluents include ethanol, glycerol and water.

[0226] The term "carrier" refers to a component that can be natural, synthetic, organic, inorganic, in which the active ingredients are combined to promote, enhance or achieve the use of the pharmaceutical composition. The carrier used herein can be one or more compatible solid or liquid fillers, diluents or encapsulating materials suitable for application to the object. Suitable carriers include, but are not limited to: sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and particularly biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure comprises isotonic saline.

[0227] Pharmaceutically acceptable carriers, excipients 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. (AR Gennaro edit. 1985).

[0228] The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0229] Administration routes of the pharmaceutical composition

[0230] In one embodiment, the pharmaceutical composition described herein can be administered intravenously, intraarterially, subcutaneously, intradermally or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for topical or systemic administration. Systemic administration may include enteral administration involving absorption through the gastrointestinal tract, or parenteral administration. "Parenteral administration" as used herein refers to administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for systemic administration. In another preferred embodiment, systemic administration is administered intravenously.

[0231] In one embodiment, after administering the pharmaceutical composition described herein, at least a portion of the active agent (e.g., a polypeptide comprising one or more epitopes or a nucleic acid encoding the polypeptide, particularly RNA) is optionally delivered to a target cell in the form of RNA particles. In one embodiment, at least a portion of the RNA is delivered to the cytosol of the target cell. In one embodiment, the RNA is translated by the target cell to produce a polypeptide. In one embodiment, the target cell is a splenocyte. In one embodiment, the target cell is an antigen presenting cell, such as a professional antigen presenting cell in the spleen. In one embodiment, the target cell is a dendritic cell in the spleen. Therefore, the RNA particles described herein can be used to deliver RNA to such a target cell. Therefore, the present disclosure also relates to a method for delivering RNA to a target cell in an object, the method comprising administering the RNA particles described herein to the object. In one embodiment, RNA is delivered to the cytosol of a target cell. In one embodiment, RNA is an RNA encoding a polypeptide comprising one or more epitopes, and RNA is translated by the target cell to produce a polypeptide.

[0232] In one embodiment, the present disclosure is directed to targeting the lymphatic system, particularly secondary lymphoid organs, more particularly the spleen.

[0233] The "lymphatic system" is part of the circulatory system and is an important part of the immune system, containing a network of lymphatic vessels that transport lymph. The lymphatic system consists of lymphatic organs, a conducting network of lymphatic vessels, and circulating lymph. Primary or central lymphoid organs produce lymphocytes from immature progenitor cells. The thymus and bone marrow constitute the primary lymphoid organs. Secondary or peripheral lymphoid organs (including lymph nodes and spleen) maintain mature initial lymphocytes and initiate adaptive immune responses.

[0234] Uses of the pharmaceutical composition

[0235] The vaccines described herein may be used to treat cancer diseases therapeutically or prophylactically.

[0236] In one embodiment, the disclosure relates to a method for inducing an immune response in a subject comprising administering to the subject a vaccine as described herein. In an exemplary embodiment, the immune response is against cancer.

[0237] The term "disease" refers to an abnormal condition that affects an individual's body. Disease is generally interpreted as a medical condition associated with specific symptoms and signs. Disease can be caused by factors that are initially from an external source, such as infectious diseases, or disease can be caused by internal dysfunction, such as autoimmune diseases. In humans, "disease" is generally used more broadly to refer to any condition that causes pain, dysfunction, suffering, social problems or death in the sick individual or similar problems that come into contact with the individual. In this broader sense, disease sometimes includes injury, disability, disorder, syndrome, infection, isolated symptoms, abnormal behavior, and atypical changes in structure and function, while in other cases and for other purposes, these can be considered as distinguishable categories. Diseases usually affect individuals not only physically but also emotionally, because infection and experience with many diseases can change an individual's view of life and individual personality.

[0238] In the context of the present invention, the term "treatment" or "therapeutic intervention" relates to the management and care of a subject for the purpose of combating a condition, such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition suffered by a subject, such as the administration of a therapeutically effective compound to alleviate symptoms or complications, delay the progression of a disease, disorder or condition, alleviate or relieve symptoms and complications, and / or cure or eliminate a disease, disorder or condition as well as prevention of a condition, wherein prevention is understood as the management and care of an individual for the purpose of combating a disease, disorder or condition, and includes the administration of an active compound to prevent the onset of symptoms or complications.

[0239] The term "therapeutic treatment" refers to any treatment that improves health and / or prolongs (increases) the life span of an individual. The treatment may eliminate the disease in an individual, prevent or slow the onset of the disease in an individual, inhibit or slow the onset of the disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual currently suffering from or previously suffering from the disease.

[0240] The term "prophylactic treatment" or "preventive therapy" relates to any treatment intended to prevent the occurrence of a disease in an individual. The terms "prophylactic treatment" or "preventive treatment" are used interchangeably herein.

[0241] The terms "individual" and "subject" are used interchangeably herein. They refer to humans or other mammals (e.g., mice, rats, rabbits, dogs, cats, cattle, pigs, sheep, horses, or primates) that may be afflicted with a disease or condition (e.g., cancer) or are susceptible to a disease or condition (e.g., cancer) but may or may not be suffering from a disease or condition. In many embodiments, the individual is a human. Unless otherwise indicated, the terms "individual" and "subject" do not indicate a specific age, and therefore encompass adults, the elderly, children, and newborns. In some embodiments of the present disclosure, an "individual" or "subject" is a "patient".

[0242] The term "patient" refers to an individual or subject being treated, particularly an individual or subject who is ill.

[0243] In one embodiment of the present disclosure, the goal is to provide an immune response against diseased cells expressing antigens, such as cancer cells expressing tumor antigens, and treat diseases, such as cancer diseases, involving cells expressing antigens, such as tumor antigens.

[0244] Vaccines comprising polypeptides containing one or more epitopes encoded by one or more over-regulated RNA transcripts or nucleic acids encoding the polypeptides as described herein can be administered to a subject to induce an immune response in the subject to the one or more epitopes, which can be therapeutic or partially or completely protective. Those skilled in the art will know that one of the principles of immunotherapy and vaccination is based on the fact that an immunoprotective response to a disease is generated by immunizing a subject with an antigen or epitope, which is immunologically related to the disease to be treated. Therefore, the pharmaceutical compositions described herein can be used to induce or enhance an immune response. Therefore, the pharmaceutical compositions described herein can be used for preventive and / or therapeutic treatment of diseases involving antigens or epitopes.

[0245] As used herein, "immune response" refers to an integrated body response to an antigen or a cell expressing an antigen, and refers to a cellular immune response and / or a humoral immune response. A cellular immune response includes, but is not limited to, a cellular response to cells expressing an antigen and characterized by presenting the antigen with a class I or class II MHC molecule. Cellular responses are associated with T lymphocytes, which can be classified as helper T cells (also known as CD4+T cells), which play a major role in inducing apoptosis in infected cells or cancer cells by regulating immune responses or killing cells (also known as cytotoxic T cells, CD8+T cells or CTLs). In one embodiment, administering a pharmaceutical composition of the present disclosure involves stimulating an anti-tumor CD8+T cell response against cancer cells expressing one or more tumor antigens. In a specific embodiment, the tumor antigen is presented together with a class I MHC molecule.

[0246] The present disclosure contemplates an immune response that may be protective, preventative, prophylactic and / or therapeutic. As used herein, "inducing an immune response" may indicate that there was no immune response to a particular antigen prior to induction, or may indicate that there was a basal level of immune response to a particular antigen prior to induction that was enhanced after induction. Thus, "inducing an immune response" includes "enhancing an immune response."

[0247] The term "immunotherapy" relates to the treatment of a disease or condition by inducing or enhancing an immune response. The term "immunotherapy" includes antigen immunization or antigen vaccination.

[0248] The terms "immunization" or "vaccination" describe the process of administering an antigen to an individual with the purpose of inducing an immune response, for example for therapeutic or prophylactic reasons.

[0249] In one embodiment, the present disclosure contemplates such an embodiment, wherein the RNA particles described herein that target spleen tissue are administered. The RNA encodes a polypeptide comprising one or more epitopes encoded by, for example, one or more over-regulated RNA transcripts described herein. The RNA is taken up by antigen presenting cells (e.g., dendritic cells) in the spleen to express the polypeptide. After optional processing and presentation by the antigen presenting cells, an immune response to one or more epitopes can be generated, resulting in preventive and / or therapeutic treatment of cancer. In one embodiment, the immune response induced by the RNA particles described herein includes presentation of one or more epitopes by antigen presenting cells (e.g., dendritic cells and / or macrophages), and activation of cytotoxic T cells caused by the presentation. For example, the polypeptide encoded by RNA or its processed product can be presented by a major histocompatibility complex (MHC) protein expressed on an antigen presenting cell. Then, the MHC peptide complex can be recognized by immune cells (e.g., T cells or B cells), resulting in its activation.

[0250] Therefore, in one embodiment, after administration, the RNA in the RNA particles described herein is delivered to the spleen and / or expressed in the spleen. In one embodiment, the RNA particles are delivered to the spleen to activate spleen antigen presenting cells. Therefore, in one embodiment, after administration of the RNA particles, RNA delivery and / or RNA expression occur in antigen presenting cells. Antigen presenting cells can be professional antigen presenting cells or non-professional antigen presenting cells. Professional antigen presenting cells can be dendritic cells and / or macrophages, and even more preferably spleen dendritic cells and / or spleen macrophages.

[0251] Therefore, the present disclosure relates to the RNA particles described herein or pharmaceutical compositions comprising the RNA particles, for use in inducing or enhancing an immune response against cancer.

[0252] In another embodiment, the present disclosure relates to the RNA particles described herein or pharmaceutical compositions comprising the RNA particles for use in the prophylactic and / or therapeutic treatment of cancer diseases.

[0253] In another embodiment, the disclosure relates to a method for delivering one or more epitopes to antigen presenting cells (e.g., professional antigen presenting cells) in the spleen, or expressing one or more epitopes in antigen presenting cells (e.g., professional antigen presenting cells) in the spleen, comprising administering to a subject an RNA particle described herein or a pharmaceutical composition comprising the RNA particle. The one or more epitopes are preferably encoded by the RNA in the RNA particle.

[0254] In one embodiment, systemic administration of RNA particles described herein or pharmaceutical compositions comprising RNA particles results in targeting and / or accumulation of RNA particles or RNA in the spleen but not in the lungs and / or liver. In one embodiment, RNA particles release RNA in the spleen and / or enter cells in the spleen. In one embodiment, systemic administration of RNA particles described herein or pharmaceutical compositions comprising RNA particles delivers RNA to antigen presenting cells in the spleen. In a specific embodiment, the antigen presenting cells in the spleen are dendritic cells or macrophages.

[0255] In another embodiment, the disclosure relates to a method for inducing or enhancing an immune response against cancer in a subject, comprising administering to the subject an RNA particle described herein or a pharmaceutical composition comprising the RNA particle.

[0256] The term "macrophage" refers to a subgroup of phagocytes produced by differentiation of monocytes. Macrophages activated by inflammation, immune cytokines or microbial products nonspecifically engulf and kill foreign pathogens by hydrolysis and oxidation attacks in macrophages, thereby causing degradation of pathogens. Peptides from degraded proteins are displayed on the macrophage surface, where they can be recognized by T cells, and they can directly interact with antibodies on the B cell surface, thereby causing T cells and B cells to activate and further stimulate an immune response. Macrophages belong to the category of antigen presenting cells. In one embodiment, the macrophage is a spleen macrophage.

[0257] The term "dendritic cell" (DC) refers to another subtype of phagocytes, which belongs to the category of antigen presenting cells. In one embodiment, dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells are initially converted into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation potential. Immature dendritic cells constantly sample pathogens in the surrounding environment, such as viruses and bacteria. Once it contacts with presentable antigens, it is activated to become a mature dendritic cell and begins to migrate to the spleen or lymph nodes. Immature dendritic cells engulf pathogens and degrade their proteins into small pieces (piece), and after maturation, these fragments are presented at their cell surfaces using MHC molecules. At the same time, it has raised cell surface receptors that act as co-receptors in T cell activation, such as CD80, CD86 and CD40, which greatly enhances its ability to activate T cells. It also raises CCR7, which is a chemotactic receptor that induces dendritic cells to reach the spleen through the bloodstream, or to reach lymph nodes through the lymphatic system. Here it acts as an antigen presenting cell and activates helper T cells and killer T cells and B cells by presenting its antigen together with non-antigen specific co-stimulatory signals. Therefore, dendritic cells can actively induce T cell or B cell related immune responses. In one embodiment, the dendritic cell is a spleen dendritic cell.

[0258] The term "antigen presenting cell" (APC) is a cell among a variety of cells that is capable of displaying, acquiring and / or presenting at least one antigen or antigenic fragment on its cell surface (or at its surface). Antigen presenting cells can be divided into professional antigen presenting cells and non-professional antigen presenting cells.

[0259] The term "professional antigen presenting cells" refers to antigen presenting cells that constitutively express major histocompatibility complex class II (class II MHC) molecules required for interaction with naive T cells. If T cells interact with class II MHC molecule complexes on the antigen presenting cell membrane, the antigen presenting cells produce co-stimulatory molecules that induce T cell activation. Professional antigen presenting cells include dendritic cells and macrophages.

[0260] The term "non-professional antigen presenting cells" refers to antigen presenting cells that do not constitutively express class II MHC molecules, but constitutively express class II MHC molecules after being stimulated by certain cytokines such as interferon-γ. Some exemplary non-professional antigen presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic β cells or vascular endothelial cells.

[0261] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of the antigen (e.g., degradation of a protein into peptides), and to the association (e.g., by binding) of one or more of these fragments with an MHC molecule for presentation by a cell, such as an antigen presenting cell, to a specific T cell.

[0262] The term "antigen-related disease" or "epitope-related disease" refers to any disease involving an antigen or epitope, such as a disease characterized by the presence of an antigen or epitope. The antigen- or epitope-related disease may be a cancer disease or simply cancer. As described above, the antigen may be a disease-associated antigen, such as a tumor-associated antigen.

[0263] The term "cancer disease" or "cancer" refers to or describes a physiological condition in an individual that is usually characterized by unregulated cell growth. Some examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularly, some examples of such cancers include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, sex and reproductive organ cancer, Hodgkin's Disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) neoplasms, neuroectodermal cancer, spinal axis tumors, gliomas, meningiomas, and pituitary adenomas. The term "cancer" according to the present disclosure also includes cancer metastasis. Generally speaking, the terms "tumor" and "cancer" are used interchangeably herein.

[0264] The term "circulating tumor cell" or "CTC" refers to cells that break away from a primary tumor or tumor metastasis and circulate in the bloodstream. CTCs can constitute the seeds for the subsequent growth of additional tumors (metastasis) in different tissues. Circulating tumor cells are present in patients with metastatic disease at a frequency of about 1 to 10 CTC / mL of whole blood. Research methods have been developed to isolate CTCs. Several research methods have been described in the art to isolate CTCs, such as techniques that use the fact that epithelial cells typically 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 to EpCAM that has been conjugated to magnetic particles, followed by separation of the labeled cells in a magnetic field. The isolated cells are then stained with antibodies to another epithelial marker, cytokeratin, and the common leukocyte marker CD45, so that rare CTCs are distinguished from contaminating leukocytes. This robust and semi-automated method identifies CTCs with an average yield of about 1 CTC / mL and a purity of 0.1% (Allard et al., 2004: Clin Cancer Res 10, 6897-6904). The second method for isolating CTCs uses a microfluidics-based CTC capture device, which involves flowing whole blood through a chamber embedded with 80,000 microcolumns, which are functionalized by coating with antibodies against EpCAM. CTCs are then stained with secondary antibodies against cytokeratins or tissue-specific markers (e.g., PSA in prostate cancer or HER2 in breast cancer) and visualized by automatically scanning the microcolumns in multiple planes along three-dimensional coordinates. The CTC chip is able to identify cytokeratin-positive circulating tumor cells in patients with a median yield of 50 cells / ml and a purity of 1% to 80% (Nagrath et al., 2007: Nature 450, 1235-1239). Another possibility for isolating CTCs is to use the CellSearch from Veridex, LLC (Raritan, NJ). TM Circulating Tumor Cell (CTC) test, which captures, identifies and counts CTCs in blood vessels. CellSearch TM The TRANSPORT(R) system is a method approved by the US Food and Drug Administration (FDA) for enumeration of CTCs in whole blood, which is based on a combination of immunomagnetic labeling and automated digital microscopy. There are other methods described in the literature for isolating CTCs, all of which can be used in conjunction with the present invention.

[0265] Due to the synergistic effects produced, combination strategies in cancer treatment can be ideal, which can be much stronger than the effects of a single treatment method. In one embodiment, the pharmaceutical composition is administered together with an immunotherapeutic agent. "Immunotherapeutic agent" as used herein refers to any agent that can participate in activating a specific immune response and / or immune effector function. The present disclosure contemplates the use of antibodies as immunotherapeutic agents. Without wishing to be bound by theory, antibodies can achieve therapeutic effects against cancer cells through a variety of mechanisms, including inducing apoptosis, blocking components of signal transduction pathways, or inhibiting the proliferation of tumor cells. In certain embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies can induce cell death through antibody-dependent cell-mediated cytotoxicity (ADCC), or bind to complement proteins, resulting in direct cytotoxicity, known as complement-dependent cytotoxicity (CDC). Some non-limiting examples of anti-cancer antibodies and potential antibody targets (in parentheses) that can be used in combination with the present disclosure include: Abavolumab (CA-125), Abciximab (CD41), Adalimumab (EpCAM), Afutuzumab (CD20), Alacizumab pegol (VEGFR2), Atumotuzumab Pentetate (CEA), Amatuximab (MORAb-009), Maanatumomab (TAG-72), Apolizumab (HLA-DR), Acitumomab (CEA), Atezolizumab (PD-L1), Bavicizumab (phosphatidylserine), Betumumab (CD22), Belimumab (BAFF), Bevacizumab (VEGF-A), Bivatuzumab mertansine (CD44), v6), Blinatumomab (CD 19), Brentuximab (CD30 TNFRSF8), Mocantuzumab (Mucin CanAg), Recantuzumab (MUC1), Capromab Pendetide (Prostate Cancer Cells), Carlumab (CNT0888), Catumaxomab (EpCAM, CD3), Cetuximab (EGFR), Citatuzumab bogatox (EpCAM), Ciliatuzumab (IGF-1 Receptor), Claudiximab (Claudin), Clivatuzumab (Celiac), Tetanuscolide (CNT0888),tetraxetan (MUC1), situzumab (TRAIL-R2), daclizumab (CD40), dalotuzumab (insulin-like growth factor I receptor), denosumab (RANKL), demuximab (B-lymphoma cells), drozitumab (DR5), emeticholic acid (GD3 ganglioside), edrecolomab (EpCAM), elotuzumab (SLAMF7), enatuzumab (PDL192), ensituximab (NPC-1C), epratuzumab (CD22), ertuinomab (HER2 / neu, CD3), irrituzumab (integrin ανβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), fenclatuzumab (SCH 900105), Figitumumab (IGF-1 receptor), Flanvotumab (glycoprotein 75), Fresolimumab (TGF-β), Galiximab (CD80), Ganitatumab (IGF-I), Gemtuzumab ozogamicin (CD33), Gevokizumab (IL-Iβ), Girentuximab (Carbonic anhydrase 9 (CA-IX)), Glembatumumab vedotin (GPNMB), Ibritumomab tiuxetan (CD20), Icrucumab (VEGFR-1), Igovotumab (CA-125), Indatuximab ravtansine)(SDC1), Intetumumab(CD51), Intuzumab(CD22), Ipilimumab(CD 152), Iratumumab(CD30), Labetuzumab(CEA), Lexatumumab(TRAIL-R2), Rivimab(HBsAg), Lintuzumab(CD33), Lorvotuzumab mertansine(CD56), Rucamumab(CD40), Ruximumab(CD23), Mapatumumab(TRAIL-R1), Matuzumab(EGFR), Mepolizumab(IL-5), Milatuzumab(CD74), Mitumumab(GD3 ganglioside), Mogamulizumab(CCR4), Moxetumomabpasudotox)(CD22), tadalafil (C242 antigen), tadalafil (5T4), natumomab (RON), necitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4), ofatumumab (CD20), olaratumab (PDGF-R a), Onartuzumab (human scatter factor receptor kinase), Oportuzumab monatox (EpCAM), Ogovolumab (CA-125), Oxelumab (OX-40), Panitumumab (EGFR), Patritumab (HER3), Pemtumomab (MUC1), Pertuzumab (HER2 / neu), Pintumomab (adenocarcinoma antigen), Protumumab (vimentin), Racotumomab (N-hydroxyethyl) acylneuraminic acid), Radretumab (fibronectin extra domain-B), Ravelumab (rabies virus glycoprotein), Ramucirumab (VEGFR2), Rilotumumab (HGF), Rituximab (CD20), Robatumumab (IGF-1 receptor), Samalizumab (CD200), Ciroizumab (FAP), Siltuximab (IL-6), Tabalumab (BAFF), Tacilizumab (alpha-fetoprotein), Pertapumomab (CD 19), tenatumomab (tenascin C), teprotumumab (CD221), simumab (CTLA-4), tegazumab (TRAIL-R2), TNX-650 (IL-13), tositumomab (CD20), trastuzumab (HER2 / neu), TRBS07 (GD2), tesitumomab (CTLA-4), tucotuzumab celmoleukin (EpCAM), ubliximab (MS4A1), urelumab (4-1BB), voroxizumab (integrin α5β1), voruximab (tumor antigen CTAA 16.88), zatutumumab (EGFR), and zatutumumab (CD4).

[0266] In one embodiment, the immunotherapeutic agent is a PD-1 axis binding antagonist. PD-1 axis binding antagonists include, but are not limited to, PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists. Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PD-L1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PD-L2" include B7-DC, Btdc, and CD273. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PD-L1 and / or PD-L2. In another embodiment, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a specific embodiment, the PD-L1 binding partner is PD-1 and / or B7-1. In another embodiment, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In a specific embodiment, the PD-L2 binding partner is PD-1. The PD-1 binding antagonist can be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). Some examples of anti-PD-1 antibodies include, but are not limited to, MDX-1106 (nivolumab, OPDIVO), Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA), MEDI-0680 (AMP-514), PDR001, REGN2810, BGB-108, and BGB-A317.

[0267] In one embodiment, the PD-1 binding antagonist is an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region. In one embodiment, the PD-1 binding antagonist is AMP-224 (also known as B7-DCIg, which is PD-L2-Fc), which is a fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0268] In one embodiment, the PD-1 binding antagonist is an anti-PD-L1 antibody, including but not limited to YW243.55.S70, MPDL3280A (atezolizumab), MEDI4736 (durvalumab), MDX-1105, and MSB0010718C (avelumab).

[0269] In one embodiment, the immunotherapeutic agent is a PD-1 binding antagonist. In another embodiment, the PD-1 binding antagonist is an anti-PD-L1 antibody. In an exemplary embodiment, the anti-PD-L1 antibody is atezolizumab.

[0270] Citation of documents and studies cited herein is not intended to admit that any of the foregoing is relevant prior art. All statements regarding the contents of these documents are based on the information available to the applicant and do not constitute any admission that the contents of these documents are correct.

[0271] The following description is presented to enable one of ordinary skill in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the embodiments described herein will be apparent to one of ordinary skill in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the various embodiments. Therefore, the various embodiments are not intended to be limited to the embodiments described and illustrated herein, but are consistent with the scope of the claims.

[0272] Example

[0273] Example 1: Selection of differentially expressed genes in B16-F10 melanoma cell line

[0274] The cDNA derived from the mRNA of the melanoma cell line B16-F10 was sequenced (Castle, JC et al. Cancer Res. 72, 1081–1091 (2012)). Gene expression abundance was estimated using pseudoalignment software kallisto (Bray, NL et al. Nat. Biotech. 34, 525-527 (2016)) and reported as transcripts per million (TPM). The gene expression values ​​were then compared with a reference gene expression database of selected normal tissues treated in the same manner. The reference database includes samples from the Mouse ENCODE project (n = 487) (Pervouchine, DD et al. Nat. Comm. 6, 5903 (2015)), samples from the Mouse Transcriptomic BodyMap (n = 72) (Li, B. et al. Sci. Rep. 7 (1): 4200 (2017)), mouse samples from the Circadian Gene Expression Atlas (n = 96) (Zhang, R. et al. PNAS 111 (45): 16219-24 (2014)), and samples from mouse tissues sequenced in-house (n = 67). Samples were annotated with respect to tissue source and tissue category. Tissue categories included embryonic tissue (all samples were labeled "embryo"), reproductive tissue (cystic gland, uterus, ovary, placenta and testis) and normal tissue (all other tissues). For each unique tissue, the median expression of all samples was calculated. A B16-F10 target was pre-selected if it exceeded the cutoff c calculated as:

[0275] c=10*M+1,

[0276] where M equals the 95th percentile of the median value of all normal tissues. Finally, the list of potential targets was ranked by the fold change of the genes expressed in B16-F10 compared to the sum of the expression values ​​in normal tissues. Expression in reproductive organs as well as embryonic tissues was allowed (these so-called cancer germline antigens are only present in tumors, male germ cells, placenta or early stages of embryonic development).

[0277] Among the most differentially expressed genes in the mouse B16-F10 melanoma cell line, we found several well-known melanoma differentiation antigens ( Figure 1For example, among the top 10 genes ranked by our algorithm, we found the known immunogenic antigens tyrosinase-related protein 1 (Tyrp1, ranked 1, Wang, R. et al. J. Exp. Med. 183, 1131–40 (1996)), solute carrier family 45, member 2 (Slc45a2, ranked 2, Park, J. et al. Cancer Immunol. Res. 5, 618–629 (2017).), tyrosinase (Tyr, ranked 3, T.et al.Eur.J.Immunol.24,759–64(1994).), dopachrome tautomerase (Dct, ranked 5, Wang, RFet al.J.Exp.Med.184,2207–16(1996).), mlan-A (Mlana, ranked 6, Kawakami, Y.et al.J.Exp.Med.180,347–52(1994).) and premelanosome protein (Pmel, ranked 9, Kawakami, Y.et al.J.Immunol.154,3961–8(1995).). This impressively demonstrates the effectiveness of our algorithm in enriching relevant tumor antigens. Among the above targets, we selected Tyrp1, Tyr, Dct, Pmel and Fkbp6 for the first proof-of-concept study. Vaccination against all targets except Tyr caused a strong T cell immune response (Examples 2 to 5).

[0278] Example 2: Immunogenicity test of Tyrp1 RNA vaccine

[0279] C57BL / 6 mice (n=5 per group) were immunized with 40 μg of Tyrp1 antigen-encoding RNA formulated as a lipid complex or 40 μg of an irrelevant RNA on days 0, 7, and 14 as described in Kranz, LM et al. Nature 534, 396–401 (2016). Five days after the last vaccination, mouse splenocytes were isolated and 5×10 5 Recognition of two Tyrp1 peptides encoding MHC I epitopes (TAPDNLGYA, 2 μg / ml) and MHC II epitopes (CRPGWRGAACNQKI, 2 μg / ml) by 10 cells. Vaccination against Tyrp1 resulted in potent CD4 + and CD8 + T cell response ( Figure 2). Statistical significance was determined by one-way ANOVA and Dunett's multiple comparison test using GraphPad Prism6. ns: P>0.05, *P<0.05, **P<0.01, ***P<0.001.

[0280] Example 3: Immunogenicity test of Dct RNA vaccine

[0281] Similar to Example 2, Figure 3 As shown in (dashed line), C57BL / 6 mice (n=5 per group) were repeatedly vaccinated with 20 μg Dct encoding RNA lipoplexes or NaCl. CD8 against Dct (SVYDFFVWL) was measured in the blood by flow cytometry with MHC tetramer (MBL international) staining on days 5, 12, 19, 26 and 33 after the first vaccination. + T cell response (as described in Kranz, LM et al. Nature 534, 396–401 (2016)). By day 33, repeated vaccination resulted in a sustained increase in antigen-specific T cells, exceeding all peripheral CD8 + 4% of T cells. Statistical significance was determined by two-way ANOVA and Sidak's multiple comparison test using GraphPad Prism 6. ns: P>0.05, *P<0.05, **P<0.01, ***P<0.001.

[0282] Example 4: Immunogenicity test of Pmel RNA vaccine

[0283] As in Example 2, Figure 4 As shown in, C57BL / 6 mice (n=8 in each group) were repeatedly vaccinated with 20 μg Pmel encoding RNA lipid complexes. On the 27th day after the first vaccination, mouse spleen cells were detected for Pmel peptide (EGSRNQDWL, 2 μg / ml) or irrelevant peptide (VSV-NP, RGYVYQGL, 2 μg / ml) by IFNγELISpot. As determined by paired two-tailed student T tests, significant Pmel-specific CD8 + T cell response. ns: P>0.05, *P<0.05, **P<0.01, ***P<0.001.

[0284] Example 5: Immunogenicity test of Fkbp6 RNA vaccine

[0285] C57BL / 6 mice (n=3 per group) were immunized with 20 μg of Fkbp6 antigen-encoding RNA formulated as a lipoplex on days 0, 7, and 14 as described in Example 2. Five days after the last vaccination, splenocytes from mice were isolated and 5×10 5 The recognition of syngeneic bone marrow-derived dendritic cells (BMDCs) electroporated with 10 μg of Fkbp6 RNA or irrelevant RNA (control) was performed by culturing bone marrow-derived cells in the presence of GM-CSF (Lutz, MB et al. J. Immunol. Methods 223, 77–92 (1999)). Vaccination against Fkbp6 resulted in a potent T cell response ( Figure 5 ).

[0286] Example 6: Tumor control following therapeutic Tyrp1 vaccination

[0287] To test whether the selected vaccine targets have therapeutic significance, we used 3×10 5 C57BL / 6 mice (n=11 to 12 per group) were inoculated intravenously (iv) with B16-F10 tumor cells. Four days after tumor cell injection, Figure 6 As shown in , RNA lipid complex vaccination was initially performed with Tyrp1 or irrelevant control (backbone without antigen) RNA (40 μg). 26 days after the start of the experiment, the lungs of the mice were removed and the tumor nodules were counted. All mice treated with control RNA showed countless tumor nodules (more than at least 500 nodules / mouse), and most mice had to be sacrificed before day 26 due to disease. In sharp contrast, Tyrp1 vaccinated mice survived until day 26, and most mice showed no visible signs of tumors. Significance was determined using the unpaired two-tailed Mann-Whitney test (tumor nodule comparison) and the log-rank test (survival). ns: P>0.05, *P<0.05, **P<0.01, ***P<0.001.

[0288] Example 7: Tumor control after therapeutic Tyrp1 or Dct vaccination

[0289] Using a similar experimental setup that allowed for the definition of tumor growth dynamics, we confirmed the therapeutic potential of Tyrp1 vaccination and additionally tested the effect of Dct RNA treatment on tumor growth. Figure 7 As shown in 5Luciferase transgenic B16-F10 tumor cells (B16-F10-LUC) were injected iv into naive C57BL / 6 mice (n=12 per group) and vaccinated with 40 μg RNA lipid complexes. Tumor growth measured by luciferase bioluminescence was measured by IVIS bioluminescence imaging system (as described in Kranz, LMet al. Nature 534, 396–401 (2016)) at days 4, 7, 12, 17, 19, and 25. The bioluminescence signal was significantly reduced compared to the untreated group or the group treated with irrelevant RNA, indicating that the reduction of tumor burden was achieved by Tyrp1 and Dct vaccination. This was confirmed by independent readout of tumor nodule counts or lung weights 25 days after tumor inoculation. Significance was determined using two-way ANOVA and Dunnett's multiple comparison test (comparison of luciferase bioluminescence over time) or Kruskal-Wallis test followed by Dunn's test (comparison of tumor nodules or lung weights). ns: P>0.05, *P<0.05, **P<0.01, ***P<0.001.

[0290] Example 8: Identification of multiple hitherto undescribed differentially expressed genes in tumor models CT26, MC38, TRAMP-C2 and 4T1

[0291] Examples 1 to 7 demonstrate that differentially expressed genes in individual tumors can be identified by comparing RNA sequencing-based gene expression with a gene expression database of healthy tissue. Among the top-ranked B16-F10 genes, several known tumor antigens were found. We demonstrated that RNA lipid complex vaccination against four out of five candidates tested generated a strong T cell response. For two of these candidates, we demonstrated that therapeutic vaccination generated a strong anti-tumor immune response, resulting in complete tumor rejection.

[0292] Subsequently, we expanded our tumor model to test whether it is also feasible to identify differentially expressed genes in other tumor types. Therefore, we sequenced mouse colon tumor cell lines CT26 and MC38, prostate cancer cell line TRAMP-C2, and breast cancer cell line 4T1 (sequencing was described in Kreiter, S. et al. Nature 520, 692–696 (2015).), and compared the RNA gene expression of the cell line with our healthy tissue database. Multiple differentially expressed genes were identified in each cell line, one of which is shown in Figure 8. The immunogenicity study of the selected candidate is described in Example 9.

[0293] Example 9: Immunogenicity testing of selected targets in CT26, MC38 and TRAMP-C2

[0294] BALB / c mice (n=3, CT26) or C57BL / 6 mice (n=3 per group, MC38 and TRAPM-C2) were immunized with 20 μg of antigen-encoding RNA formulated as lipoplexes on days 0, 7, and 14 as described in Example 2. For most groups, each mouse was injected with two antigens. Five days after the last vaccination, splenocytes from mice were isolated and 5×10 5 The recognition of syngeneic bone marrow-derived dendritic cells (BMDC) electroporated with 10 μg of antigenic RNA or irrelevant RNA (control) was performed by culturing bone marrow-derived cells in the presence of GM-CSF (Lutz, MB et al. J. Immunol. Methods 223, 77–92 (1999)). The targets Rhox5 and Ndst4 derived from CT26 were identified ( Fig. 9 ), MC38-derived targets GM14819, Rhox2h, Gm15091, Gm15097, Dppa4, GM773, Prl2c2, Fmr1nb, and Luzp4 ( Fig.10 ) and against the TRAPM-C2 target Amotl2 ( Fig.11 ) immune response.

[0295] Example 10: Selection of differentially expressed genes for personalized cancer immunotherapy in humans

[0296] In order to identify potential targets in individual patients, the tumor cDNA derived from mRNA will be sequenced and the gene expression abundance will be estimated. The gene expression values ​​will then be compared with a reference gene expression database of selected normal tissues treated in the same manner. The reference database may include RNA-Seq data from: normal samples from the Genotype-Tissue Expression (GTEx) database, Human Protein Atlas (HPA), normal tissues from the Cancer Genome Atlas (TCGA), Blueprint Epigenome database, and other groups. The selection of highly expressed antigens will be performed according to Example 1. After selection, the potential targets will be verified before the final selection of vaccination treatment.

Claims

1. A method for producing a personalized cancer vaccine, comprising the following steps: (a) identifying one or more RNA transcripts present in a tumor specimen of a cancer patient, wherein each of the one or more RNA transcripts encodes an amino acid sequence, and wherein each of the one or more RNA transcripts is present in the tumor specimen at a copy number exceeding a predetermined expression threshold; and (b) providing a vaccine characterized in that at least one epitope is derived from an amino acid sequence encoded by said one or more RNA transcripts.

2. The method of claim 1, wherein the step of identifying one or more RNA transcripts comprises determining the copy number of RNA transcripts present in a tumor specimen of a cancer patient, and comparing the copy number of each of the RNA transcripts to a respective predetermined expression threshold.

3. The method according to claim 1 or 2, wherein the copy number of the RNA transcript in the tumor specimen is at least 10 times, preferably at least 1000 times, that in non-tumor tissue.

4. The method according to any one of claims 1 to 3, wherein the step of identifying one or more RNA transcripts comprises single cell sequencing of one or more cancer cells.

5. The method of claim 4, wherein the cancer cells are circulating tumor cells.

6. The method according to any one of claims 1 to 5, wherein the step of identifying one or more RNA transcripts comprises using next generation sequencing (NGS).

7. The method according to any one of claims 1 to 6, wherein the step of identifying one or more RNA transcripts comprises sequencing the RNA of the tumor specimen and / or a DNA library obtained from the RNA of the tumor specimen.

8. The method according to any one of claims 1 to 7, comprising the further step of determining the usability of epitopes encoded by said one or more RNA transcripts for cancer vaccination.

9. The method of any one of claims 1 to 8, wherein the vaccine comprises a polypeptide comprising one or more epitopes encoded by the one or more RNA transcripts, or a nucleic acid encoding the polypeptide.

10. The method of claim 9, wherein the polypeptide comprises at most 30 epitopes.

11. The method of claim 9 or 10, wherein the polypeptide further comprises one or more epitopes that are not encoded by the one or more RNA transcripts but are expressed by cancer cells.

12. The method of claim 11, wherein the one or more epitopes not encoded by the one or more RNA transcripts are cancer-specific neo-epitopes.

13. The method according to any one of claims 9 to 12, wherein the epitope is in its natural sequence environment so as to form a vaccine sequence.

14. The method of claim 13, wherein the vaccine sequence is about 30 amino acids in length.

15. The method according to any one of claims 9 to 14, wherein the epitopes and / or vaccine sequences are arranged head to tail.

16. The method according to any one of claims 9 to 15, wherein the epitope and / or vaccine sequence are separated by a linker.

17. The method according to any one of claims 9 to 16, wherein the epitope and / or vaccine sequence has the same amino acid sequence in the tumor specimen as in non-tumor tissue.

18. The method according to any one of claims 1 to 17, wherein the vaccine is an RNA vaccine.

19. The method according to any one of claims 1 to 18, wherein the vaccine is a prophylactic and / or therapeutic vaccine.

20. Personalized cancer vaccine obtained by the method according to any one of claims 1 to 19.

21. A personalized cancer vaccine comprising a recombinant polypeptide or a nucleic acid encoding the polypeptide, wherein the recombinant polypeptide comprises an epitope produced by the expression of an RNA transcript in a tumor specimen of a cancer patient, wherein the RNA transcript is present in the tumor specimen at a copy number exceeding a predetermined expression threshold.

22. The vaccine according to claim 21, wherein the copy number of the RNA transcript in the tumor specimen is at least 10 times, preferably at least 1000 times, that in non-tumor tissue.

23. The vaccine of claim 21 or 22, wherein the polypeptide further comprises an epitope that is not encoded by the RNA transcript but is expressed by cancer cells.

24. A method for treating a cancer patient, comprising the steps of: (a) providing a personalized cancer vaccine by the method according to any one of claims 1 to 19; as well as (b) administering the vaccine to the patient.

25. A method of treating a cancer patient comprising administering to said patient a vaccine according to any one of claims 21 to 23.

26. The method of claim 24 or 25, wherein the vaccine is administered intravenously, transdermally, intramuscularly or subcutaneously.

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