Therapeutic Combinations for Treating Cancer - Patent application

Abstract

The present invention relates to methods and kits for treating a subject, e.g., a patient, with cancer by administering to the subject an anti-cancer vaccine in combination with one or more checkpoint inhibitors.

Description

[Technical field]

[0001] The present invention relates to methods and kits for treating a subject, e.g., a patient, with cancer by administering to the subject an anti-cancer vaccine in combination with one or more checkpoint inhibitors. [Background technology]

[0002] Although cancer treatments have improved over the past several decades, particularly with early detection and diagnosis, which has significantly increased survival rates, only about 60% of patients diagnosed with cancer survive five years after diagnosis. Current cancer treatments include surgical removal of tumors and lesions, as well as radiation and cytotoxic chemotherapy agents, each of which has its own well-known advantages and disadvantages.

[0003] More recently, immunotherapy has become an alternative approach to treat cancer. Therapeutic anti-cancer vaccines represent a type of immunotherapy that works by stimulating or restoring the ability of the subject's immune system to fight cancer. In contrast to preventive or prophylactic vaccines, therapeutic anti-cancer vaccines are used to treat existing cancer. Anti-cancer vaccines can reduce or even eliminate some of the side effects seen in traditional cancer treatments.

[0004] One such type of anti-cancer vaccine has been developed by the applicant. These are polypeptides or polynucleotides encoding such polypeptides, comprising three subunits: a targeting unit that targets antigen-presenting cells (APCs), a dimerization unit, and an antigen unit that includes one or more cancer antigens. The dimerization unit allows the polypeptide to form a dimeric protein. Such anti-cancer vaccines are disclosed, for example, in WO 2004 / 076489 (A1), WO 2011 / 161244 (A1), WO 2013 / 092875 (A1) and WO 2017 / 118695 (A1).

[0005] When administered to a patient as a dimeric protein (or administered to a patient as a polynucleotide and expressed in vivo), the dimeric protein targets APCs, which results in enhanced vaccine efficacy compared to the same non-targeted antigen.

[0006] Another type of cancer immunotherapy is checkpoint inhibitors that target different immune checkpoints, i.e., important regulators of the immune system that can weaken immune response to immunological stimulation when stimulated.Some cancers can protect themselves from attack by the body's immune system by stimulating immune targets, thus impairing or even nullifying the success of anti-cancer immunotherapy treatment.Checkpoint inhibitor therapy has been used to block inhibitory checkpoints, thereby restoring immune system function and treating cancer, such as melanoma or non-small cell lung cancer patients.There is still a need for effective cancer therapy. Summary of the Invention

[0007] The present invention relates to methods and kits for treating a subject, e.g., a patient, with cancer by administering to the subject certain anti-cancer vaccines in combination with one or more checkpoint inhibitors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Thus, in a first aspect, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, e.g., a dimerization unit, and an antigen unit that comprises one or more cancer antigens or portions thereof; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a multimeric protein consisting of a plurality of polypeptides as defined in (ii), for example consisting of two polypeptides, for example a dimeric protein; and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0009] In one embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, and an antigen unit that comprises one or more cancer antigens or portions thereof; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides defined in (ii), and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0010] In another embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a dimerization unit, and an antigen unit that comprises one or more cancer antigens or portions thereof; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a dimeric protein consisting of two polypeptides defined in (ii); and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0011] As used herein, a "subject" is an animal, e.g., a mouse or a human, preferably a human. The terms "subject with cancer" and "patient" may be used interchangeably herein.

[0012] "Cancer" refers to a broad group of different diseases characterized by the uncontrolled growth of abnormal cells in the body. "Cancer" or "cancerous tissue" includes tumors, and as used herein encompasses both solid tumors and tumor cells found in bodily fluids such as blood, including metastatic cancers. Uncontrolled cell division and growth leads to the formation of malignant tumors that can invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. After metastasis, the distant tumor can be said to be "derived" from the pre-metastatic tumor.

[0013] "Cancer antigen" as described herein includes neoantigens, shared cancer antigens present in a patient, or shared cancer antigens.

[0014] "Portion" refers to a portion / fragment of a cancer antigen, ie a portion / fragment of the amino acid sequence of the cancer antigen or the nucleotide sequence encoding same, e.g. an epitope.

[0015] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0016] Anti-cancer vaccines The anti-cancer vaccine for use in the method according to the invention may be a personalized anti-cancer vaccine or a non-personalized anti-cancer vaccine, i.e. an "off-the-shelf" anti-cancer vaccine (referred to as a "shared anti-cancer vaccine"). These two types of anti-cancer vaccines differ in their antigenic units, as detailed below.

[0017] The anti-cancer vaccine for use in the method of the present invention can be described as a polypeptide with an N-terminal start and a C-terminal end (shown in FIG. 1). The elements and units of the polypeptide (targeting unit (TU), multimerization unit, e.g., dimerization unit (DimU) in this figure, and antigen unit) can be arranged in the polypeptide such that the antigen unit is located at the C-terminal end of the polypeptide (FIG. 1a) or at the N-terminal start of the polypeptide (FIG. 1b). Preferably, the antigen unit is located at the C-terminal end of the polypeptide. A unit linker (UL) can connect the multimerization unit, e.g., the dimerization unit, and the antigen unit. FIG. 1 shows an antigen unit with four neoepitopes (neo1, neo2, neo3, neo4) separated by linkers (SUL1, SUL2, SUL3). An alternative way of describing the arrangement of neoepitopes neo1-neo4 is that they are arranged in three antigenic subunits, each of which contains a neoepitope and a subunit linker (SUL1, SUL2, SUL3) and a terminal neoepitope (neo4) that is closest to the C-terminal end or N-terminal start of the polypeptide. The subunits are indicated in the figure by square brackets. Thus, an antigenic unit containing n neoepitopes contains n-1 subunits, each of which contains a neoepitope and a subunit linker. As described herein, the four neoepitopes may be the same or different neoepitopes, and the three linkers / subunit linkers may be the same or different. The order and orientation of the above units and elements is the same in proteins and polynucleotides.

[0018] In the following, various units and elements of anti-cancer vaccines are discussed in detail.These units and elements exist in polynucleotides as nucleic acid sequences that code for the units, while they exist in polypeptides or multimeric proteins as amino acid sequences.For ease of reading, in the following, the units of anti-cancer vaccines are mainly described in relation to polypeptides / multimeric proteins, i.e., based on the amino acid sequences of such vaccines.

[0019] Antigenic unit Antigen units for personalized anti-cancer vaccines The personalized anti-cancer vaccine for use in the method according to the invention comprises an antigenic unit that is specifically designed only for the patient who is or will be treated with the vaccine. The antigenic unit of such a vaccine thus comprises one or more cancer antigens that are patient-specific antigens or parts thereof, such antigens including neo-antigens or shared cancer antigens present in the patient.

[0020] "Shared cancer antigen present in a patient" is used herein to describe a shared cancer antigen or shared tumor antigen that has been identified as being present in the patient's tumor cells.

[0021] "Neo-antigens or patient-specific cancer antigens" are used herein to describe cancer antigens or tumor antigens found in a patient's tumor cells that contain one or more mutations compared to normal (i.e., healthy, non-cancerous) cells of the same patient.

[0022] A "shared cancer epitope present in a patient" is used herein to describe an amino acid sequence contained in, or a nucleotide sequence encoding, a shared cancer antigen present in a patient that contains one or more immunogenic mutations, i.e., mutations known to be immunogenic or predicted to be immunogenic.

[0023] "Neoepitope or patient-specific cancer epitope" is used herein to describe an amino acid sequence contained in, or a nucleotide sequence encoding, a neoantigen or patient-specific cancer antigen that contains one or more immunogenic mutations, i.e., mutations that are predicted to be immunogenic.

[0024] Thus, in one embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, e.g., a dimerization unit, and an antigen unit, the antigen unit comprising one or more patient-specific cancer antigens or a portion thereof, the polynucleotide comprising: (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a multimeric protein consisting of a plurality of polypeptides as defined in (ii), for example consisting of two polypeptides, for example a dimeric protein; and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0025] In one embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigen unit that targets an antigen-presenting cell, wherein the antigen unit comprises one or more patient-specific cancer antigens or a portion thereof; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides defined in (ii), and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0026] In another embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigen unit that targets an antigen-presenting cell, wherein the antigen unit comprises one or more patient-specific cancer antigens or a portion thereof; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a dimeric protein consisting of two polypeptides defined in (ii); and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0027] In one embodiment, the patient-specific cancer antigen is a shared cancer antigen present in a patient.Therefore, in such an embodiment, the antigen unit comprises a shared cancer antigen present in one or more patients or a part thereof, for example, a shared cancer antigen present in one patient or one or more parts of the shared cancer antigen present in such one patient, for example, one or more epitopes, or a shared cancer antigen present in several patients or one or more parts of the shared cancer antigen present in several patients, for example, one or more epitopes.

[0028] In another embodiment, the patient-specific cancer antigen is a neoantigen. Thus, in such an embodiment, the antigenic unit comprises one or more neoantigens or parts thereof, such as one neoantigen or one or more parts of such one neoantigen, such as one or more neoepitopes, or several neoantigens or one or more parts of several neoantigens, such as one or more neoepitopes.

[0029] In yet another embodiment, the antigen unit comprises any combination of the above embodiments, i.e. any combination of shared cancer antigens or portions thereof present in one or more patients and any combination of one or more neo-antigens or portions thereof as above.

[0030] Antigenic units of personalized anti-cancer vaccines comprising one or more neo-antigens or parts thereof Cancer develops from a patient's normal tissues by one or a few cells that start abnormal and uncontrolled proliferation of cells due to mutations. Although cancer cells are mutated, most of the genome in these cells is intact and identical to the rest of the patient's cells. One approach to attack tumors is based on the knowledge that any tumor in any patient is unique: patient-specific mutations lead to the expression of patient-specific mutant proteins, i.e., patient-specific cancer antigens, i.e., neo-antigens, that are unique to a particular patient. These neo-antigens are not identical to any proteins in the patient's normal cells. Such neo-antigens are therefore suitable targets for therapeutic anti-cancer vaccines that are specifically and exclusively manufactured for that patient, i.e., personalized anti-cancer vaccines.

[0031] The mutation may be any mutation that results in a change in at least one amino acid. Thus, the mutation may be one of the following: Nonsynonymous mutations resulting in an amino acid change Mutations that result in a frameshift, thereby resulting in a completely different open reading frame in the orientation following the mutation Read-through mutations in which the stop codon is altered or deleted, resulting in a longer protein with a tumor-specific epitope Splicing variants resulting in unique tumor-specific protein sequences Chromosomal rearrangements resulting in chimeric proteins with a tumor-specific epitope at the junction of two proteins. When the mutation is due to a chromosomal rearrangement, the tumor-specific epitope can result from the change of at least one amino acid or from the combination of two in-frame coding sequences.

[0032] In one embodiment, the antigenic unit comprises one or more neo-antigens or parts thereof, preferably one or more neo-epitopes, more preferably several neo-epitopes. Such neo-epitopes may be selected for inclusion in the antigenic unit according to their predicted therapeutic efficacy, see WO 2017 / 118695(A1), the disclosure of which is incorporated herein by reference.

[0033] Thus, in one embodiment, the invention provides a method for treating a subject (e.g., a patient) with cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, e.g., a dimerization unit, and an antigen unit, the antigen unit comprising one or more neo-antigens or portions thereof, the polynucleotide comprising: (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a multimeric protein consisting of a plurality of polypeptides as defined in (ii), for example consisting of two polypeptides, for example a dimeric protein; and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0034] In one embodiment, the invention provides a method for treating a subject (e.g., a patient) having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigen unit that targets an antigen-presenting cell, the antigen unit comprising one or more neo-antigens or a portion thereof; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides defined in (ii), and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0035] In another embodiment, the invention provides a method for treating a subject (e.g., a patient) having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigen unit that targets an antigen-presenting cell, the antigen unit comprising one or more neo-antigens or a portion thereof; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a dimeric protein consisting of two polypeptides defined in (ii); and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0036] In one embodiment, an antigenic unit comprises one or more parts of one neo-antigen or one or more parts of several neo-antigens, preferably one or more neo-epitopes. In a preferred embodiment, in the antigenic unit, the neo-epitopes are separated by a linker. An alternative way to describe the separation of all neo-epitopes by a linker is that all but the terminal neo-epitopes, i.e. the neo-epitopes at the N-terminal start of the polypeptide or at the C-terminal end of the polypeptide, are located in the antigenic subunit, each subunit comprising a neo-epitope and a subunit linker. The separation of the neo-epitopes by a linker allows each neo-epitope to be presented to the immune system in an optimal way.

[0037] Thus, an antigenic unit comprising n neoepitopes comprises n-1 antigenic subunits, each subunit comprising a neoepitope and a subunit linker, and further comprising a terminal neoepitope. In one embodiment, n is an integer from 1 to 50, e.g., 3 to 50 or 15 to 40 or 10 to 30 or 10 to 25 or 10 to 20 or 15 to 30 or 15 to 25 or 15 to 20. In a preferred embodiment, the antigenic subunit comprises a neoepitope and a subunit linker.

[0038] Thus, in one embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, e.g., a dimerization unit, and an antigen unit that targets an antigen-presenting cell, wherein the antigen unit comprises (A) n-1 antigen subunits, each subunit comprising a neoepitope and a subunit linker, and (B) a terminal neoepitope, where n is the number of neoepitopes in the antigen unit and n is an integer between 1 and 50; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a multimeric protein consisting of a plurality of polypeptides as defined in (ii), for example consisting of two polypeptides, for example a dimeric protein; and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0039] In one embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigen unit that targets an antigen-presenting cell, the antigen unit comprising (A) n-1 antigen subunits, each subunit comprising a neoepitope and a subunit linker, and (B) a terminal neoepitope, where n is the number of neoepitopes in the antigen unit and n is an integer between 1 and 50; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides defined in (ii), and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0040] In another embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigen unit that targets an antigen-presenting cell, the antigen unit comprising (A) n-1 antigen subunits, each subunit comprising a neoepitope and a subunit linker, and (B) a terminal neoepitope, where n is the number of neoepitopes in the antigen unit and n is an integer between 1 and 50; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a dimeric protein consisting of two polypeptides defined in (ii); and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0041] The neoepitope preferably has a length suitable for presentation by HLA (human leukocyte antigen) molecules. Thus, in a preferred embodiment, the neoepitope has a length of 7 to 30 amino acids. More preferred are neoepitopes having a length of 7 to 10 amino acids or 13 to 30 amino acids, for example, 20 to 30 amino acids, for example, 27 amino acids.

[0042] HLA is the major histocompatibility complex (MHC) in humans. There are two major classes of MHC molecules, MHC class 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, and the terms HLA allele and HLA molecule are also used interchangeably herein.

[0043] Preferably, the antigenic unit comprises multiple neoepitopes, i.e. several neoepitopes. In one embodiment, the several neoepitopes are different neoepitopes. In another embodiment, the antigenic unit comprises multiple copies of the same neoepitope. In yet another embodiment, the antigenic unit comprises several different neoepitopes and multiple copies of the same neoepitope.

[0044] Therefore, a preferred approach is to include as many neoepitopes as possible (i.e., different and / or multiple copies of the same neoepitope) in an antigen unit, thereby efficiently attacking the cancer without compromising the ability of the vaccine to activate T cells against the desired neoepitopes due to the effect of dilution of the desired T cells. Furthermore, to ensure that all neoepitopes are efficiently loaded into the same antigen-presenting cell, the nucleotide sequences encoding all neoepitopes are included in a continuous polynucleotide chain that results in the expression of a protein containing all the neoepitopes, instead of expressing each neoepitope as a separate peptide.

[0045] To design the antigenic unit, the patient's tumor exome is analyzed to identify neo-antigens. Preferably, the most immunogenic neo-epitope sequences from one or more neo-antigens are selected for inclusion in the antigenic unit.

[0046] The antigenic unit comprises at least one neoepitope, preferably at least three neoepitopes, more preferably at least five neoepitopes, such as seven neoepitopes. In a more preferred embodiment, the antigenic unit comprises at least 10 neoepitopes. In another more preferred embodiment, the antigenic unit comprises at least 15 neoepitope sequences, such as at least 20 or at least 25 or at least 30 or at least 35 or at least 40 or at least 45 neoepitope sequences.

[0047] Antigenic units of personalized anti-cancer vaccines comprising shared cancer antigens or parts thereof present in one or more patients Shared tumor antigens are expressed by many tumors, either across patients with the same cancer type, or across patient types and cancer types. One example is the HPV16 antigen, a viral antigen that is expressed in about 50% of all patients with squamous cell carcinoma of the head and neck, but also in patients with other cancers, such as cervical cancer and vulvar squamous cell carcinoma. Many of these shared antigens have been previously characterized as immunogenic and / or are known, i.e., their immunogenicity has been confirmed by appropriate methods, and the results have been published, for example, in scientific publications. Others have already been predicted, for example, by algorithms known in the art, to be presented on specific HLA class I or class II alleles, and their predicted immunogenicity has been published, for example, in scientific publications, without confirming their immunogenicity by appropriate methods.

[0048] In one embodiment, the antigenic unit comprises a shared cancer antigen or portion thereof present in one or more patients, e.g., a shared cancer epitope present in the patient, that is known to be immunogenic, has a known expression pattern, and / or is known or already predicted to bind specific HLA class I and class II alleles.

[0049] T cells specific for shared cancer antigens present in the patient can migrate to the tumor and influence the tumor microenvironment, thus increasing the likelihood that additional tumor-specific T cells can attack the cancer.

[0050] Thus, in one embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, e.g., a dimerization unit, and an antigen unit, wherein the antigen unit comprises a shared cancer antigen or a portion thereof present in one or more patients; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a multimeric protein consisting of a plurality of polypeptides as defined in (ii), for example consisting of two polypeptides, for example a dimeric protein; and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0051] In one embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigen unit that targets an antigen-presenting cell, wherein the antigen unit comprises a shared cancer antigen or a portion thereof present in one or more patients; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides defined in (ii), and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0052] In another embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigen unit that targets an antigen-presenting cell, wherein the antigen unit comprises a shared cancer antigen or a portion thereof present in one or more patients; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a dimeric protein consisting of two polypeptides defined in (ii); and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0053] In one embodiment, the shared cancer antigen present in the patient is selected from the group consisting of overexpressed cellular proteins, aberrantly expressed cellular proteins, cancer-testis antigens, viral antigens, differentiation antigens, mutated cancer genes and mutated tumor suppressor genes, carcinoembryonic antigens, shared fusion antigens, shared intron-retained antigens, dark matter antigens, and shared antigens caused by spliceosomal or frameshift mutations.

[0054] In one embodiment, the shared cancer antigen present in the patient is an overexpressed or aberrantly expressed human cellular protein, i.e., a cellular protein found at increased levels in tumors compared to normal healthy cells and tissues. Examples of such overexpressed or aberrantly expressed cellular proteins include tumor proteins D52, Her-2 / neu, hTERT (telomerase), and survivin.

[0055] In another embodiment, the shared cancer antigen present in the patient is a cancer-testis antigen that is normally expressed in male germ cells in the testis, but not in adult somatic tissues. In some cases, such antigens are also expressed in ovaries and trophoblasts. In malignant tumors, this gene regulation is disrupted, resulting in antigen expression in a percentage of tumors of various types. 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.

[0056] In yet another embodiment, the shared cancer antigen present in the patient is a differentiation antigen, e.g., tyrosinase.

[0057] In yet another embodiment, the shared antigen present in the patient 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).

[0058] In yet another embodiment, the shared cancer antigen present in the patient is a mutated cancer gene. Examples of mutated cancer genes include KRAS, CALR, and TRP-2.

[0059] In yet another embodiment, the shared cancer antigen present in the patient is a mutated tumor suppressor gene, examples include mutated p53, mutated pRb, mutated BCL2, and mutated SWI / SNF.

[0060] In yet another embodiment, the shared cancer antigen present in the patient is a carcinoembryonic antigen, such as alpha-fetoprotein or carcinoembryonic antigen.

[0061] In yet another embodiment, the shared antigen present in the patient is a shared intron-retained antigen or a shared antigen caused by a frameshift mutation, such as CDX2 or CALR.

[0062] In yet another embodiment, the shared antigen present in the patient is a shared antigen caused by a spliceosome mutation. An example is an antigen caused by a mutation such as SF3B1 mut.

[0063] Generally speaking, for any cancer antigen, when a patient presents with cancer, immune tolerance is likely to have occurred. Anti-cancer vaccines should specifically induce immune responses against the antigens incorporated into the vaccine. Peripheral immune tolerance to the selected antigen may be weak or strong. By incorporating a shared cancer antigen present in such a patient or one or more parts thereof into the antigen unit (alone or together with a shared cancer antigen or part thereof and / or neo-antigens or neo-epitopes present in other patients), the vaccine containing such antigen unit will induce a sufficiently strong and broad immune response to affect the tumor microenvironment and change the patient's immune response against the tumor from a suppressive / tolerant type to a pro-inflammatory type. This may help to break the tolerance to some other antigens, thus showing considerable clinical benefit for the patient. The aforementioned concept may be referred to as cancer immune set point shifting.

[0064] In one embodiment, the antigenic unit comprises a shared cancer antigen, or a part thereof, present in one or more patients, which is a human cellular protein, preferably an overexpressed or aberrantly expressed human cellular protein or a differentiation antigen.

[0065] Shared cancer antigens present in patients include: sequencing the patient's genome or exome and optionally searching the whole genome / exome-sequence data, e.g. by tailor-made software, to identify, e.g., mutated cancer genes or mutated tumor suppressor genes; Immunohistochemistry of patient tumor tissue, for example to detect the presence of mutant proteins; RT-PCR, for example to detect the presence of known mutations in viral antigens or cancer genes; For example, ELISA using antibodies against mutant tumor proteins in serum samples; RNA-seq of tumor tissue and comparison with healthy tissue, e.g. to detect expression / overexpression of shared cancer antigens; - searching, for example by tailor-made software, to identify intron-retaining antigens in raw RNA-seq data; - searching, for example by tailor-made software, in whole genome sequence data to identify transposable elements that are components of dark matter antigens; Detection of short repeats, e.g., to identify dark matter antigens in raw whole-exome / RNA-seq data; RNA-seq data, e.g., to identify shared viral antigens, as well as It can be detected in the patient's tissues or body fluids by methods known in the art, including comparing RNA-seq of a patient's tumor sample with either the patient's own healthy tissue or a cohort / database (e.g., TCGA) against consensus transcript expression such as GTEX / HPA gene expression data.

[0066] In a preferred embodiment, the antigenic unit comprises a shared cancer antigen present in one or more patients, or a portion of such an antigen, that is known to be immunogenic, e.g., has previously been described to elicit an immune response in other patients, or is predicted to bind to the patient's HLA class I and / or class II alleles.

[0067] In another preferred embodiment, the antigenic unit comprises one or more portions of a shared cancer antigen present in one or more patients, e.g., one or more epitopes known to be immunogenic or predicted to bind to the patient's HLA class I and / or class II alleles.

[0068] In one embodiment, the antigenic unit comprises a shared cancer epitope present in one or more patients. In a preferred embodiment, such an epitope has a length suitable for presentation by the patient's HLA alleles.

[0069] In one embodiment, the antigenic unit comprises a shared cancer epitope present in one or more patients that has a length suitable for specific presentation on HLA class I or HLA class II. In one embodiment, the epitope has a length of 7-11 amino acids for HLA class I presentation. In another embodiment, the epitope has a length of 13-30 amino acids for HLA class II presentation.

[0070] In one embodiment, the antigenic unit comprises a shared cancer epitope present in one or more patients having a length of 7 to 30 amino acids, such as 7 to 10 amino acids (e.g. 7, 8, 9 or 10 amino acids) or 13 to 30 amino acids (e.g. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids), such as 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.

[0071] The antigenic unit may comprise a shared cancer antigen present in one or more patients, either full length or one or more portions thereof.

[0072] In one embodiment, the antigenic unit comprises a shared cancer antigen present in one patient that is full length. In another embodiment, the antigenic unit comprises a shared cancer antigen present in several patients, each of which is full length.

[0073] In yet another embodiment, the antigenic unit comprises one or more portions of a shared cancer antigen present in a patient, e.g., a shared cancer epitope present in one or more patients. In yet another embodiment, the antigenic unit comprises one or more portions of a shared cancer antigen present in several patients, e.g., one or more epitopes of a shared cancer antigen present in several patients.

[0074] In yet another embodiment, the antigenic unit comprises a full length shared antigen present in one or more patients and one or more portions of a shared cancer antigen present in one or more patients. an antigenic unit comprising a full-length shared antigen present in one patient and one or more epitopes of a shared cancer antigen present in one patient, an antigenic unit comprising a shared cancer antigen present in several patients, each of which is full length, and one or more epitopes of a shared cancer antigen present in one patient, an antigenic unit comprising a full-length shared antigen present in one patient and one or more epitopes of a shared cancer antigen present in several patients; and · An antigenic unit that comprises a shared cancer antigen present in several patients (each of which is full length) and one or more epitopes of a shared cancer antigen present in several patients.

[0075] In a preferred embodiment, the epitope is already known to be immunogenic (e.g., described in the literature as immunogenic) or already predicted to bind to the patient's HLA class I and class II alleles (e.g., as described in the literature), preferably already predicted to bind to the patient's HLA class I alleles. In another preferred embodiment, the immunogenicity of the epitope is predicted, e.g., the binding of the epitope to one or more of the patient's HLA class I and / or HLA class II molecules is predicted by methods known in the art, such as the methods disclosed in WO 2021 / 205027(A1), the disclosure of which is incorporated herein by reference, or the methods described in the section "Methods for designing antigen units of personalized anti-cancer vaccines for use in the present invention" contained herein.

[0076] In one embodiment, the antigenic unit comprises between 1 and 10 full length shared antigens present in the patient.

[0077] In another embodiment, the antigenic unit comprises 1-30 portions of a shared antigen present in one or more patients, which portions comprise one or more epitopes predicted to bind to HLA class I or class II alleles of the patients. In yet another embodiment, the antigenic unit comprises shared cancer epitopes present in 1-50 patients, optionally epitopes predicted to bind to HLA class I or class II alleles of the patients.

[0078] Antigenic unit of a personalized anti-cancer vaccine comprising a shared cancer antigen or a part thereof present in one or more patients and one or more neo-antigens or parts thereof Such an antigen unit is essentially a combination of a) all of the above embodiments relating to an antigen unit comprising a shared cancer antigen present in one or more patients or a part thereof, and b) all of the above embodiments relating to an antigen unit comprising one or more neo-antigens or a part thereof.

[0079] Thus, in one embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, e.g., a dimerization unit, and an antigen unit, wherein the antigen unit comprises a shared cancer antigen or a portion thereof present in one or more patients, and one or more neo-antigens or portions thereof; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a multimeric protein consisting of a plurality of polypeptides as defined in (ii), for example consisting of two polypeptides, for example a dimeric protein; and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0080] In one embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigen unit that targets an antigen-presenting cell, the antigen unit comprising a shared cancer antigen or a portion thereof present in one or more patients and one or more neo-antigens or portions thereof; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides defined in (ii), and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0081] In another embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigen unit that targets an antigen-presenting cell, the antigen unit comprising a shared cancer antigen or a portion thereof present in one or more patients and one or more neo-antigens or portions thereof; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a dimeric protein consisting of two polypeptides defined in (ii); and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0082] Methods for designing antigenic units of personalized anti-cancer vaccines for use in the present invention The shared cancer antigens and neo-antigens present in a particular patient identified are preferably further processed to find those antigens that, when included in an antigenic unit, will make the vaccine most effective. The manner and order in which such processing is performed will depend on how the antigens were identified, i.e., the data that forms the basis of such processing.

[0083] In one embodiment, the processing and selection of antigens to be included in the antigenic unit is carried out as follows. 1) A search is performed in the literature and / or one or more databases to retrieve information about the shared cancer antigens and their sequences, and preferably information about their expression patterns, immunogenicity or predicted immunogenicity, epitopes and / or HLA presentation. Such a search is also performed to determine whether the identified antigen is a shared cancer antigen or a neo-antigen present in the patient. 2) If the identified antigen is determined to be a shared cancer antigen present in the patient, its sequence is studied to identify epitopes, preferably all epitopes, that are predicted to bind to the patient's HLA class I / II alleles.Prediction can be performed by using prediction tools known in the art, for example prediction software known in the art, such as NetMHCpan and similar software. 3) The most likely sequences of the shared cancer antigens present in the patient that are or are predicted to be the most immunogenic are selected for inclusion in the antigen unit, i.e., sequences that show predicted binding to one or more of the patient's HLA class I / II alleles. In one embodiment, for example, if only a few likely epitopes are identified in step 2, or if longer stretches of non-immunogenic sequences exist between epitopes, the smallest epitopes are selected. In another embodiment, longer sequences are selected that include several epitopes that bind to the patient's specific HLA alleles. In yet another embodiment, the full-length sequence of the antigen is selected for inclusion in the antigen unit. 4) The most promising portions of neo-antigenic sequences, e.g., neo-epitopes, are selected for inclusion into the antigenic unit based on predicted immunogenicity and binding of such sequences to the patient's HLA class I / II alleles.

[0084] Tumor mutations are discovered by sequencing tumor and normal tissues and comparing the sequences obtained from tumor tissues with those of normal tissues. A variety of methods are available to detect the presence of specific mutations or alleles in a patient's DNA or RNA. These methods include dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMan system, and various DNA "chip" technologies such as the Affymetrix SNP chip. Alternatively, mutations can be identified by direct protein sequencing.

[0085] From the possibly hundreds or thousands of mutations in the tumor exome, the most promising sequences are selected in silico based on a predictive HLA binding algorithm. The intention is to identify all relevant epitopes and determine the sequences to be included in the antigenic unit after ranking or scoring. Methods known in the art may be suitable for scoring, ranking and selecting neoepitopes, including those disclosed in WO 2020 / 065023(A1) and WO 2020 / 221 / 783(A1).

[0086] Moreover, any suitable algorithm for such scoring and ranking may be used, including the following: Free software analysis of peptide-MHC binding (IEDB and NetMHCpan) available for download from the following websites: www.iedb.org www.cbs.dtu.dk / services / NetMHC Commercially available sophisticated software for predicting optimal sequences for vaccine design can be found, for example, in: www.oncoimmunity.com / omictools.com / t-cell-epitopes-category github.com / griffithlab / pVAC-Seq crdd.osdd.net / raghava / cancertope / help.php www.epivax.com / tag / neoantigen / Each mutation is scored for its antigenic or immunogenicity, and the most antigenic or immunogenic neoepitopes are selected and optimally positioned in the antigenic unit, for example as described herein.

[0087] Antigenic units of non-individualized anti-cancer vaccines Non-personalized or "off-the-shelf" anti-cancer vaccines (also called shared anti-cancer vaccines) comprise antigenic units that include one or more shared cancer antigens or parts thereof.

[0088] "Shared cancer antigen" or "shared tumor antigen" is used herein to describe an antigen that has been described to be expressed by many tumors, either across patients with the same cancer type, or across patient types and cancer types.

[0089] "Shared cancer epitope" is used herein to describe an amino acid sequence contained in a shared cancer antigen that is known or predicted to be immunogenic.

[0090] In one embodiment, the antigenic units of such non-personalized anti-cancer vaccines comprise one or more shared cancer antigens or portions thereof, e.g., shared cancer epitopes, that are known to be immunogenic, have known expression patterns, and / or are known or predicted to bind to specific HLA class I and class II molecules.

[0091] Thus, in one embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, e.g., a dimerization unit, and an antigen unit, the antigen unit comprising one or more shared cancer antigens or portions thereof, the polynucleotide comprising: (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a multimeric protein consisting of a plurality of polypeptides as defined in (ii), for example consisting of two polypeptides, for example a dimeric protein; and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0092] In one embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigen unit that targets an antigen-presenting cell, wherein the antigen unit comprises one or more shared cancer antigens or portions thereof; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides defined in (ii), and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0093] In another embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigen unit that targets an antigen-presenting cell, wherein the antigen unit comprises one or more shared cancer antigens or portions thereof; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a dimeric protein consisting of two polypeptides defined in (ii); and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0094] Examples of shared cancer antigens include those disclosed under the heading "Antigen units of personalized anti-cancer vaccines comprising shared cancer antigens or parts thereof present in one or more patients" and scFvs derived from monoclonal Igs produced by myelomas or lymphomas, also called myeloma / lymphoma M component, in patients with B-cell lymphoma or multiple myeloma, telomerase, e.g. hTERT, HIV-derived sequences, e.g. sequences such as gp120 or Gag-derived sequences, tyrosinase, tyrosinase-related protein (TRP)-1 or TRP-2, melanoma antigens, prostate-specific antigen and idiotype, HPV antigens, e.g. any one selected from the group consisting of E1, E2, E6, E7, L1 and L2, e.g. E6 and / or E7 of HPV16 and / or HPV18.

[0095] Any shared cancer antigen sequence of sufficient length that contains a specific epitope can be used as an antigen unit.Thus, in one embodiment, the antigen unit comprises an amino acid sequence of at least 7 amino acids, for example at least 8 amino acids, which corresponds to at least about 21 nucleotides, for example at least 24 nucleotides in the nucleic acid sequence that codes for such antigen unit.

[0096] In yet another embodiment, the antigenic unit comprises one or more portions of a shared cancer antigen, e.g., one or more shared cancer epitopes. In yet another embodiment, the antigenic unit comprises one or more portions of several shared cancer antigens, e.g., one or more epitopes of several shared cancer antigens. In yet another embodiment, the antigenic unit comprises one or more full-length shared antigens and one or more portions of one or more shared cancer antigens. Examples include: an antigenic unit comprising one full-length shared antigen and one or more epitopes of one shared cancer antigen, and an antigenic unit comprising several shared cancer antigens, each of which is full length, and one or more epitopes of one shared cancer antigen, and an antigenic unit comprising one full-length shared antigen and one or more epitopes of several shared cancer antigens, and · Several shared cancer antigens (each of which is full length) and antigenic units that contain one or more epitopes of several shared cancer antigens.

[0097] Examples of shared anti-cancer vaccines that include shared HPV antigens are disclosed in WO 2013 / 092875(A1), which is incorporated herein by reference.

[0098] Methods for designing antigenic units of shared anti-cancer vaccines for use in the methods of the present invention Also, for shared anti-cancer vaccines for use in the methods of the invention, the antigenic units are designed to contain sequences that are likely to make the vaccine effective in a variety of patients (e.g., patients with a particular cancer type).

[0099] In one embodiment, the selection of antigens to be included in the antigen unit is performed by performing a search in the literature and / or one or more databases to search for information about shared cancer antigens and their sequences, and preferably their expression patterns, immunogenicity or predicted immunogenicity, epitopes and / or HLA presentation.Then, epitopes that are known or predicted to bind to various HLA class I / II alleles of many patients, or epitopes that bind to a specific subset of HLA class I / II alleles that are prevalent in a specific cancer indication and / or a specific patient population across different cancer indications, are identified.Preferably, the most promising, i.e., the sequences of the shared cancer antigens that are most immunogenic or predicted to be most immunogenic, are selected for inclusion in the antigen unit.

[0100] Further embodiments of antigenic units The following applies generally to antigenic units in anti-cancer vaccines for use in the methods of the present invention, i.e. antigenic units contained in individualized anti-cancer vaccines and antigenic units contained in shared anti-cancer vaccines.

[0101] The term antigen is used in this section of the application for neoantigens, neoepitopes, shared cancer antigens present in a patient, parts of shared cancer antigens present in a patient such as shared cancer epitopes present in a patient, shared cancer antigens, and parts of shared cancer antigens such as shared cancer epitopes.

[0102] In one embodiment, the antigenic unit contains only one copy of each antigen, thus, for example, if 10 different antigens are contained in an antigenic unit, a vaccine containing the antigenic unit will elicit a cell-mediated immune response against all 10 different antigens and thus be able to efficiently attack cancer.

[0103] However, if only a small number of antigens are included in the antigen unit, the antigen unit may contain at least two copies of a particular antigen in order to enhance the immune response to the antigen.

[0104] Such a situation may arise in the design of antigen units for personalized anti-cancer vaccines, when only a few neoantigens / neoepitopes that are predicted to be / are predicted to be sufficiently immunogenic to bind to the patient's HLA alleles can be identified in the patient. In such patients, when shared cancer antigens present in the patient in addition to the neoantigens / neoepitopes are identified, it is preferable to include in the antigen unit the shared cancer antigen sequence or a part thereof present in such one or more patients, rather than including multiple copies of the same neoantigen / neoepitope.

[0105] The length of an antigen unit is determined by the length and number of antigens contained therein. In one embodiment, an antigen unit contains about 21 to about 2000 amino acids, preferably about 30 to about 1500 amino acids, more preferably about 50 to about 1000 amino acids, such as about 100 to about 500 amino acids, about 100 to about 400 amino acids, or about 100 to about 300 amino acids.

[0106] In another embodiment, the antigenic unit contains up to 3500 amino acids, for example, 60 to 3500 amino acids, for example, about 80 or about 100 or about 150 amino acids to about 3000 amino acids, for example, about 200 to about 2500 amino acids, for example, about 300 to about 2000 amino acids, or about 400 to about 1500 amino acids, or about 500 to about 1000 amino acids.

[0107] To enhance the immune response, for a personalized anti-cancer vaccine, the antigens may be arranged in antigen subunits as described below: An antigen unit may be described as a polypeptide with an N-terminal start and a C-terminal end. The antigen unit is preferably linked to the dimerization unit via a unit linker. The antigen unit is located at either the COOH-terminal end or the NH2-terminal end of the polypeptide / dimeric protein. The antigen unit is preferably at the COOH-terminal end of the polypeptide / dimeric protein. In one embodiment, the antigens, preferably epitopes, are arranged in order from more antigenic to less antigenic in the direction from the N-terminal start of the antigen unit to the C-terminal end of the antigen unit. Alternatively, it is preferred that the most hydrophobic antigen is located substantially in the middle of the antigen unit and the most hydrophilic antigen is located at the N-terminal start and / or C-terminal end of the antigen unit, especially when hydrophilicity / hydrophobicity varies greatly between antigens.

[0108] Since precise positioning of the antigenic unit in the center is only possible when the antigenic unit contains an odd number of antigens, the term "substantially" in this context refers to an antigenic unit containing an even number of antigens, in which the most hydrophobic antigen is positioned as close to the center as possible.

[0109] As an example, an antigenic unit may include five antigenic subunits, each of which includes a different epitope, e.g., a different neoepitope, as follows: 1-2-3 * -4-5 are placed (1, 2, 3 * , 4 and 5 are different neoepitopes, - is a subunit linker, *indicates the most hydrophobic neoepitope, which is located in the center of the antigenic unit).

[0110] In another example, the antigenic unit comprises six antigenic subunits, each of which comprises a different epitope, e.g., a different neoepitope, as follows: 1-2-3 * -4-5-6, or alternatively: 1-2-4-3 * -5-6 are placed (1, 2, 3 * , 4, 5 and 6 are different neoepitopes, - is a subunit linker, * represents the most hydrophobic neoepitope, which is located essentially in the center of the antigenic unit).

[0111] Alternatively, the antigen subunits may be arranged to alternate between hydrophilic and hydrophobic antigens.

[0112] Optionally, GC-rich sequences encoding antigens (e.g., GC-rich sequences encoding neoepitopes or epitopes) are positioned to avoid GC clusters. In one embodiment, GC-rich sequences encoding antigens are positioned such that there is at least one non-GC-rich sequence between them.

[0113] In one embodiment, the antigen unit comprises one or more linkers. In another embodiment, the antigen unit comprises multiple antigens, e.g., multiple epitopes, e.g., neoepitopes, and the antigens are separated by linkers. In yet another embodiment, the antigen unit comprises multiple antigens, and each antigen is separated from the other antigens by a linker. An alternative way to describe the separation of each antigen from the other antigens by a linker is that all but the terminal antigens, i.e., the antigens at the N-terminal start of the polypeptide or the C-terminal end of the polypeptide (i.e., located at the end of the antigen unit that is not linked to the dimerization unit), are arranged in antigen subunits, and each subunit comprises or consists of an antigen, e.g., a neoepitope, and a subunit linker.

[0114] Thus, an antigen unit containing n antigens contains n-1 antigen subunits, each subunit containing an antigen and a subunit linker, and further containing a terminal antigen. In one embodiment, n is an integer from 1 to 50, for example, 3 to 50, or 15 to 40, or 10 to 30, or 10 to 25, or 10 to 20, or 15 to 30, or 15 to 25, or 15 to 20.

[0115] Separation of the antigens by a linker allows each antigen to be presented to the immune system in an optimal manner.

[0116] Thus, in one embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, e.g., a dimerization unit, and an antigen unit that targets an antigen-presenting cell, wherein the antigen unit comprises (A) n-1 antigen subunits, each of which comprises a subunit linker and an antigen selected from the group consisting of a neoantigen, a neoepitope, a shared cancer antigen present in a patient, a portion of a shared cancer antigen present in a patient, and a shared cancer epitope present in a patient, or selected from the group consisting of a shared cancer antigen, a portion of a shared cancer antigen, and a shared cancer epitope, and (B) a terminal antigen, wherein n is the number of antigens in the antigen unit and n is an integer of 1 to 50; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a multimeric protein consisting of a plurality of polypeptides as defined in (ii), for example consisting of two polypeptides, for example a dimeric protein; and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0117] In one embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigen unit that targets an antigen-presenting cell, wherein the antigen unit comprises (A) n-1 antigen subunits, each of which comprises a subunit linker and an antigen selected from the group consisting of a neoantigen, a neoepitope, a shared cancer antigen present in a patient, a portion of a shared cancer antigen present in a patient, and a shared cancer epitope present in a patient, or an antigen selected from the group consisting of a shared cancer antigen, a portion of a shared cancer antigen, and a shared cancer epitope, and (B) a terminal antigen, wherein n is the number of antigens in the antigen unit and n is an integer of 1 to 50; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides defined in (ii), and an anti-cancer vaccine comprising (b) administering to the subject one or more checkpoint inhibitors.

[0118] In another embodiment, the present invention provides a method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigen unit that targets an antigen-presenting cell, wherein the antigen unit comprises (A) n-1 antigen subunits, each of which comprises a subunit linker and an antigen selected from the group consisting of a neoantigen, a neoepitope, a shared cancer antigen present in a patient, a portion of a shared cancer antigen present in a patient, and a shared cancer epitope present in a patient, or selected from the group consisting of a shared cancer antigen, a portion of a shared cancer antigen, and a shared cancer epitope, and (B) a terminal antigen, wherein n is the number of antigens in the antigen unit and n is an integer of 1 to 50; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) an anti-cancer vaccine comprising a dimeric protein consisting of two polypeptides defined in (ii); (b) administering to the subject one or more checkpoint inhibitors.

[0119] Linker in antigen unit The linker in the antigen unit separates the antigens, e.g., epitopes, contained therein. As mentioned above, all antigens, e.g., neoepitopes, can be located in the subunit, separated from each other by linkers. In the following, the terms subunit linker and linker are used interchangeably, both referring to the linker in the antigen unit.

[0120] In one embodiment, the subunit linker is designed to be non-immunogenic.It can be a rigid linker, meaning that it does not allow the two amino acid sequences that it links to move substantially freely relative to each other.Alternatively, it can be a flexible linker, i.e., it allows the two amino acid sequences that it links to move substantially freely relative to each other.

[0121] Both types of linkers are useful, in one embodiment, the subunit linker is a flexible linker, which allows antigens to be presented to T cells in an optimal manner, even when the antigen unit contains multiple antigens.

[0122] In one embodiment, the subunit linker is a peptide of 4 to 40 amino acids, such as 35, 30, 25 or 20 amino acids, such as 5 to 20 amino acids or 5 to 15 amino acids or 8 to 20 amino acids or 8 to 15 amino acids, 10 to 15 amino acids or 8 to 12 amino acids. In another embodiment, the subunit linker consists of 10 amino acids.

[0123] In one embodiment, for example, in an antigen unit that contains a neoepitope, the subunit linker is the same in all antigen subunits.However, if one or more of the antigens contain a sequence similar to that of the linker, it may be advantageous to replace adjacent subunit linkers with linkers of different sequences.Also, if it is predicted that the antigen-subunit linker junction constitutes an immunogenic epitope by itself, linkers of different sequences may be used.

[0124] In one embodiment, the subunit linker is a flexible linker, preferably a flexible linker comprising small non-polar (e.g., glycine, alanine or leucine) or polar (e.g., serine or threonine) amino acids. The small size of these amino acids provides flexibility and allows mobility of the connected amino acid sequence. The incorporation of serine or threonine can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thus reducing unfavorable interactions between the linker and the antigen. In one embodiment, the flexible linker is a serine (S) and / or glycine (G) rich linker, i.e., a linker comprising several serine and / or several glycine residues. Preferred examples are GGGGS (SEQ ID NO:24), GGGSS (SEQ ID NO:25), GGGSG (SEQ ID NO:26), GGSGG (SEQ ID NO:27), SGSSGS (SEQ ID NO:28) or several variants thereof such as GGGGSGGGGS (SEQ ID NO:29), (GGGGS)m (SEQ ID NO:30), (GGGS)m (SEQ ID NO:31), (GGSGG)m (SEQ ID NO:32), (GGGSG)m (SEQ ID NO:33) or (SGSSGS)m (SEQ ID NO:34), where m is an integer from 1 to 5, for example 1, 2, 3, 4 or 5. In a preferred embodiment, m is 2. In another preferred embodiment, the serine and / or glycine rich linker further comprises at least one leucine (L) residue, for example at least one or at least two or at least three leucine residues, for example 1, 2, 3 or 4 leucine residues.

[0125] In one embodiment, the subunit linker comprises or consists of LGGGS (SEQ ID NO:35), GLGGS (SEQ ID NO:36), GGLGS (SEQ ID NO:37), GGGLS (SEQ ID NO:38), or GGGGL (SEQ ID NO:39). In another embodiment, the subunit linker comprises or consists of LGGSG (SEQ ID NO:40), GLGSG (SEQ ID NO:41), GGLSG (SEQ ID NO:42), GGGLG (SEQ ID NO:43), or GGGSL (SEQ ID NO:44). In yet another embodiment, the subunit linker comprises or consists of LGGGSS (SEQ ID NO:45), GLGSS (SEQ ID NO:46), or GGLSS (SEQ ID NO:47).

[0126] In yet another embodiment, the subunit linker comprises or consists of LGLGS (SEQ ID NO:48), GLGLS (SEQ ID NO:49), GLLGS (SEQ ID NO:50), LGGLS (SEQ ID NO:51) or GLGGL (SEQ ID NO:52). In yet another embodiment, the subunit linker comprises or consists of LGLSG (SEQ ID NO:53), GLLSG (SEQ ID NO:54), GGLSL (SEQ ID NO:55), GGLLG (SEQ ID NO:56) or GLGSL (SEQ ID NO:57). In yet another embodiment, the subunit linker comprises or consists of LGLSS (SEQ ID NO:58) or GGLLS (SEQ ID NO:59).

[0127] In another embodiment, the subunit linker is a serine-glycine linker having a length of 10 amino acids and containing one or two leucine residues.

[0128] In one embodiment, the subunit linker comprises or consists of LGGGSGGGGS (SEQ ID NO:60), GLGGSGGGGS (SEQ ID NO:61), GGLGSGGGGS (SEQ ID NO:62), GGGLSGGGGS (SEQ ID NO:63), or GGGGLGGGGS (SEQ ID NO:64). In another embodiment, the subunit linker comprises or consists of LGGSGGGGSG (SEQ ID NO:65), GLGSGGGGSG (SEQ ID NO:66), GGLSGGGGSG (SEQ ID NO:67), GGGLGGGGSG (SEQ ID NO:68), or GGGSLGGGSG (SEQ ID NO:69). In yet another embodiment, the subunit linker comprises or consists of LGGSSGGGSS (SEQ ID NO:70), GLGSSGGGSS (SEQ ID NO:71), GGLSSGGGSS (SEQ ID NO:72), GGGLSGGGSS (SEQ ID NO:73), or GGGSLGGGSS (SEQ ID NO:74).

[0129] In one embodiment, the subunit linker comprises or consists of LGGGSLGGGS (SEQ ID NO:75), GLGGSGLGGS (SEQ ID NO:76), GGLGSGGLGS (SEQ ID NO:77), GGGLSGGGLS (SEQ ID NO:78) or GGGGLGGGGL (SEQ ID NO:79). In a further embodiment, the subunit linker comprises or consists of LGGSGLGGSSG (SEQ ID NO:80), GLGSGLSGSSG (SEQ ID NO:81), GGLSGGGLSG (SEQ ID NO:82), GGGLGGGGLG (SEQ ID NO:83) or GGGSLGGGSL (SEQ ID NO:84). In yet another embodiment, the subunit linker comprises or consists of LGGSSLGGSS (SEQ ID NO:85), GLGSSGLGSS (SEQ ID NO:86) or GGLSSGGLSS (SEQ ID NO:87).

[0130] In yet another embodiment, the subunit linker comprises or consists of GSGGGA (SEQ ID NO: 88), GSGGGAGSGGGA (SEQ ID NO: 89), GSGGGAGSGGGAGSGGGA (SEQ ID NO: 90), GSGGGAGSGGGAGSGGGAGSGGGA SEQ ID NO: 91), or GENLYFQSGG (SEQ ID NO: 92). In yet another embodiment, the subunit linker comprises or consists of SGGGSSGGGS (SEQ ID NO: 93), SSGGGSSGGG (SEQ ID NO: 94), GGSGGGGSGG (SEQ ID NO: 95), GSGSGSGSGS (SEQ ID NO: 96), GGGSSGGGSG (amino acids 121-130 of SEQ ID NO: 1), GGGSSS (SEQ ID NO: 97), GGGSSGGGSSGGGSS (SEQ ID NO: 98), or GLGGLAAA (SEQ ID NO: 99).

[0131] In another embodiment, the subunit linker is a rigid linker. Such rigid linkers can be useful to efficiently separate (larger) antigens and prevent them from interfering with each other. In one embodiment, the subunit linker comprises or consists of KPEPKPAPAPKP (SEQ ID NO: 100), AEAAAKEAAAKA (SEQ ID NO: 101), (EAAAK)m (SEQ ID NO: 102), PSRLEEELRRRLTEP (SEQ ID NO: 103) or SACYCELS (SEQ ID NO: 104).

[0132] In yet another embodiment, the subunit linker comprises or consists of TQKSLSLSPGKGLGGL (SEQ ID NO: 105). In yet another embodiment, the subunit linker comprises or consists of SLSLSPGKGLGGL (SEQ ID NO: 106).

[0133] In yet another embodiment, the subunit linker comprises or consists of GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 107), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 108), or ELKTPLGDTTHT (amino acids 94-105 of SEQ ID NO:1), or EPKSCDTPPPCPRCP (amino acids 106-120 of SEQ ID NO:1).

[0134] In yet another embodiment, the subunit linker is a cleavable linker, e.g., a linker that contains one or more recognition sites for endopeptidases, e.g., furin, caspases, cathepsins, etc. Cleavable linkers can be introduced to release free functional protein domains (e.g., encoded by larger antigens), which can overcome steric hindrance between such domains, such as reduced biological activity, altered biodistribution, or other disadvantages due to interference of such domains.

[0135] Further examples of sequences encoded by subunit linkers are disclosed in paragraphs

[0098] to

[0099] and the sequences listed in WO 2020 / 176797(A1), which are incorporated by reference herein, and in paragraphs

[0135] to

[0139] of U.S. Patent Application Publication No. 2019 / 0022202(A1), which are incorporated by reference herein.

[0136] Unit Linker The antigen unit is preferably linked to the dimerization unit by a unit linker. Thus, in one embodiment, the anti-cancer vaccine for use in the method of the present invention comprises a nucleotide sequence that encodes a unit linker by which the antigen unit is linked to the dimerization unit.

[0137] The unit linker may include restriction sites to facilitate construction of the polynucleotide. In one embodiment, the unit linker is GLGGL (SEQ ID NO:52) (the nucleotide sequence of SEQ ID NO:10) or GLSGL (SEQ ID NO:109). In another embodiment, the unit linker comprises or consists of GGGGS (SEQ ID NO:24), GGGGSGGGGS (SEQ ID NO:29), (GGGGS)m (SEQ ID NO:30), EAAAK (SEQ ID NO:110), (EAAAK)m (SEQ ID NO:102), (EAAKG)mS (SEQ ID NO:111), GPSRLEEELRRRLTEPG (SEQ ID NO:112), AAY, or HEYGAEALERAG (SEQ ID NO:114).

[0138] Targeting Unit Anti-cancer vaccines for use in the methods of the present invention include targeting units that target antigen-presenting cells (APCs). APCs include dendritic cells (DCs) and subsets thereof.

[0139] The term "targeting unit" as used herein refers to a unit that delivers a polypeptide / dimeric protein (encoded by a polynucleotide as defined above) to an antigen-presenting cell for presentation to MHC class I restricted CD4+ T cells or to provide cross-presentation to CD8+ T cells with MHC class I restriction.

[0140] Due to the targeting unit, the anti-cancer vaccine for use in the method of the present invention attracts DC, neutrophils and other immune cells. Thus, the anti-cancer vaccine not only targets the antigen unit contained therein to specific cells, but also promotes a response amplification effect (adjuvant effect) by recruiting specific immune cells to the administration site of the anti-cancer vaccine. This unique mechanism is very important in clinical practice: the anti-cancer vaccine for use in the method of the present invention can be administered to patients without any adjuvant, since the vaccine itself provides the adjuvant effect.

[0141] The targeting unit is designed to target the anti-cancer vaccine for use in the methods of the present invention to a surface molecule expressed on APCs (e.g., a molecule expressed exclusively on a subset of DCs).

[0142] Examples of such surface molecules on APCs are HLA, cluster of differentiation 14 (CD14), cluster of differentiation 40 (CD40), CLEC9A, chemokine receptors and Toll-like receptors (TLRs). Chemokine receptors include CC motif chemokine receptor 1 (CCR1), CC motif chemokine receptor 3 (CCR3), CC motif chemokine receptor 4 (CCR4), CC motif chemokine receptor 5 (CCR5), CC motif chemokine receptor 6 (CCR6), CC motif chemokine receptor 7 (CCR7), CC motif chemokine receptor 8 (CCR8) and XCR1. Toll-like receptors include TLR-2, TLR-4 and TLR-5. Targeting units are or include moieties that interact with these surface molecules.

[0143] Thus, in one embodiment, the targeting unit comprises or consists of an antibody binding region, such as an antibody variable domain (VL and VH) with specificity for HLA, CD14, CD40, CLEC9A or a Toll-like receptor, hi another embodiment, the targeting unit comprises or consists of a synthetic or natural ligand. Examples include soluble CD40 ligand, natural ligands such as chemokines, including their human forms, e.g., chemokine ligand 5, also called CC motif ligand 5 (CCL5 or RANTES), mouse CCL3 (or MIP-1α), and macrophage proinflammatory protein alpha and its isoforms, including the human isoforms hCCL3, hCCL3L1, hCCL3L2 and hCCL3L3, chemokine ligand 4 (CCL4) and its isoforms CCL4L, chemokine ligand 19 (CCL19), chemokine ligand 20 (CCL20), chemokine ligand 21 (CCL21), chemokine motif ligand 1 or 2 (XCL1 or XCL2), and bacterial antigens, e.g., flagellin.

[0144] In one embodiment, the targeting unit has affinity for MHC class II proteins. Thus, in one embodiment, the targeting unit comprises or consists of an antibody binding region, such as an antibody variable domain (VL and VH) with specificity for an MHC class II protein, such as one selected from the group consisting of anti-HLA-DP, anti-HLA-DR and anti-pan-HLA class II.

[0145] In another embodiment, the targeting unit has affinity for a surface molecule selected from the group consisting of CD14, CD40, TLR-2, TLR-4 and TLR-5. Thus, in one embodiment, the targeting unit comprises or consists of an antibody binding region, e.g., an antibody variable domain (VL and VH) with specificity for CD14, CD40, TLR-2, TLR4 or TLR-5, such as anti-CD14, anti-CD40, anti-TLR-2, anti-TLR-4 or anti-TLR-5. In yet another embodiment, the targeting unit comprises or consists of a flagellin with affinity for TLR-5. In yet another embodiment, the targeting unit comprises or consists of an antibody binding region with specificity for CLEC9A, such as anti-CLEC9A or a variant thereof, e.g., anti-CLEC9A Fv, or the targeting unit comprises or consists of a CLEC9 ligand, e.g., a CLEC9 ligand comprising or consisting of a nucleic acid sequence having SEQ ID NO: 115 or an amino acid sequence encoded by said nucleic acid sequence.

[0146] Preferably, the targeting unit has affinity for a chemokine receptor selected from CCR1, CCR3, CCR5 and CCR7, more preferably for a chemokine receptor selected from CCR1, CCR3 and CCR5. In one embodiment, the targeting unit has affinity for the chemokine receptor CCR7. In another embodiment, the targeting unit comprises or consists of CCL19, such as CCL19 comprising or consisting of the nucleotide sequence of SEQ ID NO: 121 or an amino acid sequence encoded by said nucleotide sequence, or CCL21, such as the human form of CCL19 or CCL21.

[0147] Preferably, the targeting unit comprises or consists of the chemokine human macrophage proinflammatory protein alpha (also called human MIP-1α (hMIP-1α) variant, LD78β or CCL3L1), which binds to its cognate receptors, including CCR1 and CCR5, expressed on the cell surface of APCs. Binding of the targeting unit to its cognate receptor results in internalization of the anti-cancer vaccine into APCs and degradation of the protein into small peptides that are loaded onto MHC molecules and presented to CD4+ and CD8+ T cells to induce a cancer-specific immune response. Once stimulated, the CD8+ T cells, with help from activated CD4+ T cells, target and kill cancer cells expressing the same antigen.

[0148] In a preferred embodiment, the targeting unit comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence 24-93 of SEQ ID NO:1, for example comprising amino acid sequence 26-93 of SEQ ID NO:1, or comprising amino acid sequence 28-93 of SEQ ID NO:1.

[0149] In a further preferred embodiment, the targeting unit comprises an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to amino acid sequence 24-93 of SEQ ID NO: 1. In an even further preferred embodiment, the targeting unit comprises amino acid sequence 24-93 of SEQ ID NO: 1.

[0150] In a more preferred embodiment, the targeting unit consists of an amino acid sequence having at least 80% sequence identity to amino acid sequence 24 to 93 of SEQ ID NO:1, for example, the targeting unit consists of amino acid sequence 26 to 93 of SEQ ID NO:1 or the targeting unit consists of amino acid sequence 28 to 93 of SEQ ID NO:1.

[0151] In a further preferred embodiment, the targeting unit consists of an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to amino acid sequence 24-93 of SEQ ID NO: 1. In yet another preferred embodiment, the targeting unit consists of amino acid sequence 24-93 of SEQ ID NO: 1.

[0152] In a preferred embodiment, the targeting unit comprises the amino acid sequence 24-93 of SEQ ID NO:1, except that up to 6 amino acids, such as up to 5 amino acids, such as up to 4 amino acids, such as up to 3 amino acids, such as up to 2 amino acids or such as up to 1 amino acid have been substituted, deleted or inserted. An embodiment of such a targeting unit is one that comprises the amino acid sequence 26-93 of SEQ ID NO:1, or one that comprises the amino acid sequence 28-93 of SEQ ID NO:1.

[0153] In another preferred embodiment, the targeting unit consists of the amino acid sequence 24-93 of SEQ ID NO:1, except that up to 6 amino acids, such as up to 5 amino acids, such as up to 4 amino acids, such as up to 3 amino acids, such as up to 2 amino acids or such as up to 1 amino acid are substituted, deleted or inserted. An embodiment of such a targeting unit is one that consists of the amino acid sequence 26-93 of SEQ ID NO:1, or one that consists of the amino acid sequence 28-93 of SEQ ID NO:1.

[0154] In a preferred embodiment, the targeting unit comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence having SEQ ID NO:5.

[0155] In a further preferred embodiment, the targeting unit comprises a nucleic acid sequence having at least 85% sequence identity, such as at least 86%, or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a nucleic acid sequence having SEQ ID NO: 5. In an even further preferred embodiment, the targeting unit comprises the nucleic acid sequence of SEQ ID NO: 5.

[0156] In a more preferred embodiment, the targeting unit consists of a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence having SEQ ID NO:5.

[0157] In a further preferred embodiment, the targeting unit consists of a nucleic acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 5. In yet another preferred embodiment, the targeting unit has the nucleic acid sequence of SEQ ID NO:5.

[0158] Multimerization Unit / Dimerization Unit The anti-cancer vaccines for use in the methods of the present invention include a multimerization unit, such as a dimerization unit.

[0159] As used herein, the term "multimerization unit" refers to the sequence of nucleotides or amino acids between the antigen unit and the targeting unit. In addition to linking the antigen unit and the targeting unit, the multimerization unit facilitates / links the multimerization of multiple polypeptides (e.g., two, three, four or more polypeptides) into a multimeric protein (e.g., a dimeric protein, a trimeric protein or a tetrameric protein). Furthermore, the multimerization unit also provides flexibility to the multimeric protein, allowing optimal binding of the targeting unit to surface molecules on the APC, even if they are located at variable distances. The multimerization unit can be any unit that meets one or more of these requirements.

[0160] Multimerization units that promote / link multimerization of more than two polypeptides In one embodiment, the multimerization unit is a trimerization unit, such as a collagen-derived trimerization unit, such as a human collagen-derived trimerization domain, such as a human collagen-derived XVIII trimerization domain (see, for example, A. Alvarez-Cienfuegos et al., Sci Rep 6, 28643 (2016)) or a human collagen XV trimerization domain. Thus, in one embodiment, the multimerization unit is a trimerization unit that comprises or consists of a nucleic acid sequence having SEQ ID NO: 116, or an amino acid sequence encoded by said nucleic acid sequence. In another embodiment, the trimerization unit is the C-terminal domain of T4 fibritin. Thus, in one embodiment, the multimerization unit is a trimerization unit that comprises or consists of an amino acid sequence having SEQ ID NO: 117.

[0161] In another embodiment, the multimerization unit is a tetramerization unit, such as a domain derived from p53, optionally further comprising a hinge region as described below. Thus, in one embodiment, the multimerization unit is a tetramerization unit comprising or consisting of a nucleic acid sequence having SEQ ID NO: 113, or an amino acid sequence encoded by said nucleic acid sequence, optionally further comprising a hinge region as described below.

[0162] Dimerization Unit As used herein, the term "dimerization unit" refers to the sequence of nucleotides or amino acids between the antigen unit and the targeting unit. In addition to linking the antigen unit and the targeting unit, the dimerization unit promotes / links two monomeric polypeptides to dimerize into a dimeric protein. Furthermore, the dimerization unit also provides flexibility to the dimeric protein to allow optimal binding of the targeting unit to surface molecules on the APC, even if they are located at variable distances. The dimerization unit can be any unit that meets one or more of these requirements.

[0163] Thus, in one embodiment, the anti-cancer vaccine for use in the method of the present invention comprises a dimerization unit comprising a hinge region. In another embodiment, the dimerization unit comprises a hinge region and another domain that promotes dimerization. In yet another embodiment, the dimerization unit comprises a hinge region, a dimerization unit linker, and another domain that promotes dimerization, the dimerization unit linker being located between the hinge region and the other domain that promotes dimerization. In one embodiment, the dimerization unit linker is a glycine-serine rich linker, preferably GGSSGGGSG (SEQ ID NO: 118), i.e., the dimerization unit comprises a glycine-serine rich dimerization unit linker, preferably the dimerization unit linker GGSSGGGSG (SEQ ID NO: 118).

[0164] The term "hinge region" refers to an amino acid sequence contained in a dimerization unit that contributes to the binding of two polypeptides, i.e., to the formation of a dimeric protein. In the context of a multimerization unit that promotes / links more than two polypeptides, the term "hinge region" refers to an amino acid sequence contained in such a multimerization unit that contributes to the linking of more than two polypeptides, for example, three or four polypeptides, and / or functions as a flexible spacer, allowing the targeting unit of the multimeric protein to simultaneously bind to multiple surface molecules on an APC, even if these surface molecules are located at variable distances.

[0165] Moreover, the hinge region functions as a flexible spacer, allowing the two targeting units of the dimeric protein to simultaneously bind to two surface molecules on the APC, even if they are located at variable distances. The hinge region can be from Ig, such as from IgG, e.g., IgG2 or IgG3. In one embodiment, the hinge region is from IgM, and comprises or consists of, e.g., a nucleotide sequence having SEQ ID NO: 119 or an amino acid sequence encoded by said nucleic acid sequence. The hinge region can contribute to dimerization through the formation of a covalent bond, e.g., a disulfide bridge between cysteine ​​residues. Thus, in one embodiment, the hinge region has the ability to form one or more covalent bonds. Preferably, the covalent bond is a disulfide bridge.

[0166] In one embodiment, the dimerization unit comprises or consists of hinge exon h1 and hinge exon h4 (human hinge region 1 and human hinge region 4) having an amino acid sequence having at least 80% sequence identity to amino acid sequence 94 to 120 of SEQ ID NO:1.

[0167] In a preferred embodiment, the dimerization unit comprises or consists of hinge exon h1 and hinge exon h4 having an amino acid sequence having at least 85% sequence identity to amino acid sequence 94-120 of SEQ ID NO:1, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98% or such as at least 99% sequence identity.

[0168] In a preferred embodiment, the dimerization unit comprises or consists of hinge exon h1 and hinge exon h4 having the amino acid sequence 94 to 120 of SEQ ID NO:1.

[0169] In a preferred embodiment, the dimerization unit comprises or consists of the amino acid sequence 94-120 of SEQ ID NO:1, except that up to four amino acids, such as up to three amino acids, such as up to two amino acids, or for example up to one amino acid, have been substituted, deleted or inserted.

[0170] In a preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence having SEQ ID NO:8.

[0171] In a further preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 85% sequence identity to a nucleic acid sequence having SEQ ID NO:8, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.

[0172] In an even more preferred embodiment, the dimerization unit comprises or consists of the nucleic acid sequence of SEQ ID NO:8.

[0173] In another embodiment, the dimerization unit comprises another domain that promotes dimerization. Preferably, the other domain is an immunoglobulin domain, such as an immunoglobulin constant domain (C domain), such as a CH1 domain, a CH2 domain or a carboxy-terminal C domain (i.e., a CH3 domain), or a sequence that is substantially identical to such a C domain or a variant thereof. Preferably, the other domain that promotes dimerization is a carboxy-terminal C domain derived from an IgG. More preferably, the other domain that promotes dimerization is a carboxy-terminal C domain derived from an IgG3.

[0174] In one embodiment, the dimerization unit comprises or consists of a carboxy-terminal C domain derived from an IgG3 having an amino acid sequence having at least 80% sequence identity to amino acids 131 to 237 of SEQ ID NO:1.

[0175] In a preferred embodiment, the dimerization unit comprises or consists of a carboxy-terminal C domain derived from IgG3 having an amino acid sequence having at least 85% sequence identity to the amino acid sequence 131 to 237 of SEQ ID NO: 1, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98% or such as at least 99% sequence identity.

[0176] In a preferred embodiment, the dimerization unit comprises or consists of a carboxy-terminal C domain derived from IgG3 having the amino acid sequence 131-237 of SEQ ID NO:1.

[0177] In a preferred embodiment, the dimerization unit comprises or consists of the amino acid sequence 131-237 of SEQ ID NO:1, except that up to 16 amino acids, such as up to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, have been substituted, deleted or inserted.

[0178] In a preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence having SEQ ID NO:10.

[0179] In a further preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a nucleic acid sequence having SEQ ID NO: 10. In an even further preferred embodiment, the dimerization unit comprises or consists of the nucleic acid sequence of SEQ ID NO: 10.

[0180] Immunoglobulin domains contribute to dimerization through non-covalent interactions, such as hydrophobic interactions. Thus, in one embodiment, an immunoglobulin domain has the ability to form a dimer through non-covalent interactions. Preferably, the non-covalent interactions are hydrophobic interactions.

[0181] It is preferred that if a dimerization unit comprises a CH3 domain, it does not comprise a CH2 domain, and vice versa.

[0182] In a preferred embodiment, the dimerization unit comprises a hinge exon h1, a hinge exon h4, a dimerization unit linker and a CH3 domain of human IgG3. In a further preferred embodiment, the dimerization unit comprises a polypeptide consisting of a hinge exon h1, a hinge exon h4, a dimerization unit linker and a CH3 domain of human IgG3.

[0183] In another preferred embodiment, the dimerization unit consists of a polypeptide consisting of hinge exon h1, hinge exon h4, a dimerization unit linker and the CH3 domain of human IgG3.

[0184] In one embodiment, the dimerization unit comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence 94-237 of SEQ ID NO:1.

[0185] In a preferred embodiment, the dimerization unit comprises an amino acid sequence having at least 85% sequence identity to amino acid sequence 94-237 of SEQ ID NO:1, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98% or such as at least 99% sequence identity.

[0186] In a more preferred embodiment, the dimerization unit consists of an amino acid sequence having at least 80%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, for example at least 97%, for example at least 98% or such as at least 99% sequence identity to the amino acid sequence 94 to 237 of SEQ ID NO:1.

[0187] In an even more preferred embodiment, the dimerization unit consists of amino acids 94 to 237 of SEQ ID NO:1.

[0188] In a preferred embodiment, the dimerization unit comprises the amino acid sequence 94-237 of SEQ ID NO:1, except that up to 22 amino acids, such as up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, have been substituted, deleted or inserted.

[0189] In a preferred embodiment, the dimerization unit consists of the amino acid sequence 94-237 of SEQ ID NO:1, except that up to 22 amino acids, for example up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, are substituted, deleted, or inserted.

[0190] In a preferred embodiment, the dimerization unit comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence having SEQ ID NO:12.

[0191] In a further preferred embodiment, the dimerization unit comprises a nucleic acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a nucleic acid sequence having SEQ ID NO: 12. In an even further preferred embodiment, the dimerization unit comprises the nucleic acid sequence of SEQ ID NO: 12.

[0192] In another preferred embodiment, the dimerization unit consists of a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence having SEQ ID NO:12.

[0193] In a further preferred embodiment, the dimerization unit consists of a nucleic acid sequence having at least 85% sequence identity to a nucleic acid sequence having SEQ ID NO: 12, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.

[0194] In an even more preferred embodiment, the dimerization unit has the nucleic acid sequence of SEQ ID NO:12.

[0195] In one embodiment, the dimerization unit comprises or consists of a dHLX protein, for example a dHLX protein comprising or consisting of a nucleic acid sequence having SEQ ID NO: 120 or an amino acid sequence encoded by said nucleic acid sequence.

[0196] In the polypeptide encoded by the nucleotide sequence of the anti-cancer vaccine for use in the method of the present invention, the multimerization unit, for example the dimerization unit, can have any orientation with respect to the antigen unit and the targeting unit. In one embodiment, the antigen unit is linked to the C-terminal end of the multimerization / dimerization unit (e.g., via a unit linker), and the targeting unit is linked to the N-terminal end of the multimerization / dimerization unit. In another embodiment, the antigen unit is linked to the N-terminal end of the multimerization / dimerization unit (e.g., via a unit linker), and the targeting unit is linked to the C-terminal end of the dimerization unit. It is preferred that the antigen unit is linked to the C-terminal end of the multimerization / dimerization unit, preferably via a unit linker, and the targeting unit is linked to the N-terminus of the multimerization / dimerization unit.

[0197] Signal peptide In one embodiment, the anti-cancer vaccine for use in the method of the present invention is a polynucleotide further comprising a nucleotide sequence encoding a signal peptide.The signal peptide is located at either the N-terminal end of the targeting unit or the C-terminal end of the targeting unit, depending on the orientation of the targeting unit in the polypeptide.The signal peptide is designed to allow the secretion of the polypeptide encoded by the polynucleotide in the cell transfected with the polynucleotide.

[0198] Any suitable signal peptide can be used. Examples of suitable peptides are Ig VH signal peptides, such as SEQ ID NO:2, human TPA signal peptides, such as SEQ ID NO:3, and human MIP-1α signal peptide.

[0199] In one embodiment, the anti-cancer vaccine for use in the methods of the present invention is a polynucleotide comprising a nucleotide sequence encoding a signal peptide comprising an amino acid sequence having at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98% or such as at least 99% sequence identity to amino acid sequence 1-23 of SEQ ID NO:1.

[0200] In another preferred embodiment, the anti-cancer vaccine for use in the methods of the present invention is a polynucleotide comprising a nucleotide sequence encoding a signal peptide comprising amino acid sequence 1 to 23 of SEQ ID NO:1, except that up to four amino acids, such as up to three amino acids, such as up to two amino acids or for example up to one amino acid, have been substituted, deleted or inserted.

[0201] In another preferred embodiment, the anti-cancer vaccine for use in the methods of the present invention is a polynucleotide comprising a nucleotide sequence encoding a signal peptide comprising amino acid sequence 1 to 23 of SEQ ID NO:1.

[0202] In a more preferred embodiment, the anti-cancer vaccine for use in the method of the present invention is a polynucleotide comprising a nucleotide sequence encoding a signal peptide consisting of an amino acid sequence having at least 80% sequence identity to amino acid sequence 1 to 23 of SEQ ID NO:1, preferably at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98% or such as at least 99% sequence identity.

[0203] In another preferred embodiment, the anti-cancer vaccine for use in the methods of the present invention is a polynucleotide comprising a nucleotide sequence encoding a signal peptide consisting of amino acid sequence 1 to 23 of SEQ ID NO:1, except that up to four amino acids, such as up to three amino acids, such as up to two amino acids, or for example up to one amino acid, are substituted, deleted or inserted.

[0204] In another preferred embodiment, the anti-cancer vaccine for use in the method of the present invention is a polynucleotide comprising a nucleotide sequence encoding a signal peptide having amino acid sequence 1 to 23 of SEQ ID NO:1.

[0205] In a preferred embodiment, the anti-cancer vaccine for use in the methods of the present invention is a polynucleotide comprising a nucleotide sequence encoding a signal peptide, the nucleotide sequence having at least 80% sequence identity to a nucleic acid sequence having SEQ ID NO:4.

[0206] In a further preferred embodiment, the anti-cancer vaccine for use in the methods of the present invention is a polynucleotide comprising a nucleotide sequence encoding a signal peptide, the nucleotide sequence having at least 85% sequence identity, such as at least 86%, or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the nucleic acid sequence having SEQ ID NO:4.

[0207] In an even more preferred embodiment, the anti-cancer vaccine for use in the methods of the present invention is a polynucleotide comprising a nucleotide sequence encoding a signal peptide, wherein the nucleotide sequence is SEQ ID NO:4.

[0208] Sequence identity Sequence identity can be determined as follows: a high level of sequence identity indicates the likelihood that the second sequence is derived from the first sequence. Amino acid sequence identity requires identical amino acid sequences between two aligned sequences. Thus, 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 can be determined with the aid of computer analysis, such as, but not limited to, 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 proposed therein. This program is used with its default settings to align the mature (biologically active) portions of the query and reference polypeptides. The number of completely conserved residues is counted and divided by the length of the reference polypeptide. By doing so, any tag or fusion protein sequences that form part of the query sequence are ignored in the alignment and subsequent sequence identity determination.

[0209] The ClustalW algorithm can similarly be used to align nucleotide sequences. Sequence identity can be calculated in a similar manner as shown for amino acid sequences.

[0210] Another preferred mathematical algorithm used for comparing sequences 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 a global alignment. Align0 does not penalize gaps at the ends of the sequences. When utilizing the ALIGN and Align0 programs to compare amino acid sequences, the BLOSUM50 substitution matrix with gap opening / extension penalties of -12 / -2 is preferably used.

[0211] Amino acid sequence variants can be prepared by introducing appropriate changes into the nucleotide sequence encoding the anti-cancer vaccine or by peptide synthesis. Such modifications include, for example, deletion from and / or insertion into and / or substitution of residues within the amino acid sequence. When used herein with respect to amino acid sequences and sequence identity, the terms substituted / substitution, deleted / deletion and inserted / inserted are well known and clear to those skilled in the art. Any combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final construct has the desired characteristics. For example, deletion, insertion or substitution of amino acid residues may produce a silent change and may produce a functionally equivalent peptide / polypeptide.

[0212] Deliberate amino acid substitutions can be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues, so long as the secondary binding activity of the agent is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0213] Included herein are conservative substitutions, i.e., homogeneous substitutions, such as basic for basic, acidic for acidic, polar for polar, etc., and non-conservative substitutions, i.e., substitutions of one class of residue for another class of residue, or substitutions involving the inclusion of unnatural amino acids, such as ornithine, diaminobutyric acid ornithine, norleucine, ornithine, pyriylalanine, thienylalanine, naphthylalanine, and phenylglycine. Conservative substitutions that may be made are, for example, within the groups of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), aliphatic amino acids (alanine, aline, leucine, isoleucine), polar amino acids (glutamine, asparagine, serine, threonine), aromatic amino acids (phenylalanine, tryptophan, tyrosine), hydroxyl amino acids (serine, threonine), large amino acids (phenylalanine, tryptophan), and small amino acids (glycine, alanine).

[0214] Substitutions may also be made with non-natural amino acids, the replacement residues including: alpha * and alpha-disubstituted * Amino acids, N-alkyl amino acids * , lactic acid * , halide derivatives of natural amino acids, e.g. trifluorotyrosine * , p-CI-phenylalanine * , p-Br-phenylalanine * , pI-phenylalanine* , L-allylglycine * , β-alanine * , La-aminobutyric acid * , Ly-aminobutyric acid * , La-aminoisobutyric acid * , Le-aminocaproic acid * , 7-aminoheptanoic acid * , L-methionine sulfone * , L-norleucine * , L-Norvaline * , p-nitro-L-phenylalanine * , L-hydroxyproline * , L-thioproline * , methyl derivatives of phenylalanine (Phe), such as 4-methylPhe * , Pentamethyl-Phe * , L-Phe(4-amino)#, L-Tyr(methyl) * , L-Phe(4-isopropyl) * , L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) * , L-diaminopropionic acid * and L-Phe(4-benzyl) * .

[0215] In the above paragraph, * indicates the hydrophobicity of the substituted residue, while # indicates the hydrophilicity of the substituted residue, * indicates the amphipathic nature of the substituted residue. The variant amino acid sequence may include an amino acid spacer, for example a suitable spacer group that may be inserted between any two amino acid residues of the sequence, including alkyl groups such as methyl, ethyl or propyl groups, in addition to glycine or β-alanine residues. A further form of variation involves the presence of one or more amino acid residues in peptoid form.

[0216] Polynucleotides The anti-cancer vaccine for use in the methods of the present invention can be in the form of a polynucleotide, such as DNA or RNA (either double-stranded or single-stranded), including genomic DNA, cDNA, and mRNA. In one embodiment, the anti-cancer vaccine is a DNA vaccine, i.e., the polynucleotide is DNA.

[0217] In one embodiment, the polynucleotide is optimized for the species of subject to which it will be administered. For administration to humans, in one embodiment the polynucleotide sequence is human codon optimized.

[0218] VB10.NEO In one embodiment, the anti-cancer vaccine is a personalized DNA anti-cancer vaccine comprising VB10.NEO, a DNA polynucleotide comprising a nucleotide sequence encoding a signal peptide, a human MIP-1α targeting unit targeting an antigen-presenting cell, a human IgG3 dimerization unit, a glycine-rich linker unit, and an antigen unit comprising one or more neoepitopes. When the antigen unit comprises more than one neoepitopes, the neoepitopes are separated by a linker. A description of VB10.NEO can be found, for example, in International Application No. PCT / EP2017 / 050206, filed January 5, 2017, and published as WO 2017 / 118695(A1).

[0219] VB10.NEO consists of a constant portion including a signal peptide, a targeting unit that targets antigen presenting cells, a dimerization unit, and a unit linker, an antigenic unit, and a variable portion that is specifically designed for each patient to whom VB10.NEO is administered and that includes one or more neoepitopes identified in the tumor of the patient to be treated with VB10.NEO. Thus, the antigenic unit is specifically and only designed for the patient to be treated or to be treated with the vaccine and includes one or more cancer antigens that are patient-specific antigens or parts thereof, such antigens including neoantigens or shared cancer antigens present in the patient. After patient-specific, tumor-specific mutations are identified, an antigenic peptide that includes a neoepitope is selected and a nucleic acid encoding the neoepitope is included in the antigenic unit of VB10.NEO.

[0220] VB10.NEO comprises a nucleotide sequence encoding a signal peptide. The signal peptide is human MIP-1α signal peptide, and VB10.NEO comprises a nucleotide sequence encoding amino acids 1 to 23 of SEQ ID NO: 1. VB10.NEO comprises a signal peptide consisting of the nucleic acid sequence of SEQ ID NO: 4.

[0221] VB10.NEO comprises a nucleotide sequence encoding a targeting unit for human MIP-1α (LD78β, CCL3L1), i.e. comprising or consisting of amino acids 24 to 93 of SEQ ID NO: 1. VB10.NEO comprises a human MIP-1α targeting unit consisting of the nucleic acid sequence of SEQ ID NO:5.

[0222] VB10.NEO comprises a nucleotide sequence encoding a dimerization unit comprising hinge exon h1, hinge exon h4, a dimerization unit linker and the CH3 domain of human IgG3. VB10.NEO comprises a nucleotide sequence encoding a dimerization unit comprising or consisting of amino acid sequence 94-237 of SEQ ID NO: 1. VB10.NEO comprises a dimerization unit having the nucleic acid sequence of SEQ ID NO: 12.

[0223] VB10.NEO comprises a nucleotide sequence encoding a unit linker as described herein, for example, as described under the heading "Unit Linkers". VB10.NEO comprises a nucleotide sequence encoding a unit linker that comprises, or consists of, GLGGL (SEQ ID NO:52) (the nucleotide sequence having SEQ ID NO:10) or GLSGL (SEQ ID NO:109).

[0224] VB10.NEO comprises a nucleotide sequence encoding a polypeptide consisting of amino acid sequence 1-242 of SEQ ID NO: 1, i.e., the constant portion of VB10.NEO consists of a nucleotide sequence encoding a polypeptide consisting of amino acid sequence 1-242 of SEQ ID NO: 1. VB10.NEO comprises the nucleic acid sequence of SEQ ID NO: 23, i.e., the constant portion of VB10.NEO consists of the nucleic acid sequence of SEQ ID NO: 23.

[0225] In one embodiment, VB10.NEO is comprised in a vector suitable for expression of the polypeptide or dimeric protein encoded by the polynucleotide, i.e., an expression vector, e.g., a DNA plasmid. The term "VB10.NEO" may be used for said DNA polynucleotide, but may also be used for an expression vector, e.g., a DNA plasmid, that comprises said DNA polynucleotide.

[0226] Polypeptides and multimeric / dimeric proteins The anti-cancer vaccine for use in the methods of the invention may be in the form of a polypeptide encoded by a polynucleotide as described above. The polypeptide may be expressed in vitro for production of the anti-cancer vaccine, or the polypeptide may be expressed in vivo as a result of administration of the polynucleotide to a subject, as described above.

[0227] The presence of the multimerization / dimerization unit results in the formation of a multimeric / dimeric protein when the polypeptide is expressed.

[0228] The multimeric / dimeric protein may be a homomultimer or a heteromultimer, for example, if the protein is a dimeric protein, the dimeric protein may be a homodimer, i.e., a dimeric protein in which the two polypeptide chains are identical and therefore contain identical units and therefore antigenic sequences, or the dimeric protein may be a heterodimer comprising two polypeptide chains, where polypeptide chain 1 contains a different antigenic sequence in its antigenic unit than polypeptide chain 2. The latter may be relevant when the number of antigens to be included in an antigenic unit exceeds the upper size limit of the antigenic unit. Preferably, the dimeric protein is a homodimeric protein.

[0229] vector The polynucleotide sequence of the anti-cancer vaccine may be a DNA polynucleotide contained in a vector, i.e. an expression vector, e.g. a DNA plasmid, suitable for transfecting a host cell and expressing the polypeptide or multimeric / dimeric protein encoded by the polynucleotide. In another embodiment, the vector is suitable for transfecting a host cell and expressing the mRNA encoding the polypeptide / multimeric protein.

[0230] In one embodiment, the vector allows for easy exchange of the various units mentioned above, particularly the antigenic unit in the case of personalized anti-cancer vaccines.

[0231] In one embodiment, the vector is a pUMVC4a vector or a vector comprising the NTC9385R vector backbone. The antigen unit may be exchanged with an antigen unit cassette restricted by an SfiI restriction enzyme cassette, the 5' site incorporates a nucleotide sequence encoding the GLGGL (SEQ ID NO:52) / GLSGL (SEQ ID NO:109) unit linker, and the 3' site is included after the stop codon in the vector.

[0232] Accordingly, the present invention provides a method for treating a subject (e.g., a patient) with cancer, comprising: (a) an anti-cancer vaccine comprising a vector comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, e.g., a dimerization unit, and an antigen unit that comprises one or more cancer antigens; (b) administering to the subject one or more checkpoint inhibitors.

[0233] In one embodiment, the invention provides a method for treating a subject (e.g., a patient) having cancer, comprising: (a) an anti-cancer vaccine comprising a vector comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, and an antigen unit that includes one or more cancer antigens; (b) administering to the subject one or more checkpoint inhibitors.

[0234] In another embodiment, the invention provides a method for treating a subject (e.g., a patient) having cancer, comprising: (a) an anti-cancer vaccine comprising a vector comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a dimerization unit, and an antigen unit comprising one or more cancer antigens; (b) administering to the subject one or more checkpoint inhibitors.

[0235] In a preferred embodiment, the vector is a DNA plasmid and the polynucleotide is DNA.

[0236] vaccine Anti-cancer vaccines for use in the methods of the present invention may include a pharma- ceutically acceptable carrier or diluent, including, but not limited to, saline, buffered saline such as PBS, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer, and combinations thereof.

[0237] In one embodiment, the pharma- ceutically acceptable carrier or diluent is an aqueous buffer solution. In another embodiment, the aqueous buffer solution is Tyrode's buffer, e.g., Tyrode's buffer containing 140 mM NaCl, 6 mM KCl, 3 mM CaCl2, 2 mM MgCl2, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) pH 7.4, and 10 mM glucose.

[0238] The anti-cancer vaccine may further comprise an adjuvant. In particular, for anti-cancer vaccines comprising polypeptides / proteins, pharma- ceutically acceptable adjuvants include poly-ICLC, 1018 ISS, aluminum salts, 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 50 V, Montanide These include, but are not limited to, ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, vadimezan, and / or AsA404 (DMXAA). However, as detailed above, due to the presence of the targeting unit, the anti-cancer vaccine for use in the method of the present invention can be administered without additional adjuvants, and thus in some embodiments, the anti-cancer vaccine does not include an adjuvant.

[0239] In some particular embodiments, the anti-cancer vaccine may comprise a pharma- ceutically acceptable amphiphilic block copolymer comprising blocks of poly(ethylene oxide) and poly(propylene oxide).

[0240] As used herein, an "amphiphilic block copolymer" is a linear or branched copolymer that comprises or consists of blocks of poly(ethylene oxide) ("PEO") and blocks of poly(propylene oxide) ("PPO"). Typical examples of useful PEO-PPO amphiphilic block copolymers have the general structures PEO-PPO-PEO (poloxamer), PPO PEO PPO, (PEO PPO-)4ED (poloxamine), and (PPO PEO-)4ED (reverse poloxamine), where "ED" is an ethylenediaminyl group.

[0241] "Poloxamer" refers to a linear amphiphilic block copolymer composed of one block of poly(ethylene oxide) bonded to one block of poly(propylene oxide) bonded to one block of PEO, i.e., a structure of the formula EOa-poB-EOa, where EO is ethylene oxide, PO is propylene oxide, a is an integer between 2 and 130, and b is an integer between 15 and 67. Poloxamers are usually named by using a three-digit identifier, where the first two digits are multiplied by 100 to give the approximate molecular weight of the PPO content, and the last digit is multiplied by 10 to indicate the approximate percentage of PEO content. For example, "Poloxamer 188" refers to a polymer containing a PPO block of about 1800 molecular weight (corresponding to b being about 31 PPO) and about 80% (w / w) PEO (corresponding to a being about 82). However, values ​​are known to vary to some extent, and commercial products such as research grade Lutrol® F68 and clinical grade Kolliphor® P188, both of which are Poloxamer 188 according to the manufacturer's datasheet, show a large variation in molecular weight (7,680-9,510), with the values ​​of a and b provided for these particular products being approximately 79 and 28, respectively. This reflects the heterogeneous nature of the block copolymers, and means that the values ​​of a and b are averages found in the final formulation.

[0242] "Poloxamine" or "sequential poloxamine" (sold under the name Tetronic®) is an X-shaped block copolymer having four PEO-PPO arms linked to a central ethylenediamine moiety via bonds between the free OH groups in the PEO-PPO arms and primary amine groups in the ethylenediamine moiety. Reverse poloxamine is similarly an X-shaped block copolymer having four PPO-PEO arms linked to a central ethylenediamine moiety via bonds between the free OH groups in the PPO-PEO arms and primary amine groups in the ethylenediamine moiety.

[0243] Preferred amphiphilic block copolymers are poloxamers or poloxamines. Poloxamers 407 and 188 are preferred, especially poloxamer 188. Preferred poloxamines are sequential poloxamines of formula (PEO-PPO)4-ED. Particularly preferred poloxamines are those commercially available under the registered trademarks Tetronic® 904, 704, and 304, respectively. The characteristics of these poloxamines are as follows: Tetronic® 904 has a total average molecular weight of 6700, a total average weight of PPO units of 4020, and a PEO percentage of about 40%. Tetronic® 704 has a total average molecular weight of 5500, a total average weight of PPO units of 3300, and a PEO percentage of about 40%. Tetronic® 304 has a total average molecular weight of 1650, a total average weight of PPO units of 990, and a PEO percentage of about 40%.

[0244] In one embodiment, the anti-cancer vaccine comprises an amphiphilic block copolymer in an amount of from 0.2% w / v to 20% w / v, such as from 0.2% w / v to 18% w / v, from 0.2% w / v to 16% w / v, from 0.2% w / v to 14% w / v, from 0.2% w / v to 12% w / v, from 0.2% w / v to 10% w / v, from 0.2% w / v to 8% The anti-cancer vaccine may comprise an amount of amphiphilic block copolymer of 2% w / v to 5% w / v, 0.2% w / v to 6% w / v, 0.2% w / v to 4% w / v, 0.4% w / v to 18% w / v, 0.6% w / v to 18% w / v, 0.8% w / v to 18% w / v, 1% w / v to 18% w / v, 2% w / v to 18% w / v, 1% w / v to 5% w / v, or 2% w / v to 4% w / v. An amount in the range of 0.5% w / v to 5% w / v is particularly preferred. In another embodiment, the anti-cancer vaccine comprises the amphiphilic block copolymer in an amount of 2% w / v to 5% w / v, for example about 3% w / v.

[0245] For anti-cancer vaccines that include a polynucleotide, the vaccine may further include a molecule that facilitates transfection of cells.

[0246] The anti-cancer vaccines may be formulated in any manner suitable for administration to a subject, e.g., a patient, such as a liquid formulation for injection, e.g., intradermal or intramuscular injection.

[0247] Anti-cancer vaccines for use in the methods of the present invention may be administered in any manner suitable for administration of either polypeptide / protein or polynucleotide vaccines to a subject, e.g., a patient, such as by intradermal, intramuscular, intranodal or subcutaneous injection, or by mucosal or epithelial application (e.g., intranasal, oral, enteral or intravesical (to the bladder) administration).

[0248] In a preferred embodiment, the anti-cancer vaccine comprises a polynucleotide as described herein, preferably a polynucleotide, and a pharma- ceutically acceptable carrier, and is administered by intramuscular or intradermal injection.

[0249] Anti-cancer vaccines for use in the methods of the present invention typically contain in the range of 0.3 mg to 6 mg, for example about 2 mg of polynucleotide, and in the range of 5 μg to 5 mg of polypeptide / protein.

[0250] Methods for preparing anti-cancer vaccines for use in the methods of the present invention Suitable methods are described in WO 2004 / 076489(A1), WO 2011 / 161244(A1), WO 2013 / 092875(A1), WO 2017 / 118695(A1) and WO 2021 / 205027(A1), in particular the section "Construction of Vaccibodies" on page 15, lines 10-13 and on page 17 of WO 2004 / 076489(A1), on page 10, lines 10-14 and Example 1 of WO 2011 / 161244(A1), on page 15 and Example 1 of WO 2013 / 092875(A1), and in the section "Methods for preparing the These are disclosed in the "vaccine" section and Example 1, as well as page 26, line 17 to page 27, line 38, page 30, line 23 to page 31, line 27, and Example 4 of No. 2021 / 205027(A1).

[0251] Checkpoint inhibitors The methods of the invention include administration of one or more checkpoint inhibitors. Checkpoint inhibitors suitable for use in the methods provided herein include antibodies, such as monoclonal antibodies, that target PD-1, PD-L1, CTLA-4, or TIGIT.

[0252] Exemplary anti-PD1 inhibitors, such as anti-PD1 antibodies, include, but are not limited to, pembrolizumab, nivolumab, and cemiplimab. Exemplary anti-PD-L1 inhibitors, such as anti-PD-L1 antibodies, include, for example, atezolizumab, avelumab, and durvalumab. Exemplary anti-CTLA-4 inhibitors, such as anti-CTLA-4 antibodies, include, for example, ipilimumab or tremelimumab. Exemplary anti-TIGIT inhibitors, such as anti-TIGIT antibodies, include, for example, tiragolumab, BMS-986207, ociperlimab, COM902, AB154, EOS884448, IBI939, SGNTNT, and etigilimab.

[0253] Preferably, the checkpoint inhibitor is selected from the group consisting of an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-CTLA-4 antibody, and an anti-PD1 antibody.

[0254] In one embodiment, the checkpoint inhibitor is an anti-PD-L1 antibody, for example, atezolizumab.

[0255] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence and that determine antigen-binding specificity, e.g., the "complementarity determining regions" ("CDRs"). Generally, antibodies contain six CDRs: three in the heavy chain variable region (VH) (CDR-H1, CDR-H2, CDR-H3) and three in the light chain variable region (VL) (CDR-L1, CDR-L2, CDR-L3).

[0256] In some embodiments, the anti-PD-L1 antibody is (a) the heavy chain amino acid sequence of SEQ ID NO: 13, and (b) atezolizumab comprising a light chain amino acid sequence of SEQ ID NO: 14.

[0257] In some embodiments, the anti-PD-L1 antibody is (a) the HVR-H1, HVR-H2, and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 15), AWISPYGGSTYYADSVKG (SEQ ID NO: 16), and RHWPGGFDY (SEQ ID NO: 17), respectively; and (b) comprising the HVR-L1, HVR-L2, and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 18), SASFLYS (SEQ ID NO: 19), and QQYLYHPAT (SEQ ID NO: 20), respectively.

[0258] The HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 sequences of atezolizumab are SEQ ID NOs: 15 to 20, respectively.

[0259] In some embodiments, the anti-PD-L1 antibody is (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 21; and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 22.

[0260] The VH of atezolizumab comprises SEQ ID NO:21 and the VL of atezolizumab comprises SEQ ID NO:22.

[0261] Atezolizumab has been approved in many countries as a monotherapy for the treatment of certain cancers. For example, atezolizumab has been approved in the United States or Europe for the following indications: treatment of adult patients with locally advanced or metastatic urothelial carcinoma (UC) after prior platinum-containing chemotherapy, or treatment of adult patients who are deemed cisplatin-ineligible and whose tumors have ≥ 5% PD-L1 expression, treatment of adult patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) after prior chemotherapy, treatment of patients with locally advanced or metastatic UC who are ineligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 (PD-L1 staining IC covers ≥ 5% of the tumor area) or who are ineligible for any platinum-containing chemotherapy regardless of the level of tumor PD-L1 expression, or who have disease progression during or after any platinum-containing chemotherapy or within 12 months of neoadjuvant or adjuvant chemotherapy, and treatment of patients with metastatic NSCLC who have disease progression during or after platinum-containing chemotherapy. Atezolizumab is also being developed as a monotherapy and in combination with other targeted and cytotoxic agents for the treatment of patients with multiple solid and hematological tumors, including lung, renal, colorectal, and breast cancer.

[0262] All currently approved indications for atezolizumab are approved at a dose of 1200 mg given as an intravenous (IV) infusion every three weeks (Q3W) until disease progression or unacceptable toxicity.

[0263] In another embodiment, the checkpoint inhibitor is an anti-TIGIT antibody. In yet another embodiment, the checkpoint inhibitor is an anti-CTLA-4 antibody. In yet another embodiment, the checkpoint inhibitor is an anti-PD 1 antibody.

[0264] In one embodiment, the methods of the invention comprise administration of two checkpoint inhibitors, for example administration of an anti-PD-L1 antibody and an anti-TIGIT antibody, or administration of an anti-PD-L1 antibody and an anti-CTLA-4 antibody, or administration of an anti-PD-1 antibody and an anti-TIGIT antibody, or administration of an anti-PD-1 antibody and an anti-CTLA-4 antibody, or administration of an anti-TIGIT antibody and an anti-CTLA-4 antibody, or administration of an anti-PD-L1 antibody and an anti-PD-1 antibody.

[0265] In another embodiment, the methods of the invention include administration of three checkpoint inhibitors, for example administration of an anti-PD-L1 antibody, an anti-PD-1 antibody and either an anti-CTLA-4 antibody or an anti-TIGIT antibody, or administration of an anti-PD-L1 antibody, an anti-CTLA-4 antibody and an anti-TIGIT antibody, or administration of an anti-PD-1 antibody, an anti-CTLA-4 antibody and an anti-TIGIT antibody.

[0266] In yet another embodiment, the methods of the invention include administration of four checkpoint inhibitors, for example, an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CTLA-4 antibody and an anti-TIGIT antibody.

[0267] In a preferred embodiment, the method of the invention comprises administration of an anti-TIGIT antibody or an anti-PD-L1 antibody or an anti-CTLA-4 antibody. In another preferred embodiment, the method of the invention comprises administration of an anti-TIGIT antibody and an anti-PD-L1 antibody, or an anti-CTLA-4 antibody and an anti-PD-L1 antibody.

[0268] In one embodiment, the checkpoint inhibitor, e.g., each checkpoint inhibitor, is contained in a composition suitable for injection. Such a composition may be a liquid composition suitable for injection, e.g., infusion injection, including, for example, sterile water, saline, e.g., isotonic saline, sugar solutions for intravenous administration, e.g., dextrose solutions, electrolyte solutions for intravenous administration, e.g., lactated Ringer's solution or acetate Ringer's solution, or aqueous buffer solutions.

[0269] In another embodiment, several checkpoint inhibitors, for example two, three or four checkpoint inhibitors, are included in a composition suitable for injection, for example a liquid composition suitable for injection.

[0270] The composition may comprise a checkpoint inhibitor or a number of checkpoint inhibitors, the checkpoint inhibitors being administered at a dose of about 0.1 mg / kg to about 1 mg / kg, e.g., about 0.2 mg / kg to about 0.9 mg / kg, about 0.3 mg / kg to about 0.8 mg / kg, about 0.4 mg / kg to about 0.7 mg / kg, about 0.5 mg / kg to about 0.6 mg / kg, or about 1 mg / kg to about 1000 mg / kg, e.g., about 2 mg / kg to about 900 mg / kg, about 3 mg / kg to about 800 mg / kg, about 4 mg / kg to about 900 mg / kg, about 5 mg / kg to about 1000 mg / kg, e.g., about 1 mg / kg to about 200 mg / kg, about 2 mg / kg to about 300 mg / kg, about 3 mg / kg to about 400 mg / kg, about 4 mg / kg to about 500 mg / kg, about 5 mg / kg to about 600 mg / kg, about 6 mg / kg to about 700 mg / kg, about 7 mg / kg to about 800 mg / kg, about 8 mg / kg to about 900 mg / kg, about 9 mg / kg to about 1000 mg / kg, about 10 mg / kg to about 1500 mg / kg, about 15 mg / kg to about 200 mg / kg, about 10 mg / kg to about 250 mg / kg, about 10 mg / kg to about 350 mg / kg, about 15 mg / kg to about 400 mg / kg, about 10 mg / kg to about 500 mg / kg, about 15 mg / kg to about 250 mg / kg, about 10 mg / kg to about 350 mg / kg, about 15 mg / kg to about 400 mg / The checkpoint inhibitors may include a total amount of about 5 mg / kg to about 700 mg / kg, about 5 mg / kg to about 600 mg / kg, about 6 mg / kg to about 550 mg / kg, about 7 mg / kg to about 500 mg / kg, about 8 mg / kg to about 450 mg / kg, about 9 mg / kg to about 400 mg / kg, about 5 mg / kg to about 200 mg / kg, about 2 mg / kg to about 150 mg / kg, about 5 mg / kg to about 100 mg / kg, about 10 mg / kg to about 100 mg / kg, and about 10 mg / kg to about 60 mg / kg. Further guidance may be provided based on the current standard of care for administration of checkpoint inhibitors in cancer therapy.

[0271] In one embodiment, a composition comprising a checkpoint inhibitor (e.g., a composition as described above) is administered via injection, including subcutaneous, intravenous, intraarterial, intratumoral, intralymphatic, intraperitoneal, intracardiac, intrathecal, and intramuscular injections, as well as infusion injections.

[0272] Administration In one embodiment, the subject treated according to the method of the invention has undergone one or more anti-cancer therapies prior to being treated according to the method of the invention. Such anti-cancer therapies include surgery, radiation therapy, chemotherapy, and immunotherapy. In another embodiment, the subject is treatment naive. In some embodiments, the cancer is non-metastatic. In some embodiments, the cancer is metastatic. In one embodiment, the subject has undergone a previous therapy to treat the cancer, and the cancer treated by the method of the invention is recurrent, relapsed, or refractory to the previous therapy.

[0273] With regard to the frequency and schedule of administering the anti-cancer vaccine and the checkpoint inhibitor in the methods of the present invention, one of skill in the art can determine the appropriate frequency and schedule for each.

[0274] For example, in a treatment cycle, the clinician may decide to administer the anti-cancer vaccine either as a single dose or as repeated doses, ie, a series of doses, for example over the course of several days, weeks or months.

[0275] The anti-cancer vaccine may be the same for each administration, or different anti-cancer vaccines may be administered, e.g., different anti-cancer vaccines in different treatment cycles. As an example, a personalized anti-cancer vaccine comprising a polynucleotide encoding 20 neoepitopes (neoepitopes 1-20 having sequences 1-20) or comprising a polypeptide / protein comprising said 20 neoepitopes is administered to a patient in a series of doses, e.g., over a period of days, weeks or months. Neoepitopes 1-20 are a subgroup of a total of 60 different neoepitopes identified in said patient, of which neoepitopes 1-20 were selected for inclusion in the vaccine by the methods and based on the criteria described earlier in this application. In subsequent treatment cycles, another personalized anti-cancer vaccine comprising a polynucleotide encoding the 20 neoepitopes (neoepitopes 21-40 having sequences 21-40) or comprising a polypeptide / protein comprising said 20 neoepitopes is administered to the same patient in a series of doses, e.g., over a period of days, weeks or months. Neoepitopes 21-40 are also a subgroup of the total of 60 different neoepitopes identified in the patient.

[0276] In one embodiment, the checkpoint inhibitor is administered in parallel with the anti-cancer vaccine, e.g., on the same day or at the same time on the same day, either as a single dose or as repeated doses, i.e., a series of doses, e.g., over a period of days, weeks, or months. Thus, the first administration of one or more checkpoint inhibitors and the first administration of the anti-cancer vaccine are administered in parallel, e.g., on the same day or at the same time on the same day.

[0277] In another embodiment, the checkpoint inhibitor is administered prior to the anti-cancer vaccine, either as a single dose or as multiple doses, i.e., a series of doses, for example over a period of days, weeks or months, such that a first administration of one or more checkpoint inhibitors precedes a first administration of the anti-cancer vaccine.

[0278] In yet another embodiment, the anti-cancer vaccine is administered either as a single dose or as repeated doses, i.e., a series of doses, e.g., over a period of days, weeks, or months, prior to one or more checkpoint inhibitors. Thus, the first administration of the anti-cancer vaccine is administered prior to the first administration of one or more checkpoint inhibitors.

[0279] In one embodiment, the method of the present invention further comprises one or more of the following steps: identifying neoepitopes in a patient and / or shared antigens present in the patient, designing antigen units for the patient, and producing an individualized anti-cancer vaccine for the patient, all of which steps are performed prior to administration of the individualized anti-cancer vaccine.

[0280] In another embodiment, the method of the present invention includes a vaccination induction period during which the anti-cancer vaccine is administered more frequently and / or in higher doses, and a vaccination maintenance period during which the anti-cancer vaccine is administered less frequently and / or in lower doses.

[0281] The actual dose of each of the anti-cancer vaccine and checkpoint inhibitor to be administered in the methods of the present invention will vary and depend on the age, weight, and general condition of the subject, the severity of the cancer being treated, the judgment of the medical professional, and the specific nature and characteristics of each of the anti-cancer vaccine and checkpoint inhibitor.

[0282] Exemplary time periods associated with treatment methods according to the invention include about 1 to 24 weeks, or about 6 to 12 months, or about 1 to 5 years.

[0283] The treatment method according to the present invention can be continued as long as the clinician supervising the patient's care considers the method to be effective and that treatment is necessary. Non-limiting parameters that indicate that the method is effective include any one or more of the following: reduction in disease progression or stable disease, i.e., the cancer progresses at a slower rate or does not progress. This includes tumors growing at a slower rate or not growing and / or metastasizing slower or not metastasizing, for example not metastasizing to lymph nodes or not forming metastatic foci, and / or not becoming more aggressive. Other non-limiting parameters that indicate that the method is effective include tumor shrinkage (in terms of weight and / or volume), reduction in the number of individual tumor colonies, tumor elimination, and progression-free survival.

[0284] Response to the treatment methods according to the invention can be assessed at any suitable time during treatment, for example after one round of treatment, after two to three cycles of treatment, etc., and by any of a number of suitable methods, including assessment of tumor shrinkage (partial response), i.e., tumor disappearance, reduction in disease progression or stable disease, and, where appropriate, analysis of one or more tumor test markers.

[0285] In another embodiment, a method according to the invention is effective to inhibit accumulation of regulatory T cells in a subject, or to stimulate activity of T cells and / or NK cells and / or proliferation of such cells in a subject.

[0286] The method of determining whether a patient responds to the method of the present invention is known in the art.The change in tumor size can be determined by any suitable method, for example, imaging.Various diagnostic imaging tests can be used, for example, computed tomography (CT scan), dual-energy CDT, positron emission tomography and MRI.Other secondary indicators of successful treatment include adequate response by suitable tumor markers (if applicable), increase in NK (natural killer) cell number, increase in T cell number or decrease in regulatory T cell number.

[0287] The method according to the invention may be the primary method of treating a patient's cancer or it may be an adjuvant method. For example, the primary treatment method may be surgery to remove a tumor, and the method of the invention is used as an adjuvant treatment before and / or after such surgery, either alone or in combination with other treatment methods such as chemotherapy or radiation therapy.

[0288] The method according to the present invention is for treating cancer patients.For example, the patient may be responsive to anti-cancer vaccine alone or checkpoint inhibitor alone, but more responsive to the administration of the combination.As a further example, the patient may be only slightly responsive to anti-cancer vaccine, or only slightly responsive to checkpoint inhibitor, but responsive or more responsive to the combination.

[0289] The method according to the present invention can be used to treat patients with solid or liquid cancer. An example of a solid cancer is a cancer that forms a solid mass, such as a tumor. An example of a liquid cancer is a cancer that exists in a body fluid, such as lymphoma or blood cancer. Examples of cancers that can be treated by the method according to the present invention are breast cancer, ovarian cancer, colon cancer, prostate cancer, bone cancer, colorectal cancer, gastric 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 carcinoma.

[0290] Also disclosed herein is a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (i) an anti-cancer vaccine comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, e.g. a dimerization unit, and an antigen unit comprising one or more cancer antigens, or (ii) a polypeptide encoded by the polynucleotide defined in (i), or (iii) a multimeric protein, e.g. a dimeric protein, consisting of a plurality of polypeptides, e.g. consisting of two polypeptides, as defined in (ii), and one or more checkpoint inhibitors for use in treating a subject having cancer, wherein the anti-cancer vaccine and the one or more checkpoint inhibitors are administered to the subject.

[0291] Further disclosed herein is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: (i) an anti-cancer vaccine comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, and an antigen unit comprising one or more cancer antigens, or (ii) a polypeptide encoded by the polynucleotide defined in (i), or (iii) a multimeric protein consisting of a plurality of the polypeptides defined in (ii), and one or more checkpoint inhibitors for use in treating a subject having cancer, wherein the anti-cancer vaccine and the one or more checkpoint inhibitors are administered to the subject.

[0292] Further disclosed herein is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: (i) an anti-cancer vaccine comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a dimerization unit, and an antigen unit comprising one or more cancer antigens, or (ii) a polypeptide encoded by the polynucleotide defined in (i), or (iii) a dimeric protein consisting of two polypeptides defined in (ii), and one or more checkpoint inhibitors for use in treating a subject having cancer, wherein the anti-cancer vaccine and the one or more checkpoint inhibitors are administered to the subject.

[0293] The anti-cancer vaccine, the one or more checkpoint inhibitors, and their respective administration to a subject, have been described in detail earlier in this application.

[0294] Further disclosed herein is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: The present invention relates to a method for treating cancer, the method comprising the steps of: (a) (i) an anti-cancer vaccine comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, e.g. a dimerization unit, and an antigen unit comprising one or more cancer antigens; or (ii) a polypeptide encoded by the polynucleotide defined in (i); or (iii) a multimeric protein, e.g. a dimeric protein, consisting of a plurality of polypeptides, e.g. consisting of two polypeptides, as defined in (ii); and (b) one or more checkpoint inhibitors for the manufacture of a medicament for the treatment of a subject having cancer, wherein the medicament is administered to the subject.

[0295] Further disclosed herein is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: The present invention relates to a method for treating a cancer, comprising the steps of: (a) administering to a subject a cancer vaccine comprising: (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, and an antigen unit comprising one or more cancer antigens; or (ii) a polypeptide encoded by the polynucleotide defined in (i); or (iii) a multimeric protein consisting of a plurality of polypeptides defined in (ii); and (b) one or more checkpoint inhibitors for the manufacture of a medicament for the treatment of a subject having cancer, wherein the medicament is administered to the subject.

[0296] Further disclosed herein is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: The present invention relates to a method for treating cancer, the method comprising the steps of: (a) administering to a subject a cancer vaccine comprising: (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a dimerization unit, and an antigen unit comprising one or more cancer antigens; or (ii) a polypeptide encoded by the polynucleotide defined in (i); or (iii) a dimeric protein consisting of two polypeptides defined in (ii); and (b) one or more checkpoint inhibitors for the manufacture of a medicament for the treatment of a subject having cancer, wherein the medicament is administered to the subject.

[0297] The anti-cancer vaccine, the one or more checkpoint inhibitors, and their respective administration to a subject, have been described in detail earlier in this application.

[0298] Further disclosed herein is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: The present invention relates to a method for treating a cancer patient comprising the steps of: (a) (i) an anti-cancer vaccine comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, e.g. a dimerization unit, and an antigen unit comprising one or more cancer antigens; or (ii) a polypeptide encoded by the polynucleotide defined in (i); or (iii) a multimeric protein, e.g. a dimeric protein, consisting of a plurality of polypeptides, e.g. consisting of two polypeptides, as defined in (ii); and (b) one or more checkpoint inhibitors for treating a subject having cancer.

[0299] Further disclosed herein is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: The present invention relates to a method for treating a cancer patient comprising the steps of: (a) using an anti-cancer vaccine comprising (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, and an antigen unit comprising one or more cancer antigens, or (ii) a polypeptide encoded by the polynucleotide defined in (i), or (iii) a multimeric protein consisting of a plurality of polypeptides defined in (ii); and (b) using one or more checkpoint inhibitors to treat a subject having cancer.

[0300] Further disclosed herein is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: The present invention relates to a method for treating a cancer patient comprising the steps of: (a) using an anti-cancer vaccine comprising (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a dimerization unit, and an antigen unit comprising one or more cancer antigens, or (ii) a polypeptide encoded by the polynucleotide defined in (i), or (iii) a dimeric protein consisting of two polypeptides defined in (ii); and (b) using one or more checkpoint inhibitors to treat a subject having cancer.

[0301] The anti-cancer vaccines, one or more checkpoint inhibitors and treatments have been described in detail earlier in this application.

[0302] Further disclosed herein is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: (i) an anti-cancer vaccine comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, e.g. a dimerization unit, and an antigen unit comprising one or more cancer antigens, or (ii) a polypeptide encoded by the polynucleotide defined in (i), or (iii) a multimeric protein, e.g. a dimeric protein, consisting of a plurality of polypeptides, e.g. consisting of two polypeptides, as defined in (ii), and one or more checkpoint inhibitors when used in the treatment of cancer.

[0303] Further disclosed herein is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: (i) an anti-cancer vaccine comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, and an antigen unit comprising one or more cancer antigens, or (ii) a polypeptide encoded by the polynucleotide defined in (i), or (iii) a multimeric protein consisting of a plurality of polypeptides defined in (ii), and one or more checkpoint inhibitors when used in the treatment of cancer.

[0304] Further disclosed herein is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: (i) an anti-cancer vaccine comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a dimerization unit, and an antigen unit comprising one or more cancer antigens, or (ii) a polypeptide encoded by the polynucleotide defined in (i), or (iii) a dimeric protein consisting of two polypeptides defined in (ii), and one or more checkpoint inhibitors when used in the treatment of cancer.

[0305] The anti-cancer vaccines, one or more checkpoint inhibitors and treatments have been described in detail earlier in this application.

[0306] Further disclosed herein is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: The present invention relates to a method for treating cancer comprising the steps of: (a) an anti-cancer vaccine comprising: (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, e.g., a dimerization unit, and an antigen unit comprising one or more cancer antigens; (ii) a polypeptide encoded by the polynucleotide defined in (i); or (iii) a multimeric protein, e.g., a dimeric protein, consisting of a plurality of the polypeptides defined in (ii); and (b) one or more checkpoint inhibitors for the treatment of cancer.

[0307] Further disclosed herein is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: The present invention relates to a method for treating cancer, the method comprising the steps of: (a) using an anti-cancer vaccine comprising: (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, and an antigen unit comprising one or more cancer antigens; (ii) a polypeptide encoded by the polynucleotide defined in (i); or (iii) a multimeric protein consisting of a plurality of the polypeptides defined in (ii); and (b) using one or more checkpoint inhibitors for the treatment of cancer.

[0308] Further disclosed herein is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: The present invention relates to a method for treating cancer using an anti-cancer vaccine comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a dimerization unit, and an antigen unit comprising one or more cancer antigens; (ii) a polypeptide encoded by the polynucleotide defined in (i); or (iii) a dimeric protein consisting of two polypeptides defined in (ii); and (b) one or more checkpoint inhibitors for the treatment of cancer.

[0309] The anti-cancer vaccines, one or more checkpoint inhibitors and treatments have been described in detail earlier in this application.

[0310] Further disclosed herein is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: A medicament for treating cancer in a subject having cancer by administering to the subject: (a) an anti-cancer vaccine comprising: (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, e.g., a dimerization unit, and an antigen unit comprising one or more cancer antigens; (ii) a polypeptide encoded by the polynucleotide defined in (i); or (iii) a multimeric protein, e.g., a dimeric protein, consisting of a plurality of polypeptides, e.g., two polypeptides, defined in (ii); and (b) one or more checkpoint inhibitors.

[0311] Further disclosed herein is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: A pharmaceutical for treating cancer in a subject having cancer, by administering to the subject: (a) an anti-cancer vaccine comprising: (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, and an antigen unit comprising one or more cancer antigens; (ii) a polypeptide encoded by the polynucleotide defined in (i); or (iii) a multimeric protein consisting of a plurality of the polypeptides defined in (ii); and (b) one or more checkpoint inhibitors.

[0312] Further disclosed herein is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: A medicament for treating cancer in a subject having cancer by administering to the subject: (a) an anti-cancer vaccine comprising: (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a dimerization unit, and an antigen unit comprising one or more cancer antigens; (ii) a polypeptide encoded by the polynucleotide defined in (i); or (iii) a dimeric protein consisting of two polypeptides defined in (ii); and (b) one or more checkpoint inhibitors.

[0313] The anti-cancer vaccine, one or more checkpoint inhibitors, their respective administration to a subject, and treatments have been described in detail earlier in this application.

[0314] kit In another aspect, the present invention provides a method for producing a composition comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, e.g., a dimerization unit, and an antigen unit that comprises one or more cancer antigens; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a multimeric protein consisting of a plurality of polypeptides as defined in (ii), for example consisting of two polypeptides, for example a dimeric protein; and an anti-cancer vaccine comprising (b) one or more checkpoint inhibitors.

[0315] In one embodiment, the present invention provides (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, and an antigen unit that comprises one or more cancer antigens; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides defined in (ii), and an anti-cancer vaccine comprising (b) one or more checkpoint inhibitors.

[0316] In another embodiment, the present invention provides (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a dimerization unit, and an antigen unit that comprises one or more cancer antigens; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a dimeric protein consisting of two polypeptides defined in (ii); and an anti-cancer vaccine comprising (b) one or more checkpoint inhibitors.

[0317] In yet another embodiment, the present invention provides a method for producing (a) an anti-cancer vaccine contained in one or more containers, (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, e.g., a dimerization unit, and an antigen unit that comprises one or more cancer antigens or portions thereof; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) A multimeric protein consisting of a plurality of polypeptides as defined in (ii), for example consisting of two polypeptides, for example a dimeric protein, Anti-cancer vaccines, (b) one or more checkpoint inhibitors contained in one or more second containers.

[0318] The kit may further comprise one or more third containers comprising a pharma- ceutically acceptable carrier or diluent, such as carriers and diluents for reconstituting a dose of (a) and / or (b).

[0319] In yet another embodiment, the present invention provides a method for producing (a) an anti-cancer vaccine contained in one or more first containers, (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, and an antigen unit that comprises one or more cancer antigens or portions thereof; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) A multimeric protein consisting of multiple polypeptides defined in (ii), and an anti-cancer vaccine comprising: (b) one or more checkpoint inhibitors contained in one or more second containers.

[0320] The kit may further comprise one or more third containers comprising a pharma- ceutically acceptable carrier or diluent, such as carriers and diluents for reconstituting a dose of (a) and / or (b).

[0321] In yet another embodiment, the present invention provides a method for producing (a) an anti-cancer vaccine contained in one or more first containers, (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a dimerization unit, and an antigen unit that comprises one or more cancer antigens or portions thereof; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a dimeric protein consisting of two polypeptides defined in (ii); and an anti-cancer vaccine comprising: (b) one or more checkpoint inhibitors contained in one or more second containers.

[0322] The kit may further comprise one or more third containers comprising a pharma- ceutically acceptable carrier or diluent, such as carriers and diluents for reconstituting a dose of (a) and / or (b).

[0323] The kit is for use in a method for treating a subject, e.g., a patient, having cancer, the method comprising administration of an anti-cancer vaccine included in the kit and administration of one or more checkpoint inhibitors to the subject.

[0324] The kit may further comprise instructions for use, including instructions for reconstitution of doses of (a) and (b), instructions for administration, and / or instructions for determining suitable doses of (a) and (b), and / or instructions for administration of (a) and (b), and / or the frequency and schedule for administering (a) and (b).

[0325] Anti-cancer vaccine contained in the kit according to the present invention The anti-cancer vaccines contained in the kit are described in detail earlier in this application.

[0326] In the first embodiment of the kit, the anti-cancer vaccine included in the kit is provided in a first container containing the polynucleotide / polypeptide / dimeric protein and a second container containing a pharma- ceutically acceptable carrier or diluent, including but not limited to saline, buffered saline, such as PBS, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer, and combinations thereof. The kit further includes instructions on how to combine the contents of the first and second containers prior to administration to reconstitute a dose of the anti-cancer vaccine for such administration.

[0327] In one embodiment, the kit comprises one such first container and one such second container for reconstituting a single dose of anti-cancer vaccine. In another embodiment, the kit comprises several such first containers and several such second containers for reconstituting several doses of anti-cancer vaccine for repeated administration. In yet another embodiment, the kit comprises several such first containers and one such second container for reconstituting several doses of anti-cancer vaccine for repeated administration, the second container comprising sufficient contents for reconstituting the anti-cancer vaccine contained in the several first containers. The repeated doses may be a series of doses for use over a period of, for example, several days, weeks or months.

[0328] The container included in the kit of the first embodiment may be any container suitable for housing an anti-cancer vaccine or a pharma- ceutically acceptable carrier.

[0329] In the second embodiment of the kit, the anti-cancer vaccine contained in the kit is provided in a container, i.e., a single container, containing a composition comprising a polynucleotide / polypeptide / dimeric protein and a pharma- ceutically acceptable carrier or diluent, including but not limited to saline, buffered saline, such as PBS, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer, and combinations thereof. Thus, the anti-cancer vaccine may be in a ready-to-administer form, or may need to be further diluted with a pharma- ceutically acceptable carrier or diluent, for example, just before it is administered, which pharma- ceutically acceptable carrier or diluent may be an additional component in the kit.

[0330] The kit of the second embodiment may include one or more containers, e.g., one container for a single dose, or several containers for repeated doses, e.g., a series of doses, e.g., for use over a period of days, weeks, or months.

[0331] The container included in the kit of the second embodiment may be any container suitable for housing the anti-cancer vaccine and a pharma- ceutically acceptable carrier.

[0332] The pharma- ceutically acceptable carrier or diluent of the first embodiment or the anti-cancer vaccine of the second embodiment may further comprise an adjuvant. In particular, for anti-cancer vaccines comprising a polypeptide / protein, pharma- ceutically acceptable adjuvants include poly-ICLC, 1018 ISS, aluminum salts, 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 50 V, Montanide These include, but are not limited to, ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, vadimesan, and / or AsA404 (DMXAA).

[0333] For anti-cancer vaccines that include a polynucleotide, the pharma- ceutically acceptable carrier or diluent or the anti-cancer vaccine may include a molecule that facilitates transfection of cells.

[0334] The kits of the first and second embodiments preferably include instructions for administration of the anti-cancer vaccine to a subject, eg, a patient, in need thereof.

[0335] In one embodiment, a first container of the kit of the first embodiment or a single container of the kit of the second embodiment comprises an anti-cancer vaccine comprising a polynucleotide, and a second container of the kit of the first embodiment or a single container of the kit of the second embodiment comprises a pharma- ceutically acceptable carrier. In a further preferred embodiment, the kits of the first and second embodiments comprise instructions for intramuscular or intradermal injection of the anti-cancer vaccine.

[0336] A first container of the kit of the first embodiment or a single container of the kit of the second embodiment typically contains 0.3 mg to 6 mg, e.g., about 2 mg, when the anti-cancer vaccine is a polynucleotide, and 5 μg to 5 mg when the anti-cancer vaccine is a polypeptide / dimeric protein.

[0337] Checkpoint inhibitors contained in the kit according to the present invention The one or more checkpoint inhibitors included in the kit are described in detail above in this application.

[0338] In one embodiment, the one or more checkpoint inhibitors included in the kit are provided in one or more containers, e.g., each container containing one checkpoint inhibitor, or one container containing all of the checkpoint inhibitors. The kit further comprises one or more containers containing a liquid suitable for injection, e.g., infusion injection, including, e.g., sterile water, saline, e.g., isotonic saline, sugar solutions for intravenous administration, e.g., dextrose solutions, electrolyte solutions for intravenous administration, e.g., lactated Ringer's solution or acetate Ringer's solution, or aqueous buffer solutions.

[0339] The kit preferably further comprises instructions on how to combine the contents of the container containing the checkpoint inhibitor with the contents of a container containing a liquid suitable for injection, including an infusion injection, to obtain a composition comprising the checkpoint inhibitor that is ready to be administered.

[0340] The composition may comprise a checkpoint inhibitor or a number of checkpoint inhibitors, the checkpoint inhibitors being administered at a dose of about 0.1 mg / kg to about 1 mg / kg, e.g., about 0.2 mg / kg to about 0.9 mg / kg, about 0.3 mg / kg to about 0.8 mg / kg, about 0.4 mg / kg to about 0.7 mg / kg, about 0.5 mg / kg to about 0.6 mg / kg, or about 1 mg / kg to about 1000 mg / kg, e.g., about 2 mg / kg to about 900 mg / kg, about 3 mg / kg to about 800 mg / kg, about 4 mg / kg to about 900 mg / kg, about 5 mg / kg to about 1000 mg / kg, e.g., about 1 mg / kg to about 200 mg / kg, about 2 mg / kg to about 300 mg / kg, about 3 mg / kg to about 400 mg / kg, about 4 mg / kg to about 500 mg / kg, about 5 mg / kg to about 600 mg / kg, about 6 mg / kg to about 700 mg / kg, about 7 mg / kg to about 800 mg / kg, about 8 mg / kg to about 900 mg / kg, about 9 mg / kg to about 1000 mg / kg, about 10 mg / kg to about 1500 mg / kg, about 15 mg / kg to about 200 mg / kg, about 10 mg / kg to about 250 mg / kg, about 10 mg / kg to about 350 mg / kg, about 15 mg / kg to about 400 mg / kg, about 10 mg / kg to about 500 mg / kg, about 15 mg / kg to about 250 mg / kg, about 10 mg / kg to about 350 mg / kg, about 15 mg / kg to about 400 mg / The amount of the compound may be about 5 mg / kg to about 700 mg / kg, about 5 mg / kg to about 600 mg / kg, about 6 mg / kg to about 550 mg / kg, about 7 mg / kg to about 500 mg / kg, about 8 mg / kg to about 450 mg / kg, about 9 mg / kg to about 400 mg / kg, about 5 mg / kg to about 200 mg / kg, about 2 mg / kg to about 150 mg / kg, about 5 mg / kg to about 100 mg / kg, about 10 mg / kg to about 100 mg / kg, and about 10 mg / kg to about 60 mg / kg.

[0341] The kit can include several containers suitable for single doses of one or more checkpoint inhibitors, or several containers suitable for repeated doses, e.g., a series of doses, e.g., for use over a period of days, weeks, or months, of one or more checkpoint inhibitors.

[0342] In another embodiment, the kit comprises one or more containers comprising one or more checkpoint inhibitors in a composition suitable for injection, e.g., infusion injection, e.g., each container comprises one checkpoint inhibitor in a composition suitable for injection, or one container comprises all checkpoint inhibitors in a composition suitable for injection. Compositions suitable for injection comprise a suitable pharma- ceutically acceptable carrier or diluent, e.g., sterile water, saline, e.g., isotonic saline, a sugar solution for intravenous administration, e.g., dextrose solution, an electrolyte solution for intravenous administration, e.g., lactated Ringer's solution or acetate Ringer's solution, or an aqueous buffer solution.

[0343] The composition may comprise a checkpoint inhibitor or a number of checkpoint inhibitors, the checkpoint inhibitors being administered at a dose of about 0.1 mg / kg to about 1 mg / kg, e.g., about 0.2 mg / kg to about 0.9 mg / kg, about 0.3 mg / kg to about 0.8 mg / kg, about 0.4 mg / kg to about 0.7 mg / kg, about 0.5 mg / kg to about 0.6 mg / kg, or about 1 mg / kg to about 1000 mg / kg, e.g., about 2 mg / kg to about 900 mg / kg, about 3 mg / kg to about 800 mg / kg, about 4 mg / kg to about 900 mg / kg, about 5 mg / kg to about 1000 mg / kg, e.g., about 1 mg / kg to about 200 mg / kg, about 2 mg / kg to about 300 mg / kg, about 3 mg / kg to about 400 mg / kg, about 4 mg / kg to about 500 mg / kg, about 5 mg / kg to about 600 mg / kg, about 6 mg / kg to about 700 mg / kg, about 7 mg / kg to about 800 mg / kg, about 8 mg / kg to about 900 mg / kg, about 9 mg / kg to about 1000 mg / kg, about 10 mg / kg to about 1500 mg / kg, about 15 mg / kg to about 200 mg / kg, about 10 mg / kg to about 250 mg / kg, about 10 mg / kg to about 350 mg / kg, about 15 mg / kg to about 400 mg / kg, about 10 mg / kg to about 500 mg / kg, about 15 mg / kg to about 250 mg / kg, about 10 mg / kg to about 350 mg / kg, about 15 mg / kg to about 400 mg / The amount of the compound may be about 5 mg / kg to about 700 mg / kg, about 5 mg / kg to about 600 mg / kg, about 6 mg / kg to about 550 mg / kg, about 7 mg / kg to about 500 mg / kg, about 8 mg / kg to about 450 mg / kg, about 9 mg / kg to about 400 mg / kg, about 5 mg / kg to about 200 mg / kg, about 2 mg / kg to about 150 mg / kg, about 5 mg / kg to about 100 mg / kg, about 10 mg / kg to about 100 mg / kg, and about 10 mg / kg to about 60 mg / kg.

[0344] The kit may include several containers suitable for single doses of one or more checkpoint inhibitors, or several such containers suitable for repeated doses, e.g., a series of doses, e.g., for use over a period of days, weeks, or months, of one or more checkpoint inhibitors.

[0345] In one embodiment, the kit further comprises instructions for administering the checkpoint inhibitors, e.g., instructions for administering them via injection, e.g., subcutaneous, intravenous, intraarterial, intratumoral, intralymphatic, intraperitoneal, intracardiac, intrathecal, or intramuscular injection, or infusion injection. [Brief description of the drawings]

[0346] [Figure 1a] 1 shows an embodiment of an anti-cancer vaccine for use in the methods of the invention, described as a polypeptide having an N-terminal start and a C-terminal end. [Figure 1b] 1 shows an embodiment of an anti-cancer vaccine for use in the methods of the invention, described as a polypeptide having an N-terminal start and a C-terminal end.

[0347] [Diagram 2] The results of Example 2 show the synergistic effect of administering the checkpoint inhibitors anti-PD-L1 antibody, anti-TIGIT antibody, or anti-PD-L1 and anti-TIGIT antibody in combination therapy with the anti-cancer vaccine VB4085 to mice challenged with CT26 tumor cells. The therapeutic efficacy of the combination therapy (measured as tumor volume) was compared to that of monotherapy, i.e., the anti-cancer vaccine VB4085 or anti-PD-L1 antibody or anti-TIGIT antibody or anti-PD-L1 and anti-TIGIT antibody, as well as the negative control, VB1026.

[0348] [Diagram 3] The results of Example 2 show the synergistic effect of administering the checkpoint inhibitors anti-PD-L1 antibody, anti-TIGIT antibody, or anti-PD-L1 and anti-TIGIT antibody in combination therapy with the anti-cancer vaccine VB4085 to mice challenged with CT26 tumor cells. The therapeutic efficacy of the combination treatment (measured as % survival days after tumor challenge) was compared to the therapeutic efficacy of monotherapy, i.e., the anti-cancer vaccine VB4085 or anti-PD-L1 antibody or anti-TIGIT antibody or anti-PD-L1 and anti-TIGIT antibody, as well as the negative control, VB1026.

[0349] [Figure 4] The results of Example 3 show the synergistic effect of administering a combination treatment, a checkpoint inhibitor anti-CTLA-4 antibody, and an anti-cancer vaccine, VB4085, to mice challenged with CT26 tumor cells. The therapeutic efficacy of the combination treatment (measured as tumor volume) was compared to the therapeutic efficacy of monotherapy, i.e., the anti-cancer vaccine, VB4085 or anti-CTLA-4, and the negative control, VB1026.

[0350] [Diagram 5]The results of Example 3 are shown, namely, the synergistic effect of administering a combination treatment, a checkpoint inhibitor anti-CTLA-4 antibody and an anti-cancer vaccine VB4085, to mice challenged with CT26 tumor cells. The therapeutic efficacy of the combination treatment (measured as % survival days after tumor challenge) was compared to the therapeutic efficacy of monotherapy, namely, the anti-cancer vaccine VB4085 or the anti-CTLA-4 antibody, and the negative control VB1026. EXAMPLES

[0351] The foregoing written specification is believed to be sufficient to enable one skilled in the art to practice the invention. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. Example 1: Anti-cancer vaccine design Two DNA constructs were designed containing nucleotide sequences encoding the following units / elements: [Table 1] Anti-cancer vaccine VB4085: Exome and RNA sequencing of the previously described mouse colon cancer cell line CT26 revealed hundreds to thousands of tumor-specific nonsynonymous mutations. Using an in silico-based method, potential immunogenic epitopes, i.e. epitopes containing mutations, were identified, and 20 of them (Table 1) were selected for inclusion in the antigenic unit of VB4085. Each of the epitopes consists of 27 amino acids, and the epitopes are separated from each other by linkers. Thus, all epitopes except the terminal epitopes are placed in subunits, each of which consists of one epitope and one flexible glycine-serine linker (GGGGSGGGGS (SEQ ID NO: 29)). VB4085 is a model of (a) a personalized DNA anti-cancer vaccine that contains several patient-specific epitopes, e.g., several neoepitopes and / or shared cancer epitopes present in several patients (the shared cancer antigens present in the patients are shared cancer antigens present in the patients that have been mutated), and (b) an off-the-shelf DNA anti-cancer vaccine that contains several shared cancer epitopes (the shared cancer antigens are mutated shared cancer antigens).

[0352] VB4085 consists of a DNA sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:1. [Table 2] Negative control VB1026: This construct is identical to VB4085, but does not contain a linker or antigen unit. It serves as a negative control. VB1026 consists of a DNA sequence encoding a polypeptide having the amino acid sequence 1-237 of SEQ ID NO:1.

[0353] All epitope gene sequences were ordered from Genescript (New Jersey, US) and cloned into the expression vector pUMVC4a, which contains the nucleotide sequences of the targeting and dimerization units described above.

[0354] Example 2: Embodiments of a method of treatment with an anti-cancer vaccine according to the present invention and checkpoint inhibitors anti-TIGIT and / or anti-PD-L1 in CT26 tumor-challenged mice compared to treatment with an anti-cancer vaccine or checkpoint inhibitor alone The treatment schedule for this study is shown in Table 2 below. [Table 3]

[0355] Anti-PD-L1 antibody (also referred to as anti-PD-L1 in this Example section) was purchased from Bio X Cell, USA. This monoclonal antibody reacts with mouse PD-L1 (anti-mouse PD-L1 antibody). Anti-TIGIT antibody (also referred to as anti-TIGIT in this Example section) was purchased from Absolute Antibody, USA. This monoclonal antibody reacts with mouse TIGIT (anti-mouse TIGIT antibody).

[0356] Treatment of Balb / c mice: Each of groups A to H contained 10 mice, and these mice were inoculated with 5 × 10 4 CT26 tumor cells were inoculated on day 0 by injecting 10 ... Tumor volume was calculated by the formula: Tumor volume = length (mm) x width (mm) x height (mm) / 2000. Treatment was terminated on day 43.

[0357] Treatment results: Statistical significance was determined by two-way ANOVA statistical test with Tukey's multiple comparison test.

[0358] Tumors in the group treated with VB4085 anticancer vaccine alone (Group B) appeared to grow slower compared to the VB1026 negative control group (Group A). ​​However, due to intragroup variability, the difference in tumor growth was not statistically different between these two groups.

[0359] The difference in therapeutic efficacy (tumor growth) between the groups treated according to one embodiment of the method of the present invention (groups F to H) and the VB1026 negative control group was statistically significant from day 26 throughout the experiment (F: p=0.01, G: p=0.005, H: p=0.003, p is the p-value) (Figure 2).

[0360] The difference in therapeutic efficacy (tumor growth) between group F receiving VB4085 anti-cancer vaccine in combination with anti-TIGIT antibody (an embodiment of the method of the present invention) and VB4085 anti-cancer vaccine monotherapy group B was statistically significant from day 34 onwards (p=0.0004) (Figure 2).

[0361] The difference in therapeutic efficacy (tumor growth) between group G receiving VB4085 anti-cancer vaccine in combination with anti-PD-L1 antibody (an embodiment of the method of the present invention) and VB4085 anti-cancer vaccine monotherapy group B was statistically significant from day 28 onwards (p=0.047) (Figure 2).

[0362] The difference in therapeutic efficacy (tumor growth) between group H receiving VB4085 anti-cancer vaccine in combination with anti-TIGIT antibody and anti-PD-L1 antibody (an embodiment of the method of the present invention) and VB4085 monotherapy group B was statistically significant from day 27 onwards (p=0.032) (Figure 2).

[0363] Treatment efficacy (as determined by tumor growth) in groups treated according to one embodiment of the method of the present invention (Groups FH) was significantly better in each group compared to their respective antibody monotherapy groups (Groups C-E) from day 26 onwards (Figure 2).

[0364] The tumors in mice in group H (an embodiment of the method of the present invention) never even grew to a large size, and some of the tumors were only observed at a single time point.

[0365] Furthermore, survival (measured as deaths / mice per group) of mice treated according to one embodiment of the method of the present invention (Groups FH) was significantly better than that of mice treated with anti-cancer vaccine or checkpoint inhibitor alone (Groups B-E), as shown in Table 3 below and in FIG. 3. [Table 4] conclusion Tumor growth in mice vaccinated with the VB4085 anticancer vaccine as monotherapy (group B) was slower on average compared to tumor growth in the negative control group treated with VB1026 (group A). ​​This is consistent with previous CT26 tumor challenge studies using mice vaccinated with VB4085, demonstrating that this DNA anticancer vaccine induces an immune response against CT26 tumor cells that results in reduced tumor growth.

[0366] Other researchers have shown that PD-L1 is expressed by CT26 cells and expression is increased in response to IFN-γ exposure, suggesting that CT26 cells may evade immune surveillance via the PD-1 / PD-L1 axis (Lau et al., Nat. Commun 8, 2017, 1-11). The results of this study showed that blockade of PD-L1 by administration of anti-PD-L1 antibodies in combination with the anti-cancer vaccine VB4085 resulted in increased anti-tumor efficacy with further reduction in tumor growth compared to VB4085 anti-cancer vaccine monotherapy.

[0367] Previously, treatment of mice inoculated with CT26 tumor cells with anti-TIGIT antibodies was shown to reduce tumor growth (Johnston et al., Cancer Cell 26(6), 2014, 923-937). The results of this study showed that blocking the TIGIT signaling pathway by administering anti-TIGIT antibodies in combination with the anti-cancer vaccine VB4085 reduced tumor growth to a greater extent than VB4085 anti-cancer vaccine monotherapy.

[0368] Combined targeting of TIGIT and PD-1 / PD-L1 has been shown to be beneficial in CT26 tumor mouse models compared to targeting either checkpoint protein individually (Johnston et al., Cancer Cell 26(6), 2014, 923-937). These findings demonstrate that the mouse model evades immune surveillance through multiple pathways. In this study, we have shown that when using a combination of anti-TIGIT and anti-PD-L1 antibodies, the inclusion of anti-cancer vaccine VB4085 in such treatment can clearly enhance the immune response against tumor cells. Such combined treatment according to embodiments of the method of the present invention showed better therapeutic efficacy (determined by tumor growth and survival) compared to any of the three treatments alone.

[0369] Example 3: An embodiment of a method of treatment with an anti-cancer vaccine according to the present invention and the checkpoint inhibitor anti-CTLA-4 in CT26 tumor-challenged mice compared to treatment with either the anti-cancer vaccine or the checkpoint inhibitor anti-CTLA-4 alone Constructs VB4085 and VB1026, described in Example 1, were used in this study. The treatment schedule for this trial is shown in Table 4 below. [Table 5]

[0370] Anti-CTLA-4 (CD152) antibody (also referred to as anti-CTLA-4 in this Example section) was purchased from Bio X Cell, USA. This monoclonal antibody reacts with mouse CTLA-4 (anti-mouse CTLA-4 antibody).

[0371] Treatment of Balb / c mice: Each of groups A to D contained 10 mice, and these mice were administered 5 × 10 4 Mice were inoculated with CT26 tumor cells on day 0 by subcutaneous injection of 10 tumor cells in the left thigh. On days 0, 8, 14, and an additional 28, mice were vaccinated intramuscularly with a total of 50 μg (25 μg for boost) of construct VB4085 or negative control construct VB1026, 25 μg in each tibialis anterior muscle (25 μg in the right tibialis anterior muscle for boost), followed by electroporation. Anti-CTLA-4 antibody administration was performed on days 11, 18, 26, and 32 by intraperitoneal injection of 200 μg / mouse. Tumor size was measured using calipers. Tumors were measured in two dimensions, length and width, with the height set equal to the width. Tumor volume was calculated by the formula: tumor volume = length (mm) x width (mm) x height (mm) / 2000. The experiment was terminated on day 39.

[0372] Treatment results: Statistical significance was determined by two-way ANOVA statistical test with Tukey's multiple comparison test.

[0373] The difference in therapeutic efficacy (tumor growth) between VB1026 anticancer vaccine monotherapy (Group B) compared to the VB4085 negative control group (Group A) was statistically significant from day 26 onwards (p=0.0067) (Figure 4).

[0374] The difference in therapeutic efficacy (tumor growth) between anti-CTLA-4 antibody monotherapy (Group C) compared to the VB1026 negative control group (Group A) was statistically significant from day 32 onwards (p=0.038) (Figure 4).

[0375] The difference in therapeutic efficacy (tumor growth) between group D (an embodiment of the method of the present invention) receiving the VB4085 anti-cancer vaccine in combination with an anti-CTLA-4 antibody and the VB1026 negative control group (group A) was statistically significant from day 23 onwards (p=0.025) (Figure 4).

[0376] The difference in therapeutic efficacy (tumor growth) between the group treated according to one embodiment of the method of the present invention (Group D) and VB4085 anti-cancer vaccine monotherapy Group B was not statistically significant (p>0.05) (Figure 4).

[0377] The difference in therapeutic efficacy (tumor growth) between group D receiving VB4085 anti-cancer vaccine in combination with anti-CTLA-4 antibody (an embodiment of the method of the present invention) and anti-CTLA-4 antibody monotherapy group C was statistically significant from day 32 onwards (p=0.032) (Figure 4).

[0378] Furthermore, survival (measured as deaths / mice per group) of mice treated according to one embodiment of the method of the present invention (Group D) was significantly better than mice treated with anti-cancer vaccine or checkpoint inhibitor alone (Groups B and C), as shown in Table 5 below and in FIG. 5. [Table 6] * One animal in the VB4085 group developed spontaneous rectal prolapse on day 32 and had to be euthanized. The animal was tumor free on day 32.

[0379] conclusion Tumor growth in mice vaccinated with VB4085 anti-cancer vaccine as monotherapy (group B) had significantly slower tumor growth compared to the negative control group treated with VB1026 (group A). ​​This is consistent with previous CT26 tumor challenge studies using mice vaccinated with VB4085, demonstrating that this DNA anti-cancer vaccine induces an immune response against CT26 tumor cells that results in reduced tumor growth.

[0380] Building on previous reports, others have shown anti-cancer efficacy in CT26 tumor-sensitized mice treated with anti-CTLA-4 antibody monotherapy (reviewed in Pedersen et al., Cancer Lett 235(2), 2006, 229-238; Grosso et al., Cancer Immun 13(1), 2013, 5).

[0381] The results of the present study show that blockade of the CTLA-4 signaling pathway by administration of anti-CTLA-4 antibody as monotherapy elicited partial antitumor responses, while in mice treated with anti-CTLA-4 antibody in combination with the VB4085 anticancer vaccine, a clearly enhanced immune response against tumor cells was observed, with 9 out of 10 mice being tumor-free at the end of the study (day 39).

[0382] Such combination treatment according to embodiments of the method of the present invention demonstrated better therapeutic efficacy (as determined by tumor growth and survival) compared to two different monotherapies.

[0383] Example 4: An embodiment of a method of treatment according to the present invention comprising administration of a personalized DNA anti-cancer vaccine comprising VB10.NEO in combination with atezolizumab Adult patients will be treated with a personalized DNA anti-cancer vaccine containing VB10.NEO in combination with atezolizumab. Patients have locally advanced and metastatic tumors that have progressed after at least one available standard therapy, or for whom standard therapy has proven ineffective or intolerable or is deemed inappropriate, or for whom clinical trials of investigational agents are recognized as standard of care. In one embodiment, the patient has at least one of the following tumors: melanoma, NSCLC, RCC, UC, HNSCC, TNBC, gastric / GEJ cancer, cervical cancer, anal cancer, or an MSI-high tumor. In one embodiment, VB10.NEO (3 mg in a pharma- ceutically acceptable carrier) is administered by intramuscular injection over the induction course Q3W (4 doses), followed by maintenance doses Q6W (6 doses) and Q12W (5 doses). Atezolizumab 1200 mg is administered by intravenous infusion on day 1 of a 21-day cycle.

[0384] In another embodiment, VB10.NEO (6 mg in a pharma- ceutically acceptable carrier) is administered by intramuscular injection over an induction course Q3W (4 doses), followed by maintenance doses Q6W (6 doses) and Q12W (5 doses). Atezolizumab 1200 mg is administered by intravenous infusion on day 1 of a 21-day cycle.

[0385] During and after treatment (from baseline to 27 months), the primary endpoints were: Incidence and severity of adverse events Changes in the following vital signs measured before and after the first VB10.NEO injection of each cycle: systolic blood pressure (mmHg), diastolic blood pressure (mmHg), pulse rate (bpm), respiratory rate (breaths / min), and body temperature (℃). · Changes in clinical laboratory parameters analyzed locally before and after the first VB10.NEO injection of each cycle (including hematology, chemistry panel, coagulation, thyroid function tests, C-reactive protein, urinalysis and serology). Secondary endpoints during and after treatment are: Assessment of antigen-specific immune responses induced by VB10.NEO administered in combination with atezolizumab (from baseline to month 25): number and magnitude of antigen-specific T cell responses before and after the start of treatment; Objective response rate (from baseline to 27 months): Complete or partial response Duration of response (from baseline to 27 months): the time from the first occurrence of a documented objective response to disease progression or death from any cause (whichever occurs first); Progression-free survival (from baseline to 27 months): the time from first treatment to the first occurrence of disease progression or death from any cause (whichever occurs first); Overall survival (from baseline to 27 months): time from first treatment to death from any cause; To determine serum concentrations of atezolizumab at specific time points (from baseline to 25 months); Prevalence of anti-drug antibodies (ADA) to atezolizumab at baseline and incidence of ADA to atezolizumab (from baseline to 25 months). In one embodiment, the method of treatment comprises administering to a patient in need thereof an effective amount of a personalized DNA anti-cancer vaccine comprising VB10.NEO in combination with atezolizumab.

[0386] In some embodiments, the patient has at least one of the following tumor types: melanoma, NSCLC, RCC, UC, HNSCC, TNBC, gastric / GEJ cancer, cervical cancer, anal cancer, or MSI-high tumors. In some embodiments, the patient has a locally advanced or metastatic tumor that has progressed after at least one available standard therapy, and / or the standard therapy has proven ineffective or intolerable or is deemed inappropriate, and / or a clinical trial of an investigational agent is recognized as the standard of care. In some embodiments, the effective amount is VB10.NEO (3 mg in a pharma- ceutically acceptable carrier) administered by intramuscular injection over an induction course Q3W (4 doses), followed by maintenance doses Q6W (6 doses) and Q12W (5 doses), and atezolizumab 1200 mg administered by intravenous infusion on day 1 of a 21 day cycle. In some embodiments, VB10.NEO (6 mg in a pharma- ceutically acceptable carrier) is administered by intramuscular injection over an induction course Q3W (4 doses), followed by maintenance doses Q6W (6 doses) and Q12W (5 doses), and tezolizumab 1200 mg is administered by intravenous infusion on day 1 of a 21 day cycle.

[0387] In some embodiments, the method of treatment results in improved overall response rate (ORR) and / or improved duration of response (DOR) and / or improved progression free survival (PFS) and / or improved overall survival (OS) compared to a reference population. In some embodiments, the reference population is a population treated with standard of care (SOC) for the tumor.

[0388] array SEQ ID NO:1 VB4085 (amino acids 1–972) / VB1026 (amino acids 1–237). M 1 QVSTAALAVLLCTMALCNQVLS 23 A 24 PLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSA 93 E 94 LKTPLGDTTHT 105 E 106 PKSCDTPPPCPRCP 120 G 121 GGSSGGGSG 130 G 131 QPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK 237 G 238 LGGL 242STMLYIRALKNPSLYGFSSGLNKDGIEGGGGSGGGGSGALKKLIYAAKLNTSLKALEGERNQVYGGGGSGGGGSDLVCDFQSFKYYAHATSLAGHLVSCPLGGGGSGGGGSEKLRNPCPNKEKAYQPPFAFRHVLKLTGGGGSGGGGSDTLSAMSNPRAMQVLLQIQQGLQTLATGGGGGSGG GGSGDVKIHAHKVVLANISPYFKAMFTGNLGGGGSGGGGSEVIQTSKYYMRDVIAIESAWLLELAPHGGGGSGGGGSFFSFFTHRFGHHVSPQVHIILANLYLLGGGGSGGGGSVILPQAPSGPSYATYLQPAQAQMLTPPGGGGSGGGGSLWVYLRPVPRPATIYLQILRLKPLTGEGGGGS GGGGSFVSPMAHYVPGIMAIESVVARFQFIVPGGGGSGGGGSTLAFLVLSTPAMFNRALKPFLKSCHFQGGGGSGGGGSFLERPMDMPYMIFYPNNPLMTGQLLGSGGGGSGGGGSIPREVGDGTRVDPFPPVQTWMRLPKLVGGGGSGGGGSGSLFGSRVQYVVNPAVKIVFLNIDPSGG GGSGGGGSNNLQKYIEIYVQKINPSRLPVVIGGLLGGGGSGGGGSAEYGDYQPEVHGVPYFRLEHYLPARVMGGGGSGGGGSTPLRKHTVHAIRKFYLEFKGSSPPPRLGGGGSGGGGSKIYEFDYHLYGQNITMIMTSVSGHLLAGGGGSGGGGSKSWIHCWKYLSVQSQLFRGSSLLFRRV SEQ ID NO:2 Signal peptide MNFGLRLIFLVLTLKGVQC SEQ ID NO:3 Signal peptide MDAMKRGLCCVLLLCGAVFVSP SEQ ID NO:4 Nucleotide sequence encoding amino acids 1 to 23 of SEQ ID NO:1 ATGCAGGTCTCCACTGCTGCCCTTGCCGTCCTCCTCTGCACCATGGCTCTCTGCAACCAGGTCCTCTCT SEQ ID NO:5: Nucleotide sequence encoding amino acids 24 to 93 of SEQ ID NO:1 GCACCACTTGCTGCTGACACGCCGACCGCCTGCTGCTTCAGCTACACCTCCCGACAGATTCCACAGAATTTCATAGCTGACTACTTTGAGACGAGCAGCCAGTGCTCCAAGCCCAGTGTCATCTTCCTAACCAAGAGAGGCCGGCAGGTCTGTGCTGACCCCAGTGAGGAGTGGGTCCAGAAATACGTCAGTGACCTGGAGCTGAGTGCC SEQ ID NO:6: Nucleotide sequence encoding amino acids 94 to 105 of SEQ ID NO:1 GAGCTCAAACCCCACTTGGTGACACAACTCACACA SEQ ID NO:7: Nucleotide sequence encoding amino acids 106 to 120 of SEQ ID NO:1 GAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCA SEQ ID NO:8 Nucleotide sequence encoding amino acids 94 to 120 of SEQ ID NO:1 GAGCTCAAAACCCCACTTGGTGACACAACTCACACAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCA SEQ ID NO:9: Nucleotide sequence encoding amino acids 121 to 130 of SEQ ID NO:1 GGCGGTGGAAGCAGCGGAGGTGGAAGTGGA SEQ ID NO:10: Nucleotide sequence encoding amino acids 131 to 237 of SEQ ID NO:1 GGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACACCA CGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA SEQ ID NO:11: Nucleotide sequence encoding amino acids 238 to 242 of SEQ ID NO:1 GGCCTCGGTGGCCTG SEQ ID NO:12: Nucleotide sequence encoding amino acids 94 to 237 of SEQ ID NO:1 GAGCTCAAAACCCCACTTGGTGACACAACTCACACAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGGCGGTGGAAGCAGCGGAGGTGGAAGTGGAGGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGC GACATCGCCGTGGAGTGGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA SEQ ID NO:13: Atezolizumab heavy chain amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO:14: Atezolizumab light chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:15: Atezolizumab HVR-H1 GFTFSDSWIH SEQ ID NO:16: Atezolizumab HVR-H21 AWISPYGGSTYYADSVKG SEQ ID NO:17: Atezolizumab HVR-H3 RHWPGGFDY SEQ ID NO:18: Atezolizumab HVR-L1 RASQDVSTAVA SEQ ID NO:19: Atezolizumab HVR-L2 SASFLYS SEQ ID NO:20: Atezolizumab HVR-L3 QQYLYHPAT SEQ ID NO:21: Atezolizumab HV EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS SEQ ID NO:22 Atezolizumab HL DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR SEQ ID NO:23 Nucleic acid sequence of VB10.NEO ATGCAGGTCTCCACTGCTGCCCTTGCCGTCCTCCTCTGCACCATGGCTCTCTGCAACCAGGTCCTCTCTGCACCACTTGCTGCTGACAGCCGACCGCCTGCTGCTTCAGCTACACCTCCCGACAGATTCCACAGAATTTCATAGCTGACTACTTTGAGACGAGCAGCCAGTGCTCCAAGC CCAGTGTCATCTTCCTAACCAAGAGAGGCCGGCAGGTCTGTGCTGACCCCAGTGAGGAGTGGGTCCAGAAATACGTCAGTGACCTGGAGCTGAGTGCCGAGCTCAAAACCCCACTTGGTGACACAACTCACAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGGC GGTGGAAGCAGCGGAGGTGGAAGTGGAGGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCGGGGAGGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAGCGGGCAGCCGGAGAACAACTACA ACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGCCTCGGTGGCCTG sequence number 113 p53 AAGCCTCTGGACGGAGAGTATTTCACTCTCCAGATCCGGGGCCCCGAAAGGTTCGAAATGTTCCGGGAGCTTAACGAGGCCTTGGAGCTGAAAGACGCACAGGCCGGAAAGGAACCG sequence number 114 HEYGAEALERAG sequence number 115 Nucleic acid sequence CLEC9 リガンド TGGCCCAGGTTCCACAGCAGCGTGTTCCACACCCAC SEQ ID NO:116 Nucleic acid sequence of the trimerization domain from human collagen GCTGGGCAGGTGAGGATCTGGGCCACATACCAGACCATGCTGGACAAGATCCGGGAGGTGCCGGAGGGCTGGCTCATCTTTGTGGCCGAGAGGGAAGAGCTCTATGTACGCGTTAGAAATGGCTTCCGGAAGGTGCTGCTGGAGGCCCGGACAGCCCTCCCGAGAGGCACGGGCAATGAG SEQ ID NO:117 The trimerization unit is the C-terminal domain of T4 fibritin GYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO:119 Nucleic acid sequence of the hinge region from IgM GCCGAACTCCCGCCCAAGGTGTCCGTGTTCGTCCCTCCCCGCGATGGGTTCTTCGGCAATCCACGAAAATCCAAACTGATTTGTCAGGCCACCGGCTTCTCCCCCCGACAGATCCAGGTGAGTTGGCTACGAGAGGGTAAACAGGTGGGGAGCGGAGTGACCACTG ACCAGGTGCAGGCCGAGGCCAAGGAAAGCGGACCCACAACATACAAAGTGACAAGCACTCTGACGATTAAGGAGTCAGACTGGCTCGGCCAATCCATGTTTACATGCCGGGTTGATCACAGAGGGTTGACCTTCCAACAGAACGCATCCAGTATGTGCGTTCCAGAT SEQ ID NO:120 Nucleic acid sequence of dHLX protein GGAGAACTGGAGGAATTACTTAAACATCTCAAGGAGTTGCTCAAAGGCCCTAGGAAGGGAGAACTGGAGGAACTCCTCAAACATCTCAAGGAGTTACTAAAGGGA SEQ ID NO:121 mCCL19 ATGGCCCCGTGTGACCCCACTCCTGGCCTTCAGCCTGCTGGTTCTCTGGACCTTCCCAGCCCCAACTCTGGGGGGTGCTAATGATGCGGAAGACTGCTGCCTGTCTGTGACCCAGCGCCCCATCCCTGGGAACATCGTGAAAGCCTTCCGCTACCTTCTT AATGAAGATGGCTGCAGGGTGCCTGCTGTTGTGTTCACCACACTAAGGGGCTATCAGCTCTGTGCACCTCCTGACCAGCCCTGGGTGGATCGCATCATCCGAAGACTGAAGAAGTCTTCTGCCAAGAACAAAGGCAACAGCACCAGAAGGAGCCCTGTGTCT Embodiment Embodiment 1. A method for treating a subject having cancer, comprising: (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, e.g., a dimerization unit, and an antigen unit that comprises one or more cancer antigens or portions thereof; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a multimeric protein consisting of a plurality of polypeptides as defined in (ii), for example consisting of two polypeptides, for example a dimeric protein; and an anti-cancer vaccine comprising (b) administering to a subject one or more checkpoint inhibitors.

[0389] Embodiment 2. The method of embodiment 1, wherein the anti-cancer vaccine is a personalized anti-cancer vaccine.

[0390] Embodiment 3. The method of embodiment 2, wherein the antigenic unit comprises one or more neo-antigens or portions thereof.

[0391] Embodiment 4. The method of embodiment 3, wherein the antigenic unit comprises one or more portions of one or more neo-antigens.

[0392] Embodiment 5. The method of embodiment 4, wherein the moiety is a neoepitope.

[0393] Embodiment 6. The method of embodiment 5, wherein the antigenic unit comprises several neoepitopes, e.g. several neoepitopes separated from each other by linkers.

[0394] Embodiment 7. The method of embodiment 5 or 6, wherein the antigenic unit comprises n-1 antigenic subunits, each subunit comprising a neoepitope and a subunit linker, and a terminal neoepitope, where n is the number of neoepitopes in the antigenic unit, and n is an integer between 1 and 50.

[0395] Embodiment 8. The method of any of embodiments 5 to 7, wherein the neoepitope has a length of 7 to 30 amino acids, such as 7 to 10 amino acids (e.g., 7, 8, 9 or 10 amino acids) or 13 to 30 amino acids (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids), such as 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.

[0396] Embodiment 9. The method of any of embodiments 3 to 8, wherein the antigen unit further comprises a shared cancer antigen or a portion thereof present in one or more patients.

[0397] Embodiment 10. The method of embodiment 9, wherein the antigenic unit further comprises one or more portions of a shared cancer antigen present in one or more patients.

[0398] Embodiment 11 The method of embodiment 10, wherein the moiety is an epitope.

[0399] Embodiment 12 The method of embodiment 11, wherein the antigenic unit further comprises several epitopes.

[0400] Embodiment 13. The method of any of embodiments 9 to 12, wherein the shared cancer antigen present in the patient is selected from the group consisting of overexpressed or aberrantly expressed human cellular proteins, cancer-testis antigens, differentiation antigens, viral antigens, mutated cancer genes, mutated tumor suppressor genes, carcinoembryonic antigens, shared intron-retained antigens, shared antigens caused by frameshift mutations, dark matter antigens, and shared antigens caused by spliceosome mutations.

[0401] Embodiment 14 The method of embodiment 2, wherein the antigenic unit comprises a shared cancer antigen or a portion thereof present in one or more patients.

[0402] Embodiment 15. The method of embodiment 14, wherein the antigenic unit comprises one or more portions of a shared cancer antigen present in one or more patients.

[0403] Embodiment 16 The method of embodiment 15, wherein the moiety is an epitope.

[0404] Embodiment 17 The method of embodiment 16, wherein the antigenic unit comprises several epitopes.

[0405] Embodiment 18. The method according to any of embodiments 14 to 17, wherein the shared cancer antigen present in the patient is selected from the group consisting of overexpressed or aberrantly expressed human cellular proteins, cancer-testis antigens, differentiation antigens, viral antigens, mutated cancer genes, mutated tumor suppressor genes, carcinoembryonic antigens, shared intron-retained antigens, shared antigens caused by frameshift mutations, dark matter antigens, and shared antigens caused by spliceosome mutations.

[0406] Embodiment 19. The method of embodiment 1, wherein the anti-cancer vaccine is a non-personalized anti-cancer vaccine.

[0407] Embodiment 20. The method of embodiment 19, wherein the antigenic unit comprises one or more shared cancer antigens or portions thereof.

[0408] Embodiment 21. The method of embodiment 20, wherein the antigenic unit comprises one or more portions of one or more shared cancer antigens.

[0409] Embodiment 22 The method of embodiment 21, wherein the moiety is an epitope.

[0410] Embodiment 23 The method of embodiment 22, wherein the antigenic unit comprises several epitopes.

[0411] Embodiment 24. The method according to any one of embodiments 20 to 23, wherein the shared cancer antigen is selected from the group consisting of overexpressed or aberrantly expressed human cellular proteins, cancer-testis antigens, differentiation antigens, viral antigens, mutated cancer genes, mutated tumor suppressor genes, carcinoembryonic antigens, shared intron-retained antigens, shared antigens caused by frameshift mutations, dark matter antigens, and shared antigens caused by spliceosome mutations, scFvs derived from monoclonal Igs produced by myelomas or lymphomas, telomerase, HIV antigens, tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2, melanoma antigens, prostate-specific antigens, and HPV antigens.

[0412] Embodiment 25. The method according to any one of embodiments 1 to 24, wherein the antigen unit comprises a maximum of 3500 amino acids, for example about 21 to about 2000 amino acids or about 60 to 3500 amino acids, for example about 80 or about 100 or about 150 amino acids to about 3000 amino acids, for example about 200 to about 2500 amino acids, for example about 300 to about 2000 amino acids or about 400 to about 1500 amino acids or about 500 to about 1000 amino acids.

[0413] Embodiment 26 The method of any of embodiments 1 to 25, wherein the targeting unit is or comprises a moiety that interacts with a surface molecule on an antigen-presenting cell.

[0414] Embodiment 27. The method of embodiment 26, wherein the surface molecule is selected from the group consisting of HLA, CD14, CD40, CLEC9A, a chemokine receptor, such as CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8 or XCR1, and a Toll-like receptor, such as TLR-2, TLR-4 or TLR-5.

[0415] Embodiment 28. The method of embodiments 26 and 27, wherein the targeting unit comprises or consists of soluble CD40 ligand, CCL4 and its isoforms, CCL5, CCL19, CCL20, CCL21, macrophage proinflammatory protein alpha including its isoforms, such as mouse CCL3, human CCL3, human CCL3L1, human CCL3L2 and human CCL3L3, XCL1, XCL2, flagellin, anti-HLA-DP, anti-HLA-DR, anti-pan HLA class II anti-CD40, anti-TLR-2, anti-TLR-4, anti-TLR-5 or anti-CLEC9A.

[0416] Embodiment 29. The method of embodiment 28, wherein the targeting unit comprises or consists of human MIP-1α (LD78β, CCL3L1).

[0417] Embodiment 30. The method of embodiment 29, wherein the targeting unit comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence 24-93 of SEQ ID NO:1, for example comprising amino acid sequence 26-93 of SEQ ID NO:1, or comprising amino acid sequence 28-93 of SEQ ID NO:1.

[0418] Embodiment 31. The method of embodiment 30, wherein the targeting unit consists of an amino acid sequence having at least 80% sequence identity to amino acid sequence 24 to 93 of SEQ ID NO:1, for example consisting of amino acid sequence 26 to 93 of SEQ ID NO:1, or consisting of amino acid sequence 28 to 93 of SEQ ID NO:1.

[0419] Embodiment 32. The method of embodiment 31, wherein the targeting unit consists of amino acid sequence 24 to 93 of SEQ ID NO:1.

[0420] Embodiment 33. The method of any one of embodiments 1 to 32, wherein the multimerization unit, e.g., the dimerization unit, comprises a hinge region, e.g., hinge exon h1 and hinge exon h4.

[0421] Embodiment 34 The method of embodiment 33, wherein the hinge region has the ability to form one or more covalent bonds.

[0422] Embodiment 35 The method of embodiment 33 or 34, wherein the hinge region is from an Ig.

[0423] Embodiment 36 The method of any one of embodiments 33 to 35, wherein the anti-cancer vaccine comprises a dimerization unit, the dimerization unit further comprising another domain that promotes dimerization.

[0424] Embodiment 37 The method of embodiment 36, wherein the other domain is an immunoglobulin domain, preferably an immunoglobulin constant domain.

[0425] Embodiment 38 The method according to embodiment 36 or 37, wherein the other domain is the carboxy-terminal C domain from an IgG, preferably an IgG3.

[0426] Embodiment 39. The method of any one of embodiments 36 to 38, wherein the dimerization unit further comprises a dimerization unit linker, such as a glycine-serine rich linker, such as GGGSSGGGSG (SEQ ID NO: 118).

[0427] Embodiment 40 The method of embodiment 39, wherein the dimerization unit linker connects the hinge region and another domain that promotes dimerization.

[0428] Embodiment 41. The method of any of embodiments 36 to 40, wherein the dimerization unit comprises hinge exon h1 and hinge exon h4, a dimerization unit linker, and the CH3 domain of human IgG3.

[0429] Embodiment 42. The method of embodiment 41, wherein the dimerization unit comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence 94 to 237 of SEQ ID NO:1.

[0430] Embodiment 43. The method according to embodiment 42, wherein the dimerization unit consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence 94 to 237 of SEQ ID NO:1.

[0431] Embodiment 44. The method according to embodiment 43, wherein the dimerization unit consists of the amino acid sequence 94 to 237 of SEQ ID NO:1.

[0432] Embodiment 45. The method according to any one of embodiments 1 to 44, wherein the anti-cancer vaccine further comprises a unit linker linking the antigen unit to a multimerization unit, such as a dimerization unit, wherein the unit linker is a non-immunogenic linker and / or a flexible or rigid linker.

[0433] Embodiment 46. The method of any one of embodiments 1 to 45, wherein the anti-cancer vaccine is a polynucleotide, preferably RNA or DNA.

[0434] Embodiment 47. The method of embodiment 46, wherein the polynucleotide further comprises a nucleotide sequence encoding a signal peptide.

[0435] Embodiment 48. The method of embodiment 47, wherein the signal peptide is selected from the group consisting of an Ig VH signal peptide, a human TPA signal peptide, and a human MIP-1α signal peptide.

[0436] Embodiment 49. The method of embodiment 47 or 48, wherein the signal peptide comprises an amino acid sequence having at least 85% sequence identity to amino acid sequence 1 to 23 of SEQ ID NO:1.

[0437] Embodiment 50. The method according to embodiment 49, wherein the signal peptide consists of an amino acid sequence having at least 85% sequence identity to amino acid sequence 1 to 23 of SEQ ID NO:1.

[0438] Embodiment 51. The method according to embodiment 50, wherein the signal peptide consists of amino acid sequence 1 to 23 of SEQ ID NO:1.

[0439] Embodiment 52. The method of any one of embodiments 46 to 51, wherein the polynucleotide is DNA.

[0440] Embodiment 53 The method of embodiment 52, wherein the polynucleotide is DNA contained in a vector.

[0441] Embodiment 54. The method of any one of embodiments 1 to 53, wherein the anti-cancer vaccine further comprises a pharma- ceutically acceptable carrier or diluent.

[0442] Embodiment 55. The method of any one of embodiments 1 to 45, wherein the anti-cancer vaccine is a polypeptide or a multimeric protein, such as a dimeric protein.

[0443] Embodiment 56 The method of embodiment 55, wherein the anti-cancer vaccine further comprises a pharma- ceutically acceptable carrier or diluent.

[0444] Embodiment 57 The method of embodiment 56, wherein the anticancer vaccine further comprises an adjuvant.

[0445] Embodiment 58. The one or more check point inhibitors are selected from the group consisting of an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-CTLA-4 antibody, and an anti-PD1 antibody, e.g., the check point inhibitor is an anti-PD-L1 antibody or an anti-TIGIT antibody or an anti-CTLA-4 antibody or an anti-PD1 antibody, or the check point inhibitor is an anti-PD-L1 antibody and an anti-TIGIT antibody or an anti-PD-L1 antibody and an anti-CTLA-4 antibody or an anti-PD-1 antibody and an anti-TIGIT antibody or an anti-PD-1 antibody and an anti-CTLA-4 antibody or an anti-TIGIT antibody and and an anti-CTLA-4 antibody or an anti-PD-L1 antibody and an anti-PD-1 antibody; or the checkpoint inhibitor is an anti-PD-L1 antibody, an anti-PD-1 antibody and an anti-CTLA-4 antibody or an anti-PD-L1 antibody, an anti-PD-1 antibody and an anti-TIGIT antibody or an anti-PD-L1 antibody, an anti-CTLA-4 antibody and an anti-TIGIT antibody or an anti-PD-1 antibody, an anti-CTLA-4 antibody and an anti-TIGIT antibody; or the checkpoint inhibitor is an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-CTLA-4 antibody and an anti-PD1 antibody.

[0446] Embodiment 59. The method of embodiment 58, wherein the checkpoint inhibitor is an anti-PD-L1 antibody.

[0447] Embodiment 60. The method of embodiment 58, wherein the checkpoint inhibitor is an anti-TIGIT antibody.

[0448] Embodiment 61. The method of embodiment 58, wherein the checkpoint inhibitors are an anti-PD-L1 antibody and an anti-TIGIT antibody.

[0449] Embodiment 62. The method of embodiment 58, wherein the checkpoint inhibitor is an anti-CTLA-4 antibody.

[0450] Embodiment 63. The method of any of embodiments 1 to 62, wherein the one or more checkpoint inhibitors are contained in a composition suitable for injection, e.g., infusion injection.

[0451] Embodiment 64. The method of any one of embodiments 1 to 63, wherein the one or more checkpoint inhibitors are administered in parallel with an anti-cancer vaccine.

[0452] Embodiment 65. The method of any of embodiments 1-62, wherein the one or more checkpoint inhibitors are administered prior to the first administration of the anti-cancer vaccine.

[0453] Embodiment 66 The method of any of embodiments 1-62, wherein the anti-cancer vaccine is administered prior to the first administration of the one or more checkpoint inhibitors.

[0454] Embodiment 67. The method of any one of embodiments 1 to 66, wherein each of the anti-cancer vaccine and the one or more checkpoint inhibitors is administered in multiple doses.

[0455] Embodiment 68. The method of any one of embodiments 1 to 67, wherein the cancer is a solid cancer or a liquid cancer.

[0456] Embodiment 69. The method of any one of embodiments 1 to 68, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, bone cancer, colorectal cancer, gastric 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 adrenocortical carcinoma.

[0457] EMBODIMENT 70. (a) (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, e.g., a dimerization unit, and an antigen unit that comprises one or more cancer antigens; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a multimeric protein consisting of a plurality of polypeptides as defined in (ii), for example consisting of two polypeptides, for example a dimeric protein; and an anti-cancer vaccine comprising (b) one or more checkpoint inhibitors.

[0458] Embodiment 71 The kit of embodiment 70, wherein the anti-cancer vaccine is a personalized anti-cancer vaccine.

[0459] Embodiment 72. The kit according to embodiment 71, wherein the antigen unit comprises one or more neo-antigens or parts thereof.

[0460] Embodiment 73. The kit of embodiment 72, wherein the antigen unit comprises one or more portions of one or more neo-antigens.

[0461] Embodiment 74. The kit of embodiment 73, wherein the moiety is a neoepitope.

[0462] Embodiment 75. A kit according to embodiment 74, wherein the antigenic unit comprises several neoepitopes, for example several neoepitopes separated from each other by linkers.

[0463] Embodiment 76. A kit according to embodiments 74 and 75, wherein the antigenic unit comprises n-1 antigenic subunits, each subunit comprising a neoepitope and a subunit linker, and a terminal neoepitope, n being the number of neoepitopes in the antigenic unit, and n being an integer between 1 and 50.

[0464] Embodiment 77. A kit according to any of embodiments 74 to 76, wherein the neoepitope has a length of 7 to 30 amino acids, such as 7 to 10 amino acids (e.g. 7, 8, 9 or 10 amino acids) or 13 to 30 amino acids (e.g. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids), such as 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.

[0465] Embodiment 78. A kit according to any of embodiments 72 to 77, wherein the antigen unit further comprises a shared cancer antigen or a part thereof present in one or more patients.

[0466] Embodiment 79. The kit of embodiment 78, wherein the antigen unit further comprises one or more portions of a shared cancer antigen present in one or more patients.

[0467] Embodiment 80. The kit of embodiment 79, wherein the moiety is an epitope.

[0468] Embodiment 81. The kit according to embodiment 80, wherein the antigenic unit further comprises several epitopes.

[0469] Embodiment 82. A kit described in any of embodiments 78 to 81, wherein the shared cancer antigen present in the patient is selected from the group consisting of overexpressed or aberrantly expressed human cellular proteins, cancer-testis antigens, differentiation antigens, viral antigens, mutated cancer genes, mutated tumor suppressor genes, carcinoembryonic antigens, shared intron-retained antigens, shared antigens caused by frameshift mutations, dark matter antigens, and shared antigens caused by spliceosome mutations.

[0470] Embodiment 83. The kit of embodiment 71, wherein the antigen unit comprises a shared cancer antigen or a part thereof present in one or more patients.

[0471] Embodiment 84. The kit of embodiment 83, wherein the antigen unit comprises one or more portions of a shared cancer antigen present in one or more patients.

[0472] Embodiment 85. The kit of embodiment 84, wherein the moiety is an epitope.

[0473] Embodiment 86. The kit according to embodiment 85, wherein the antigenic unit comprises several epitopes.

[0474] Embodiment 87. A kit described in any of embodiments 83 to 86, wherein the shared cancer antigen present in the patient is selected from the group consisting of overexpressed or aberrantly expressed human cellular proteins, cancer-testis antigens, differentiation antigens, viral antigens, mutated cancer genes, mutated tumor suppressor genes, carcinoembryonic antigens, shared intron-retained antigens, shared antigens caused by frameshift mutations, dark matter antigens, and shared antigens caused by spliceosome mutations.

[0475] Embodiment 88. The kit of embodiment 70, wherein the anti-cancer vaccine is a non-personalized anti-cancer vaccine.

[0476] Embodiment 89. The kit of embodiment 88, wherein the antigen unit comprises one or more shared cancer antigens or parts thereof.

[0477] Embodiment 90. The kit of embodiment 89, wherein the antigen unit comprises one or more portions of one or more shared cancer antigens.

[0478] Embodiment 91. The kit of embodiment 90, wherein the moiety is an epitope.

[0479] Embodiment 92. The kit according to embodiment 91, wherein the antigen unit comprises several epitopes.

[0480] Embodiment 93. The kit according to any one of embodiments 89 to 92, wherein the shared cancer antigen is selected from the group consisting of overexpressed or aberrantly expressed human cell proteins, cancer-testis antigens, differentiation antigens, viral antigens, mutated cancer genes, mutated tumor suppressor genes, carcinoembryonic antigens, shared intron-retained antigens, shared antigens caused by frameshift mutations, dark matter antigens, and shared antigens caused by spliceosome mutations, scFvs derived from monoclonal Igs produced by myeloma or lymphoma, telomerase, HIV antigens, tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2, melanoma antigens, prostate-specific antigens, and HPV antigens.

[0481] Embodiment 94. A kit according to any of embodiments 70 to 93, wherein the antigen unit comprises up to 3500 amino acids, for example about 21 to about 2000 amino acids or about 60 to 3500 amino acids, for example about 80 or about 100 or about 150 amino acids to about 3000 amino acids, for example about 200 to about 2500 amino acids, for example about 300 to about 2000 amino acids or about 400 to about 1500 amino acids or about 500 to about 1000 amino acids.

[0482] Embodiment 95. A kit according to any of embodiments 70 to 96, wherein the targeting unit is or comprises a moiety that interacts with a surface molecule on an antigen-presenting cell.

[0483] Embodiment 96. The kit of embodiment 95, wherein the surface molecule is selected from the group consisting of HLA, CD14, CD40, CLEC9A, a chemokine receptor, such as CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8 or XCR1, and a Toll-like receptor, such as TLR-2, TLR-4 or TLR-5.

[0484] Embodiment 97. The kit according to embodiments 95 and 96, wherein the targeting unit comprises or consists of soluble CD40 ligand, CCL4 and its isoforms, CCL5, CCL19, CCL20, CCL21, macrophage proinflammatory protein alpha including its isoforms, such as mouse CCL3, human CCL3, human CCL3L1, human CCL3L2 and human CCL3L3, XCL1, XCL2, flagellin, anti-HLA-DP, anti-HLA-DR, anti-pan HLA class II anti-CD40, anti-TLR-2, anti-TLR-4, anti-TLR-5 or anti-CLEC9A.

[0485] Embodiment 98. The kit according to embodiment 97, wherein the targeting unit comprises or consists of human MIP-1α (LD78β, CCL3L1).

[0486] Embodiment 99. The kit described in embodiment 98, wherein the targeting unit comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence 24 to 93 of SEQ ID NO:1.

[0487] Embodiment 100. The kit described in embodiment 99, wherein the targeting unit consists of an amino acid sequence having at least 80% sequence identity to amino acid sequence 24 to 93 of SEQ ID NO:1.

[0488] Embodiment 101. The kit described in embodiment 100, wherein the targeting unit consists of amino acid sequence 24 to 93 of SEQ ID NO:1.

[0489] Embodiment 102. A kit according to any of embodiments 70 to 101, wherein the multimerization unit, e.g., the dimerization unit, comprises a hinge region, e.g., hinge exon h1 and hinge exon h4.

[0490] Embodiment 103. The kit of embodiment 102, wherein the hinge region has the ability to form one or more covalent bonds.

[0491] Embodiment 104. The kit of embodiment 102 or 103, wherein the hinge region is from an Ig.

[0492] Embodiment 105. A kit according to any one of embodiments 102 to 104, wherein the anti-cancer vaccine comprises a dimerization unit, which further comprises another domain that promotes dimerization.

[0493] Embodiment 106. The kit of embodiment 105, wherein the other domain is an immunoglobulin domain, preferably an immunoglobulin constant domain.

[0494] Embodiment 107. The kit according to embodiment 105 or 106, wherein the other domain is the carboxy-terminal C domain derived from IgG, preferably IgG3.

[0495] Embodiment 108. A kit according to any of embodiments 105 to 107, wherein the dimerization unit further comprises a dimerization unit linker, such as a glycine-serine rich linker, such as GGGSSGGGSG (SEQ ID NO: 118).

[0496] Embodiment 109. The kit according to embodiment 108, wherein the dimerization unit linker connects the hinge region and another domain that promotes dimerization.

[0497] Embodiment 110. A kit according to any of embodiments 105 to 109, wherein the dimerization unit comprises hinge exon h1 and hinge exon h4, a dimerization unit linker, and the CH3 domain of human IgG3.

[0498] Embodiment 111. The kit according to embodiment 110, wherein the dimerization unit comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence 94 to 237 of SEQ ID NO:1.

[0499] Embodiment 112. The kit according to embodiment 111, wherein the dimerization unit consists of an amino acid sequence having at least 80% sequence identity to amino acid sequence 94 to 237 of SEQ ID NO:1.

[0500] Embodiment 113. The kit according to embodiment 112, wherein the dimerization unit consists of amino acid sequence 94 to 237 of SEQ ID NO:1.

[0501] Embodiment 114. A kit according to any of embodiments 70 to 113, wherein the anticancer vaccine further comprises a unit linker linking the antigen unit to a multimerization unit, for example a dimerization unit, wherein the unit linker is a non-immunogenic linker and / or a flexible or rigid linker.

[0502] Embodiment 115. A kit according to any of embodiments 70 to 114, wherein the anticancer vaccine is a polynucleotide, preferably RNA or DNA.

[0503] Embodiment 116. The kit according to embodiment 115, wherein the polynucleotide further comprises a nucleotide sequence encoding a signal peptide.

[0504] Embodiment 117. The kit according to embodiment 116, wherein the signal peptide is selected from the group consisting of an Ig VH signal peptide, a human TPA signal peptide and a human MIP-1α signal peptide.

[0505] Embodiment 118. The kit according to embodiment 116 or 117, wherein the signal peptide comprises an amino acid sequence having at least 85% sequence identity to amino acid sequence 1 to 23 of SEQ ID NO:1.

[0506] Embodiment 119. The kit described in embodiment 118, wherein the signal peptide consists of an amino acid sequence having at least 85% sequence identity to amino acid sequence 1 to 23 of SEQ ID NO:1.

[0507] Embodiment 120. The kit according to embodiment 119, wherein the signal peptide consists of amino acid sequence 1 to 23 of SEQ ID NO:1.

[0508] Embodiment 121. A kit according to any one of embodiments 115 to 120, wherein the polynucleotide is DNA.

[0509] Embodiment 122. The kit of embodiment 121, wherein the polynucleotide is a DNA contained in a vector.

[0510] Embodiment 123. A kit according to any of embodiments 70 to 114, wherein the anticancer vaccine is a polypeptide or a multimeric protein, such as a dimeric protein.

[0511] Embodiment 124. A kit according to any of embodiments 70 to 123, wherein the kit further comprises one or more pharma- ceutically acceptable carriers or diluents.

[0512] Embodiment 125. A kit according to embodiment 124, comprising a first container comprising an anti-cancer vaccine and a second container comprising a pharma- ceutically acceptable carrier or diluent.

[0513] Embodiment 126. The kit according to embodiment 125, wherein the anticancer vaccine is a polypeptide or a multimeric protein, such as a dimeric protein, and the pharma- ceutically acceptable carrier or diluent further comprises an adjuvant.

[0514] Embodiment 127. A kit according to embodiment 125 or 126, wherein the kit comprises several such first containers and one or several such second containers.

[0515] Embodiment 128. The kit according to embodiment 124, comprising a container containing an anti-cancer vaccine and a pharma- ceutically acceptable carrier or diluent.

[0516] Embodiment 129. A kit according to embodiment 128, comprising several such containers.

[0517] Embodiment 130. The one or more check point inhibitors are selected from the group consisting of an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-CTLA-4 antibody, and an anti-PD1 antibody, e.g., the check point inhibitor is an anti-PD-L1 antibody or an anti-TIGIT antibody or an anti-CTLA-4 antibody or an anti-PD1 antibody, or the check point inhibitor is an anti-PD-L1 antibody and an anti-TIGIT antibody or an anti-PD-L1 antibody and an anti-CTLA-4 antibody or an anti-PD-1 antibody and an anti-TIGIT antibody or an anti-PD-1 antibody and an anti-CTLA-4 antibody or an anti-TIGIT antibody and The kit according to any of embodiments 70 to 129, wherein the checkpoint inhibitor is an anti-CTLA-4 antibody or an anti-PD-L1 antibody and an anti-PD-1 antibody; or the checkpoint inhibitor is an anti-PD-L1 antibody, an anti-PD-1 antibody and an anti-CTLA-4 antibody or an anti-PD-L1 antibody, an anti-PD-1 antibody and an anti-TIGIT antibody or an anti-PD-L1 antibody, an anti-CTLA-4 antibody and an anti-TIGIT antibody or an anti-PD-1 antibody, an anti-CTLA-4 antibody and an anti-TIGIT antibody; or the checkpoint inhibitor is an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-CTLA-4 antibody and an anti-PD1 antibody.

[0518] Embodiment 131. The kit of embodiment 130, wherein the checkpoint inhibitor is an anti-PD-L1 antibody.

[0519] Embodiment 132. The kit of embodiment 130, wherein the checkpoint inhibitor is an anti-TIGIT antibody.

[0520] Embodiment 133. The kit of embodiment 130, wherein the checkpoint inhibitors are an anti-PD-L1 antibody and an anti-TIGIT antibody.

[0521] Embodiment 134. The kit of embodiment 130, wherein the checkpoint inhibitor is an anti-CTLA-4 antibody.

[0522] Embodiment 135. A kit according to any of embodiments 70 to 134, comprising a container containing a checkpoint inhibitor and a container containing a liquid suitable for injection into a subject, e.g., for infusion injection into a subject.

[0523] Embodiment 136. A kit according to embodiment 135, comprising several containers each containing a checkpoint inhibitor, and a container containing a liquid suitable for injection into a subject, e.g., for infusion injection into a subject.

[0524] Embodiment 137. A kit according to embodiment 135, comprising several containers each containing a checkpoint inhibitor and several containers containing a liquid suitable for injection into a subject, for example for infusion injection into a subject.

[0525] Embodiment 138. A kit according to any of embodiments 70 to 134, comprising a container containing a checkpoint inhibitor and a liquid suitable for injection into a subject, e.g., for infusion injection into a subject.

[0526] Embodiment 139. A kit according to embodiment 138, comprising several containers.

[0527] Embodiment 140. A kit according to any one of embodiments 70 to 139, wherein the kit further comprises instructions for use.

[0528] Embodiment 141. The kit of embodiment 140, wherein the instructions for use include instructions for reconstitution of (a) and (b), and / or instructions for determining suitable doses of (a) and (b), and / or instructions for administration of (a) and (b), and / or instructions for the frequency and schedule for administering (a) and (b).

[0529] Embodiment 142. A kit described in any of embodiments 70 to 141 for use in a method of treating a subject having cancer, the method comprising administering to the subject an anti-cancer vaccine and one or more checkpoint inhibitors contained in the kit.

[0530] EMBODIMENT 143. (a) an anti-cancer vaccine contained in one or more first containers, (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, e.g., a dimerization unit, and an antigen unit that comprises one or more cancer antigens or portions thereof; or (ii) a polypeptide encoded by a polynucleotide defined in (i); or (iii) a multimeric protein consisting of a plurality of polypeptides as defined in (ii), for example consisting of two polypeptides, for example a dimeric protein; and an anti-cancer vaccine comprising: (b) one or more checkpoint inhibitors contained in one or more second containers.

[0531] Embodiment 144. The kit of embodiment 143, wherein the anti-cancer vaccine is a personalized anti-cancer vaccine.

[0532] Embodiment 145. The kit according to embodiment 144, wherein the antigen unit comprises one or more neo-antigens or parts thereof.

[0533] Embodiment 146. The kit according to embodiment 145, wherein the antigen unit comprises one or more portions of one or more neo-antigens.

[0534] Embodiment 147. The kit of embodiment 146, wherein the moiety is a neoepitope.

[0535] Embodiment 148. The kit according to embodiment 147, wherein the antigenic unit comprises several neoepitopes, for example several neoepitopes separated from each other by linkers.

[0536] Embodiment 149. A kit according to embodiments 147 and 148, wherein the antigenic unit comprises n-1 antigenic subunits, each subunit comprising a neoepitope and a subunit linker, and a terminal neoepitope, where n is the number of neoepitopes in the antigenic unit, and n is an integer from 1 to 50.

[0537] Embodiment 150. The kit according to any of embodiments 147 to 149, wherein the neoepitope has a length of 7 to 30 amino acids, such as 7 to 10 amino acids (e.g. 7, 8, 9 or 10 amino acids) or 13 to 30 amino acids (e.g. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids), such as 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.

[0538] Embodiment 151. A kit according to any of embodiments 145 to 150, wherein the antigen unit further comprises a shared cancer antigen or a part thereof present in one or more patients.

[0539] Embodiment 152. The kit of embodiment 151, wherein the antigen unit further comprises one or more portions of a shared cancer antigen present in one or more patients.

[0540] Embodiment 153. The kit of embodiment 152, wherein the moiety is an epitope.

[0541] Embodiment 154. The kit according to embodiment 153, wherein the antigenic unit further comprises several epitopes.

[0542] Embodiment 155. A kit described in any of embodiments 151 to 154, wherein the shared cancer antigen present in the patient is selected from the group consisting of overexpressed or aberrantly expressed human cellular proteins, cancer-testis antigens, differentiation antigens, viral antigens, mutated cancer genes, mutated tumor suppressor genes, carcinoembryonic antigens, shared intron-retained antigens, shared antigens caused by frameshift mutations, dark matter antigens, and shared antigens caused by spliceosome mutations.

[0543] Embodiment 156. The kit of embodiment 144, wherein the antigen unit comprises a shared cancer antigen or a part thereof present in one or more patients.

[0544] Embodiment 157. The kit of embodiment 156, wherein the antigen unit comprises one or more portions of a shared cancer antigen present in one or more patients.

[0545] Embodiment 158. The kit of embodiment 157, wherein the moiety is an epitope.

[0546] Embodiment 159. The kit according to embodiment 158, wherein the antigen unit comprises several epitopes.

[0547] Embodiment 160. A kit described in any of embodiments 156 to 159, wherein the shared cancer antigen present in the patient is selected from the group consisting of overexpressed or aberrantly expressed human cellular proteins, cancer-testis antigens, differentiation antigens, viral antigens, mutated cancer genes, mutated tumor suppressor genes, carcinoembryonic antigens, shared intron-retained antigens, shared antigens caused by frameshift mutations, dark matter antigens, and shared antigens caused by spliceosome mutations.

[0548] Embodiment 161 The kit of embodiment 143, wherein the anticancer vaccine is a non-personalized anticancer vaccine.

[0549] Embodiment 162. The kit of embodiment 161, wherein the antigen unit comprises one or more shared cancer antigens or parts thereof.

[0550] Embodiment 163. The kit of embodiment 162, wherein the antigen unit comprises one or more portions of one or more shared cancer antigens.

[0551] Embodiment 164. The kit of embodiment 163, wherein the moiety is an epitope.

[0552] Embodiment 165. The kit according to embodiment 164, wherein the antigen unit comprises several epitopes.

[0553] Embodiment 166. The kit according to any one of embodiments 162 to 165, wherein the shared cancer antigen is selected from the group consisting of overexpressed or aberrantly expressed human cell proteins, cancer-testis antigens, differentiation antigens, viral antigens, mutated cancer genes, mutated tumor suppressor genes, carcinoembryonic antigens, shared intron-retained antigens, shared antigens caused by frameshift mutations, dark matter antigens, and shared antigens caused by spliceosome mutations, scFvs derived from monoclonal Igs produced by myelomas or lymphomas, telomerase, HIV antigens, tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2, melanoma antigens, prostate-specific antigens, and HPV antigens.

[0554] Embodiment 167. A kit according to any of embodiments 143 to 166, wherein the antigen unit comprises up to 3500 amino acids, for example about 21 to about 2000 amino acids or about 60 to 3500 amino acids, for example about 80 or about 100 or about 150 amino acids to about 3000 amino acids, for example about 200 to about 2500 amino acids, for example about 300 to about 2000 amino acids or about 400 to about 1500 amino acids or about 500 to about 1000 amino acids.

[0555] Embodiment 168. A kit according to any of embodiments 143 to 167, wherein the targeting unit is or comprises a moiety that interacts with a surface molecule on an antigen-presenting cell.

[0556] Embodiment 169. The kit of embodiment 168, wherein the surface molecule is selected from the group consisting of HLA, CD14, CD40, CLEC9A, chemokine receptors, such as CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8 or XCR1, and Toll-like receptors, such as TLR-2, TLR-4 or TLR-5.

[0557] Embodiment 170. The kit according to embodiments 168 and 169, wherein the targeting unit comprises or consists of soluble CD40 ligand, CCL4 and its isoforms, CCL5, CCL19, CCL20, CCL21, macrophage proinflammatory protein alpha including its isoforms, such as mouse CCL3, human CCL3, human CCL3L1, human CCL3L2 and human CCL3L3, XCL1, XCL2, flagellin, anti-HLA-DP, anti-HLA-DR, anti-pan HLA class II anti-CD40, anti-TLR-2, anti-TLR-4, anti-TLR-5 or anti-CLEC9A.

[0558] Embodiment 171. The kit according to embodiment 170, wherein the targeting unit comprises or consists of human MIP-1α (LD78β, CCL3L1).

[0559] Embodiment 172. The kit described in embodiment 171, wherein the targeting unit comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence 24 to 93 of SEQ ID NO:1.

[0560] Embodiment 173. The kit described in embodiment 172, wherein the targeting unit consists of an amino acid sequence having at least 80% sequence identity to amino acid sequence 24 to 93 of SEQ ID NO:1.

[0561] Embodiment 174. The kit described in embodiment 173, wherein the targeting unit consists of amino acid sequence 24 to 93 of SEQ ID NO:1.

[0562] Embodiment 175. A kit according to any of embodiments 143 to 174, wherein the multimerization unit, e.g. the dimerization unit, comprises a hinge region, e.g. hinge exon h1 and hinge exon h4.

[0563] Embodiment 176. The kit of embodiment 175, wherein the hinge region is capable of forming one or more covalent bonds.

[0564] Embodiment 177. The kit of embodiment 175 or 176, wherein the hinge region is from an Ig.

[0565] Embodiment 178. A kit according to any of embodiments 175 to 177, wherein the anticancer vaccine comprises a dimerization unit, which further comprises another domain that promotes dimerization.

[0566] Embodiment 179. The kit according to embodiment 178, wherein the other domain is an immunoglobulin domain, preferably an immunoglobulin constant domain.

[0567] Embodiment 180. The kit according to embodiment 178 or 179, wherein the other domain is the carboxy-terminal C domain derived from IgG, preferably IgG3.

[0568] Embodiment 181. A kit according to any of embodiments 178 to 180, wherein the dimerization unit further comprises a dimerization unit linker, such as a glycine-serine rich linker, such as GGGSSGGGSG (SEQ ID NO: 118).

[0569] Embodiment 182. The kit according to embodiment 181, wherein the dimerization unit linker connects the hinge region and another domain that promotes dimerization.

[0570] Embodiment 183. A kit according to any of embodiments 178 to 182, wherein the dimerization unit comprises hinge exon h1 and hinge exon h4, a dimerization unit linker, and the CH3 domain of human IgG3.

[0571] Embodiment 184. The kit according to embodiment 183, wherein the dimerization unit comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence 94 to 237 of SEQ ID NO:1.

[0572] Embodiment 185. The kit according to embodiment 184, wherein the dimerization unit consists of an amino acid sequence having at least 80% sequence identity to amino acid sequence 94 to 237 of SEQ ID NO:1.

[0573] Embodiment 186. The kit according to embodiment 185, wherein the dimerization unit consists of amino acid sequence 94 to 237 of SEQ ID NO:1.

[0574] Embodiment 187. A kit according to any of embodiments 143 to 186, wherein the anticancer vaccine further comprises a unit linker linking the antigen unit to a multimerization unit, for example a dimerization unit, wherein the unit linker is a non-immunogenic linker and / or a flexible or rigid linker.

[0575] Embodiment 188. A kit according to any of embodiments 143 to 187, wherein the anticancer vaccine is a polynucleotide, preferably RNA or DNA.

[0576] Embodiment 189. The kit of embodiment 188, wherein the polynucleotide further comprises a nucleotide sequence encoding a signal peptide.

[0577] Embodiment 190. The kit of embodiment 189, wherein the signal peptide is selected from the group consisting of an Ig VH signal peptide, a human TPA signal peptide, and a human MIP-1α signal peptide.

[0578] Embodiment 191. The kit according to embodiment 189 or 190, wherein the signal peptide comprises an amino acid sequence having at least 85% sequence identity to amino acid sequence 1 to 23 of SEQ ID NO:1.

[0579] Embodiment 192. The kit according to embodiment 191, wherein the signal peptide consists of an amino acid sequence having at least 85% sequence identity to amino acid sequence 1 to 23 of SEQ ID NO:1.

[0580] Embodiment 193. The kit according to embodiment 192, wherein the signal peptide consists of amino acid sequence 1 to 23 of SEQ ID NO:1.

[0581] Embodiment 194. A kit according to any one of embodiments 188 to 193, wherein the polynucleotide is DNA.

[0582] Embodiment 195. The kit of embodiment 194, wherein the polynucleotide is a DNA contained in a vector.

[0583] Embodiment 196. A kit according to any of embodiments 143 to 195, wherein the anticancer vaccine is a polypeptide or a multimeric protein, such as a dimeric protein.

[0584] Embodiment 197. The one or more checkpoint inhibitors are selected from the group consisting of an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-CTLA-4 antibody, and an anti-PD1 antibody, e.g., the checkpoint inhibitor is an anti-PD-L1 antibody or an anti-TIGIT antibody or an anti-CTLA-4 antibody or an anti-PD1 antibody, or the checkpoint inhibitor is an anti-PD-L1 antibody and an anti-TIGIT antibody or an anti-PD-L1 antibody and an anti-CTLA-4 antibody or an anti-PD-1 antibody and an anti-TIGIT antibody or an anti-PD-1 antibody and an anti-CTLA-4 antibody or an anti-TIGIT antibody and an anti-PD-1 antibody. The kit according to any of embodiments 143 to 196, wherein the checkpoint inhibitor is an anti-PD-L1 antibody, an anti-PD-1 antibody and an anti-CTLA-4 antibody or an anti-PD-L1 antibody, an anti-PD-1 antibody and an anti-TIGIT antibody or an anti-PD-L1 antibody, an anti-CTLA-4 antibody and an anti-TIGIT antibody or an anti-PD-1 antibody, an anti-CTLA-4 antibody and an anti-TIGIT antibody, or the checkpoint inhibitor is an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-CTLA-4 antibody and an anti-PD-1 antibody.

[0585] Embodiment 198. The kit of embodiment 197, wherein the checkpoint inhibitor is an anti-PD-L1 antibody.

[0586] Embodiment 199. The kit of embodiment 197, wherein the checkpoint inhibitor is an anti-TIGIT antibody.

[0587] Embodiment 200. The kit of embodiment 197, wherein the checkpoint inhibitors are an anti-PD-L1 antibody and an anti-TIGIT antibody.

[0588] Embodiment 201. The kit of embodiment 197, wherein the checkpoint inhibitor is an anti-CTLA-4 antibody.

[0589] Embodiment 202. A kit according to any of embodiments 143 to 201, wherein the anti-cancer vaccine further comprises a pharma- ceutically acceptable carrier or diluent, and the one or more checkpoint inhibitors are contained in a composition suitable for injection, e.g., infusion injection.

[0590] Embodiment 203. A kit according to any of embodiments 143 to 202, wherein the kit further comprises one or more third containers comprising a pharma- ceutically acceptable carrier or diluent.

[0591] Embodiment 204. A kit described in any of embodiments 143 to 203, wherein the kit further comprises instructions for use, e.g., instructions for reconstituting the doses of (a) and (b), and / or instructions for determining suitable doses of (a) and (b), and / or instructions for administration of (a) and (b), and / or instructions for the frequency and schedule for administering (a) and (b). Embodiment 205. A kit described in any of embodiments 143 to 204 for use in a method of treating a subject having cancer, the method comprising administering to the subject an anti-cancer vaccine and one or more checkpoint inhibitors contained in the kit.

[0592] Embodiment 205. A method for treating a subject having cancer, comprising: (a) a personalized DNA anti-cancer vaccine, (i) a DNA polynucleotide, e.g., VB10.NEO, comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a dimerization unit, and an antigen unit that comprises one or more neoepitopes; Individualized DNA anti-cancer vaccines and (b) administering to the subject an anti-PD-L1 antibody, such as atezolizumab.

[0593] Embodiment 206 The method of embodiment 205, wherein the targeting unit is or comprises a moiety that interacts with a surface molecule on an antigen-presenting cell.

[0594] Embodiment 207. The method of embodiment 206, wherein the surface molecule is selected from the group consisting of HLA, CD14, CD40, CLEC9A, a chemokine receptor, such as CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8 or XCR1, and a Toll-like receptor, such as TLR-2, TLR-4 or TLR-5.

[0595] Embodiment 208. The method according to embodiments 206 and 207, wherein the targeting unit comprises or consists of soluble CD40 ligand, CCL4 and its isoforms, CCL5, CCL19, CCL20, CCL21, macrophage proinflammatory protein alpha including its isoforms, such as mouse CCL3, human CCL3, human CCL3L1, human CCL3L2 and human CCL3L3, XCL1, XCL2, flagellin, anti-HLA-DP, anti-HLA-DR, anti-pan HLA class II anti-CD40, anti-TLR-2, anti-TLR-4, anti-TLR-5 or anti-CLEC9A.

[0596] Embodiment 209. The method of embodiment 208, wherein the targeting unit comprises or consists of human MIP-1α (LD78β, CCL3L1).

[0597] Embodiment 210. The method of embodiment 209, wherein the targeting unit comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence 24 to 93 of SEQ ID NO:1, for example comprising amino acid sequence 26 to 93 of SEQ ID NO:1, or comprising amino acid sequence 28 to 93 of SEQ ID NO:1.

[0598] Embodiment 211. The method described in embodiment 210, wherein the targeting unit consists of an amino acid sequence having at least 80% sequence identity to amino acid sequence 24 to 93 of SEQ ID NO: 1, for example consisting of amino acid sequence 26 to 93 of SEQ ID NO: 1 or consisting of amino acid sequence 28 to 93 of SEQ ID NO: 1.

[0599] Embodiment 212. The method of embodiment 211, wherein the targeting unit consists of amino acid sequence 24 to 93 of SEQ ID NO:1.

[0600] Embodiment 213. The method of any of embodiments 205 to 212, wherein the dimerization unit comprises a hinge region, such as hinge exon h1 and hinge exon h4.

[0601] Embodiment 214 The method of embodiment 213, wherein the hinge region has the ability to form one or more covalent bonds.

[0602] Embodiment 215. The method of embodiment 213 or 214, wherein the hinge region is from an Ig.

[0603] Embodiment 216 The method of any of embodiments 213 to 215, wherein the dimerization unit further comprises another domain that promotes dimerization.

[0604] Embodiment 217. The method of embodiment 216, wherein the other domain is an immunoglobulin domain, preferably an immunoglobulin constant domain.

[0605] Embodiment 218. The method according to embodiment 216 or 217, wherein the other domain is the carboxy-terminal C domain derived from an IgG, preferably an IgG3.

[0606] Embodiment 219. The method of any of embodiments 216 to 218, wherein the dimerization unit further comprises a dimerization unit linker, such as a glycine-serine rich linker, such as GGGSSGGGSG (SEQ ID NO: 118).

[0607] Embodiment 220 The method of embodiment 219, wherein the dimerization unit linker connects the hinge region and another domain that promotes dimerization.

[0608] Embodiment 221. The method of any of embodiments 216 to 220, wherein the dimerization unit comprises hinge exon h1 and hinge exon h4, a dimerization unit linker, and the CH3 domain of human IgG3.

[0609] Embodiment 222. The method of embodiment 221, wherein the dimerization unit comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence 94 to 237 of SEQ ID NO:1.

[0610] Embodiment 223. The method according to embodiment 222, wherein the dimerization unit consists of an amino acid sequence having at least 80% sequence identity to amino acid sequence 94 to 237 of SEQ ID NO:1.

[0611] Embodiment 224. The method according to embodiment 223, wherein the dimerization unit consists of the amino acid sequence 94 to 237 of SEQ ID NO:1.

[0612] Embodiment 225. The method according to any one of embodiments 205 to 224, wherein the anti-cancer vaccine further comprises a unit linker linking the antigen unit to the dimerization unit, wherein the unit linker is a non-immunogenic linker and / or a flexible or rigid linker.

[0613] Embodiment 226. The method of any of embodiments 205 to 225, wherein the DNA further comprises a nucleotide sequence encoding a signal peptide.

[0614] Embodiment 227. The method of embodiment 226, wherein the signal peptide is selected from the group consisting of an Ig VH signal peptide, a human TPA signal peptide, and a human MIP-1α signal peptide.

[0615] Embodiment 228. The method according to embodiment 226 or 227, wherein the signal peptide comprises an amino acid sequence having at least 85% sequence identity to amino acid sequence 1 to 23 of SEQ ID NO:1.

[0616] Embodiment 229. The method according to embodiment 228, wherein the signal peptide consists of an amino acid sequence having at least 85% sequence identity to amino acid sequence 1 to 23 of SEQ ID NO:1.

[0617] Embodiment 230. The method of embodiment 229, wherein the signal peptide consists of amino acid sequence 1 to 23 of SEQ ID NO:1.

[0618] Embodiment 231. The method of any one of embodiments 205 to 23, wherein the DNA polynucleotide comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 80% sequence identity with amino acid sequence 1 to 242 of SEQ ID NO:1, for example a polypeptide comprising amino acid sequence 1 to 242 of SEQ ID NO:1.

[0619] Embodiment 232. The method of embodiment 231, wherein the DNA polynucleotide comprises a nucleotide sequence encoding a polypeptide consisting of an amino acid sequence having at least 80% sequence identity to amino acid sequence 1 to 242 of SEQ ID NO:1, for example a polypeptide consisting of amino acid sequence 1 to 242 of SEQ ID NO:1.

[0620] Embodiment 233. A method according to any of embodiments 205 to 232, wherein the DNA polynucleotide comprises a constant portion and a variable portion, the constant portion consisting of a nucleotide sequence encoding a polypeptide consisting of amino acid sequence 1 to 242 of SEQ ID NO:1. Embodiment 234. The method of any one of embodiments 205 to 233, wherein the DNA polynucleotide is contained in a vector.

[0621] Embodiment 235. The method of any of embodiments 205 to 234, wherein the anti-cancer vaccine further comprises a pharma- ceutically acceptable carrier or diluent.

[0622] Embodiment 236 The method of any one of embodiments 205 to 235, wherein the anti-PD-L1 antibody is comprised in an aqueous liquid composition for injection, e.g., for infusion injection.

[0623] Embodiment 237. The method of any one of embodiments 205-236, wherein the anti-PD-L1 antibody is administered by infusion injection.

[0624] Embodiment 238. The method of any one of embodiments 205 to 237, wherein the anticancer vaccine is administered intramuscularly.

[0625] Embodiment 239. The method of any one of embodiments 205 to 237, wherein the cancer is a solid tumor.

[0626] Embodiment 240. A method for treating a subject having a solid tumor, comprising: (a) administering intramuscularly a personalized anti-cancer vaccine comprising a pharma- ceutically acceptable carrier and a DNA polynucleotide, e.g., VB10.NEO, wherein the DNA polynucleotide comprises a constant portion consisting of a nucleotide sequence encoding a polypeptide consisting of amino acid sequence 1-242 of SEQ ID NO:1, and a variable portion consisting of a nucleotide sequence encoding a polypeptide consisting of one or more neoepitopes separated by a linker; and (b) administering to the subject an anti-PD-L1 antibody, such as atezolizumab, by infusion injection. Embodiment 241. A method for treating a patient having cancer, comprising administering to the patient an effective amount of a personalized DNA anti-cancer vaccine comprising VB10.NEO in combination with atezolizumab; The patient has at least one of the following tumor types: melanoma, NSCLC, RCC, UC, HNSCC, TNBC, gastric / GEJ cancer, cervical cancer, anal cancer, or MSI-high tumor; Patients have locally advanced or metastatic tumors that have progressed after at least one available standard therapy, and / or standard therapy has proven ineffective or intolerable or is deemed inappropriate, and / or a clinical trial of an investigational agent is recognized as the standard of care; the effective amount is a) VB10.NEO (3 mg in a pharma- ceutical acceptable carrier) administered by intramuscular injection over an induction course Q3W (4 doses), followed by maintenance doses Q6W (6 doses) and Q12W (5 doses), and 1200 mg atezolizumab administered by intravenous infusion on day 1 of a 21 day cycle; or b) VB10.NEO (6 mg in a pharma-ceutical acceptable carrier) administered by intramuscular injection over an induction course Q3W (4 doses), followed by maintenance doses Q6W (6 doses) and Q12W (5 doses), and 1200 mg atezolizumab administered by intravenous infusion on day 1 of a 21 day cycle; The method, wherein the treatment method results in improved overall response rate (ORR) and / or improved duration of response (DOR) and / or improved progression free survival (PFS) and / or improved overall survival (OS) compared to a reference population, optionally wherein the reference population is a population treated with standard of care (SOC) for the tumor.

Claims

1. A pharmaceutical composition in the form of an anticancer vaccine for the treatment of cancer, comprising: (i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets an antigen-presenting cell, a multimerization unit, e.g., a dimerization unit, and an antigen unit that comprises one or more cancer antigens or portions thereof; or (ii) a polypeptide encoded by the polynucleotide defined in (i); or (iii) A pharmaceutical composition comprising a multimeric protein, e.g. a dimeric protein, consisting of a plurality of polypeptides, e.g. consisting of two polypeptides, as defined in (ii), which is administered in combination with one or more checkpoint inhibitors.

2. 2. The pharmaceutical composition according to claim 1, wherein the anti-cancer vaccine is an individualized anti-cancer vaccine, preferably wherein the antigenic unit comprises one or more neo-antigens or parts thereof, e.g. one or more neo-epitopes, more preferably wherein the antigenic unit comprises several neo-epitopes separated from each other by linkers.

3. The pharmaceutical composition of claim 2, wherein the antigenic unit comprises a shared cancer antigen or a part thereof, such as an epitope, such as several epitopes, present in one or more patients.

4. The pharmaceutical composition according to claim 1, wherein the anti-cancer vaccine is a non-personalized anti-cancer vaccine, preferably wherein the antigenic units comprise one or more shared cancer antigens or parts thereof, such as epitopes, such as several epitopes.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the one or more checkpoint inhibitors are selected from the group consisting of an anti-PD-L1 antibody, an anti-TIGIT antibody, and an anti-CTLA-4 antibody.

6. The pharmaceutical composition according to claim 5, wherein the checkpoint inhibitor is an anti-PD-L1 antibody and an anti-TIGIT antibody.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein said targeting unit comprises or consists of soluble CD40 ligand, CCL4 and its isoforms, CCL5, CCL19, CCL20, CCL21, macrophage proinflammatory protein alpha including its isoforms, such as murine CCL3, human CCL3, human CCL3L1, human CCL3L2 and human CCL3L3, XCL1, XCL2, flagellin, anti-HLA-DP, anti-HLA-DR, anti-pan HLA class II anti-CD40, anti-TLR-2, anti-TLR-4, anti-TLR-5 or anti-CLEC9A.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the anti-cancer vaccine comprises a polynucleotide which is RNA or DNA, preferably DNA, more preferably DNA contained in a vector, and preferably the polynucleotide further comprises a nucleotide sequence encoding a signal peptide.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the anti-cancer vaccine comprises a polynucleotide encoding a human MIP-1α signal peptide, a human MIP-1α targeting unit, a dimerization unit comprising a hinge region and an immunoglobulin domain, preferably a dimerization unit comprising hinge exon h1, hinge exon h4, a dimerization unit linker and the CH3 domain of human IgG3, and a unit linker, preferably wherein the polynucleotide is DNA.

10. The pharmaceutical composition described in claim 9, wherein the signal peptide comprises or consists of the amino acid sequence 1 to 23 of SEQ ID NO:1, the targeting unit comprises or consists of the amino acid sequence 24 to 93 of SEQ ID NO:1, and the dimerization unit comprises or consists of the amino acid sequence 94 to 237 of SEQ ID NO:

1.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the check point inhibitor is an anti-PD-L1 antibody, an anti-TIGIT antibody, or an anti-CTLA4 antibody, or the check point inhibitor is an anti-PD-L1 antibody and an anti-TIGIT antibody.

12. 12. The pharmaceutical composition of any one of claims 1 to 11, wherein the anti-cancer vaccine further comprises a pharma- ceutically acceptable carrier or diluent, and wherein the one or more checkpoint inhibitors are comprised in a composition suitable for injection, e.g. by infusion injection.

13. 13. The pharmaceutical composition of any one of claims 1 to 12, wherein the one or more check point inhibitors are administered in parallel with the anti-cancer vaccine or prior to the first administration of the anti-cancer vaccine, or the anti-cancer vaccine is administered prior to administration of the one or more check point inhibitors.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the cancer is a solid cancer or a liquid cancer, such as a cancer selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, bone cancer, colorectal cancer, gastric 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 adrenocortical carcinoma.

15. A kit comprising: (a) a pharmaceutical composition in the form of an anti-cancer vaccine according to any one of claims 1 to 12 contained in one or more first containers; and (b) one or more check point inhibitors contained in one or more second containers, characterized in that the one or more check point inhibitors are preferably selected from the group consisting of anti-PD-L1 antibodies, anti-TIGIT antibodies, and anti-CTLA-4 antibodies.

Images