Personalized therapeutic anti-cancer vaccines

Abstract

The present invention relates to personalized therapeutic anti-cancer vaccines, methods for treating cancer in which such anti-cancer vaccines are used, and methods for manufacturing such vaccines. [Solution] An immunologically effective amount, (i) a polynucleotide comprising nucleotide sequences encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented covalent antigen sequence or one or more portions thereof; or (ii) A polypeptide encoded by the polynucleotide as defined in (i); or (iii) A dimeric protein consisting of two polypeptides encoded by the polynucleotide defined in (i); and Medicinally acceptable carriers Personalized therapeutic anti-cancer vaccines, including [specific ingredient / feature].

Description

Technical Field

[0001] The present invention relates to an individualized therapeutic anticancer vaccine, a method for treating cancer in which such an anticancer vaccine is used, and a method for producing the vaccine.

Background Art

[0002] The treatment of cancer has improved over the past few decades, particularly due to early detection and diagnosis, which have significantly increased survival. However, only about 60% of patients diagnosed with cancer survive for 5 years after diagnosis. Most of the cancer treatments used are surgical procedures, radiation, and cytotoxic chemotherapeutic drugs, all of which have severe side effects. In the past few years, cancer immunotherapy, i.e., anticancer vaccines, which target cancer cells with the help of the patient's own immune system, has attracted attention because such therapies can reduce or even eliminate some of the side effects seen in traditional cancer treatments.

[0003] The basis of immunology is based on self-nonself discrimination. Most pathogens that induce infectious diseases contain molecular signatures that can be recognized by the host and trigger an immune response. However, tumor cells are derived from normal cells and generally do not express any molecular signatures, making it more difficult to distinguish them from normal cells. Nevertheless, most tumor cells express different types of tumor antigens, which can be shared tumor antigens, i.e., antigens expressed by the same type of tumor in multiple individuals or by various tumors in multiple individuals, or patient-specific antigens found in a particular patient.

[0004] Shared tumor antigens include mutations in overexpressed or abnormally expressed cellular proteins, oncogenes, or tumor suppressor genes, as well as viral antigens. Patient-specific tumor antigens can arise from one or more mutations in the tumor genome that lead to changes in the amino acid sequence of the protein in question. These include non-synonymous mutations, frameshift mutations, fusion antigens, and intron-retaining antigens. [Overview of the Initiative]

[0005] The inventors have discovered that personalized anti-cancer vaccines that induce an immune response to a shared tumor antigen present in the patient in whom the vaccine is designed and produced, as well as an additionally patient-specific tumor antigen, enhance the anti-cancer immune response necessary to control or inhibit the proliferation of tumor cells.

[0006] definition In the context herein, the term "tumor" is used to refer to both solid tumors and tumor cells found in bodily fluids, such as blood.

[0007] Patient-specific tumor antigens, patient-specific cancer antigens, and patient-specific antigens are used interchangeably herein for tumor antigens found in a specific individual / patient, and the tumor antigens include one or more mutations found in tumor cells compared to the patient's normal cells.

[0008] Patient-specific tumor epitopes, patient-specific cancer epitopes, patient-specific epitopes, and neoepitopes are peptides contained in patient-specific tumor antigens and are used interchangeably herein for peptides containing one or more immunogenic mutations.

[0009] Patient-specific tumor epitope sequences, patient-specific cancer epitope sequences, patient-specific epitope sequences, and neoepitope sequences are interchangeable herein to describe nucleic acid sequences encoding the epitope / neoepitope or amino acid sequences containing the epitope / neoepitope.

[0010] Co-tumor antigens, co-cancer antigens, and co-antigens are used interchangeably herein to describe tumor antigens expressed by the same type of tumor in multiple individuals, or by various tumors in multiple individuals.

[0011] The co-tumor antigen sequence, co-cancer antigen sequence, and co-antigen sequence are used interchangeably herein to describe a nucleic acid sequence that encodes part or all of a co-antigen, or an amino acid sequence containing said part or all of said.

[0012] Patient-present shared tumor antigens, patient-present shared cancer antigens, and patient-present shared antigens are used interchangeably herein to describe shared tumor antigens that are present in or have been identified as present in the patient.

[0013] Patient-presented shared tumor antigen sequences, patient-presented shared cancer antigen sequences, and patient-presented shared antigen sequences are used interchangeably in this specification to describe nucleic acid sequences that encode part or all of a patient-presented shared antigen, or amino acid sequences that include said part or all of it.

[0014] Personalized therapeutic anti-cancer vaccines are used to describe vaccines designed and produced for specific individuals / patients with the aim of stimulating an immune response that can recognize tumor cells already present in an individual and control or inhibit the proliferation of such tumor cells.

[0015] Individuals and patients are used interchangeably in this specification and represent specific human beings who have cancer or are suspected of having cancer.

[0016] Detailed description of the invention Cancer develops from a patient's normal tissue due to one or a few cells that, as a result of a mutation, begin abnormal, uncontrolled proliferation. While cancer cells are mutated, most of their genome remains intact and identical to the rest of the patient's cells. One approach to attacking tumors is based on the knowledge that any tumor in an individual / patient is unique, and patient-specific mutations lead to the expression of patient-specific mutant proteins (tumor-specific antigens) unique to a particular patient. These tumor-specific and patient-specific antigens are not identical to any proteins in the patient's normal cells. Therefore, such patient-specific antigens should be suitable targets for therapeutic anti-cancer vaccines produced specifically for the patient in question, i.e., personalized therapeutic anti-cancer vaccines. The challenge with this type of anti-cancer vaccine is that while patient-specific antigens are selected for inclusion in the vaccine according to their predicted therapeutic efficacy, some exhibit the expected therapeutic efficacy in clinical contexts, while not all do. There is a need for improvement.

[0017] Co-occurring tumor antigens (CTAs) have been found to be expressed by many tumors, either among patients with the same type of cancer or between patients and cancer types. One example is the HPV16 antigen, which is expressed in approximately 50% of all head and neck squamous cell carcinoma (SCCHN) patients, but is also expressed in patients with different cancers, such as cervical cancer and vulvar squamous cell carcinoma (vSCC). Many of these CTAs have been previously characterized as immunogenic and / or presented on specific HLA class I or class II alleles. CTAs can be included in over-the-counter therapeutic anti-cancer vaccines used in many patients (see, for example, International Publication No. 2013 / 092875). However, this type of anti-cancer vaccine shows clinical efficacy in some patients but little to no therapeutic efficacy in others.

[0018] T-cell responses depend on the processing and presentation of cancer epitopes on each patient's HLA molecule. Due to the diversity of HLA class I and HLA class II molecules, general-purpose vaccines used in multiple patients typically contain large, full-length antigens to optimize the likelihood that the antigen will contain epitopes present on a wide range of HLA molecules. However, patients only need shorter sequences of antigens containing epitopes that match their specific HLA molecule. Therefore, it is not possible to design and manufacture a general-purpose anti-cancer vaccine that covers all tumor antigens in every patient.

[0019] Furthermore, over-the-counter therapeutic anti-cancer vaccines that target only one tumor antigen ignore the tumor heterogeneity commonly found in tumors, and immune pressure against a single antigen can result in tumor clones with different mutations.

[0020] This invention presents a personalized therapeutic anti-cancer vaccine that targets patient-presented shared antigens and optionally additionally targets patient-specific antigens. This increases therapeutic efficacy compared to anti-cancer vaccines containing only patient-specific antigens or to general anti-cancer vaccines containing shared antigens.

[0021] Patient-presented shared antigens included in the vaccine of the present invention may have known immunogenicity, known expression patterns, and known binding to specific HLA class I and / or class II molecules. T cells specific to patient-presented shared antigens can migrate to tumors and influence the tumor microenvironment, thereby increasing the likelihood that additional tumor-specific T cells can attack tumor cells. Tumors are heterogeneous to varying degrees and may therefore consist of tumor cells expressing different subsets of patient-presented shared antigens included in the vaccine. Including patient-presented shared antigens and optionally patient-specific antigens in the personalized vaccine according to the present invention increases the likelihood of recognition and killing of multiple or all tumor cells due to a shift in the immunomicroenvironment when activated T cells that migrate to tumors reach a threshold.

[0022] The vaccine according to the present invention utilizes the normal adaptive immune system to provide immunity against tumor cells. The adaptive immune system is specific in that any antigen specifically induces an immune response to that antigen through the recognition of the specific antigen during a process called antigen presentation. The cells of the adaptive immune system are lymphocytes, particularly B cells and T cells. B cells are involved in humoral immune responses, while T cells are involved in cell-mediated immune responses.

[0023] In particular, the vaccine according to the present invention is designed to induce a cell-mediated immune response through the activation of T cells against tumor antigens. T cells recognize epitopes when they are processed and presented in complex with MHC molecules, as discussed below.

[0024] The patient-presented shared antigen sequences of the shared antigens and optionally patient-specific antigens contained in the vaccine according to the present invention and optionally the patient-specific antigen sequences are designed to be presented as MHC-peptide complexes in the peptide-binding grooves of MHC molecules. There are two major classes of major histocompatibility complex (MHC) molecules, MHC I and MHC II. The terms MHC (class) I and MHC (class) II are used interchangeably herein with HLA (class) I and HLA (class) II. Human leukocyte antigen (HLA) is the major histocompatibility complex in humans.

[0025] MHC I is found on the cell surface of all nucleated cells in the body. One function of MHC I is to present peptides of non-self proteins from within the cell to cytotoxic T cells. The MHC I peptide complex is inserted into the plasma membrane of the cell presenting the peptide to the cytotoxic T cell, thereby triggering the activation of the cytotoxic T cell against a specific MHC-peptide complex. Since the peptide is placed in the groove of the MHC I molecule, peptides are allowed to be about 8-10 amino acids in length.

[0026] MHC II molecules are a family of molecules that are normally found only on antigen-presenting cells such as dendritic cells, monocytes, some endothelial cells, thymic epithelial cells, and B cells.

[0027] Unlike MHC I, the antigens presented by MHC class II molecules are derived from extracellular proteins. The extracellular proteins undergo endocytosis, are digested in lysosomes, and the resulting peptides are loaded onto MHC class II molecules and then presented on the cell surface. The antigen-binding groove of the MHC class II molecule is open at both ends and can present longer peptides, generally 15-24 amino acid residues in length.

[0028] MHC class I molecules are recognized by CD8 and co-receptors on T cells, usually called CD8+ T cells (or CD8+ cells), while MHC class II molecules are recognized by CD4 and co-receptors on T cells, usually called CD4+ T cells (or CD4+ cells).

[0029] The individualized anti-cancer vaccine of the present invention comprises a polynucleotide encoding a polypeptide comprising three units, namely a targeting unit, a dimerization unit and an antigenic unit. Due to the dimerization unit, the polypeptide forms a dimeric protein, a so-called vaccibody.

[0030] The genes encoding the three units are genetically engineered to be expressed as one gene. When expressed in vivo, the polypeptide / dimeric protein targets antigen-presenting cells (APCs), which results in enhanced vaccine efficacy compared to the same non-targeted antigen.

[0031] In a first aspect, the present invention relates to a polynucleotide comprising nucleotide sequences encoding a targeting unit, a dimerization unit and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented shared antigen sequence or one or more portions thereof, and optionally one or more patient-specific antigen sequences or one or more portions thereof.

[0032] In a second aspect, the present invention relates to a polypeptide encoded by a polynucleotide comprising nucleotide sequences encoding a targeting unit, a dimerization unit and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented shared antigen sequence or one or more portions thereof, and optionally one or more patient-specific antigen sequences or one or more portions thereof.

[0033] In a third aspect, the present invention relates to a dimer protein comprising two polypeptides encoded by polynucleotides, each having a nucleotide sequence encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented covalent antigen sequence or one or more portions thereof, and optionally one or more patient-specific antigen sequences or one or more portions thereof.

[0034] In a fourth embodiment, the present invention provides an immunologically effective amount of (i) a polynucleotide comprising nucleotide sequences encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented covalent antigen sequence or one or more portions thereof, and optionally one or more patient-specific antigen sequences or one or more portions thereof; or (ii) A polypeptide encoded by a polynucleotide as defined in (i), or (iii) A dimeric protein consisting of two polypeptides encoded by a polynucleotide as defined in (i); and Medicinally acceptable carriers This includes, for example, personalized therapeutic anti-cancer vaccines.

[0035] In one embodiment, the present invention provides an immunologically effective amount of (a) a polynucleotide comprising nucleotide sequences encoding a targeting unit, a dimerizing unit and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented covalent antigen sequence or one or more portions thereof, and optionally one or more patient-specific antigen sequences or one or more portions thereof; and (b) Medicinally acceptable carriers This includes, for example, personalized therapeutic anti-cancer vaccines.

[0036] In another embodiment, the present invention provides an immunologically effective amount of (a) A dimeric protein or polypeptide encoded by a polynucleotide comprising nucleotide sequences encoding a targeting unit, a dimerizing unit and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented co-antigen sequence or one or more portions thereof, and optionally one or more patient-specific antigen sequences or one or more portions thereof; and (b) Medicinally acceptable carriers This includes, for example, personalized therapeutic anti-cancer vaccines.

[0037] The antigenic units contained in the polynucleotides, polypeptides, dimeric proteins, and personalized therapeutic anticancer vaccines according to the present invention include at least one patient-presented co-antigen sequence or one or more portions thereof. In one embodiment, the patient-presented co-antigen is a co-antigen selected from the group consisting of overexpressed cellular proteins, abnormally expressed cellular proteins, oncotesticular antigens, viral antigens, differentiation antigens, mutant oncogenes, mutant tumor suppressor genes, carcinoembryonic antigens, co-fusion antigens, co-intron-holding antigens, dark matter antigens, co-antigens caused by spliceosome mutations, and co-antigens caused by frameshift mutations.

[0038] In one embodiment, the patient-presented shared antigen is a human cell protein that is overexpressed or abnormally expressed, i.e., a cell protein found in tumors at increased levels compared to normal healthy cells and tissues. Examples of such overexpressed or abnormally expressed cell proteins include tumor protein D52, Her-2 / neu, hTERT (telomerase), and Survivin. In another embodiment, the patient-presented shared antigen is a cancer-testis antigen whose expression occurs in normal testicular tissue as well as in human malignant tumors. Examples of cancer-testis antigens include MAGE-A, MAGE-B, GAGE, PAGE-1, SSX, HOM-MEL-40 (SSX2), NY-ESO-1, LAGE-1, and SCP-1. In yet another embodiment, the patient-presented shared antigen is a differentiation antigen, such as tyrosinase and TRP-2. In yet another embodiment, the patient-presented shared antigen is a viral antigen. Examples of viral antigens include human papillomavirus (HPV), hepatitis B virus (HBV), Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), Merkel cell polyomavirus (MCV or MCPyV), human cytomegalovirus (HCMV), and human T lymphotropic virus (HTLV). In yet another embodiment, the patient-presented shared antigen is a mutant oncogene. Examples of mutant oncogenes include RAS mutations, PIK3CA mutations, and EGFR mutations, including KRAS. In yet another embodiment, the patient-presented shared antigen is a mutant tumor suppressor gene. Examples include mutant p53, mutant pRB, mutant BCL2, and mutant SWI / SNF. In yet another embodiment, the patient-presented shared antigen is a carcinoembryonic antigen, such as alpha-fetoprotein or carcinoembryonic antigen. In yet another embodiment, the patient-presented co-antigen is a co-intron-holding antigen or a co-antigen caused by a frameshift mutation, such as CDX2 or CALR. In yet another embodiment, the patient-presented co-antigen is a co-antigen caused by a spliceosome mutation. One example is an antigen caused by a mutation such as the SF3B1 mutation.

[0039] For any given cancer antigen, immune tolerance may be occurring when a patient develops cancer. Anti-cancer vaccines should specifically elicit an immune response to the antigen incorporated into the vaccine. Peripheral immune tolerance to a selected antigen may be weak or strong. Patients are more likely to establish central immune tolerance to covalent antigens, i.e., human cell proteins, that are also expressed in normal tissues, such as overexpressed and differentiated antigens. By incorporating such covalent antigen sequences or portions thereof into an antigenic unit (alone or with other patient-presented covalent antigen sequences and optionally with patient-specific antigen sequences), vaccines according to the present invention containing the antigenic unit may be able to induce a sufficiently strong and broad immune response to influence the tumor microenvironment and shift the patient's immune response against the tumor from a suppressive / tolerant immune response to a pro-inflammatory immune response. This may be a significant clinical benefit for the patient, as it may help break tolerance to several other antigens. The above concept may be referred to as cancer immune setpoint tipping.

[0040] Therefore, in one embodiment, at least one patient-presented co-antigen sequence is a co-antigen that is a human cell protein, preferably an overexpressed or abnormally expressed human cell protein, or a differentiation antigen.

[0041] At least one patient-presented shared antigen may be detected in the patient's tissue or bodily fluids by methods known in the art, including: • Sequencing of the patient's genome or exome, and tailor-made software search of whole-genome / exome-seq data for selective identification of, for example, mutant oncogenes or mutant tumor suppressor genes; • Immunohistochemistry of patient tumor tissue to detect the presence of mutant proteins; RT-PCR for detecting the presence of known mutations in viral antigens or oncogenes; • ELISA using antibodies against mutant tumor proteins in serum samples; • Comparison with healthy tissue to detect RNA-seq and covalent antigen expression / overexpression in tumor tissue; • Tailor-made software search in raw RNA sequence data to identify intron-retaining antigens; • Tailor-made software search in whole-genome-seq data to identify translocation elements, which are components of dark matter antigens; • Detection of short repeats in raw whole exome / RNA sequence data for the identification of dark matter antigens; • RNA-seq data for identifying co-viral antigens; and Comparison of RNA-seq from patient tumor samples with either the patient's own healthy tissue or GTEX / HPA gene expression data from a cohort / database (e.g., TCGA).

[0042] In a preferred embodiment, the antigenic unit comprises at least one patient-presented co-antigen sequence, or a portion of such an antigen sequence known to be immunogenic, for example, having previously shown an immunogenic response in another patient, being described as inducing an immune response in another patient, or being predicted to bind to the HLA class I and / or HLA class II alleles of a particular patient. In another preferred embodiment, the antigenic unit comprises one or more portions of at least one patient-presented co-antigen sequence, for example, one or more epitopes known to be immunogenic or being predicted to bind to the HLA class I and / or HLA class II alleles of a particular patient. In a further preferred embodiment, the antigenic unit comprises one or more portions of at least one patient-presented co-antigen sequence, for example, one or more epitopes known to be immunogenic or being predicted to bind to the HLA class I allele of a particular patient.

[0043] In one embodiment, the antigenic unit comprises a patient-presented co-antigen sequence or one or more portions thereof having a length suitable for presentation by a specific patient's HLA allele. Therefore, in one embodiment, the patient-presented co-antigen sequence or portion thereof is 7 to 30 amino acids long. In another embodiment, the patient-presented co-antigen sequence or portion thereof is 7 to 10 amino acids long or 13 to 30 amino acids long, for example, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long, for example, 9 amino acids long.

[0044] An antigenic unit may include the full length of at least one patient-presented shared antigen sequence, or one or more portions thereof. In one embodiment, the antigenic unit includes one portion of a patient-presented shared antigen sequence. In another embodiment, the antigenic unit includes several portions of a patient-presented shared antigen sequence. The antigenic unit may include more than one patient-presented shared antigen sequence, i.e., sequences of several patient-presented antigens, each in its full length, or one or more portions of each such antigen. In one embodiment, the antigenic unit includes the full length of one patient-presented shared antigen sequence and one or more portions of sequences of one or more other patient-presented shared antigens, for example, one portion of one other patient-presented shared antigen sequence, or several portions of one other patient-presented shared antigen sequence, or one portion of each sequence of several other patient-presented shared antigens, or a portion of several other patient-presented shared antigens, or several portions of each sequence. In a preferred embodiment, the antigenic unit comprises sequences of several patient-presented co-antigens, for example, several portions of sequences of several patient-presented co-antigens, more preferably several epitopes of several patient-presented co-antigens, which are known to be immunogenic or are expected to bind to the HLA class I and / or HLA class II alleles of a particular patient, preferably to the HLA class I allele of a particular patient.

[0045] In yet another embodiment, the antigenic unit comprises the full length of one or more patient-presented shared antigens and one or more portions of one or more patient-presented shared cancer antigens. Examples include: • Antigenic units comprising the full length of one patient-presented shared antigen and one or more epitopes of one patient-presented shared cancer antigen; and • Several patient-presented shared cancer antigens, each in its full length, and antigenic units containing one or more epitopes of a single patient-presented shared cancer antigen; and • Antigenic units comprising the full length of one patient-presented shared antigen and one or more epitopes of several patient-presented shared cancer antigens; and • Several patient-presented shared cancer antigens, each in its full length, and antigenic units containing one or more epitopes of several patient-presented shared cancer antigens.

[0046] In yet another embodiment, the antigenic unit comprises the full length of at least one patient-presented co-antigen sequence or the full length of one or more patient-presented co-antigen sequences. In one embodiment, the antigenic unit comprises the full length of 1 to 10 patient-presented co-antigen sequences. In another embodiment, the antigenic unit comprises 1 to 30 portions of the patient-presented co-antigen sequence in the form of a long peptide sequence, e.g., a peptide sequence about 28 to 100 amino acids in length, or a nucleic acid sequence encoding such a long peptide sequence, wherein the long peptide sequence comprises a plurality of epitopes that are expected to bind to the patient's HLA class I and / or HLA class II alleles. In yet another embodiment, the antigenic unit comprises 1 to 50 portions of the patient-presented co-antigen sequence in the form of a short peptide sequence / epitope that is expected to bind to the patient's HLA class I and / or HLA class II alleles, or a nucleic acid sequence encoding such a short peptide sequence / epitope.

[0047] In one embodiment, 3 to 50 patient-presented shared antigen sequences are included in the antigenic unit, which are, for example, 3 to 30 sequences, 3 to 20 sequences, 3 to 15 sequences, or 3 to 10 sequences.

[0048] In another embodiment, 5 to 50 patient-presented shared antigen sequences are included in the antigenic unit, which are, for example, 5 to 30 sequences, 5 to 25 sequences, 5 to 20 sequences, 5 to 15 sequences, or 5 to 10 sequences.

[0049] In a further embodiment, 10 to 50 patient-presented shared antigen sequences are included in the antigenic unit, which are, for example, 10 to 40 sequences, for example 10 to 30 sequences, for example 10 to 25 sequences, for example 10 to 20 sequences, or for example 10 to 15 sequences.

[0050] It is preferable to include sequences of multiple different patient-presented co-antigens and optionally multiple different patient-specific antigens in the antigenic unit to prevent the tumor from escaping the immune system by, for example, shutting down the expression of antigens that are targets of anti-cancer vaccines. Generally, the more genes that a tumor needs to shut down to escape the immune system, the less likely it is that the tumor will actually have the ability to shut them all down while still being able to grow or even survive. Furthermore, tumors can be heterogeneous in that not all of each and every patient-presented co-antigen or patient-specific antigen is expressed by all tumor cells.

[0051] Therefore, in a preferred embodiment, the approach is to include as many patient-presented shared antigen sequences and optionally patient-specific antigen sequences as possible in the antigenic unit of the vaccine of the present invention in order to efficiently attack the tumor by activating T cells that can recognize more tumor antigens expressed by tumor cells. Furthermore, in order to ensure that all patient-presented shared antigen sequences and optionally patient-specific antigen sequences are efficiently taken up by the same antigen-presenting cells, they are configured to be a single amino acid chain instead of separate peptides, or to encode a single amino acid chain (i.e., the antigenic unit). However, as stated above, the objective of the vaccine of the present invention is to activate the patient's T cells against the patient-presented shared antigen sequences and optionally patient-specific antigen sequences contained in the antigenic unit, and including too many such sequences in the antigenic unit may result in dilution of T cells. Therefore, it is important to select the optimal patient-presented shared antigen sequences and optionally patient-specific antigen sequences to be included in the antigenic unit.

[0052] The optimal patient-presented shared antigen sequence is one that is known to be immunogenic. In a preferred embodiment, the antigenic unit comprises one or more portions of at least one patient-presented shared antigen sequence, for example, one or more epitopes that are known to be immunogenic or that are predicted to bind to a specific patient's HLA allele, preferably the patient's HLA class I allele.

[0053] It is preferable to "maximize the use" of the antigenic unit, i.e., the antigenic unit contains the minimum possible number of amino acids / sequences that do not contribute to the immunogenicity of the antigenic unit. For example, including a full-length patient-presented co-antigen sequence in the antigenic unit is less preferable if such a sequence contains only a few epitopes that are known to be immunogenic or are predicted to bind to a particular patient's HLA allele, and the remainder of the sequence does not contribute to the immunogenicity of the antigenic unit. On the other hand, including the full-length patient-presented co-antigen sequence may be reasonable if such a sequence contains several or many such epitopes that are close together.

[0054] The antigenic unit may further contain one or more patient-specific antigen sequences. Patient-specific antigens may be identified by sequencing the genome or exome of the patient's tumor. Compared to the exome of the patient's normal tissue, such sequences contain one or more mutations. The mutations may be any mutations leading to a change in at least one amino acid. Therefore, the mutations may be one of the following: Non-synonymous mutations that lead to changes in amino acids • Frameshift and subsequent mutations that lead to open reading frames with completely different orientations after the mutation. • Read-through mutations in which a stop codon is modified or deleted, leading to a longer protein with tumor-specific epitopes. • Splice mutations that lead to unique tumor-specific protein sequences • Chromosomal rearrangement that produces a chimeric protein with a tumor-specific epitope at the junction of two proteins.

[0055] The antigenic unit may comprise one or more patient-specific antigen sequences or one or more portions thereof. In one embodiment, the antigenic unit comprises one or more (several) patient-specific antigen sequences. In another embodiment, the antigenic unit comprises one or more portions of such one or more patient-specific antigen sequences, preferably one or more patient-specific epitopes.

[0056] The epitope preferably has a length suitable for presentation by the MHC molecules discussed above. Therefore, in a preferred embodiment, the epitope is 7 to 30 amino acids long. More preferably, the epitope sequence is 7 to 10 amino acids long or 13 to 30 amino acids long, for example, an epitope sequence having an amino acid length of 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0057] In a preferred embodiment, the antigenic unit comprises at least one patient-specific epitope, or at least five patient-specific epitopes, or at least ten patient-specific epitopes. In another preferred embodiment, the antigenic unit comprises at least fifteen patient-specific epitopes, for example, at least twenty patient-specific epitopes.

[0058] In one embodiment, the antigenic unit contains 3 to 50 patient-specific antigen sequences, which are, for example, 3 to 30 sequences, 3 to 20 sequences, 3 to 15 sequences, or 3 to 10 sequences. In a preferred embodiment, such sequences are epitopes.

[0059] In another embodiment, 5 to 50 patient-specific antigen sequences are included in the antigenic unit, which are, for example, 5 to 30 sequences, 5 to 25 sequences, 5 to 20 sequences, 5 to 15 sequences, or 5 to 10 sequences. In a preferred embodiment, such sequences are epitopes.

[0060] In a further embodiment, 10 to 50 patient-specific antigen sequences may be included in the antigenic unit, which may be, for example, 10 to 40 sequences, 10 to 30 sequences, 10 to 25 sequences, 10 to 20 sequences, or 10 to 15 sequences. In a preferred embodiment, such sequences are epitopes.

[0061] In particular, if the patient-specific antigen sequence included in the antigenic unit is a short epitope, for example, only a few amino acids long, the short epitope is included in the antigenic unit so as to be flanked on both sides by amino acid sequences. Preferably, the short epitope is positioned essentially centrally between two flanking sequences to ensure that the epitope is presented by antigen-presenting cells after processing. The flanking sequences are preferably amino acid sequences adjacent to the epitope in the antigen.

[0062] The following applies to patient-presented shared antigen sequences and their portions, and, where present, to patient-specific antigen sequences and their portions. Therefore, hereafter, the term "antigen sequence" is used and intended to cover both patient-presented shared antigen sequences and their portions, as well as patient-specific antigen sequences and their portions.

[0063] In one embodiment, the antigenic unit contains one copy of each antigen sequence, and as a result, if, for example, 10 different such sequences are contained in the antigenic unit, the vaccine containing the antigenic unit induces a cell-mediated immune response to all 10 different antigen sequences.

[0064] However, if only a few antigen sequences are included in an antigenic unit, either because only a few antigens have been identified, or because only a few of the identified antigens are known or predicted to be sufficiently immunogenic / binding to the patient's HLA alleles, the antigenic unit may include at least two copies of the specific antigen sequence to enhance the immune response to the antigen.

[0065] The length of an antigenic unit is determined primarily by the number of antigen sequences it contains, in addition to the length of those sequences. In one embodiment, an antigenic unit contains 7 to 2000 amino acids, for example 21 to 2000 amino acids, preferably about 30 to about 1500 amino acids, more preferably about 50 to about 1000 amino acids, for example about 100 to about 500 amino acids or about 100 to about 400 amino acids or about 100 to about 300 amino acids.

[0066] While a reasonable immune response to a tumor can be obtained when antigen sequences are randomly organized within antigenic subunits, it is preferable to follow at least one of the following methods for organizing them within antigenic units to enhance the immune response:

[0067] The antigenic unit may be described as a polypeptide having an N-terminal start and a C-terminal termination. The antigenic unit is preferably connected to the dimerizing unit via a unit linker. The antigenic unit is located at either the COOH terminus or the NH2 terminus of the polypeptide / dimer protein. Preferably, the antigenic unit is located at the COOH terminus of the polypeptide / dimer protein.

[0068] The antigenic sequences are preferably separated by linkers. In one embodiment, all antigenic sequences except for terminal antigenic sequences, i.e., antigenic sequences located at the ends of antigenic units that are not connected to dimerizing units, are organized within antigenic subunits, each subunit consisting of an antigenic sequence and a subunit linker. Due to the separation of antigenic sequences by linkers, each antigen is presented to the immune system in an optimal manner.

[0069] In one embodiment, the antigen sequence is organized in a direction from the N-terminal start of the antigenic unit to the C-terminal end of the antigenic unit, preferably from the dimerized unit to the C-terminal end of the antigenic unit, from the highest antigenicity to the lowest antigenicity.

[0070] In another embodiment, particularly when the hydrophilicity / hydrophobicity varies greatly among the antigenic sequences, it is preferable that the most hydrophobic antigenic sequence is positioned substantially in the center of the antigenic unit, and the most hydrophilic antigenic sequence is positioned at the start and / or end of the antigenic unit.

[0071] Since truly central placement of an antigenic unit is only possible when the antigenic unit contains an odd number of antigen sequences, the term "substantially" in this context refers to an antigenic unit containing an even number of antigen sequences in which the most hydrophobic antigen sequence is positioned as close to the center as possible. For example, an antigenic unit contains five antigenic subunits, each antigenic subunit containing different antigen sequences organized as follows: 1-2-3*-4-5; each of 1, 2, 3*, 4, and 5 is a different antigen sequence, - is a linker, and * indicates the most hydrophobic antigen sequence positioned in the center of the antigenic unit.

[0072] In another example, an antigenic unit comprises six antigenic subunits, each antigenic subunit containing a different antigenic sequence organized as follows: 1-2-3*-4-5-6 or alternatively as follows: 1-2-4-3*-5-6; each of 1, 2, 3*, 4, 5, and 6 is a different antigenic sequence, - is a linker, and * indicates the most hydrophobic antigenic sequence located substantially in the center of the antigenic unit.

[0073] Alternatively, the antigen sequence may be arranged in alternating groups of hydrophilic and hydrophobic antigen sequences.

[0074] Furthermore, GC-rich antigen sequences should not be arranged adjacent to each other in order to avoid GC clustering. In a preferred embodiment, one GC-rich antigen sequence is followed by at least one non-GC-rich antigen sequence before a second GC-rich antigen sequence follows.

[0075] In one embodiment, the antigenic unit comprises an antigen sequence in the following order: E7|linker|NY-ESO-1|linker|E6. In a preferred embodiment, the antigenic unit comprises SEQ ID NO: 14.

[0076] In another preferred embodiment, the antigenic unit includes SEQ ID NO: 14 and SEQ ID NO: 15. SEQ ID NO: 15 contains the antigen sequence in the following order: T1D320|Linker|T1D814|Linker|T1D182|Linker|T1D689|Linker|E7|Linker|T1D339|Linker|T1D428|Linker|NY-ESO-1|Linker|T1D572|Linker|T1D359|Linker|T1D488|Linker|T1D554|Linker|T1D272|Linker|T1D210|Linker|T1D849|Linker|T1D4|Linker|T1D77|Linker|T1D717|Linker|T1D586|Linker|E6 Includes in.

[0077] In one embodiment, the antigenic unit comprises an antigen sequence in the following order: E6|linker|NY-ESO-1|linker|E7. In a preferred embodiment, the antigenic unit comprises SEQ ID NO: 16.

[0078] In another preferred embodiment, the antigenic unit includes SEQ ID NOs: 16 and 17. SEQ ID NOs: 17 includes the antigenic sequence in the following order: E6|linker|T1D323|linker|T1D506|linker|T1D12|linker|T1D315|linker|T1D302|linker|T1D700|linker|NY-ESO-1|linker|T1D535|linker|T1D358|linker|T1D670|linker|T1D294|linker|T1D336|linker|T1D499|linker|T1D425|linker T1D491|linker|T1D314|linker|T1D430|linker|E7|linker|T1D582.

[0079] In one embodiment, the antigenic unit comprises an antigen sequence in the following order: NY-ESO-1|linker|E7|linker|E6. In a preferred embodiment, the antigenic unit comprises SEQ ID NO: 18.

[0080] In another preferred embodiment, the antigenic unit includes SEQ ID NOs: 18 and 19. SEQ ID NOs: 19 includes the antigenic sequence in the following order: T1D223|linker|T1D164|linker|T1D56|linker|T1D36|linker|T1D129|linker|T1D274|linker|T1D62|linker|T1D5|linker|T1D144|linker|T1D441|linker|T1D368|linker|NY-ESO-1|linker|T1D234|linker|T1D162|linker|T1D39|linker|T1D272|linker|E7|linker|T1D328|linker|T1D188|linker|E6.

[0081] The antigenic unit may further include one or more linkers that separate one antigen sequence from other antigen sequences, and a linker (hereinafter also called a unit linker) that connects the antigenic unit to a dimerization unit. The one or more linkers ensure that each antigen sequence is presented to the immune system in an optimal manner and that the antigenic unit, when included in the vaccine of the present invention, increases the efficacy of the vaccine.

[0082] One or more linkers are preferably designed to be non-immunogenic and preferably flexible, thereby enabling the antigenic sequence to be presented to the T cell in an optimal manner, even if the antigenic unit contains a large number of antigen sequences.

[0083] Preferably, the length of one or more linkers is 4 to 20 amino acids to ensure flexibility. In another preferred embodiment, the length of one or more linkers is 8 to 20 amino acids, for example 8 to 15 amino acids, for example 8 to 12 amino acids, or for example 10 to 15 amino acids. In a particular embodiment, the length of one or more linkers is 10 amino acids.

[0084] In a specific embodiment, the antigenic unit comprises 10 antigen sequences, and the linkers between these sequences have a length of 8 to 20 amino acids, for example, 8 to 15 amino acids, for example, 8 to 12 amino acids, or for example, 10 to 15 amino acids. In a particular embodiment, the antigenic unit comprises 10 antigen sequences, and the linkers between these sequences have a length of 10 amino acids.

[0085] Preferably, one or more linkers all have the same nucleotide or amino acid sequence. However, if one or more antigen sequences contain amino acid motifs similar to the linkers, it may be advantageous to replace the linkers adjacent to those antigen sequences with linkers of different sequences. Furthermore, if the antigen sequence / linker junction is expected to constitute an immunogenic epitope within itself, linkers of different sequences may be used.

[0086] One or more linkers are preferably serine(S)-glycine(G) linkers, or serine-glycine amino acid sequences, such as GGGGS, GGGSS, GGGSG, GGGGS, or multiple variants thereof, such as GGGGSGGGGS or (GGGGS). m (GGGSS) m (GGGSG) m It contains or consists of a nucleotide encoding m, where m is an integer between 1 and 5, 1 and 4, or 1 and 3. In a preferred embodiment, m is 2.

[0087] In a preferred embodiment, the serine-glycine linker further comprises at least one leucine(L), for example, at least two or at least three leucines. The serine-glycine linker may, for example, contain one, two, three or four leucines. Preferably, the serine-glycine linker contains one or two leucines.

[0088] In one embodiment, one or more linkers include or consist of the sequences LGGGS, GLGGS, GGLGS, GGGLS, or GGGGL. In another embodiment, one or more linkers include or consist of the sequences LGGSG, GLGSG, GGLSG, GGGLG, or GGGSL. In yet another embodiment, one or more linkers include or consist of the sequences LGGSS, GLGSS, GGLSS, GGGLS, or GGGSL.

[0089] In yet another embodiment, one or more linkers include or consist of the sequence LGLGS, GLGLS, GLLGS, LGGLS, or GLGGL. In yet another embodiment, one or more linkers include or consist of the sequence LGLSG, GLLSG, GGLSL, GGGLLG, or GLGSL. In yet another embodiment, one or more linkers include or consist of the sequence LGLSS, GLGLS, GGLLS, GLGSL, or GLGSL.

[0090] In another embodiment, one or more linkers are serine-glycine linkers having a length of 10 amino acids and containing one or two leucines.

[0091] In one embodiment, one or more linkers include or consist of the sequences LGGGSGGGGS, GLGGSGGGGS, GGLGSGGGGS, GGGLSGGGGS, or GGGGLGGGGS. In another embodiment, one or more linkers include or consist of the sequences LGGSG GGGSG, GLGSGGGGSG, GGLSGGGGSG, GGGLGGGGSG, or GGGSLGGGSG. In yet another embodiment, one or more linkers include or consist of the sequences LGGSSGGGSS, GLGSSGGGSS, GGLSSGGGSS, GGGLSGGGSS, or GGGSLGGGSS.

[0092] In a further embodiment, one or more linkers include or consist of the sequences LGGGSLGGGS, GLGGSGLGGS, GGLGSGGLGS, GGGLSGGGLS, or GGGGLGGGGL. In another embodiment, one or more linkers include or consist of the sequences LGGSGLGGSG, GLGSGGLGSG, GGLSGGGLSG, GGGLGGGGLG, or GGGSLGGGSL. In yet another embodiment, one or more linkers include or consist of the sequences LGGSSLGGSS, GLGSSGLGSS, GGLSSGGLSS, GGGLSGGGLS, or GGGSLGGGSL.

[0093] In one embodiment, the antigenic unit comprises 10 antigen sequences separated by 9 linkers, i.e., the terminal sequence is an antigen sequence, not a linker. In another embodiment, the antigenic unit comprises 15 antigen sequences separated by 14 linkers or 20 antigen sequences separated by 19 linkers.

[0094] In another embodiment, the antigenic unit comprises 10 to 20 or 10 to 25 antigen sequences separated by a linker. Preferably, the linker has a length of 10 amino acids. The linker may also have any length as defined herein above, for example, 5 to 12 amino acids.

[0095] Alternatively, one or more linkers may be selected from the group consisting of GSAT linkers, i.e., linkers containing one or more glycine, serine, alanine, and threonine residues, and SEG linkers, i.e., linkers containing one or more serine, glutamic acid, and glycine residues, or multiple variants thereof.

[0096] The antigenic unit and the dimerizing unit are preferably connected by a unit linker. The unit linker may include a restriction site to facilitate the construction of the polynucleotide. The unit linker is preferably a GLGGL linker or a GLSGL linker.

[0097] The vaccine of the present invention contains a targeting unit that targets antigen-presenting cells. Due to the targeting unit, the polypeptide / dimeric protein / vaccine of the present invention leads to the attraction of dendritic cells (DCs), neutrophils, and other immune cells. Therefore, the polypeptide / dimeric protein / vaccine containing the targeting unit not only targets the antigenic unit to specific cells but also additionally promotes a response amplification effect (adjuvant effect) by recruiting specific immune cells to the administration site of the polynucleotide / polynucleotide / dimeric protein / vaccine. This unique mechanism has great utility in clinical settings, and since the vaccine itself provides the adjuvant effect, patients can be given the vaccine of the present invention without any additional adjuvants.

[0098] When used herein, the term “targeting unit” refers to a unit that delivers a polypeptide / dimeric protein / vaccine having its antigenic unit to an antigen-presenting cell for MHC class I-restricted presentation to CD4+ T cells or for cross-presentation to CD8+ T cells via MHC class I restriction.

[0099] The targeting unit is connected to the antigenic unit via a dimerizing unit, the latter located at either the COOH or NH2 terminus of the polypeptide / dimer protein. Preferably, the antigenic unit is located at the COOH terminus of the polypeptide / dimer protein.

[0100] The targeting unit is designed to target the polypeptide / dimeric protein / vaccine of the present invention to surface molecules expressed on appropriate antigen-presenting cells, such as molecules exclusively expressed on a subset of dendritic cells (DCs).

[0101] Examples of such target surface molecules on APCs include HLA, differentiation cluster 14 (CD14), differentiation cluster 40 (CD40), chemokine receptors, and Toll-like receptors (TLRs). Examples of chemokine receptors include CC-motif chemokine receptor 1 (CCR1), CC-motif chemokine receptor 3 (CCR3), CC-motif chemokine receptor 5 (CCR5), and XCR1. Toll-like receptors may include, for example, TLR-2, TLR-4, and / or TLR-5.

[0102] The polypeptide / dimeric protein / vaccine of the present invention can be targeted to the surface molecule by a targeting unit comprising, for example, one or more antibody-binding regions specific to HLA, CD14, CD40, or Toll-like receptors; ligands, such as soluble CD40 ligand; natural ligands such as chemokines, such as chemokine ligand 5, also called CC motif ligand 5 (CCL5 or RANTES), or macrophage inflammatory protein alpha (CCL3 or MIP-1a / MIP1-α); chemokine motif ligand 1 or 2 (XCL1 or XCL2); or a bacterial antigen such as flagellin.

[0103] In one embodiment, the targeting unit has affinity for MHC class II proteins. Therefore, in one embodiment, the nucleotide sequence encoding the targeting unit encodes antibody variable domains (VL and VH) that are specific to MHC class II proteins selected from the group consisting of anti-HLA-DP, anti-HLA-DR, and anti-pan-HLA class II proteins.

[0104] In another embodiment, the targeting unit has affinity for a surface molecule selected from the group consisting of CD40, TLR-2, TLR-4, and TLR-5. Therefore, in one embodiment, the nucleotide sequence encoding the targeting unit encodes antibody variable domains (VL and VH) that are specific to anti-CD40, anti-TLR-2, anti-TLR-4, and anti-TLR-5. In one embodiment, the nucleotide sequence encoding the targeting unit encodes flagellin, which has affinity for TLR-5.

[0105] Preferably, the targeting unit has affinity for a chemokine receptor selected from CCR1, CCR3, and CCR5. More preferably, the nucleotide sequence encoding the targeting unit encodes chemokine human macrophage inflammatory protein alpha (hMIP-1 alpha; also known as LD78 beta, and hereafter referred to as (h)MIP1α and LD78β), which binds to its cognitive receptors, CCR1 and CCR5, expressed on the cell surface of the APC.

[0106] The binding of the polypeptide / dimeric protein / vaccine of the present invention to its cognitive receptor leads to internal translocation in the APC and degradation of the protein into small peptides, which are loaded onto MHC molecules and presented to CD4+ and CD8+ T cells to induce a tumor-specific immune response. Upon stimulation, CD8+ T cells, with the help of activated CD4+ T cells, target and kill tumor cells expressing the same antigen.

[0107] In one embodiment of the present invention, the targeting unit includes an amino acid sequence having at least 80% sequence identity to amino acid sequences 24-93 of SEQ ID NO: 1. In a preferred embodiment, the targeting unit includes an amino acid sequence having at least 85% sequence identity to amino acid sequences 24-93 of SEQ ID NO: 1, for example, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. In one embodiment, the targeting unit includes amino acid sequences 24-93 of SEQ ID NO: 1.

[0108] In a more preferred embodiment, the targeting unit consists of an amino acid sequence having at least 80% sequence identity to amino acid sequences 24-93 of SEQ ID NO: 1, for example, at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, for example at least 100% sequence identity to amino acid sequences 24-93 of SEQ ID NO: 1.

[0109] The term "dimerizing unit," as used herein, refers to a sequence of nucleotides or amino acids between an antigenic unit and a targeting unit. Thus, the dimerizing unit serves to connect the antigenic unit and the targeting unit, and facilitates the dimerization of two monomeric polypeptides into a dimerized protein. Furthermore, the dimerizing unit also provides flexibility in the polypeptide / dimerized protein, enabling optimal binding of the targeting unit to surface molecules on the APC, even if they are located at variable distances. The dimerizing unit may be any unit that fulfills these requirements.

[0110] Therefore, in one embodiment, the dimerization unit may include a hinge region. In another embodiment, the dimerization unit includes another domain that promotes dimerization. In yet another embodiment, the dimerization unit includes a hinge region and another domain that promotes dimerization. In one embodiment, the hinge region and the other domains may be connected through a linker (dimerization unit linker). In yet another embodiment, the dimerization unit includes a hinge region, a dimerization unit linker and another domain that promotes dimerization, with the dimerization unit linker located between the hinge region and the other domain that promotes dimerization.

[0111] The term "hinge region" refers to the peptide sequence of a dimerized protein that promotes dimerization. In other words, the term "hinge region" refers to the amino acid sequence contained in the dimerization unit that contributes to the joining of two polypeptides, i.e., to the formation of a dimerized protein.

[0112] Furthermore, the hinge region functions as a flexible spacer between units, allowing them to simultaneously bind to two target molecules on the APC, even if the two targeting units of the dimer protein are expressed at a variable distance from each other. The hinge region may be derived from Ig, for example from IgG3. The hinge region may comprise one or more portions of an Ig-derived hinge region. The hinge region may contribute to dimerization through the formation of covalent bonds, such as disulfide bridges between cysteine ​​molecules. Thus, in one embodiment, the hinge region has the ability to form one or more covalent bonds, which can be, for example, disulfide bridges.

[0113] In one embodiment, the dimerization unit includes hinge exons h1 and h4 (human hinge region 1 and human hinge region 4) having amino acid sequences that have at least 80% sequence identity with amino acid sequences 94-120 of SEQ ID NO: 2.

[0114] In a preferred embodiment, the dimerization unit comprises hinge exon h1 and hinge exon h4 having amino acid sequences with at least 85% sequence identity to amino acid sequences 94-120 of SEQ ID NO: 2, for example, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0115] In one embodiment, the other domain that promotes dimerization is an immunoglobulin domain, such as a carboxy-terminal C domain (C domain), such as a CH1 domain, CH2 domain, or carboxy-terminal C domain (i.e., a CH3 domain), or a sequence substantially identical to the C domain or a variant thereof. Preferably, the other domain that promotes dimerization is a carboxy-terminal C domain derived from IgG. More preferably, the other domain that promotes dimerization is a carboxy-terminal C domain derived from IgG3.

[0116] In one embodiment, the dimerization unit includes a carboxy-terminal C domain derived from IgG3 having an amino acid sequence that has at least 80% sequence identity with amino acid sequences 131-237 of SEQ ID NO: 2.

[0117] In a preferred embodiment, the dimerization unit includes a carboxy-terminal C domain derived from IgG3 having an amino acid sequence with at least 85% sequence identity to amino acid sequences 131-237 of SEQ ID NO: 2, for example, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0118] Immunoglobulin domains contribute to dimerization through non-covalent interactions, such as hydrophobic interactions. Therefore, in one embodiment, immunoglobulin domains have the ability to form dimers via non-covalent interactions. Preferably, the non-covalent interaction is a hydrophobic interaction.

[0119] If the dimerization unit contains a CH3 domain, it is preferable that it does not contain a CH2 domain. Furthermore, if the dimerization unit contains a CH2 domain, it is preferable that it does not contain a CH3 domain.

[0120] In a preferred embodiment, the dimerization unit comprises hinge exon h1 and hinge exon h4, a third linker (or dimerization unit linker), and a polypeptide consisting of the CH3 domain of human IgG3.

[0121] In one embodiment of the present invention, the dimerization unit includes an amino acid sequence having at least 80% sequence identity with amino acid sequences 94-237 of SEQ ID NO: 2. In a preferred embodiment, the dimerization unit includes an amino acid sequence having at least 85% sequence identity with amino acid sequences 94-237 of SEQ ID NO: 2, for example, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0122] In a more preferred embodiment, the dimerization unit consists of an amino acid sequence having at least 80% sequence identity with respect to amino acid sequences 94-237 of SEQ ID NO: 2, for example, at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, for example at least 100% sequence identity with respect to amino acid sequences 94-237 of SEQ ID NO: 2.

[0123] In a more preferred embodiment, the dimerization unit consists of amino acid sequences 94-237 of SEQ ID NO: 2.

[0124] In one embodiment, a linker (dimerization unit linker) connecting the hinge region to other domains is present within the dimerization unit. In another embodiment, a linker is present, and it is a G3S2G3SG linker. In an alternative embodiment, the dimerization unit linker is a glycine-serine rich linker, preferably GGGSSGGGSG, i.e., the dimerization unit comprises a glycine-serine rich dimerization unit linker, and preferably the dimerization unit linker is GGGSSGGGSG. It should be understood that the dimerization unit may have any orientation relative to the antigenic unit and the targeting unit. In one embodiment, the antigenic unit is at the COOH terminus of the dimerization unit (e.g., via a unit linker), and the targeting unit is at the N terminus of the dimerization unit. In another embodiment, the antigenic unit is at the N terminus of the dimerization unit, and the targeting unit is at the COOH terminus of the dimerization unit. It is preferable that the antigenic unit is at the COOH terminus of the dimerization unit.

[0125] In a preferred embodiment, the polynucleotide of the present invention further comprises a nucleotide sequence encoding a signal peptide. The signal peptide is located either at the N-terminus or the C-terminus of the targeting unit, depending on the orientation of the targeting unit in the polypeptide. The signal peptide is constructed to enable the secretion of the polypeptide encoded by the polynucleotide in cells transfected with the polynucleotide.

[0126] Any suitable signal peptide may be used. Examples of suitable peptides include Ig VH signal peptides, e.g., SEQ ID NO: 9a, and human TPA signal peptides, e.g., SEQ ID NO: 10 and signal peptides having an amino acid sequence having at least 80% sequence identity to amino acid sequences 1-23 of SEQ ID NO: 1. In an alternative embodiment, the signal peptide is a human MIP1-α signal peptide.

[0127] In a preferred embodiment, the signal peptide comprises an amino acid sequence having at least 85%, for example, at least 86%, for example, at least 87%, for example, at least 88%, for example, at least 89%, for example, at least 90%, for example, at least 91%, for example, at least 92%, for example, at least 93%, for example, at least 94%, for example, at least 95%, for example, at least 96%, for example, at least 97%, for example, at least 98%, for example, at least 99%, for example, 100% sequence identity with amino acid sequences 1 to 23 of SEQ ID NO: 1. In an alternative embodiment, the signal peptide comprises amino acid sequences 1 to 23 of SEQ ID NO: 1.

[0128] In a more preferred embodiment, the signal peptide consists of an amino acid sequence having at least 80%, preferably at least 85%, for example, at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, for example at least 100% sequence identity with amino acid sequences 1 to 23 of SEQ ID NO: 1. In an alternative embodiment, the signal peptide consists of amino acid sequences 1 to 23 of SEQ ID NO: 1.

[0129] Sequence identity may be determined as follows: a high level of sequence identity indicates the possibility that the second sequence originates from the first sequence. Amino acid sequence identity requires identical amino acid sequences between two aligned sequences. Therefore, a candidate sequence that shares 70% amino acid identity with a reference sequence requires that, after alignment, 70% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence. Identity may be determined with the aid of computer analysis, without limitation, for example, the ClustalW computer alignment program (Higgins D., Thompson J., Gibson T., Thompson JD, Higgins DG, Gibson TJ, 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680), and the default parameters suggested therein. This program, along with its default settings, aligns the mature (bioactive) portions of the query and reference polypeptides. The number of perfectly conserved residues is counted and divided by the length of the reference polypeptide. In doing so, any tags or fusion protein sequences that form portions of the query sequence are ignored in the subsequent determination of alignment and sequence identity.

[0130] The ClustalW algorithm may also be used to align nucleotide sequences. Sequence identity may be calculated in a similar manner to that indicated for amino acid sequences.

[0131] Another preferred mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the FASTA sequence alignment software package (Pearson WR, Methods Mol Biol, 2000, 132:185-219). Align calculates sequence identity based on global alignment. Align0 does not penalize gaps at the ends of sequences. When using the ALIGN and Align0 programs to compare amino acid sequences, a BLOSUM50 substitution matrix with a -12 / -2 gap opening / extension penalty is preferably used.

[0132] The vaccine of the present invention may contain the polynucleotides described above. The polynucleotides may include double-stranded or single-stranded DNA nucleotide sequences or RNA nucleotide sequences, such as genomic DNA, cDNA, and RNA sequences.

[0133] The polynucleotide is preferably optimized for the species in order to express the polypeptide according to the present invention; that is, the polynucleotide sequence is preferably optimized for human codons.

[0134] The vaccine of the present invention may further comprise a polypeptide encoded by the polynucleotide sequence defined above. The polypeptide may be expressed in vitro for the production of the vaccine according to the present invention, or the polypeptide may be expressed in vivo as a result of administration of the polynucleotide defined above to an individual / patient.

[0135] The presence of a dimerizing unit leads to the formation of a dimeric protein when a polypeptide is expressed. The dimeric protein may be a homodimer, i.e., a homodimer in which the two polypeptide chains are identical and consequently contain the same antigenic sequence, or it may be a heterodimer containing two different monomeric polypeptides encoded within the antigenic unit. The latter may be appropriate when the amount of antigenic sequence exceeds the upper limit size for the antigenic unit. However, it is preferable that the dimeric protein be a homodimeric protein.

[0136] In a fifth aspect, the present invention relates to a vector comprising a polynucleotide including nucleotide sequences encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented co-antigen sequence or one or more portions thereof, and optionally one or more patient-specific antigen sequences or one or more portions thereof.

[0137] The vector is for transfection into host cells and expression of polypeptide / dimeric proteins encoded by the polynucleotides described above, i.e., an expression vector, such as a DNA plasmid.

[0138] The vector preferably allows for easy exchange of the various units described above, particularly the antigenic unit. In one embodiment, the vector may be a vector comprising a pUMVC4a vector or an NTC9385R vector skeleton. The antigenic unit may be replaced with an antigenic unit cassette in which the 5' site is incorporated in a GLGGL / GLSGL linker and the 3' site is restricted by an SfiI restriction enzyme cassette included after the stop codon in the vector.

[0139] In a sixth aspect, the present invention relates to a host cell comprising a polynucleotide including nucleotide sequences encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented covalent antigen sequence or one or more portions thereof, and optionally one or more patient-specific antigen sequences or one or more portions thereof.

[0140] In a seventh aspect, the present invention relates to a host cell comprising a vector containing a polynucleotide comprising nucleotide sequences encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented co-antigen sequence or one or more portions thereof, and optionally one or more patient-specific antigen sequences or one or more portions thereof.

[0141] Suitable host cells include prokaryotes, yeasts, insects, or higher eukaryotic cells. In a preferred embodiment, the host cells are human cells, preferably cells from a cancer patient, more preferably cells from the same cancer patient from which at least one patient-presented covalent antigen sequence or one or more portions thereof, and optionally one or more patient-specific antigen sequences or one or more portions thereof, are derived.

[0142] The vaccine according to the present invention is a personalized therapeutic anti-cancer vaccine in which at least one patient-presented shared antigen sequence and one or more patient-specific antigen sequences are identified in the patient receiving the vaccine, for example, in the patient's tumor tissue or bodily fluids, such as blood.

[0143] Therefore, in the eighth embodiment, the present invention relates to a method for preparing a personalized therapeutic anti-cancer vaccine comprising an immunologically effective amount of the above-defined dimeric protein or polypeptide by producing the polypeptide in vitro.

[0144] The in vitro synthesis of polypeptides and proteins may be carried out by any suitable method known to those skilled in the art, for example, by peptide synthesis or by the expression of polypeptides in any of the suitable expression systems known in the art, followed by purification.

[0145] Therefore, in one embodiment, the present invention provides an immunologically effective amount of (i) A dimeric protein comprising two polypeptides encoded by polynucleotides, the polynucleotides comprising nucleotide sequences encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented covalent antigen sequence or one or more portions thereof, and optionally one or more patient-specific antigen sequences or one or more portions thereof; or (ii) A polypeptide encoded by a polynucleotide comprising nucleotide sequences encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented covalent antigen sequence or one or more portions thereof, and optionally one or more patient-specific antigen sequences or one or more portions thereof. A method for preparing a personalized therapeutic anti-cancer vaccine, comprising the production of a dimeric protein or polypeptide in vitro, (a) Transfecting cells with polynucleotides; (b) Culturing cells; (c) Collecting and purifying dimeric proteins or polypeptides expressed from cells, (d) Mixing the dimeric protein or polypeptide obtained from step c) with a pharmaceutically acceptable carrier. This provides a method that includes [something].

[0146] In a preferred embodiment, the dimeric protein or polypeptide from step c) is dissolved in the pharmaceutically acceptable carrier.

[0147] The pharmaceutically acceptable carrier is preferably an aqueous pharmaceutically acceptable carrier, such as water or a buffer solution. In one embodiment, the vaccine further comprises an adjuvant.

[0148] Purification may be carried out according to any preferred method, such as chromatography, centrifugation, or differential solubility.

[0149] In a ninth embodiment, the present invention relates to a method for in vitro preparing a personalized therapeutic anti-cancer vaccine comprising an immunologically effective amount of the polynucleotide defined above.

[0150] Therefore, in one embodiment, the present invention provides a method for preparing a personalized therapeutic anti-cancer vaccine comprising an immunologically effective amount of polynucleotides comprising nucleotide sequences encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented co-antigen sequence or one or more portions thereof, and optionally one or more patient-specific antigen sequences or one or more portions thereof, the method is a. Preparation of polynucleotides; b. Selective cloning of polynucleotides into an expression vector and c. Mix the polynucleotide from step a) or the vector (form) from step b) with a pharmaceutically acceptable carrier. Includes.

[0151] Polynucleotides may be prepared by any suitable method known to those skilled in the art. For example, polynucleotides may be prepared by chemical synthesis using an oligonucleotide synthesizer.

[0152] In particular, smaller nucleotide sequences, such as those encoding portions of targeting units, dimerizing units, and / or antigenic units, may be synthesized individually and then ligated to produce a final polynucleotide for inclusion in the vector skeleton.

[0153] Prior to designing the antigenic units to be included in personalized therapeutic anti-cancer vaccines, a method is used to identify the antigenic sequences to be included in the antigenic units (i.e., patient-presented shared antigen sequences and optionally, patient-specific antigen sequences) before the vaccine preparation method is developed.

[0154] Patient-presented shared antigens may be identified in the patient's (tumor) tissue or bodily fluids by methods known in the art (obtained by methods known in the art), including: • Sequencing of the patient's genome or exome, and tailor-made software search of whole-genome / exome-seq data for selective identification of, for example, mutant oncogenes or mutant tumor suppressor genes; • Immunohistochemistry of patient tumor tissue to detect the presence of mutant proteins; RT-PCR for detecting the presence of known mutations in viral antigens or oncogenes; • ELISA using antibodies against mutant tumor proteins in serum samples; • Comparison with healthy tissue to detect RNA-seq and covalent antigen expression / overexpression in tumor tissue; • Tailor-made software search in raw RNA sequence data to identify intron-retaining antigens; • Tailor-made software search in whole-genome-seq data to identify translocation elements, which are components of dark matter antigens; • Detection of short repeats in raw whole exome / RNA sequence data for the identification of dark matter antigens; • RNA-seq data for identifying co-viral antigens; and Comparison of RNA-seq from patient tumor samples with either the patient's own healthy tissue or GTEX / HPA gene expression data from a cohort / database (e.g., TCGA).

[0155] In a preferred embodiment, the antigenic unit comprises at least one patient-presented co-antigen sequence known to be immunogenic. In another preferred embodiment, the antigenic unit comprises one or more portions of at least one patient-presented co-antigen sequence, for example, one or more epitopes known to be immunogenic or predicted to bind to an HLA allele of a particular patient. If a patient-specific antigen sequence is included in the antigenic unit, the antigenic unit preferably comprises a patient-specific antigen sequence having predicted immunogenicity.

[0156] Therefore, the identified patient-presented shared antigens and patient-specific antigens may be further processed to find sequences that, when included in the antigenic unit, make the vaccine of the present invention most effective. Methods and sequences for such processing depend on how the antigens were identified, i.e., the data forming the basis for such processing. In one embodiment, processing and selection of antigen sequences to be included in the vaccine of the present invention are carried out as follows: 1) A search is performed in the literature and / or one or more databases to obtain information on co-antigens and their sequences, and preferably information on their expression patterns, immunogenicity, epitopes, and HLA presentation. Such searches are performed to determine whether the identified antigen is a patient-presented co-antigen or a patient-specific antigen. 2) If the identified antigen is determined to be a patient-presented co-antigen, its sequence is studied to identify epitopes, preferably all, that are predicted to bind to patient-specific HLA class I and / or class II alleles. The patient's HLA class I and / or II alleles are determined, for example, by sequencing normal tissue, such as blood cells. The prediction may be performed using prediction tools known in the art, i.e., prediction software known in the art, such as NetMHCpan and similar tools. 3) The most promising, i.e., most immunogenic, sequences from the patient-presented shared antigens that show predicted binding to one or more of the patient's HLA class I / II alleles are selected for inclusion in the antigenic unit. In one embodiment, for example, if only a few promising epitopes are identified in step 2, or if longer stretches of non-immunogenic sequences exist between the epitopes, a number of smallest epitopes are selected. In another embodiment, longer sequences containing several epitopes that bind to the patient's specific HLA alleles are selected. In yet another embodiment, a full-length sequence is selected for inclusion in the antigenic unit. 4) The most promising patient-specific antigenic sequences, e.g., epitopes, are selected to be included in the antigenic unit based on the predicted immunogenicity of such sequences and their binding to the patient's HLA class I and / or class II alleles.

[0157] If patient-specific antigen sequences are included in the antigenic unit, once such antigens are identified and the patient's HLA class I and / or II alleles are determined, the next step is to select the most promising sequence, e.g., an epitope, based on the predicted immunogenicity of such sequences and their binding to the patient's HLA class I and / or class II alleles.

[0158] Tumor mutations are detected by sequencing tumor and normal tissues and comparing the resulting sequences. Various methods are available to detect the presence of specific mutations or alleles in an individual's DNA or RNA. For example, in addition to techniques including dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, and the TaqMan system, various DNA "chip" technologies, such as the Affymetrix SNP chip, may be applied. Alternatively, methods for identifying mutations by direct protein sequencing may be performed.

[0159] From among possibly hundreds or thousands of mutations in tumor exomes, the most promising sequences are selected in silico based on a predictive HLA-binding algorithm. The intention is to identify all valid epitopes and, after ranking or scoring, determine which sequences will be included in the antigenic unit.

[0160] For example, any suitable algorithm from the following may be used: Peptide-MHC binding compatibility can be analyzed using available free software (IEDB and NetMHCpan) from the following website: http: / / www.iedb.org / http: / / www.cbs.dtu.dk / services / NetMHC /

[0161] Commercially available, advanced software for predicting optimal sequences for vaccine design can be found below: http: / / www.oncoimmunity.com / https: / / omictools.com / t-cell-epitopes-category https: / / github.com / griffithlab / pVAC-Seq http: / / crdd.osdd.net / raghava / cancertope / help.php http: / / www.epivax.com / tag / neoantigen /

[0162] Each mutation is scored for its antigenicity, and the most antigenic epitope is selected and optimally organized within the antigenic unit.

[0163] Therefore, in one embodiment, the present invention is a method for preparing a personalized therapeutic anti-cancer vaccine, a) Step of identifying at least one patient-presented shared antigen in the patient's tumor tissue or body fluids. b) The step of determining the patient's HLA class I and / or class II alleles. c) A step of predicting the immunogenicity of at least one identified antigen or one or more parts thereof by their predicted binding affinity to the patient's HLA class I and / or II alleles. d) A step of selecting at least one antigen or one or more parts thereof based on their immunogenicity predicted in step c); and e) A step of preparing a polynucleotide sequence comprising an antigenic unit containing a nucleotide sequence encoding at least one antigen or one or more portions thereof selected in step d). This provides a method that includes [something].

[0164] In another embodiment, the present invention relates to a method for preparing a personalized therapeutic anti-cancer vaccine, a) Identifying at least one patient-presented co-antigen in the patient's tumor tissue or body fluids, and identifying one or more patient-specific antigens in the patient's tumor tissue; b) A step of determining the patient's HLA class I and / or class II alleles; c) A step of predicting the immunogenicity of at least one identified patient-presented shared antigen or portion thereof and one or more identified patient-specific antigens or portions thereof by their predicted binding affinity to the patient's HLA class I and / or II alleles; d) A step of selecting at least one patient-specific co-antigen or one or more parts thereof and one or more patient-specific antigens or one or more parts thereof based on their immunogenicity predicted in step c); and e) A step of preparing a polynucleotide sequence comprising an antigenic unit comprising at least one patient-specific covalent antigen or one or more portions thereof selected in step d) and a nucleotide sequence encoding one or more patient-specific antigens or one or more portions thereof. This provides a method that includes [something].

[0165] In one embodiment, the polynucleotide sequence prepared in step e) further comprises a nucleotide sequence encoding a targeting unit and a dimerizing unit as described herein.

[0166] In a preferred embodiment, the prepared polynucleotide sequence is cloned into an expression vector. In yet another preferred embodiment, the polynucleotide sequence from step e) is cloned into an expression vector containing nucleotide sequences encoding dimerization units and targeting units.

[0167] In yet another embodiment, the polynucleotide or vector is mixed with a pharmaceutically acceptable carrier.

[0168] The final vaccine will be released next, as follows: - Polynucleotides as defined above - Polypeptides encoded by the polynucleotides defined above - A dimeric protein containing a polypeptide chain encoded by the polynucleotides defined above. It is manufactured to contain one of the following.

[0169] The vaccine further comprises a pharmaceutically acceptable carrier and may further comprise other pharmaceutically acceptable excipients, such as stabilizers, adjuvants, buffers and the like.

[0170] Examples of pharmaceutically acceptable carriers include, but are not limited to, saline solutions, buffered saline solutions such as PBS, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffers, and combinations thereof.

[0171] In particular, for vaccines containing polypeptides / proteins, pharmaceutically acceptable excipients include poly-ICLC, 1018 ISS, aluminum salt, Amplivax, AS 15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact EV1 P321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, and Montanide This includes, but is not limited to, ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM, vector systems, PLGA microparticles, reciquimod, SRL172, virosoms and other virus-like particles, YF-17D, VEGF traps, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, bajimezan, and / or AsA404 (DMXAA).

[0172] In particular, with respect to vaccines containing polynucleotides in an expression vector, the vaccine may also contain an adjuvant in the form of a plasmid containing a nucleotide sequence encoding a molecule that facilitates cell transfection and / or a chemokine or cytokine to enhance the immune response.

[0173] The vaccine is formulated into any suitable formulation for administration to the patient, such as a liquid formulation for intradermal or intramuscular injection.

[0174] The vaccine may be administered in any suitable form for either a polypeptide / protein vaccine or a polynucleotide vaccine, for example, intradermally, intramuscularly, or subcutaneously by injection, or applied to the mucous membrane or epithelium, for example, intranasally, or orally, enterally, or into the bladder.

[0175] In particular, if the vaccine is a polynucleotide vaccine, it is preferably administered intramuscularly or intradermally.

[0176] In one embodiment, the vaccine is administered by intranodular injection. As used herein, the term “intranodular injection” means that the vaccine is injected into a lymph node.

[0177] The personalized therapeutic anti-cancer vaccine of the present invention, prepared by the method described above, may be obtained within 12 weeks, for example, within 9 weeks, 8 weeks, 6 weeks, or 4 weeks.

[0178] The cancer being treated may be any cancer, for example, a cancer in which cancer cells have modifications resulting in shared cancer antigens and, optionally, patient-specific cancer antigens. The cancer may be a primary tumor, a metastatic tumor, or both. The tumor being examined for modifications may be a primary tumor or a metastatic tumor. In one embodiment, the cancer being treated is a cancer known to have a high antigen burden, for example, melanoma, lung cancer, kidney, head and neck, or bladder cancer. In another embodiment, the cancer being treated may be a solid tumor or a humoral tumor. An example of a solid tumor is a cancer that forms a solid mass, for example, a tumor. An example of a humoral tumor is a cancer that exists in body fluids, for example, lymphoma or hematological cancer. Examples of cancers that can be treated with the vaccine of the present invention include breast cancer, ovarian cancer, colon cancer, prostate cancer, bone cancer, colorectal cancer, stomach cancer, lymphoma, malignant melanoma, liver cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancer, kidney cancer, bile duct cancer, brain cancer, cervical cancer, bladder cancer, esophageal cancer, Hodgkin's disease, and adrenal cortical cancer.

[0179] In a preferred embodiment, the treatment is carried out using a vaccine containing the polynucleotide described above, where the polynucleotide is, for example, DNA or RNA, and is preferably contained in a vector.

[0180] It is preferable to inject the polynucleotide vaccine of the present invention intramuscularly, for example, into large muscles, such as large muscles in the shoulder, buttocks, or thigh. It has been found that the polypeptide / dimer proteins of the present invention are produced locally, and appropriate immune cells essentially transport the polypeptide / dimer proteins internally at the site of their production, i.e., substantially no polypeptide / dimer proteins reach the bloodstream.

[0181] Any preferred method for injecting the polynucleotide vaccine may be used, for example, by the use of a jet injector or assisted by electroporation.

[0182] The vaccine may be administered as a single dose, or the administration may be repeated. If the vaccine is administered repeatedly, it is preferable that it be administered at intervals of at least 3 weeks to avoid T cell exhaustion.

[0183] Therefore, in one embodiment, the drug regimen is vaccination at weeks 0, 3, and 6, and thereafter every four weeks as long as the patient has a clinical benefit. The vaccine may be administered for up to one year.

[0184] The vaccine contains an immunologically effective amount of polynucleotide / polypeptide / dimeric protein. “Immunologically effective amount” means the amount of such compound required to induce an immune response in a patient vaccinated with the aforementioned compound. Non-limiting parameters indicating such an immune response include one or more of the following: cessation of tumor growth and / or cessation of its expansion and / or reduction of tumor size; reduction in disease progression or stabilization of the disease, i.e., cancer progressing at a slower rate or not progressing at all. This includes tumors growing at a slower rate or not growing and / or expanding at a slower rate or not expanding at all, e.g., not expanding to lymph nodes or forming metastases and / or not becoming more malignant. Other non-limiting parameters indicating such an immune response include tumor reduction (in terms of weight and / or volume); reduction in the number of individual tumor colonies; tumor elimination; and progression-free survival. Ultimately, the physician determines the dosage, which is variable and may depend on age, weight, and the overall condition of the patient being treated, the severity of the cancer being treated, the physician's judgment, and the specific properties and characteristics of the personalized vaccine of the present invention. In one embodiment, the dosage is typically in the range of 0.3 to 6 mg for DNA vaccines and in the range of 5 μg to 5 mg for polypeptide / protein vaccines.

[0185] In a tenth embodiment, the present invention relates to a method for treating cancer in a patient, comprising an immunologically effective amount, (i) a polynucleotide comprising nucleotide sequences encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one covalent antigen sequence or one or more portions thereof present in the patient and one or more antigen sequences or one or more portions thereof that are selectively specific to the patient; or (ii) A polypeptide encoded by a polynucleotide as defined in (i); or (iii) A dimeric protein consisting of two polypeptides encoded by a polynucleotide as defined in (i); and Medicinally acceptable carriers The present invention provides a method comprising administering a personalized therapeutic anti-cancer vaccine containing [specific ingredient] to a patient.

[0186] Therefore, the present invention provides a method for treating cancer in a patient, comprising administering to the patient a personalized therapeutic anti-cancer vaccine according to the present invention, which is specifically prepared for the patient.

[0187] Alternatively, the present invention relates to an immunologically effective amount for use in a method of treating cancer in a patient. (i) a polynucleotide comprising nucleotide sequences encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented covalent antigen sequence or one or more portions thereof and optionally one or more patient-specific antigen sequences or one or more portions thereof; or (ii) A polypeptide encoded by a polynucleotide as defined in (i); or (iii) A dimeric protein consisting of two polypeptides encoded by a polynucleotide as defined in (i); and Medicinally acceptable carriers The present invention provides a personalized therapeutic anti-cancer vaccine, wherein the vaccine is specifically formulated for the patient.

[0188] Furthermore, the present invention relates to the production of pharmaceuticals for the treatment of cancer in patients, (i) a polynucleotide comprising nucleotide sequences encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented covalent antigen sequence or one or more portions thereof and optionally one or more patient-specific antigen sequences or one or more portions thereof; or (ii) A polypeptide encoded by a polynucleotide as defined in (i); or (iii)(i) A dimeric protein consisting of two polypeptides encoded by a polynucleotide as defined in (iii)(i). The use of a polynucleotide, polypeptide, or dimeric protein is provided, which is specifically prepared for the patient.

[0189] The vaccine treatment according to the present invention may be combined with any other anti-cancer treatment, such as radiotherapy, chemotherapy, and surgical treatment.

[0190] The vaccine treatment according to the present invention may also be combined with checkpoint blocker treatment. [Brief explanation of the drawing]

[0191] [Figure 1] Figure 1 shows the amino acid sequences of all epitopes predicted to bind to the HLA class I allele of HPV16 E6 and patient 1. The two underlined sequences constitute the HLA class I optimized sequences for inclusion in the antigenic unit of a personalized therapeutic anti-cancer vaccine for patient 1. [Figure 2] Figure 2 shows the amino acid sequences of all epitopes predicted to bind to HPV16 E6 and the HLA class I and HLA class II alleles of patient 1. The sequences in the boxes constitute the HLA class I / HLA class II optimized sequences for inclusion in the antigenic unit of a personalized therapeutic anti-cancer vaccine for patient 1. [Figure 3] Figure 3 shows the amino acid sequences of all epitopes predicted to bind to HPV16 E7 and the HLA class I allele of patient 1. The underlined sequences constitute the HLA class I optimized sequences for inclusion in the antigenic unit of a therapeutic anti-cancer vaccine personalized for patient 1. [Figure 4]Figure 4 shows the amino acid sequences of all epitopes predicted to bind to HPV16 E7 and the HLA class I and HLA class II alleles of patient 1. The sequences in the two boxes constitute the HLA class I / HLA class II optimized sequences for inclusion in the antigenic unit of a therapeutic anti-cancer vaccine personalized for patient 1. [Figure 5] Figure 5 shows the amino acid sequences of all epitopes predicted to bind to the HLA class I alleles of HPV16 E6 and Patient 2. The underlined sequences constitute the HLA class I optimized sequences for inclusion in the antigenic unit of a personalized therapeutic anti-cancer vaccine for Patient 2. [Figure 6] Figure 6 shows the amino acid sequences of all epitopes predicted to bind to HPV16 E6 and the HLA class I and HLA class II alleles of patient 2. The sequences in the boxes constitute the HLA class I / HLA class II optimized sequences for inclusion in the antigenic unit of a personalized therapeutic anti-cancer vaccine for patient 2. [Figure 7] Figure 7 shows the amino acid sequences of all epitopes predicted to bind to the HLA class I alleles of HPV16 E7 and Patient 2. The underlined sequences constitute the HLA class I optimized sequences for inclusion in the antigenic unit of a personalized therapeutic anti-cancer vaccine for Patient 2. [Figure 8] Figure 8 shows the amino acid sequences of all epitopes predicted to bind to HPV16 E7 and the HLA class I and HLA class II alleles of patient 2. The sequences in the two boxes constitute the HLA class I / HLA class II optimized sequences for inclusion in the antigenic unit of a personalized therapeutic anti-cancer vaccine for patient 2. [Figure 9] Figure 9 shows the immunogenicity of DNA vaccines (constructs) VB4097, VB4100, and VB4105 in mice vaccinated with these constructs, measured by the IFN-γ immune response (total T cell response) from T cells, compared to the negative control VB1026. [Figure 10] Figure 10 shows the immunogenicity of DNA vaccines (constructs) VB4100, VB4101, and VB4102 in mice vaccinated with these constructs, measured by the IFN-γ immune response from CD8+ T cells compared to the negative control VB1026. [Figure 11] Figure 11 shows the immunogenicity of DNA vaccines (constructs) VB4100 and VB4102 in mice vaccinated with these constructs, by measuring the IFN-γ immune response from A) T cells (total T cell response), B) CD8+ T cells, and C) CD4+ T cells, compared to the negative control VB1026. [Figure 12] Figure 12 shows the immunogenicity of DNA vaccines (constructs) VB4118, VB4119, VB4121, VB4127, VB4128, and VB4130 in mice vaccinated with these constructs, measured by the IFN-γ immune response from T cells compared to the negative control VB1026. [Examples]

[0192] Example 1: Design of the antigenic unit contained in the polynucleotide / polypeptide / dimeric protein and vaccine of the present invention The antigenic unit may be designed with the following variations in the patient-presented shared antigen sequence: A. Overall arrangement B. HLA-optimized sequences. The selected sequences for inclusion in the antigenic unit are optimized to cover the most immunogenic epitopes, i.e., epitopes that have high binding affinity to the patient's HLA I and / or HLA II molecules. C. Epitopes with predicted binding affinity to the patient's HLA I and / or HLA II molecules. D. A+C combination E. Combinations of B and C F. Combinations of A and B

[0193] The patient-presented shared antigen sequence described above may be, for example, HPV16. In cases D and F, which include full-length sequences, case D may be, for example, a combination of HPV16 E7(A) as a full-length sequence and an epitope (C) from HPV E6, and case F may be, for example, a combination of HPV16 E7(A) as a full-length sequence and an HLA-optimized sequence (B). The above examples involve a single patient-presented shared antigen but include two different regions thereof. In another embodiment, cases D and F relate to a combination of a first patient-presented shared antigen, for example, a full-length sequence (A) of HPV16 E7, and a second patient-presented shared antigen, for example, an epitope (C) of KRAS or an HLA-optimized sequence (B).

[0194] Therefore, the antigenic unit may contain A-F from one patient-presented shared antigen, or it may contain A-F from several patient-presented shared antigens.

[0195] Sequences A-F are organized into antigenic units according to the methods provided herein. Different antigenic unit designs may be evaluated in animal models, for example, as described in Example 3, to determine the optimal antigenic unit design. The broadness, strength, and kinetics of antigen-specific immunogenicity can be determined by IFN-gamma ELISPOT analysis.

[0196] Antitumor efficacy can be tested in tumor challenge experiments.

[0197] Example 2: Construction of polynucleotides according to the present invention A polynucleotide according to the present invention has been designed and comprises the following units and components:

[0198] [Table 1]

[0199] Example 3: Selection of patient-presented shared antigen sequences for inclusion in antigenic units contained in polynucleotides / polypeptides / dimeric peptides and vaccines according to the present invention. Blood and tumor tissue samples were obtained from two patients, Patient 1 and Patient 2, who presented with squamous cell carcinoma of the head and neck. Blood samples were analyzed for exome sequencing to characterize exons in healthy cells. Tumor tissue samples were analyzed for exome sequencing to characterize exons in tumor tissue, and RNA-seq was performed to evaluate the RNA expression levels of each gene. The presence of HPV16 covalent antigens was identified using an anti-HPV16 antibody in ELISA.

[0200] To identify the most immunogenic sequences, the HLA class I and II alleles of each patient were determined by sequencing of normal tissue (blood cells). The following HLA class I and II alleles were identified:

[0201] [Table 2]

[0202] HPV has a circular double-stranded DNA genome, approximately 8kb in size, encoding eight genes, of which E6 and E7 possess transformation properties. Viral E6 and E7 proteins are known to be involved in the conversion of healthy cells into malignant cells. The ability of HPV16 E6 and E7 proteins to associate with tumor suppressor factors p53 and pRB, respectively, suggests a mechanism by which these viral proteins induce tumors. Therefore, the E6 and E7 sequences of HPV16 were selected to identify sequences within them for inclusion in an antigenic unit, as they are known covalent tumor antigens. Prediction of binding to patient HLA class I and HLA class II alleles was performed using NetMHCpan 4.0 software.

[0203] Patient 1: HPV 16 E6: A total of 16 epitopes, each 9 amino acids long, were predicted to bind to the HLA class I allele of patient 1, and a total of 16 epitopes, each 9 amino acids long, were predicted to bind to the HLA class II allele of patient 1 (Figure 2 and Table 3).

[0204] Therefore, an antigenic unit was designed to be included in a personalized therapeutic anti-cancer vaccine for patient 1, which may include: A. Full-length HPV16 E6 sequence (151 amino acids) B. An HLA class I optimized sequence containing a portion of the 16 epitopes, for example, the 12 underlined epitopes shown on the left side of Figure 1. This sequence contains 65 amino acids, or 43% of the full-length sequence. C. Two HLA class I optimized sequences, where the first sequence contains 12 of the 16 epitopes and the second sequence contains 3 of the epitopes (Figure 1). Therefore, the combined two sequences contain 15 of the 16 epitopes, totaling 92 amino acids, or 61% of the full-length sequence. D. An HLA class I / HLA class II optimized sequence containing 12 of 16 HLA class I epitopes and 9 of 16 HLA class II epitopes, thus containing 21 of 32 HLA class I / class II epitopes (Figure 2, box). This sequence contains 65 amino acids, i.e., 43% of the full-length sequence.

[0205] It is preferable to include C or D in the antigenic unit.

[0206] [Table 3]

[0207] HPV16 E7: Nine epitopes, each 9 amino acids long, were predicted to bind to the HLA class I allele of patient 1, while twelve epitopes, each 9 amino acids long, were predicted to bind to the HLA class II allele of patient 1 (Figure 4 and Table 4).

[0208] Therefore, an antigenic unit was designed to be included in a personalized therapeutic anti-cancer vaccine for patient 1, which may include: E. Full-length HPV16 E7 sequence (98 amino acids) F. An HLA class I optimized sequence containing a portion of the nine epitopes, for example, the underlined epitope shown in Figure 3. This sequence contains 56 amino acids, or 57% of the full-length sequence. G. Two HLA class I / HLA class II optimized sequences, where the first sequence contains 2 of the 9 HLA class I epitopes and 7 of the 16 HLA class II epitopes, and the second sequence contains 4 of the 9 HLA class I epitopes and 5 of the 16 HLA class II epitopes (Figure 4, box). Therefore, the combined two sequences contain 6 of the 9 HLA class I epitopes and all of the HLA class II epitopes. The combined two sequences contain 54 amino acids, i.e., 55% of the full-length sequence.

[0209] It is preferable to include F or G in the antigenic unit.

[0210] Based on the above, an antigenic unit was designed to be included in a personalized therapeutic anti-cancer vaccine for patient 1, which includes at least one of C, D, F, and G, or all of C, D, F, and G, or any combination of these between the two described extreme cases.

[0211] [Table 4]

[0212] Patient 2: HPV16 E6 A total of 14 epitopes, each 9 amino acids long, were predicted to bind to the HLA class I allele of patient 2, and a total of 14 epitopes, each 9 amino acids long, were predicted to bind to the HLA class II allele of patient 2 (Figure 6 and Table 5).

[0213] Therefore, an antigenic unit was designed to be included in a personalized therapeutic anti-cancer vaccine for patient 2, which may include the following: H. Full-length HPV16 E6 sequence (151 amino acids) I. An HLA class I optimized sequence containing a portion of the 14 epitopes, for example, the 11 underlined epitopes shown in Figure 5. This sequence contains 57 amino acids, or 38% of the full-length sequence. J. An HLA class I / HLA class II optimized sequence containing 11 of 14 HLA class I epitopes and 8 of 14 HLA class II epitopes, thus containing 19 of 28 HLA class I / class II epitopes (Figure 6, box). This sequence contains 59 amino acids, i.e., 39% of the full-length sequence.

[0214] It is preferable to include J in the antigenic unit.

[0215] [Table 5]

[0216] HPV16 E7 Ten epitopes, each 9 amino acids long, were predicted to bind to the HLA class I allele of patient 2, while eleven epitopes, each 9 amino acids long, were predicted to bind to the HLA class II allele of patient 2 (Figure 8 and Table 6).

[0217] Therefore, antigenic units can be designed to be included in a personalized therapeutic anti-cancer vaccine for patient 2, including the following: A. Full-length HPV16 E7 sequence (98 amino acids) B. An HLA class I optimized sequence containing a portion of the 10 epitopes, for example, the six underlined epitopes shown in Figure 7. This sequence contains 26 amino acids, or 27% of the full-length sequence. C. Two HLA class I / HLA class II optimized sequences, where the first sequence contains 6 of the 10 HLA class I epitopes and 6 of the 11 HLA class II epitopes, and the second sequence contains 2 of the 10 HLA class I epitopes and 5 of the 11 HLA class II epitopes (Figure 8, box). Therefore, the two combined sequences contain 8 of the 11 HLA class I epitopes and all of the HLA class II epitopes. The two combined sequences contain 45 amino acids, i.e., 45% of the full-length sequence.

[0218] It is preferable to include L or M in the antigenic unit.

[0219] Based on the above, an antigenic unit was designed to be included in a personalized therapeutic anti-cancer vaccine for patient 2, which includes at least one of J, L, and M, or all of J, L, and M, or any combination of these between the two described extreme cases.

[0220] [Table 6]

[0221] Comparing patients 1 and 2, i.e., Figures 2 and 6 and Figures 4 and 8, it is clear that the optimal sequence to be included in the vaccine according to the present invention differs considerably between the two patients.

[0222] Example 4: Comparison of vaccines containing patient-specific antigens and patient-presented shared antigens A mouse TC-2 tumor model is used to compare the efficacy of a vaccine containing only patient-specific antigen sequences with the efficacy of a vaccine according to the present invention that contains patient-presented shared antigen sequences and optionally patient-specific antigen sequences.

[0223] Co-antigens and specific antigens present in the TC-2 tumor cell line are identified, processed, and selected as described herein; i.e., co-antigen sequences for inclusion in antigenic units are selected based on their binding affinity to MHC molecules, and specific antigen sequences are selected based on additional parameters in an in silico immunogenicity prediction method. The co-antigen selected for inclusion in antigenic units is the viral antigen HPV16, and sequences encoding portions of its E6 and E7 proteins were selected.

[0224] All selected antigen sequences are ordered from a commercial supplier, e.g., Genscript (New Jersey, US), and cloned into the expression vector pUMVC4a, which contains sequences encoding the LD78 beta-targeting unit and the hIgG3 dimerizing unit.

[0225] The antigenic unit of vector 1 contains only a covalent antigen sequence, while the antigenic unit of vector 2 contains both a covalent antigen sequence and a specific antigen sequence.

[0226] To verify accurate vaccine body formation, HEK293 cells are transfected with the vector, and vaccine body proteins in the supernatant are identified by Western blotting and / or sandwich ELISA. An empty pUMVC4a vector is included as a negative control. Intact homodimer protein formation is confirmed as follows: Proteins in the supernatant from transfected cells are detected by Western blotting with an anti-hMIP-1 alpha antibody in the presence or absence of a reducing agent that results in the reduction of the dimeric protein to monomers.

[0227] Vaccines were prepared by mixing 20 μg each of vector 1 and vector 2 with aqueous buffer. The vaccine was intramuscularly injected into the tibialis anterior muscle of mice, followed by electroporation using TriGrid, Ichor, (US). On day 13, the mice were euthanized and their spleens were collected.

[0228] T cell responses are evaluated using IFN-gamma ELISpot. We observe that the vaccine according to the present invention induces a higher and broader T cell response compared to vaccines containing only specific antigen sequences.

[0229] Example 5: Individual therapeutic anti-cancer DNA vaccines according to the present invention Individual therapeutic anti-cancer DNA vaccines according to the present invention may be prepared by GMP production of a polynucleotide-containing vector according to the present invention in accordance with regulatory authority guidelines, as well as by filling and final formulation of the DNA vaccine. The vector may be formulated by dissolving it in sterile saline solution, such as PBS, at a concentration of 2-6 mg / ml. The vaccine may be administered intradermally or intramuscularly with or without subsequent electroporation, or alternatively, using a jet injector.

[0230] Example 6: Selection of patient-presented shared antigens (and patient-specific antigens) sequences for inclusion in antigenic units contained in polynucleotides / polypeptides / dimeric peptides and vaccines according to the present invention. Blood and tumor tissue samples were obtained from three patients (Patients 1, 2, and 3) with squamous cell carcinoma of the head and neck. Blood samples were analyzed for exome sequencing to characterize exons in healthy cells. Tumor tissue samples were analyzed for exome sequencing to characterize exons in tumor tissue, and RNA-seq was performed to evaluate the RNA expression levels of each gene.

[0231] The presence of HPV16 covalent antigens was identified using PCR. Ectopic expression of NY-ESO-1 was determined by ELISA using an anti-NY-ESO-1 antibody. Patient-specific antigen sequences for each patient were identified as previously described in this application and in International Publication No. 2017 / 118695 (incorporated herein by reference).

[0232] NY-ESO-1 (also known as cancer / testis antigen 1B) is a protein belonging to the family of cancer testis antigens (CTAs) that has been found to be re-expressed at the mRNA and protein levels in various malignancies, but its normal expression in adult tissues is restricted to germ cells and placental cells. Expression of NY-ESO-1 has been reported in a wide range of tumor types.

[0233] Regarding HPV, the E6 and E7 sequences of HPV16 are known shared tumor antigens, and sequences among them were selected to find sequences for inclusion in antigenic units.

[0234] To find the most immunogenic sequences, the HLA class I and II alleles of each patient were determined by sequencing normal tissue (blood cells). Prediction of the binding of HPV16 E6 / E7 sequences and NY-ESO-1 sequences to the HLA class I and HLA class II alleles of the patients was performed using NetMHCpan 4.0 software. Sequences from the IEDB database known from the literature to have induced positive T cell responses (not matching the patient's HLA alleles) were included in the analysis.

[0235] The HLA class I and II alleles found for patients 1, 2, and 3 are listed in the following table:

[0236]

Table 7

[0237] Patient 1:

[0238]

Table 8

[0239] An antigenic unit for inclusion in an individualized therapeutic anti-cancer vaccine for patient 1 was designed, which includes SEQ ID NO: 14 and the sequences described in the right column of the above table in the following order: E7|linker|NY-ESO-1|linker|E6.

[0240] A second antigenic unit was designed for inclusion in a personalized therapeutic anti-cancer vaccine for Patient 1, which includes Sequence ID No. 15 and contains the sequence listed in the right column of the table above, plus 17 additional patient-specific antigen sequences. The most hydrophobic sequence was placed substantially in the center of the antigenic unit, and the most hydrophilic sequences were placed at the beginning and end of the antigenic unit. Glycine-serine linkers were inserted between the sequences. The antigenic unit contains the sequences in the following order, where T1D represents the patient-specific antigen sequence: T1D320|Linker|T1D814|Linker|T1D182|Linker|T1D689|Linker|E7|Linker|T1D339|Linker|T1D428|Linker|NY-ESO-1|Linker|T1D572|Linker|T1D359|Linker|T1D488|Linker|T1D554|Linker|T1D272|Linker|T1D210|Linker|T1D849|Linker|T1D4|Linker|T1D77|Linker|T1D717|Linker|T1D586|Linker|E6.

[0241] [Table 9]

[0242] Patient 2:

[0243] [Table 10]

[0244] An antigenic unit was designed for inclusion in a personalized therapeutic anti-cancer vaccine for patient 2, which includes sequence number 16 and contains the sequence listed in the right column of the table above in the following order: E6|linker|NY-ESO-1|linker|E7.

[0245] A second antigenic unit was designed for inclusion in a personalized therapeutic anti-cancer vaccine for Patient 2, which includes Sequence ID No. 17 and contains the sequence listed in the right column of the table above, plus 17 additional patient-specific antigen sequences. The most hydrophobic sequence was placed substantially in the center of the antigenic unit, and the most hydrophilic sequences were placed at the beginning and end of the antigenic unit. A glycine-serine linker was inserted between the sequences. The antigenic unit contains the sequences in the following order, where T1D represents the patient-specific antigen sequence: E6|Linker|T1D323|Linker|T1D506|Linker|T1D12|Linker|T1D315|Linker|T1D302|Linker|T1D700|Linker|NY-ESO-1|Linker|T1D535|Linker|T1D358|Linker|T1D670|Linker|T1D294|Linker|T1D336|Linker|T1D499|Linker|T1D425|Linker T1D491|Linker|T1D314|Linker|T1D430|Linker|E7|Linker|T1D582.

[0246] [Table 11]

[0247] Patient 3:

[0248] [Table 12]

[0249] An antigenic unit was designed for inclusion in a personalized therapeutic anti-cancer vaccine for patient 3, which includes sequence number 18 and contains the sequence listed in the right column of the table above in the following order: NY-ESO-1|linker|E7|linker|E6.

[0250] Design a second antigenic unit for inclusion in an individualized therapeutic anti-cancer vaccine for Patient 3, which includes SEQ ID NO: 19, the sequences listed in the right column of the above table, and additionally 17 patient-specific antigen sequences. The most hydrophobic sequences were placed substantially in the center of the antigenic unit, and the most hydrophilic sequences were placed at the beginning and end of the antigenic unit. Glycine-serine linkers were inserted between the sequences. The antigenic unit includes the sequences in the following order, where T1D represents patient-specific antigen sequences: T1D223|Linker|T1D164|Linker|T1D56|Linker|T1D36|Linker|T1D129|Linker|T1D274|Linker|T1D62|Linker|T1D5|Linker|T1D144|Linker|T1D441|Linker|T1D368|Linker|NY-ESO-1|Linker|T1D234|Linker|T1D162|Linker|T1D39|Linker|T1D272|Linker|E7|Linker|T1D328|Linker|T1D188|Linker|E6.

[0251]

Table 13

[0252] Example 7: Immunogenicity of the DNA vaccine according to the invention Design of a DNA vaccine containing a shared antigen sequence and / or neoepitope: Five DNA vaccines (constructs) were designed that contain nucleotide sequences encoding the units / parts shown in Table 14:

[0253]

Table 14

[0254] DNA vaccine VB4097 containing 10 CT26 neoepitopes: This construct was selected as a model for an individualized DNA vaccine containing patient-specific antigen sequences, i.e., neoepitopes.

[0255] Previously described exome sequencing and RNA sequencing of the mouse colon cancer cell line CT26 revealed hundreds to thousands of tumor-specific nonsynonymous mutations. Using in silico methods, we identified potentially immunogenic sequences, i.e., neoepitopes, and selected 10 of them (Table 15) to be included in the antigenic unit of VB4097. Each of the 10 identified neoepitopes is a peptide consisting of 27 amino acids. All except the terminal neoepitope were organized into subunits, each subunit consisting of one neoepitope and one flexible glycine-serine linker (GGGGSGGGGS).

[0256] VB4097 consists of a DNA sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 20.

[0257] [Table 15]

[0258] DNA vaccines VB4100, VB4101, and VB4102 containing the NY-ESO-1 sequence or a portion thereof. These constructs were selected as models for personalized DNA vaccines containing patient-presented shared antigen sequences.

[0259] Human New York esophageal squamous cell carcinoma 1 (NY-ESO-1) has been shown to be a highly immunogenic oncotesticular antigen that is abnormally expressed in several cancer types. Although NY-ESO-1 is not endogenously expressed in the CT26 cancer cell line, several immunogenic sequences were predicted to bind to mouse MHC classes I and II in BALB / c mice using an in silico method. The above construct was selected as a model for a personalized DNA vaccine containing patient-presented shared antigen sequences.

[0260] Three NY-ESO-1 structures were designed for this purpose: VB4100 has an antigenic unit containing the full length of NY-ESO-1. VB4100 consists of a DNA sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 21. VB4101 has an antigenic unit containing amino acids 81-88 of the NY-ESO-1 sequence, which is predicted to be an MHC class I antigen. VB4101 consists of a DNA sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 22. VB4102 has an antigenic unit containing amino acids 81-126 of the NY-ESO-1 sequence, which contains several antigens that are predicted to be MHC class and class II antigens. VB4102 consists of a DNA sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 23.

[0261] Table 16 shows the various NY-ESO-1 sequences included in the construct.

[0262] [Table 16]

[0263] DNA vaccine VB4105 containing 10 CT26 neoepitopes and the full-length sequence of the NY-ESO-1 sequence. This construct was selected as a model for a personalized DNA vaccine containing patient-presented shared antigen sequences and patient-specific antigen sequences (neoepitopes). It contains an antigenic unit comprising the full-length sequence of NY-ESO-1 and 10 CT26 neoepitopes shown in Table 1, each of which is isolated from subsequent neoepitopes or the NY-ESO-1 sequence by a flexible glycine-serine linker (GGGGSGGGGS). VB4105 consists of a DNA sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 24.

[0264] Negative control VB1026: This construct is identical to the construct described above, but it does not contain a unit linker or an antigenic unit. It serves as a negative control. VB1026 consists of a DNA sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 25.

[0265] Construction of expression vectors containing constructs, and confirmation of the expression and secretion of intact dimeric proteins encoded by the constructs: The sequences of the antigenic units of all the above constructs were ordered from Genscript (New Jersey, USA) and cloned into the expression vector pUMVC4a; a master plasmid containing nucleotide sequences encoding the signal peptide, targeting unit, and dimerizing unit as described in Table 14 above.

[0266] HEK293 cells (ATCC) were transiently transfected with the above construct. In short, 2 × 10 5 Cells / well were plated into 24-well tissue culture plates containing 10% FBS growth medium and transfected with 1 μg of each DNA plasmid using Lipofectamine® 2000 reagent under conditions suggested by the producer (Invitrogen, Thermo Fischer Scientific). Transfected cells were then maintained at 37°C with 5% CO2 for up to 5 days. Cell supernatants were then collected for characterization of protein expression encoded by constructs using supernatant sandwich ELISA with antibodies specific to anti-hIgG (CH3 domain), hMIP-1α, and their respective antigenic units.

[0267] Evaluation of the immunogenicity of the construct: The immunogenicity of the construct was determined by measuring the in vivo induced T-cell immune response in mice vaccinated with the construct.

[0268] Six-week-old female BALB / c mice were obtained from Janvier Labs (France). All animals were housed in the animal facility at Radium Hospital (Oslo, Norway). All animal protocols were approved by the Norwegian Food Safety Authority (Oslo, Norway). Five mice were used per group for testing constructs containing antigenic units, while three mice were used per group for negative controls.

[0269] A 20 μg construct was administered intramuscularly twice, on day 0 and day 21, followed by electroporation. The spleen was collected on day 28.

[0270] Spleen was mashed in a cell strainer to obtain single-cell suspensions. For each construct to be tested, a portion of the single-cell suspension was used to purify CD4+ and CD8+ T cells using Dynabeads® depletion. Total spleen cells, CD4-depleted spleen cells, and CD8-depleted spleen cells were then tested for INF-γ production in the ELISpot assay. Spleen cells isolated from mice vaccinated with constructs VB4097 and VB4105 were restimulated using the ten neoepitopes shown in Table 15, and spleen cells isolated from mice vaccinated with the constructs indicated in Table 17 were restimulated using the NY-ESO-1 peptide shown below.

[0271] [Table 17]

[0272] Comparison of immunogenicity of constructs VB4097, VB4100, and VB4105 The constructs VB4097 (10 neoepitopes), VB4100 (full-length NY-ESO-1), and VB4105 (10 neoepitopes and full-length NY-ESO-1) were compared to the peptides in Table 15 (VB4097 and VB4105) and Table 17 (VB4100 and VB4105) for their ability to induce a T cell immune response.

[0273] As shown in Figure 9, mice vaccinated with the negative control VB1026 showed low basal immunogenicity to both the neoepitope and the NY-ESO-peptide sequence.

[0274] Both VB4097 and VB4105, which contain the same 10 CT26 neoepitopes, induce similar total T cell responses (INF-γ responses) to the 10 neoepitopes, regardless of whether the antigenic unit contains only the 10 neoepitopes (VB4097) or additionally the full-length NY-ESO-1 sequence (VB4105) (Figure 9, gray bars).

[0275] Furthermore, both VB4100 and VB4105, which contain the full-length sequence of NY-ESO-1, induce a similar total T-cell response (INF-γ response) to the peptide used for restimulation, as shown in Table 17, regardless of whether the antigenic unit contains only the full-length sequence of NY-ESO-1 (VB4100) or an additional 10 neoepitopes (VB4105) (Figure 9, black bars).

[0276] Regarding VB4105, the addition of the full-length NY-ESO-1 sequence to the antigenic unit of VB4097, which contains 10 neoepitopes, induced a higher total T-cell response compared to that induced by vaccination with VB4097, due to the addition of immunogenicity to NY-ESO-1.

[0277] These results indicate that the vaccine according to the present invention, which contains patient-presented shared antigens, can induce an immune response similar to that of a vaccine containing patient-specific antigens (neoepitopes). Furthermore, these results indicate that there is benefit in including both patient-presented shared antigens and patient-specific antigens (neoepitopes) in the antigenic unit.

[0278] Comparison of immunogenicity of constructs VB4100, VB4101, and VB4102 Using an in silico method, it was predicted that the epitope consisting of amino acids 81-88 of NY-ESO-1 would strongly bind to MHC class I and activate CD8+ T cells, while the peptide consisting of amino acids 81-126 of NY-ESO-1 was predicted to contain several MHC class I and class II antigens. Therefore, constructs VB4100 (full-length NY-ESO-1), VB4101 (amino acids 81-88 of NY-ESO-1), and VB4102 (amino acids 81-126 of NY-ESO-1) were constructed and their ability to induce a T cell immune response was compared.

[0279] First, the three constructs were compared for their ability to induce a CD8+ T cell immune response against the predicted amino acid 81–88 region. CD8+ T cells isolated from spleen cells of mice vaccinated with the constructs were restimulated using the minimal epitope of amino acids 81–88 shown in Table 16. As shown in Figure 10, the experiment confirmed that the epitope consisting of amino acids 81–88 of NY-ESO-1 is indeed a strong CD8+ T cell epitope. Furthermore, the immunogenicity of this region does not depend on whether the epitope is the sole sequence in the antigenic unit (VB4101) or whether the antigenic unit contains a longer NY-ESO-1 sequence or a full-length NY-ESO-1 sequence.

[0280] Next, VB4102 was compared with VB4100 to evaluate whether several predicted MHC class I and class II antigens in amino acid sequence 81–126 of NY-ESO-1 evoke a similar response to the full-length NY-ESO-1 sequence. CD4+ T cells and CD8+ T cells isolated from spleen cells of mice vaccinated with the construct were restimulated with the peptides shown in Table 17. As shown in Figure 11, amino acid sequence 81–126 of NY-ESO-1 evokes a stronger response than the full-length NY-ESO-1 sequence in terms of total T cell response (Figure 11A) from both isolated CD8+ (Figure 11B) and CD4+ T cells (Figure 11C).

[0281] These results suggest that by using an in silico method, shorter (shorter) sequences / epitopes of patient-presented shared antigens that are expected to induce a strong immune response can be identified. By including such sequences / epitopes instead of longer or full-length sequences of patient-presented shared antigens in the antigenic unit of the vaccine of the present invention, space is left in the antigenic unit to include sequences of other patient-presented shared antigens and / or patient-specific antigens / neoepitopes. This enhances the likelihood that patients given such individual anti-cancer vaccines will exhibit a strong immune response to the vaccine.

[0282] Example 8: Immunogenicity of the DNA vaccine according to the present invention Design of DNA vaccines containing shared antigen sequences and / or neoepitopes: We designed six DNA vaccines (constructs) containing nucleotide sequences encoding the units / parts shown in Table 18:

[0283] [Table 18]

[0284] DNA vaccine VB4118 containing 10 B16 neoepitopes: This construct was selected as a model for personalized DNA vaccines containing patient-specific antigen sequences, i.e., neoepitopes.

[0285] Previously described exome sequencing and RNA sequencing of the mouse melanoma cell line B16.F10 revealed hundreds to thousands of tumor-specific nonsynonymous mutations. Using in silico methods, potentially immunogenic sequences, i.e., neoepitopes, were identified, and 10 of them (Table 19) were selected to be included in the antigenic unit of VB4118. Each of the 10 identified neoepitopes is a peptide consisting of 27 amino acids. All except the terminal neoepitope were organized into subunits, each subunit consisting of one neoepitope and one flexible glycine-serine linker (GGGGSGGGGS).

[0286] VB4118 consists of a DNA sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 26.

[0287] [Table 19]

[0288] DNA vaccines VB4119 containing the TRP-2 sequence and VB4127 containing the frameshift antigen. These constructs were selected as models for personalized DNA vaccines containing patient-presented shared antigen sequences.

[0289] Tyrosinase-related protein 2 (TRP-2) is a normal differentiation protein in the melanocyte lineage. This co-antigen (differentiation antigen) is known to induce tumor rejection of B16 melanoma cells in C57BL / 6 mice in vivo. In the literature, the 9-amino acid length MHC class I epitope shown in Table 20 (amino acids 180-188 of TRP-2) has been identified as the immunogenic sequence responsible for the antitumor effect of TRP-2.

[0290] VB4119 consists of a DNA sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 27.

[0291] [Table 20]

[0292] Frameshift mutations are DNA mutations that occur when nucleotide indels (insertions or deletions) lead to shifts in the DNA reading frame. Consequently, the entire DNA sequence after the indel is misread, and the resulting proteins are altered. Frameshift mutations occurring in tumor cells generate novel peptide sequences that can be highly immunogenic, and furthermore, because the same frameshift antigen can occur across different patients, they are promising targets for shared antigen cancer vaccines (see Ballhausen et al., Nat. Commun. 11, 2020, 1-13). Three frameshift antigens, shown in Table 21, have been identified as immunogenic by in silico methods and are encoded in the VB4127 construct. In the antigenic unit, they are separated from each other by a flexible glycine-serine linker (GGGGSGGGGS).

[0293] VB4127 consists of a DNA sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 28.

[0294] [Table 21]

[0295] DNA vaccine containing 10 B16 neoepitopes and TRP-2 epitopes (VB4121), 10 B16 neoepitopes and 3 frameshift antigens (VB4128), or 10 B16 neoepitopes, TRP-2 epitopes and 3 frameshift antigens (VB4130). These constructs were selected as models for personalized DNA vaccines containing patient-presented co-antigen sequences and patient-specific antigen sequences (neoepitopes). Each construct contains an antigenic unit containing 10 B16 neoepitopes shown in Table 19, each of which B16 neoepitopes was isolated from subsequent neoepitopes or co-antigen sequences using a flexible glycine-serine linker (GGGGSGGGGS). The same linker was used to isolate co-antigen sequences in constructs containing some of such sequences. VB4121 consists of a DNA sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 29; VB4128 consists of a DNA sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 30; and VB4130 consists of a DNA sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 31.

[0296] Construct VB1026, described in Example 7, was used as a negative control.

[0297] Construction of an expression vector containing the construct, and confirmation of the expression and secretion of the intact dimeric protein encoded by the construct. An expression vector containing the above-mentioned construct was constructed as described in Example 7. The expression and secretion of the intact dimeric protein encoded by the construct were confirmed as described in Example 7.

[0298] Evaluation of the immunogenicity of the constructs The immunogenicity of the construct was determined by measuring the in vivo induced T-cell immune response in mice vaccinated with the construct.

[0299] Six-week-old female C57BL / 6 mice were obtained from Janvier Labs (France). All animals were housed in the animal facility at Radium Hospital (Oslo, Norway). All animal protocols were approved by the Norwegian Food Safety Authority (Oslo, Norway). Five mice were used per group for testing constructs containing antigenic units, while three mice were used per group for negative controls.

[0300] A 20 μg construct was administered intramuscularly on day 0, followed by electroporation. Spleens were collected from vaccinated mice on day 14. The collected spleens were processed as described in Example 7, and the ELISpot assay was performed.

[0301] Splenocytes isolated from mice vaccinated with constructs VB4118, VB4121, VB4128, and VB4130 were restimulated using the ten neoepitopes shown in Table 19. Splenocytes isolated from mice vaccinated with constructs VB4119, VB4121, and VB4130 were restimulated using the TRP-2 peptides shown in Table 20. Splenocytes isolated from mice vaccinated with constructs VB4127, VB4128, and VB4130 were restimulated using the frameshift peptides shown in Table 22 below.

[0302] [Table 22]

[0303] Comparison of immunogenicity of constructs VB4118, VB4119, VB4121, VB4127, VB4128, and VB4130. The constructs VB4118 (10 neoepitopes), VB4119 (TRP-2), VB4121 (10 neoepitopes and TRP-2), VB4127 (3 frameshift antigens), VB4128 (10 neoantigens and 3 frameshift antigens), and VB4130 (10 neoepitopes, 3 frameshift antigens, and TRP-2) were compared to the peptides in Tables 19, 20, and 22, where applicable, for their ability to induce a T cell immune response.

[0304] As shown in Figure 12, mice vaccinated with the negative control VB1026 showed low baseline immunogenicity to all peptides tested.

[0305] Both the vaccine model construct containing only patient-specific antigens (VB4118) and the vaccine model construct containing only patient-presented shared antigens (VB4119 and VB4127) induce an immune response in vaccinated mice.

[0306] VB4118, VB4121, VB4128, and VB4130, all containing the same 10 B16 neoepitopes, induce similar total T cell responses (INF-γ responses) to 10 neoepitopes, regardless of whether the antigenic unit contains only 10 neoepitopes (VB4118), or additionally a TRP-2 epitope (VB4121), three frameshift antigens (VB4128), or a TRP-2 epitope and three frameshift antigens (VB4130) (Figure 12, black bars).

[0307] Furthermore, Figure 12 shows that adding co-existing antigens to neoepitope constructs leads to a broader and increased total T cell response, with the highest total T cell response observed for a construct (VB4130) encoding 10 neoepitopes, 3 frameshift antigens, and a TRP-2 epitope.

[0308] Furthermore, the results of this study indicate that vaccines according to the present invention, which contain patient-presented shared antigens, can induce an immune response similar to that of vaccines containing patient-specific antigens (neoepitopes). These results also indicate the benefit of an increased and broader total T-cell response by including different types of shared antigens and patient-specific antigens (neoepitopes) in the antigenic unit.

[0309] array Sequence ID 1 CC motif chemokine 3-like 1 precursor containing signal peptides (aa 1-23) and mature peptides (hMIP1α / LD78-beta, aa 24-93): [ka] Sequence ID 2 [ka] Sequence ID 3 Linker, amino acid sequence: GLSGL Sequence ID 4 Linker, amino acid sequence: GLGGL Sequence ID 5 Hinge region 1 (Human IgG3 UH hinge), 12 amino acids: ELKTPLGDTTHT Sequence ID 6 Hinge region 4 (Human IgG3, MH hinge, 15 amino acids): EPKSCDTPPPCPRCP Sequence ID 7 Gly-Ser Linker: GGGSSGGGSG Sequence ID 8 hCH3 IgG3, amino acid sequence: [ka] Sequence ID 9 Signal peptide MNFGLRLIFLVLTLKGVQC Sequence ID 10 Signal peptide MDAMKRGLCCVLLLCGAVFVSP Sequence number 11: HPV16 E6 [ka] Sequence ID 12: HPV16 E7 [ka] Sequence ID 13: NY-ESO-1 [ka] Sequence ID No. 14 (302 amino acids) Antigenic units containing the antigen sequence in the following order: E7|linker|NY-ESO-1|linker|E6. [ka] Sequence ID 15 (924 amino acids) Antigenic units containing the antigen sequence in the following order: T1D320|linker|T1D814|linker|T1D182|linker|T1D689|linker|E7|linker|T1D339|linker|T1D428|linker|NY-ESO-1|linker|T1D572|linker|T1D359|linker|T1D488|linker|T1D554|linker|T1D272|linker|T1D210|linker|T1D849|linker|T1D4|linker|T1D77|linker|T1D717|linker|T1D586|linker|E6. [ka] Sequence ID 16 (227 amino acids) Antigenic units containing the antigen sequence in the following order: E6|linker|NY-ESO-1|linker|E7 [ka] Sequence ID No. 17 (850 amino acids) Antigenic units containing the antigen sequence in the following order: E6|linker|T1D323|linker|T1D506|linker|T1D12|linker|T1D315|linker|T1D302|linker|T1D700|linker|NY-ESO-1|linker|T1D535|linker|T1D358|linker|T1D670|linker|T1D294|linker|T1D336|linker|T1D499|linker|T1D425|linker T1D491|linker|T1D314|linker|T1D430|linker|E7|linker|T1D582. [ka] Sequence ID No. 18 (221 amino acids): The antigenic unit contains the antigen sequence in the following order: NY-ESO-1|linker|E7|linker|E6. [ka] Sequence ID No. 19 (831 amino acids) Antigenic units containing the antigen sequence in the following order: T1D223|linker|T1D164|linker|T1D56|linker|T1D36|linker|T1D129|linker|T1D274|linker|T1D62|linker|T1D5|linker|T1D144|linker|T1D441|linker|T1D368|linker|NY-ESO-1|linker|T1D234|linker|T1D162|linker|T1D39|linker|T1D272|linker|E7|linker|T1D328|linker|T1D188|linker|E6. [ka] Sequence ID 20 Amino acid sequence of VB4097 [ka] Sequence ID 21 Amino acid sequence of VB4100 [ka] Sequence ID 22 Amino acid sequence of VB4101 [ka] Sequence ID 23 Amino acid sequence of VB4102 [ka] Sequence ID 24 Amino acid sequence of VB4105 [ka] Sequence ID 25 Amino acid sequence of VB1026 [ka] Sequence ID 26 Amino acid sequence of VB4118 [ka] Sequence ID 27 Amino acid sequence of VB4119 [ka] Sequence ID 28 Amino acid sequence of VB4127 [ka] Sequence ID 29 Amino acid sequence of VB4121 [ka] Sequence ID 30 Amino acid sequence of VB4128 [ka] Sequence ID 31 Amino acid sequence of VB4130 [ka] Sequence ID 32 HPV E6 Amino Acids 6-15 QERPRKLPQ Sequence ID 33 HPV E6 Amino Acids 8-17 RPRKLPQLC Sequence ID 34 HPV E6 Amino Acids 23-32 IHDIILECV Sequence ID 35 HPV E6 Amino Acids 24-33 HDIILECVY Sequence ID 36 HPV E6 Amino Acids 26-35 IILECVYCK Sequence ID 37 HPV E6 Amino Acids 35-44 QQLLRREVY Sequence ID 38 HPV E6 Amino Acids 42-51 VYDFARRDL Sequence ID 39 HPV E6 Amino Acids 43-52 YDFARRDLC Sequence ID 40 HPV E6 Amino Acids 44-53 DFARRDLCI Sequence ID 41 HPV E6 Amino Acids 45-54 FARRDLCIV Sequence ID 42 HPV E6 Amino Acids 60-69 YAVRDKCLK Sequence ID 43 HPV E6 Amino Acids 62-71 VRDKCLKFY Sequence ID 44 HPV E6 Amino Acids 81-90 YSLYGTTLE Sequence ID 45 HPV E6 Amino Acids 125-134 FHNIRGRWT Sequence ID 46 HPV E6 Amino Acids 131-140 RWTGRCMSC Sequence ID 47 HPV E6 Amino Acids 143-152 SRTRRETQL Sequence ID 48 Amino acids 16-31 of HPV E6 CTELQTTIHDIILEC Sequence ID 49 HPV E6 Amino Acids 17-32 TELQTTIHDIILECV Sequence ID 50 HPV E6 Amino Acids 18-33 ELQTTIHDIILECVY Sequence ID 51 HPV E6 Amino Acids 19-34 LQTTIHDIILECVYC Sequence ID 52 HPV E6 Amino Acids 20-35 QTTIHDIILECVYCK Sequence ID 53 HPV E6 Amino Acids 34-49 KQQLLRREVYDFARR Sequence ID 54 HPV E6 Amino Acids 49-64 DLCIVYRDGNPYAVR Sequence ID 55 HPV E6 Amino Acids 50-65 LCIVYRDGNPYAVRD Sequence ID 56 HPV E6 Amino Acids 51-66 CIVYRDGNPYAVRDK Sequence ID 57 HPV E6 Amino Acids 78-93 HYCYSLYGTTLEQQY Sequence ID 58 HPV E6 Amino Acids 95-110 PLCDLLIRCINRQKP Sequence ID 59 HPV E6 Amino Acids 96-111 LCDLLIRCINRQKPL Sequence ID 60 HPV E6 Amino Acids 97-112 CDLLIRCINRQKPLC Sequence ID 61 HPV E6 Amino Acids 98-113 DLLIRCINRQKPLCP Sequence ID 62 HPV E6 Amino Acids 99-114 LLIRCINRQKPLCPE Sequence ID 63 HPV E6 Amino Acids 121-136 KKQRFHNIRGRWTGR Sequence ID 64 HPV E7 Amino Acids 7-16 TLHEYMLDL Sequence ID 65 HPV E7 Amino Acids 22-31 LYGYGQLND Sequence ID 66 HPV E7 Amino Acids 38-47 IDGPAGQAE Sequence ID 67 HPV E7 Amino Acids 48-57 DRAHYNIVT Sequence ID 68 HPV E7 Amino Acids 55-64 VTFCCKCDS Sequence ID 69 HPV E7 Amino Acids 66-75 RLCVQSTHV Sequence ID 70 HPV E7 Amino Acids 72-81 THVDIRTLE Sequence ID 71 HPV E7 Amino Acids 73-82 HVDIRTLED Sequence ID 72 HPV E7 Amino Acids 85-94 GTLGIVCPI Sequence ID 73 HPV E7 Amino Acids 4-19 DTPTLHEYMLDLQPE Sequence ID 74 HPV E7 Amino Acids 5-20 TPTLHEYMLDLQPET Sequence ID 75 HPV E7 Amino Acids 6-21 PTLHEYMLDLQPETT Sequence ID 76 HPV E7 Amino Acids 7-22 TLHEYMLDLQPETTD Sequence ID 77 HPV E7 Amino Acids 8-23 LHEYMLDLQPETTDL Sequence ID 78 HPV E7 Amino Acids 9-24 HEYMLDLQPETTDLY Sequence ID 79 HPV E7 Amino Acids 10-25 EYMLDLQPETTDLYG Sequence ID 80 HPV E7 Amino Acids 70-85 QSTHVDIRTLEDLLM Sequence ID 81 HPV E7 Amino Acids 71-86 STHVDIRTLEDLLMG Sequence ID 82 HPV E7 Amino Acids 72-87 THVDIRTLEDLLMGT Sequence ID 83 HPV E7 Amino Acids 73-88 HVDIRTLEDLLMGTL Sequence ID 84 HPV E7 Amino Acids 74-89 VDIRTLEDLLMGTLG Sequence ID 85 HPV E6 Amino Acids 16-25 CTELQTTIH Sequence ID 86 HPV E6 Amino Acids 42-51 VYDFARRDL Sequence ID 87 HPV E6 Amino Acids 45-54 FARRDLCIV Sequence ID 88 HPV E6 Amino Acids 52-61 IVYRDGNPY Sequence ID 89 HPV E6 Amino Acids 54-63 YRDGNPYAV Sequence ID 90 HPV E6 Amino Acids 68-77 KFYSKISEY Sequence ID 91 HPV E6 Amino Acids 73-82 ISEYRHYCY Sequence ID 92 HPV E6 Amino Acids 75-84 EYRHYCYSL Sequence ID 93 HPV E6 Amino Acids 80-89 CYSLYGTTL Sequence ID 94 HPV E6 Amino Acids 84-93 YGTTLEQQY Sequence ID 95 HPV E6 Amino Acids 88-97 LEQQYNKPL Sequence ID 96 HPV E6 amino acids 91-100 QYNKPLCDL Sequence ID 97 HPV E6 Amino Acids 11-20 RHLDKKQRF Sequence ID 98 HPV E6 Amino Acids 12-21 RFHNIRGRW Sequence ID 99 HPV E6 Amino Acids 18-33 ELQTTIHDIILECVY Sequence ID 100 HPV E6 Amino Acids 49-64 DLCIVYRDGNPYAVR Sequence ID 101 HPV E6 Amino Acids 50-65 LCIVYRDGNPYAVRD Sequence ID 102 HPV E6 Amino Acids 51-66 CIVYRDGNPYAVRDK Sequence ID 103 HPV E6 Amino Acids 74-89 SEYRHYCYSLYGTTL Sequence ID 104 HPV E6 Amino Acids 75-90 EYRHYCYSLYGTTLE Sequence ID 105 HPV E6 Amino Acids 76-91 YRHYCYSLYGTTLEQ Sequence ID 106 HPV E6 Amino Acids 77-92 RHYCYSLYGTTLEQQ Sequence ID 107 HPV E6 Amino Acids 78-93 HYCYSLYGTTLEQQY Sequence ID 108 HPV E6 Amino Acids 96-111 LCDLLIRCINRQKPL Sequence ID 109 HPV E6 Amino Acids 97-112 CDLLIRCINRQKPLC Sequence ID 110 HPV E6 Amino Acids 98-113 DLLIRCINRQKPLCP Sequence ID 111 HPV E6 Amino Acids 99-114 LLIRCINRQKPLCPE Sequence ID 112 HPV E6 Amino Acids 121-136 KKQRFHNIRGRWTGR Sequence ID 113 HPV E7 Amino Acids 3-12 GDTPTLHEY Sequence ID 114 HPV E7 Amino Acids 7-16 TLHEYMLDL Sequence ID 115 HPV E6 Amino Acids 9-18 HEYMLDLQP Sequence ID 116 HPV E6 Amino Acids 15-24 LQPETTDLY Sequence ID 117 HPV E6 Amino Acids 19-28 TTDLYGYGQ Sequence ID 118 HPV E6 Amino Acids 20-29 TDLYGYGQL Sequence ID 119 HPV E6 Amino Acids 44-53 QAEPDRAHY Sequence ID 120 HPV E6 Amino Acids 49-58 RAHYNIVTF Sequence ID 121 HPV E6 Amino Acids 71-80 STHVDIRTL Sequence ID 122 HPV E6 Amino Acids 79-88 LEDLLMGTL Sequence ID 123 HPV E6 Amino Acids 03-18 GDTPTLHEYMLDLQP Sequence ID 124 HPV E6 Amino Acids 4-19 DTPTLHEYMLDLQPE Sequence ID 125 HPV E6 Amino Acids 5-20 TPTLHEYMLDLQPET Sequence ID 126 HPV E6 Amino Acids 6-21 PTLHEYMLDLQPETT Sequence ID 127 HPV E6 Amino Acids 7-22 TLHEYMLDLQPETTD Sequence ID 128 HPV E6 Amino Acids 8-23 LHEYMLDLQPETTDL Sequence ID 129 HPV E6 Amino Acids 70-85 QSTHVDIRTLEDLLM Sequence ID 130 HPV E6 Amino Acids 71-86 STHVDIRTLEDLLMG Sequence ID 131 HPV E6 Amino Acids 72-87 THVDIRTLEDLLMGT Sequence ID 132 HPV E6 Amino Acids 73-88 HVDIRTLEDLLMGTL Sequence ID 133 HPV E6 Amino Acids 74-89 VDIRTLEDLLMGTLG Sequence ID 134 T1D320 [ka] Sequence ID 135 T1D814 [ka] Sequence ID 136 T1D182 [ka] Sequence ID 137 T1D689 [ka] Sequence ID 138 T1D339 [ka] Sequence ID 139 T1D428 [ka] Sequence ID 140 T1D572 [ka] Sequence ID 141 T1D359 [ka] Sequence ID 142 T1D488 [ka] Sequence ID 143 T1D554 [ka] Sequence ID 144 T1D272 [ka] Sequence ID 145 T1D210 [ka] Sequence ID 146 T1D849 [ka] Sequence ID 147 T1D4 [ka] Sequence ID 148 T1D77 [ka] Sequence ID 149 T1D717 [ka] Sequence ID 150 T1D586 [ka] Sequence ID 151 T11D323 [ka] Sequence ID 152 T11D506 [ka] Sequence ID 153 T11D12 [ka] Sequence ID 154 T11D315 [ka] Sequence ID 155 T11D302 [ka] Sequence ID 156 T11D700 [ka] Sequence ID 157 T11D535 [ka] Sequence ID 158 T11D358 [ka] Sequence ID 159 T11D670 [ka] Sequence ID 160 T11D294 [ka] Sequence ID 161 T11D336 [ka] Sequence ID 162 T11D499 [ka] Sequence ID 163 T11D425 [ka] Sequence ID 164 T11D491 [ka] Sequence ID 165 T11D314 [ka] Sequence ID 166 T11D430 [ka] Sequence ID 167 T11D582 [ka] Sequence ID 168 T1D223 [ka] Sequence ID 169 T1D164 [ka] Sequence ID 170 T1D56 [ka] Sequence ID 171 T1D36 [ka] Sequence ID 172 T1D129 [ka] Sequence ID 173 T1D274 [ka] Sequence ID 174 T1D62 [ka] Sequence ID 175 T1D5 [ka] Sequence ID 176 T1D144 [ka] Sequence ID 177 T1D441 [ka] Sequence ID 178 T1D368 [ka] Sequence ID 179 T1D234 [ka] Sequence ID 180 T1D162 [ka] Sequence ID 181 T1D39 [ka] Sequence ID 182 T1D272 [ka] Sequence ID 183 T1D328 [ka] Sequence ID 184 T1D188 [ka] Sequence ID 185 C-pepM1 [ka] Sequence ID 186 C-pepM6 [ka] Sequence ID 187 C-pepM8 [ka] Sequence ID 188 C-pepM29 [ka] Sequence ID 189 C-pepM31 [ka] Sequence ID 190 C-pepM43 [ka] Sequence ID 191 C-pepM89 [ka] Sequence ID 192 C-pep149 [ka] Sequence ID 193 C-pepM171 [ka] Sequence ID 194 C-pepM173 [ka] Sequence ID 195 VB 4101 Amino Acids 81-88 RGPESRLL Sequence ID 196 VB4102 Amino Acids 81-126 [ka] Sequence ID 197 NY-ESO_Pep-1 MQAEGRGTGGSTGDA Sequence ID 198 NY-ESO_Pep-2 TGGSTGDADGPGGPG Sequence ID 199 NY-ESO_Pep-3 ADGPGGPGIPDGPGG Sequence ID 200 NY-ESO_Pep-4 GIPDGPGGNAGGPGE Sequence ID 201 NY-ESO_Pep-5 GNAGGPGEAGATGGR Sequence ID 202 NY-ESO_Pep-6 EAGATGGRGPRGAGA Sequence ID 203 NY-ESO_Pep-7 RGPRGAGAARASGPG Sequence ID 204 NY-ESO_Pep-8 AARASGPGGGAPRGP Sequence ID 205 NY-ESO_Pep-9 GGGAPRGPHGGAASG Sequence ID 206 NY-ESO_Pep-10 PHGGAASGLNGCCRC Sequence ID 207 NY-ESO_Pep-11 GLNGCCRCGARGPES Sequence ID 208 NY-ESO_Pep-12 CGARGPESRLLEFYL Sequence ID 209 NY-ESO_Pep-13 RGPESRLLEFYLAMP Sequence ID 210 NY-ESO_Pep-14 SRLLEFYLAMPFATP Sequence ID 211 NY-ESO_Pep-15 LAMPFATPMEAELAR Sequence ID 212 NY-ESO_Pep-16 PMEAELARRSLAQDA Sequence ID 213 NY-ESO_Pep-17 RRSLAQDAPPLPVPG Sequence ID 214 NY-ESO_Pep-18 DAPPLPVPGVLLKEF Sequence ID 215 NY-ESO_Pep-19 APPLPVPGVLLKEFT Sequence ID 216 NY-ESO_Pep-20 GVLLKEFTVSGNILT Sequence ID 217 NY-ESO_Pep-21 TVSGNILTIRLTAAD Sequence ID 218 NY-ESO_Pep-22 TIRLTAADHRQLQLS Sequence ID 219 NY-ESO_Pep-23 DHRQLQLSISSCLQQ Sequence ID 220 NY-ESO_Pep-24 SISSCLQQLSLLMWI Sequence ID 221 NY-ESO_Pep-25 QLSLLMWITQCFLPV Sequence ID 222 NY-ESO_Pep-26 ITQCFLPVFLAQPPS Sequence ID 223 NY-ESO_Pep-27 VFLAQPPSGQRR Sequence ID 224 NY-ESO_Pep-28 RGPESRLL Sequence ID 225 B-pepM2 [ka] Sequence ID 226 B-pepM7 [ka] Sequence ID 227 B-pepM36 [ka] Sequence ID 228 B-pepM78 [ka] Sequence ID 229 B-pepM79 [ka] Sequence ID 230 B-pepM82 [ka] Sequence ID 231 B-pepM83 [ka] Sequence ID 232 B-pepM84 [ka] Sequence ID 233 B-pepM85 [ka] Sequence ID 234 B-pepM86 [ka] Sequence ID 235 VB4119 Amino Acid 180-188 SVYDFFVWL Sequence ID 236 B-pepM108 YISDHMKVHSPSPCL Sequence ID 237 B-pepM115-M122 [ka] Sequence ID 238 B-pepM141-M142 [ka] Sequence ID 239 B-pepM108 YISDHMKVHSPSPCL Sequence ID 240 B-pepM115 EGWQTCWGRSRKHWG Sequence ID 241 B-pepM116 GRSRKHWGSTWNGSA Sequence ID 242 B-pepM117 GSTWNGSARLSPGST Sequence ID 243 B-pepM118 ARLSPGSTLWVMRIC Sequence ID 244 B-pepM119 TLWVMRICLRSLGIA Sequence ID 245 B-pepM120 CLRSLGIARTWLSCR Sequence ID 246 B-pepM121 ARTWLSCRSTSRKCS Sequence ID 247 B-pepM122 RSTSRKCSPAFPASS Sequence ID 248 B-pepM141 [ka] Sequence ID 249 B-pepM142 [ka]

[0310] Embodiment A: 1. An immunologically effective amount, (i) a polynucleotide comprising nucleotide sequences encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented co-antigen sequence or one or more portions thereof, and optionally one or more patient-specific antigen sequences or one or more portions thereof; or (ii) A polypeptide encoded by a polynucleotide as defined in (i), or (iii) A dimeric protein consisting of two polypeptides encoded by a polynucleotide as defined in (i); and Medicinally acceptable carriers Personalized therapeutic anti-cancer vaccines, including [specific ingredient / feature].

[0311] 2. The vaccine according to Embodiment A1, wherein the at least one patient-presented co-antigen sequence is a co-antigen selected from the group consisting of overexpressed cellular proteins, abnormally expressed cellular proteins, oncotesticular antigens, viral antigens, differentiation antigens, mutant oncogenes and mutant tumor suppressor genes, carcinoembryonic antigens, co-fusion antigens, co-intron-holding antigens, dark matter antigens and co-antigens caused by spliceosome mutations or frameshift mutations.

[0312] 3. The vaccine according to either Embodiment A1 or A2, wherein the at least one patient-presented co-antigen sequence is a human cell protein, preferably an overexpressed or abnormally expressed human cell protein, or a co-antigen that is a differentiation antigen.

[0313] 4. The vaccine according to any one of Embodiments A1 to A3, wherein at least one patient-presented shared antigen sequence is known to be immunogenic, or one or more portions thereof are known to be immunogenic, or are predicted to bind to the patient's HLA class I or HLA class II allele, preferably the patient's HLA class I allele.

[0314] 5. The vaccine according to any one of Embodiments A1 to A4, wherein the at least one patient-presented shared antigen sequence has a length suitable for presentation by the patient's HLA allele, preferably 7 to 30 amino acids in length.

[0315] 6. The vaccine according to any of Embodiments A1 to A5, comprising one or more patient-presented shared antigen sequences or one or more portions thereof.

[0316] 7. The vaccine according to Embodiment A6, comprising a sequence of several patient-presented co-antigens or one or more portions thereof, preferably several portions of a sequence of several patient-presented co-antigens, more preferably several epitopes of several patient-presented co-antigens, wherein the epitopes are known to be immunogenic or are expected to bind to the HLA class I and HLA class II alleles of the particular patient.

[0317] 8. The vaccine according to any one of Embodiments A1 to A7, wherein the antigenic unit comprises the full length of one or more patient-presented shared antigen sequences, preferably 1 to 10 patient-presented shared antigen sequences.

[0318] 9. The vaccine according to any one of Embodiments A1 to A7, wherein the antigenic unit comprises 1 to 30 portions of a patient-presented co-antigen sequence in the form of a long peptide sequence, preferably a peptide sequence of about 28 to 100 amino acids.

[0319] 10. The vaccine according to Embodiment A9, wherein the long peptide sequence comprises a plurality of epitopes that are predicted to bind to the patient's HLA class I or HLA class II allele.

[0320] 11. The vaccine according to any one of Embodiments A1 to A7, wherein the antigenic unit comprises 1 to 50 portions of a patient-presented shared antigen sequence in the form of a short peptide sequence / epitope.

[0321] 12. The vaccine according to Embodiment A11, wherein the short peptide sequence / epitope is expected to bind to the patient's HLA class I or HLA class II allele.

[0322] 13. The vaccine according to any one of embodiments A11 to A12, wherein the short peptide sequence / epitope has a length of 7 to 30 amino acids, for example, 7 to 10 or 13 to 30 amino acids.

[0323] 14. A vaccine according to any of the preceding embodiments A1 to A13, comprising one or more patient-specific antigen sequences or one or more portions thereof.

[0324] 15. The vaccine according to Embodiment A14, comprising several patient-specific antigen sequences or one or more portions thereof.

[0325] 16. The vaccine according to Embodiment A15, comprising one or more portions of the patient-specific antigen sequence, preferably one or more patient-specific epitopes.

[0326] 17. The vaccine according to Embodiment A16, comprising one or more patient-specific epitopes having a length of 7 to 30 amino acids, for example, 7 to 10 or 13 to 30 amino acids.

[0327] 18. The vaccine according to any one of Embodiments A14 to A17, wherein the antigenic unit comprises at least 10 patient-specific epitopes, preferably at least 15 patient-specific epitopes, for example, at least 20 patient-specific epitopes.

[0328] 19. The vaccine according to any of the preceding embodiments A1 to A18, wherein the antigenic unit comprises 21 to 2000 amino acids, preferably about 30 to about 1500 amino acids, more preferably about 50 to about 1000 amino acids, for example, about 100 to about 500 amino acids, or about 100 to about 400 amino acids, or about 100 to about 300 amino acids.

[0329] 20. The vaccine according to any of the preceding embodiments A1 to A19, wherein the antigenic unit comprises one or more linkers, preferably one or more non-immunogenic and / or flexible linkers.

[0330] 21. The vaccine according to Embodiment A20, wherein the length of one or more linkers is 4 to 20 amino acids.

[0331] 22. The vaccine according to any one of embodiments A20 to A21, wherein one or more linkers separate the antigen sequences from each other.

[0332] 23. The vaccine according to any of the preceding embodiments A1 to A22, wherein the dimerization unit comprises a hinge region and another domain that optionally promotes dimerization, optionally connected via a linker.

[0333] 24. The vaccine according to Embodiment A23, wherein the hinge region is derived from Ig.

[0334] 25. The vaccine according to any one of embodiments A23 and A24, wherein the hinge region has the ability to form one or more covalent bonds, preferably in the form of disulfide crosslinks.

[0335] 26. The vaccine according to any one of Embodiments A23 to A25, wherein the other domain that promotes dimerization is an immunoglobulin domain, preferably a carboxy-terminal C domain, or a sequence substantially identical to or a variant thereof of the C domain.

[0336] 27. The vaccine according to Embodiment A26, wherein the carboxyl-terminal C domain is derived from IgG.

[0337] 28. The vaccine according to any one of embodiments A26 and A27, wherein the immunoglobulin domain of the dimerization unit has the ability to homodimerize preferably via a non-covalent interaction, more preferably via a hydrophobic interaction.

[0338] 29. The vaccine according to any one of Embodiments A23 to A28, wherein the dimerization unit does not contain a CH2 domain.

[0339] 30. The dimerization unit, through the third linker, converts human IgG3 to C H A vaccine according to any one of embodiments A23 to A29, comprising hinge exons h1 and h4 connected to three domains.

[0340] 31. The vaccine according to any one of Embodiments A23 to A30, wherein the dimerization unit comprises an amino acid sequence having at least 80% sequence identity with amino acid sequence 94 to 237 of Sequence ID No. 3.

[0341] 32. The vaccine according to any of the preceding embodiments A1 to A31, wherein the antigenic unit and the dimerizing unit are connected via a linker, preferably a linker including a restriction site.

[0342] 33. The vaccine according to any one of the prior embodiments A1 to A32, wherein the targeting unit has affinity for a chemokine receptor selected from CCR1, CCR3, and CCR5.

[0343] 34. The vaccine according to any of the preceding embodiments A1 to A33, wherein the targeting unit includes an antibody-binding region specific to CD14, CD40, or a Toll-like receptor or ligand, such as a soluble CD40 ligand, or a chemokine, such as RANTES or MIP-1a, or a bacterial antigen, such as flagellin.

[0344] 35. The vaccine according to any one of embodiments A1 to A33, wherein the targeting unit has affinity for MHC class II proteins, preferably MHC class II proteins selected from the group consisting of anti-HLA-DP, anti-HLA-DR, and anti-pan-HLA class II.

[0345] 36. The vaccine according to any one of Embodiments A1 to A33, wherein the targeting unit has an amino acid sequence having at least 80% sequence identity with amino acid sequences 24 to 93 of Sequence ID No. 1.

[0346] 37. The vaccine according to any of the prior embodiments A1 to A36, wherein the polynucleotide further encodes a signal peptide.

[0347] 38. The vaccine according to any of the preceding embodiments A1 to A37, wherein the targeting unit, dimerizing unit and antigenic unit in the peptide are in the order of targeting unit, dimerizing unit and antigenic unit from N-terminus to C-terminus.

[0348] 39. The vaccine according to any of the preceding embodiments A1 to A38, wherein the polynucleotide sequence is optimized for human codons.

[0349] 40. The vaccine according to any one of the preceding embodiments A1 to A39, wherein the polynucleotide sequence is a DNA nucleotide sequence or an RNA nucleotide sequence.

[0350] 41. A polynucleotide as defined in any of Embodiments A1 to A40.

[0351] 42. A vector comprising the polynucleotide described in Embodiment A41.

[0352] 43. A host cell comprising a polynucleotide as defined in any of Embodiments A1 to A40, or a vector as described in Embodiment A42.

[0353] 44. The polynucleotide according to Embodiment A41, formulated for administration to a patient to induce the production of a dimeric protein in the patient.

[0354] 45. A polypeptide encoded by a polynucleotide sequence as defined in any of Embodiments A1 to A40.

[0355] 46. ​​A dimeric protein comprising the polypeptide described in two embodiments A45.

[0356] 47. The dimer protein described in Embodiment A46, which is a homodimer protein.

[0357] 48. A polynucleotide according to Embodiment A41, a polypeptide according to Embodiment A45, or a dimer protein according to any one of Embodiments A46 to A47, for use as a pharmaceutical.

[0358] 49. An immunologically effective amount, (i) A dimer protein comprising two polypeptides encoded by polynucleotides, the polynucleotides comprising nucleotide sequences encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented co-antigen sequence or one or more portions thereof, and optionally one or more patient-specific antigen sequences or one or more portions thereof; or (ii) A polypeptide encoded by a polynucleotide comprising nucleotide sequences encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented covalent antigen sequence or one or more portions thereof, and optionally one or more patient-specific antigen sequences or one or more portions thereof. A method for preparing a personalized therapeutic anti-cancer vaccine, including, a) Transfecting cells with the polynucleotide; b) Culturing the cells; c) Collecting and purifying the dimeric protein or polypeptide expressed from the cells, d) Mixing the dimeric protein or polypeptide obtained from step c) with a pharmaceutically acceptable carrier. The method, including the method described above.

[0359] 50. A method for preparing a personalized therapeutic anticancer vaccine comprising an immunologically effective amount of polynucleotides comprising nucleotide sequences encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented co-antigen sequence or one or more portions thereof, and optionally one or more patient-specific antigen sequences or one or more portions thereof, and the method is a. Preparing the polynucleotide; b. Selectively cloning the polynucleotide into an expression vector and c. Mixing the polynucleotide from step a) or the vector from step b) with a pharmaceutically acceptable carrier. The method, including the method described above.

[0360] 51. The method according to Embodiment A49 or A50, comprising the steps of identifying a patient-presented co-antigen and a patient-specific antigen before preparing the polynucleotide, identifying the patient's HLA class I and HLA class II alleles, and selecting a patient-presented co-antigen sequence and a patient-specific antigen sequence arbitrarily based on immunogenicity.

[0361] 52. A method for treating cancer in a patient, comprising administering to the patient a vaccine described in any of Embodiments A1 to A40.

[0362] 53. The method according to Embodiment A52, wherein the vaccine comprises a polynucleotide and is administered intradermally or intramuscularly.

[0363] 54. The method according to Embodiment A53, wherein the polynucleotide is DNA.

[0364] 55. The method according to Embodiment A53, wherein the polynucleotide is RNA.

[0365] 56. The method according to any one of claims A52 to A55, wherein the administration is performed using a jet injector.

[0366] 57. The method according to any one of claims A52 to A56, wherein administration is assisted by electroporation.

[0367] Embodiment B 1. An immunologically effective amount, (i) a polynucleotide comprising nucleotide sequences encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented covalent antigen sequence or one or more portions thereof; or (ii) A polypeptide encoded by a polynucleotide as defined in (i); or (iii) A dimeric protein consisting of two polypeptides encoded by a polynucleotide as defined in (i); and Medicinally acceptable carriers Personalized therapeutic anti-cancer vaccines, including [specific ingredient / feature].

[0368] 2. The vaccine according to Embodiment B1, wherein the antigenic unit further comprises one or more patient-specific antigen sequences or one or more portions thereof.

[0369] 3. The vaccine according to Embodiment B1 or B2, wherein the at least one patient-presented co-antigen sequence is a sequence of a co-antigen selected from the group consisting of overexpressed cellular proteins, abnormally expressed cellular proteins, oncotesticular antigens, viral antigens, differentiation antigens, mutant oncogenes, mutant tumor suppressor genes, carcinoembryonic antigens, co-fusion antigens, co-intron-holding antigens, dark matter antigens, co-antigens caused by spliceosome mutations, and co-antigens caused by frameshift mutations.

[0370] 4. The vaccine according to any one of Embodiments B1 to B3, wherein the at least one patient-presented co-antigen sequence is a sequence of a co-antigen that is a human cell protein.

[0371] 5. The vaccine according to Embodiment B4, wherein the human cell protein is a human cell protein or differentiation antigen that is overexpressed or abnormally expressed.

[0372] 6. The vaccine according to any one of Embodiments B1 to B5, wherein at least one patient-presented shared antigen sequence or one or more portions thereof is known to be immunogenic or is predicted to bind to the patient's HLA class I and / or HLA class II alleles.

[0373] 7. The vaccine according to Embodiment B6, wherein at least one patient-presented shared antigen sequence or one or more portions thereof is expected to bind to the patient's HLA class I allele.

[0374] 8. The vaccine according to any one of embodiments B1 to B7, wherein the at least one patient-presented shared antigen sequence or one or more portions thereof has a length suitable for presentation by the patient's HLA alleles.

[0375] 9. The vaccine according to Embodiment B8, wherein at least one patient-presented shared antigen sequence or one or more portions thereof has a length of 7 to 30 amino acids.

[0376] 10. The vaccine according to any one of Embodiments B1 to B9, wherein the antigenic unit comprises one or more patient-presented shared antigen sequences or one or more portions thereof.

[0377] 11. The vaccine according to Embodiment B10, wherein the antigenic unit comprises a sequence of several patient-presented shared antigens or one or more portions thereof.

[0378] 12. The vaccine according to Embodiment B11, wherein the antigenic unit comprises several portions of sequences of several patient-presented shared antigens.

[0379] 13. The vaccine according to Embodiment B12, wherein the antigenic unit comprises several epitopes of several patient-presented co-antigens, the epitopes of which are known to be immunogenic or are expected to bind to the patient's HLA class I and / or HLA class II alleles.

[0380] 14. The vaccine according to any one of Embodiments B1 to B13, wherein the antigenic unit comprises the full length of one or more patient-presented shared antigen sequences.

[0381] 15. The vaccine according to Embodiment B14, wherein the antigenic unit comprises the full length of 1 to 10 patient-presented shared antigen sequences.

[0382] 16. The vaccine according to any one of Embodiments B1 to B15, wherein the antigenic unit comprises 1 to 30 portions of at least one patient-presented shared antigen sequence.

[0383] 17. The vaccine according to Embodiment B16, wherein the 1 to 30 portions have a length of 28 to 100 amino acids.

[0384] 18. The vaccine according to Embodiment B17, wherein the portion comprises a plurality of epitopes that are predicted to bind to the HLA class I and / or HLA class II alleles of the patient.

[0385] 19. The vaccine according to any one of Embodiments B1 to B18, wherein the antigenic unit comprises 1 to 50 patient-presented shared antigen sequences in the form of an epitope.

[0386] 20. The vaccine according to Embodiment B19, wherein the epitope is expected to bind to the HLA class I and / or HLA class II alleles of the patient.

[0387] 21. The vaccine according to any one of embodiments B19 to B20, wherein the epitope has a length of 7 to 30 amino acids.

[0388] 22. The vaccine according to any one of Embodiments B2 to B21, wherein the antigenic unit comprises several patient-specific antigen sequences or one or more portions thereof.

[0389] 23. The vaccine according to Embodiment B22, wherein the antigenic unit comprises one or more portions of the several patient-specific antigen sequences.

[0390] 24. The vaccine according to any one of Embodiments B2 to B23, wherein the antigenic unit comprises one or more patient-specific epitopes.

[0391] 25. The vaccine according to Embodiment B24, wherein one or more patient-specific epitopes have a length of 7 to 30 amino acids.

[0392] 26. The vaccine according to any one of embodiments B24 to B25, wherein the antigenic unit comprises at least five patient-specific epitopes.

[0393] 27. The vaccine according to any one of embodiments B24 to B25, wherein the antigenic unit comprises at least 10 patient-specific epitopes.

[0394] 28. The vaccine according to any one of Embodiments B24 to B25, wherein the antigenic unit comprises at least 15 patient-specific epitopes.

[0395] 29. The vaccine according to any of the preceding embodiments B1 to B28, wherein the antigenic unit comprises 7 to 2000 amino acids.

[0396] 30. The vaccine according to Embodiment B29, wherein the antigenic unit contains 30 to 1500 amino acids.

[0397] 31. The vaccine according to Embodiment B29, wherein the antigenic unit contains 50 to 1000 amino acids.

[0398] 32. The vaccine according to any of the preceding embodiments B1 to B31, wherein the antigenic unit comprises one or more linkers.

[0399] 33. The vaccine according to Embodiment B32, wherein one or more linkers are non-immunogenic and / or flexible linkers.

[0400] 34. The vaccine according to either embodiment B32 or B33, wherein the length of one or more linkers is 4 to 20 amino acids.

[0401] 35. The vaccine according to any one of embodiments B32 to B34, wherein one or more linkers separate the antigen sequences from each other.

[0402] 36. The vaccine according to any of the preceding embodiments B1 to B35, wherein the dimerization unit includes a hinge region.

[0403] 37. The vaccine according to Embodiment B36, wherein the hinge region has the ability to form one or more covalent bonds, preferably in the form of disulfide crosslinks.

[0404] 38. The vaccine according to either embodiment B36 or B37, wherein the hinge region is derived from Ig.

[0405] 39. The vaccine according to any one of embodiments B36 to B38, wherein the dimerizing unit further comprises another domain that promotes dimerization.

[0406] 40. The vaccine according to Embodiment B39, wherein the other domain is an immunoglobulin domain, preferably an immunoglobulin constant domain.

[0407] 41. The vaccine according to either Embodiment B39 or B40, wherein the other domain is a carboxy-terminal C domain derived from IgG, preferably from IgG3.

[0408] 42. The vaccine according to any one of embodiments B36 to B41, wherein the dimerization unit further comprises a linker, preferably a linker connecting the hinge region and the other domains that promote dimerization.

[0409] 43. The vaccine according to any one of embodiments B36 to B42, wherein the dimerization unit comprises hinge exons h1 and h4 connected to the CH3 domain of human IgG3 via a linker.

[0410] 44. The vaccine according to any one of Embodiments B36 to B43, wherein the dimerization unit includes an amino acid sequence having at least 80% sequence identity with amino acid sequence 94 to 237 of Sequence ID No. 3.

[0411] 45. The vaccine according to any one of embodiments B36 to B44, wherein the dimerization unit consists of amino acid sequence 94 to 237 of Sequence ID No. 3.

[0412] 46. ​​The vaccine according to any of the preceding embodiments B1 to B45, wherein the antigenic unit and the dimerizing unit are connected through a linker, preferably a linker including a restriction site.

[0413] 47. The vaccine according to any of the preceding embodiments B1 to B46, wherein the targeting unit targets antigen-presenting cells.

[0414] 48. The vaccine according to Embodiment B47, wherein the targeting unit is a portion that interacts with a surface molecule on the antigen-presenting cell, or includes such portion.

[0415] 49. The vaccine according to Embodiment B48, wherein the surface molecule is selected from the group consisting of HLA, CD14, CD40, chemokine receptors, and Toll-like receptors.

[0416] 50. The vaccine according to any one of Embodiments B47 to B49, wherein the targeting unit comprises or consists of an antibody variable domain having specificity for a soluble CD40 ligand, RANTES, MIP-1α, XCL1, XCL2, flagellin, anti-HLA-DP, anti-HLA-DR, anti-pan-HLA class II, or anti-CD40, anti-TLR-2, anti-TLR-4, or anti-TLR-5.

[0417] 51. The vaccine according to Embodiment B50, wherein the targeting unit includes or consists of MIP-1α.

[0418] 52. The vaccine according to Embodiment B51, wherein the targeting unit comprises an amino acid sequence having at least 80% sequence identity with amino acid sequences 24-93 of Sequence ID No. 1.

[0419] 53. The vaccine according to Embodiment B52, wherein the targeting unit comprises an amino acid sequence having at least 80% sequence identity with amino acid sequences 24-93 of Sequence ID No. 1.

[0420] 54. The vaccine according to Embodiment B53, wherein the targeting unit consists of amino acid sequences 24-93 of Sequence ID No. 1.

[0421] 55. A vaccine according to any of the preceding embodiments B1 to B54, comprising a polynucleotide, preferably RNA or DNA.

[0422] 56. The vaccine according to Embodiment B55, wherein the polynucleotide is optimized for human codons.

[0423] 57. The vaccine according to either embodiment B55 or B56, wherein the polynucleotide further comprises a nucleotide sequence encoding a signal peptide.

[0424] 58. The vaccine according to Embodiment B57, wherein the signal peptide is selected from a list consisting of Ig VH signal peptide, human TPA signal peptide, and human MIP1-α signal peptide.

[0425] 59. The vaccine according to any one of Embodiments B57 to B58, wherein the signal peptide comprises an amino acid sequence having at least 85% sequence identity with amino acid sequences 1 to 23 of SEQ ID NO: 1.

[0426] 60. The vaccine according to Embodiment B59, wherein the signal peptide comprises an amino acid sequence having at least 85% sequence identity with amino acid sequences 1 to 23 of SEQ ID NO: 1.

[0427] 61. The vaccine according to Embodiment B60, wherein the signal peptide consists of amino acid sequences 1 to 23 of SEQ ID NO: 1.

[0428] 62. The vaccine according to any of the preceding embodiments B1 to B61, wherein the targeting unit, dimerizing unit and antigenic unit in the polypeptide are arranged in an N-terminus to C-terminus order of targeting unit, dimerizing unit and antigenic unit, or the targeting unit, dimerizing unit and antigenic unit in the polynucleotide are arranged in a 5' to 3' order of targeting unit, dimerizing unit and antigenic unit.

[0429] 63. The vaccine according to any of the preceding embodiments B1 to B62, wherein the pharmaceutically acceptable carrier is selected from the group consisting of saline solution, buffered saline solution, PBS, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer, and combinations thereof.

[0430] 64. A method for preparing a personalized therapeutic anti-cancer vaccine according to Embodiment B1, a) Step of identifying at least one patient-presented shared antigen in the patient's tumor tissue or body fluids. b) The step of determining the HLA class I and / or class II alleles of the patient. c) A step of predicting the immunogenicity of the identified at least one antigen or one or more portions thereof by their predicted binding affinity to the patient's HLA class I and / or II alleles. d) A step of selecting at least one antigen or one or more parts thereof based on their immunogenicity predicted in step c); e) A step of preparing a polynucleotide sequence comprising an antigenic unit comprising a nucleotide sequence encoding at least one antigen or one or more portions thereof selected in step d); f) The step of cloning the polynucleotide sequence into an expression vector containing nucleotide sequences encoding targeting units and dimerizing units; and g) A step of mixing the expression vector obtained in step f with a pharmaceutically acceptable carrier. The method, including the method described above.

[0431] 65. A method for preparing a personalized anti-cancer vaccine according to Embodiment B2, wherein the method is In step a), identify one or more patient-specific antigens in the tumor tissue of the patient. In step c), predict the immunogenicity of one or more identified patient-specific antigens or one or more portions thereof by their predicted binding affinity to the patient's HLA class I and / or II alleles. In step d), select one or more patient-specific antigens or one or more parts thereof based on their immunogenicity predicted in step c). It further includes; Furthermore, the polynucleotide sequence of step e) further comprises nucleotide sequences encoding one or more patient-specific antigens or one or more portions thereof selected in step d), The aforementioned method.

[0432] 66. A polynucleotide as defined in any of Embodiments B1 to B62.

[0433] 67. A vector comprising a polynucleotide as described in Embodiment B66.

[0434] 68. A host cell comprising a polynucleotide as defined in any of Embodiments B1 to B62, or a vector as described in Embodiment B67.

[0435] 69. The polynucleotide according to Embodiment B66, formulated for administration to a patient to induce the production of a dimeric protein in the patient.

[0436] 70. A polypeptide encoded by a polynucleotide sequence as defined in any of Embodiments B1 to B62.

[0437] 71. A dimeric protein comprising two polypeptides as defined in two embodiments B70.

[0438] 72. The dimer protein described in Embodiment B71, which is a homodimer protein.

[0439] 73. A polynucleotide according to Embodiment B66, a polypeptide according to Embodiment B70, or a dimer protein according to either Embodiment B71 or B72, for use as a pharmaceutical.

[0440] 74. A method for preparing a personalized therapeutic anti-cancer vaccine comprising an immunologically effective amount of a dimeric protein or polypeptide as defined in any of Embodiments B1 to B54, a) Transfecting cells with polynucleotides as defined in any of embodiments B1 to B62; b) Culturing the cells; c) Collecting and purifying the dimeric protein or polypeptide expressed from the cells; and d) Mixing the dimeric protein or polypeptide obtained from step c) with a pharmaceutically acceptable carrier. The method, including the method described above.

[0441] 75. A method for preparing a personalized therapeutic anti-cancer vaccine comprising an immunologically effective amount of a polynucleotide as defined in any of embodiments B1 to B62, a) Preparing the polynucleotide; b) Selectively cloning the polynucleotide into an expression vector and c) Mixing the polynucleotide from step a) or the vector from step b) with a pharmaceutically acceptable carrier. The method, including the method described above.

[0442] 76. A method for treating a patient having cancer, comprising administering to the patient a vaccine according to any one of embodiments B1 to B63, which has been specifically prepared for the patient.

[0443] 77. The method according to Embodiment B76, wherein the vaccine comprises a polynucleotide and is administered intradermally or intramuscularly.

[0444] 78. The method according to Embodiment B77, wherein the polynucleotide is DNA.

[0445] 79. The method according to Embodiment B78, wherein the polynucleotide is RNA.

[0446] 80. The method according to any of embodiments B76 to B79, wherein administration is performed using a jet injector.

[0447] 81. The method according to any one of embodiments B76 to B80, wherein administration is assisted by electroporation.

[0448] 82. A vaccine according to any of embodiments B1 to B63 for use in a method of treating cancer in a patient, wherein the vaccine is specifically prepared for the patient.

[0449] 83. The use of a polynucleotide according to Embodiment B66, a polypeptide according to Embodiment B70, or a dimerized protein according to either Embodiment B71 or B72 for the production of a pharmaceutical for the treatment of cancer in a patient, wherein the polynucleotide, polypeptide, or dimerized protein is specifically prepared for the patient.

[0450] 84. A method for preparing polynucleotides according to Embodiment B66, a) Step of identifying at least one patient-presented shared antigen in the patient's tumor tissue or body fluids. b) The step of determining the HLA class I and / or class II alleles of the patient. c) A step of predicting the immunogenicity of the identified at least one antigen or one or more portions thereof by their predicted binding affinity to the patient's HLA class I and / or II alleles. d) A step of selecting at least one antigen or one or more parts thereof based on their immunogenicity predicted in step c); e) a step of preparing a polynucleotide sequence comprising an antigenic unit comprising a nucleotide sequence encoding at least one antigen or one or more portions thereof selected in step d); and f) The step of cloning the polynucleotide sequence into an expression vector containing nucleotide sequences encoding a targeting unit and a dimerizing unit. The method, including the method described above.

[0451] 85. The above method, In step a), identify one or more patient-specific antigens in the tumor tissue of the patient. In step c), predict the immunogenicity of one or more identified patient-specific antigens or one or more portions thereof by their predicted binding affinity to the patient's HLA class I and / or II alleles. In step d), select one or more patient-specific antigens or one or more parts thereof based on their immunogenicity predicted in step c). It further includes; Furthermore, the polynucleotide sequence of step e) further comprises nucleotide sequences encoding one or more patient-specific antigens or one or more portions thereof selected in step d), The method described in Embodiment B84.

Claims

1. An immunologically effective amount, (i) a polynucleotide comprising nucleotide sequences encoding a targeting unit, a dimerizing unit, and an antigenic unit, wherein the antigenic unit comprises at least one patient-presented co-antigen sequence or one or more portions thereof; or (ii) A polypeptide encoded by the polynucleotide as defined in (i); or (iii) A dimeric protein comprising two polypeptides encoded by the polynucleotide defined in (i); and Medicinally acceptable carriers Personalized therapeutic anti-cancer vaccines, including [specific ingredient / feature].

2. The vaccine according to claim 1, wherein the antigenic unit further comprises one or more patient-specific antigen sequences or one or more portions thereof.

3. The vaccine according to claim 1 or 2, wherein the at least one patient-presented co-antigen sequence is a sequence of a co-antigen selected from the group consisting of overexpressed cellular proteins, abnormally expressed cellular proteins, oncotesticular antigens, viral antigens, differentiation antigens, mutant oncogenes, mutant tumor suppressor genes, carcinoembryonic antigens, co-fusion antigens, co-intron-holding antigens, dark matter antigens, co-antigens caused by spliceosome mutations, and co-antigens caused by frameshift mutations.

4. The vaccine according to any one of claims 1 to 3, wherein the at least one patient-presented co-antigen sequence is a sequence of a co-antigen that is a human cell protein.

5. The vaccine according to any one of claims 1 to 4, wherein at least one patient-presented shared antigen sequence or one or more portions thereof is known to be immunogenic or is predicted to bind to the patient's HLA class I and / or HLA class II alleles.

6. The vaccine according to any one of claims 1 to 5, wherein the at least one patient-presented shared antigen sequence or one or more portions thereof has a length suitable for presentation by the patient's HLA allele, preferably having a length of 7 to 30 amino acids.

7. The vaccine according to any one of claims 1 to 6, wherein the antigenic unit comprises a sequence of several patient-presented shared antigens or one or more portions thereof.

8. The vaccine according to claim 7, wherein the antigenic unit comprises several portions of sequences of several patient-presented shared antigens.

9. The vaccine according to claim 8, wherein the antigenic unit comprises several epitopes of several patient-presented co-antigens, the epitopes being known to be immunogenic or predicted to bind to the patient's HLA class I and / or HLA class II alleles.

10. The vaccine according to any one of claims 1 to 9, wherein the antigenic unit comprises the full length of one or more patient-presented shared antigen sequences.

11. The vaccine according to any one of claims 1 to 10, wherein the antigenic unit comprises 1 to 30 portions of at least one patient-presented shared antigen sequence.

12. The vaccine according to claim 11, wherein the portion comprises a plurality of epitopes that are predicted to bind to the HLA class I and / or HLA class II alleles of the patient.

13. The vaccine according to any one of claims 1 to 12, wherein the antigenic unit comprises 1 to 50 patient-presented shared antigen sequences in the form of an epitope.

14. The vaccine according to claim 13, wherein the epitope is expected to bind to the HLA class I and / or HLA class II alleles of the patient.

15. The vaccine according to any one of claims 13 to 14, wherein the epitope has a length of 7 to 30 amino acids.

16. The vaccine according to any one of claims 2 to 15, wherein the antigenic unit comprises several patient-specific antigen sequences or one or more portions thereof.

17. The vaccine according to any one of claims 2 to 16, wherein the antigenic unit comprises one or more patient-specific epitopes.

18. The vaccine according to claim 17, wherein one or more patient-specific epitopes have a length of 7 to 30 amino acids.

19. The vaccine according to any one of claims 17 to 18, wherein the antigenic unit comprises at least five patient-specific epitopes.

20. The vaccine according to any one of claims 1 to 19, wherein the antigenic unit comprises 7 to 2000 amino acids.

21. The vaccine according to any one of claims 1 to 20, wherein the antigenic unit comprises one or more linkers, preferably one or more linkers that separate the antigen sequences from each other.

22. The vaccine according to any one of claims 1 to 21, wherein the dimerization unit includes a hinge region, preferably a hinge region derived from Ig.

23. The vaccine according to claim 22, wherein the dimerization unit further comprises another domain that promotes dimerization, preferably an immunoglobulin domain, more preferably an immunoglobulin constant domain.

24. The vaccine according to claim 22 or 23, wherein the other domain is a carboxy-terminal C domain derived from IgG, preferably from IgG3.

25. The vaccine according to any one of claims 22 to 24, wherein the dimerization unit further comprises a linker, preferably a linker connecting the hinge region and the other domains that promote dimerization.

26. The vaccine according to any one of claims 22 to 25, wherein the dimerization unit comprises hinge exons h1 and h4 connected to the CH3 domain of human IgG3 via a linker.

27. The vaccine according to any one of claims 1 to 26, wherein the antigenic unit and the dimerizing unit are connected via a linker, preferably a linker including a restriction site.

28. The vaccine according to any one of claims 1 to 27, wherein the targeting unit targets antigen-presenting cells, preferably the targeting unit is a portion that interacts with surface molecules on the antigen-presenting cells, or includes such portion, and preferably the surface molecules are selected from the group consisting of HLA, CD14, CD40, chemokine receptors, and Toll-like receptors.

29. The vaccine according to claim 28, wherein the targeting unit comprises or consists of an antibody variable domain having specificity for a soluble CD40 ligand, RANTES, MIP-1α, XCL1, XCL2, flagellin, anti-HLA-DP, anti-HLA-DR, anti-pan-HLA class II, or anti-CD40, anti-TLR-2, anti-TLR-4, or anti-TLR-5, and preferably comprises or consists of MIP-1α.

30. A vaccine according to any one of claims 1 to 29, comprising a polynucleotide, preferably RNA or DNA.

31. The vaccine according to claim 30, wherein the polynucleotide further comprises a nucleotide sequence encoding a signal peptide.

32. The vaccine according to any one of claims 1 to 31, wherein the targeting unit, dimerizing unit and antigenic unit in the polypeptide are arranged in an N-terminus to C-terminus order of targeting unit, dimerizing unit and antigenic unit, or the targeting unit, dimerizing unit and antigenic unit in the polynucleotide are arranged in a 5' to 3' order of targeting unit, dimerizing unit and antigenic unit.

33. The vaccine according to any one of claims 1 to 32, wherein the pharmaceutically acceptable carrier is selected from the group consisting of saline solution, buffered saline solution, PBS, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer, and combinations thereof.

34. A method for preparing a personalized therapeutic anti-cancer vaccine according to claim 1, a) Step of identifying at least one patient-presented shared antigen in the patient's tumor tissue or body fluids. b) The step of determining the HLA class I and / or class II alleles of the patient. c) A step of predicting the immunogenicity of the identified at least one antigen or one or more portions thereof based on their predicted binding affinity to the patient's HLA class I and / or II alleles. d) A step of selecting at least one antigen or one or more parts thereof based on their immunogenicity predicted in step c); e) A step of preparing a polynucleotide sequence comprising an antigenic unit comprising a nucleotide sequence encoding at least one antigen or one or more portions thereof selected in step d); f) The step of cloning the polynucleotide sequence into an expression vector containing nucleotide sequences encoding a targeting unit and a dimerizing unit; and g) A step of mixing the expression vector obtained in step f with a pharmaceutically acceptable carrier. The method, including the method described above.

35. A method according to claim 34 for preparing a personalized anti-cancer vaccine according to claim 2, wherein the method is: In step a), identify one or more patient-specific antigens in the tumor tissue of the patient. In step c), predict the immunogenicity of one or more identified patient-specific antigens or one or more portions thereof by their predicted binding affinity to the patient's HLA class I and / or II alleles. In step d), select one or more patient-specific antigens or one or more parts thereof based on their immunogenicity predicted in step c). It further includes; Furthermore, the polynucleotide sequence of step e) further comprises a nucleotide sequence encoding one or more patient-specific antigens or one or more portions thereof selected in step d), The aforementioned method.

36. A polynucleotide as defined in any one of claims 1 to 32.

37. A vector comprising the polynucleotide described in claim 36.

38. A host cell comprising a polynucleotide as defined in any one of claims 1 to 32, or a vector as described in claim 37.

39. A polypeptide encoded by a polynucleotide sequence as defined in any one of claims 1 to 32.

40. A dimeric protein comprising two polypeptides as defined in claim 39.

41. The dimer protein according to claim 40, which is a homodimer protein.

42. A polynucleotide according to claim 36, a polypeptide according to claim 39, or a dimer protein according to either claim 40 or 41, for use as a pharmaceutical.

43. A method for treating a patient having cancer, comprising administering to the patient a vaccine according to any one of claims 1 to 33, which has been specifically prepared for the patient.

44. The method according to claim 43, wherein the vaccine comprises a polynucleotide and is administered intradermally or intramuscularly.

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