TUMOR ANTIGENS, COMPOUNDS COMPRISING TUMOR ANTIGENS KRAS, TPX2 OR AURKA AND USES THEREOF

Abstract

The present invention relates to tumor antigens encoded in 5' upstream open reading frames (uORFs) within the 5' UTRs of different mRNAs. Compositions and peptides comprising such tumor antigens and viruses encoding such tumor antigens are provided. The present invention also relates to the use of such compositions, peptides and viruses in the treatment of cancer.

Description

[Technical field]

[0001] The present invention relates to the field of vaccination and immunotherapy, in particular to cancer immunotherapy. More specifically, the present invention relates to tumor antigens encoded in 5' upstream open reading frames (uORFs) within the 5' UTRs of different mRNAs. Compositions and peptides comprising such tumor antigens and viruses encoding such tumor antigens are provided. The present invention also relates to the use of such compositions, peptides and viruses in the treatment of cancer. [Background technology]

[0002] Although the immune system can recognize and eliminate tumor cells to some extent, this antitumor response is often weak and inefficient. Boosting this weak antitumor response by therapeutic vaccination has been a long-sought goal in cancer treatment. Therefore, modulating the immune system to enhance the immune response has become a promising therapeutic approach in oncology, since it can be combined with standard treatment procedures. Tumor cells generally express some antigens, such as tumor-associated antigens (TAA), viral antigens (oncoviruses) or mutation-derived antigens (neo-antigens). A variety of TAAs, viral antigens or neo-antigens expressed in cancer cells have been identified and used as targets for cancer vaccines. One approach to induce tumor-specific immune responses is peptide-based cancer vaccination, which includes administration of TAA, viral antigens or neo-antigens to treat cancer according to the nature of the tumor.

[0003] Tumor-associated antigens are usually present on normal cells, whereas tumor-specific antigens are present only on tumor cells. Tumor-specific antigens include antigens derived from mutations and antigens derived from cryptic open reading frames and / or frameshift mutations. Such antigens can be recognized as foreign antigens by the immune system, and therefore activate anti-tumor immune responses in animal tumor models and cancer patients. However, neo-antigens with tumor-specific mutations often require vaccines tailored to individual patients, which is cumbersome and costly. Recently, there has been an increased interest in so-called "dark antigens," which are peptides that are specifically expressed in tumors and are encoded in sequence regions previously considered to be "non-coding," such as the 5'UTR of mRNA. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide novel tumor antigens that are specifically expressed in the tumors of many patients. Such tumor antigens provide tumor specificity but do not require a cumbersome and costly individualized approach. It is also an object of the present invention to provide vaccines, in particular vaccines that can be used in heterologous prime-boost regimes, that combine such tumor antigens with additional advantageous features, such as multiple antigenic domains. This is particularly relevant because each human has a different set of MHC molecules, and the presentation of multiple peptides allows the presentation of the best (highest affinity) peptides in each HLA. This object is achieved by the subject matter described below and in the appended claims. The present invention is described in detail below, but it should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, which may vary. It should also be understood that the terms used herein are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0005] The components of the present invention are described below. Although these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to produce further embodiments. The various described examples and preferred embodiments should not be construed as limiting the present invention to only the explicitly described embodiments. This description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all described elements in this application should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0006] Throughout this specification and the claims that follow, unless the context requires otherwise, the term "comprise", as well as variations such as "comprises" and "comprising", will be understood to mean the inclusion of the stated members, integers or steps, but not the exclusion of other members, integers or steps that are not stated. The term "consisting of" is a specific embodiment of the term "comprising", in which any other members, integers or steps that are not specified are excluded. In the context of the present invention, the term "comprising" encompasses the term "consisting of". Thus, the term "comprising" encompasses "including" as well as "consisting", e.g., a composition "comprising" X may consist of only X, or may include something additional, e.g., X+Y.

[0007] The terms "a," "an," and "the," and similar references used in the context of describing the present invention (especially in the context of the claims) should be construed to include both the singular and the plural in scope, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention. The term "substantially" does not exclude "completely", for example a composition that is "substantially free" of Y may be completely free of Y. Where necessary, the word "substantially" may be omitted from the definition of the invention. The term "about" in reference to a number x means x±10%.

[0008] Throughout this specification, the terms "peptide", "polypeptide", "protein" and variations of these terms are used interchangeably. These terms refer to peptides, oligopeptides, or proteins, including fusion proteins, that preferably comprise at least two amino acids linked to each other by a ("normal") peptide bond. Alternatively, the amino acids may be linked to each other by modified peptide bonds, as in the case of isosteric peptides, for example. Thus, as used herein, the term "peptide" refers to relatively short (oligo)peptides as well as relatively long (polypeptides) and proteins, regardless of their length. "Classical" peptides, typically composed of amino acids selected from the 20 amino acids defined by the genetic code, linked to each other by normal peptide bonds, are preferred. However, in addition to these amino acids, peptides may also comprise or be composed of amino acids other than the 20 amino acids defined by the genetic code. In some embodiments, peptides may be composed of amino acids that have been modified by natural processes, such as post-translational maturation processes, or by chemical processes, well known to those skilled in the art. Modifications may occur anywhere in the peptide, i.e., in the peptide backbone, in the amino acid chain, or even at the carboxy or amino terminus of the peptide. Thus, the terms "peptide", "polypeptide" and "protein" also include modified peptides, polypeptides and proteins. For example, peptide modifications can include acetylation, acylation, ADP-ribosylation, amidation, covalent immobilization of a nucleotide or nucleotide derivative, covalent immobilization of a lipid or lipid derivative, covalent immobilization of phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, ceneroylation, sulfation, amino acid additions such as arginylation or ubiquitination.

[0009] As used herein (i.e. throughout the application), the term "sequence variant" refers to any alteration in a reference sequence. The term "sequence variant" includes nucleotide sequence variants and amino acid sequence variants. Preferably, the reference sequence is any of the sequences listed in the "Table of Sequences and SEQ ID NOs" (Sequence Listing), i.e. SEQ ID NO: 1 to SEQ ID NO: 51. In particular, the sequence variant has at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity with the reference sequence (over the entire length of the sequence). The sequence identity can be calculated as described below. In particular, the sequence variant usually retains certain functions of the reference sequence. In some embodiments, the amino acid sequence variant has an altered sequence in which one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acids in the reference sequence are deleted or substituted, or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acids are inserted or added to the sequence of the reference amino acid sequence. As a result of the alterations, the amino acid sequence variant has an amino acid sequence that is at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, and particularly preferably at least 99% identical to the reference sequence. For example, a variant sequence that is at least 90% identical has no more than 10 alterations, i.e., any combination of deletions, insertions or substitutions, per 100 amino acids of the reference sequence. Of course, the same applies to nucleic acid sequences as well.

[0010] For sequences that do not have an exact correspondence (amino acid or nucleic acid), the "percent identity" of the first sequence can be determined against the second sequence. In general, the two sequences to be compared can be aligned to obtain the maximum correlation between the sequences. This can include inserting "gaps" in one or both sequences to increase the degree of alignment. The percent identity can then be determined over the entire length of each of the sequences to be compared (so-called global alignment), which is particularly suitable for sequences that are identical or similar in length, or over a shorter defined length (so-called local alignment), which is more suitable for sequences that are not equal in length. Methods for comparing the identity and homology of two or more sequences are well known in the art. The percentage that two sequences are identical can be determined, for example, using a mathematical algorithm. A preferred but non-limiting example of a mathematical algorithm that can be used is the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877. Such an algorithm is incorporated into the BLAST family of programs, e.g., the BLAST or NBLAST programs (Altschul et al., 1990, J. Mol. Biol. 215, 403-410 or Altschul et al. (1997), Nucleic Acids Res, 25:3389-3402) (accessible from the NCBI home page at the worldwide website ncbi.nlm.nih.gov)) and FASTA (Pearson (1990), Methods Enzymol. 183, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci. USA 85, 2444-2448).

[0011] Generally, the substitution of one or more amino acids present in the reference amino acid sequence is preferably made conservatively. Examples of conservative substitutions include the substitution of one aliphatic residue with another aliphatic residue, such as the substitution of Ile, Val, Leu or Ala with each other, or the substitution of one polar residue with another polar residue, such as the substitution between Lys and Arg, between Glu and Asp, or between Gln and Asn. Other such conservative substitutions are also known, such as the substitution of entire regions with similar hydrophobic properties (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1):105-132). The substitution of one or more L-amino acids with one or more D-amino acids is considered as a conservative substitution in the context of the present invention. Exemplary amino acid substitutions are shown in Table 1 below.

[0012] [Table 1]

[0013] As used herein, an "antigen" is any structural entity that serves as a target for a receptor of the adaptive immune response, in particular for antibodies, T cell receptors, and / or B cell receptors. Preferably, the antigen is a peptide (including polypeptides and proteins). An "epitope", also known as an "antigenic determinant", is a part (or fragment) of an antigen that is recognized by the immune system, in particular antibodies, T cell receptors, and / or B cell receptors. Thus, an antigen has at least one epitope, i.e., a single antigen has one or more epitopes. In the context of the present invention, the term "epitope" is primarily used to denote a T cell epitope that is presented on the surface of an antigen-presenting cell, where it is bound to the major histocompatibility complex (MHC). With respect to peptide antigens, T cell epitopes presented by MHC class I molecules are typically, but not exclusively, peptides of 8-11 amino acids in length, whereas MHC class II molecules generally, but not exclusively, present longer peptides of 12-25 amino acids in length.

[0014] "CD4+ epitope" or "CD4 + The term "restricted epitope" as used herein refers to a CD4 + It refers to an epitope recognized by T cells, which consists in particular of an antigen fragment present in the groove of an MHC class II molecule. + An epitope preferably consists of about 12 to 25 amino acids, but may also consist of, for example, about 8 to 25 amino acids or about 6 to 100 amino acids. "CD8 + epitope" or "CD8 + The term "restricted epitope" as used herein refers to a CD8 + It represents an epitope recognized by T cells, which consists in particular of an antigen fragment present in the groove of the MHC class I molecule. + An epitope preferably consists of about 8 to 11 amino acids. It can also consist of, for example, about 8 to 15 amino acids or about 6 to 100 amino acids.

[0015] As used herein, a "fragment" of an antigen usually has a minimum length of 8 amino acids, i.e. the fragment comprises at least 8 consecutive amino acids of the antigen, preferably at least 10 consecutive amino acids of the antigen, more preferably at least 15 consecutive amino acids of the antigen, even more preferably at least 20 consecutive amino acids of the antigen, even more preferably at least 25 consecutive amino acids of the antigen, most preferably at least 30 consecutive amino acids of the antigen. An antigen fragment usually comprises one or more epitopes. Thus, a fragment of an antigen is typically immunogenic. A "sequence variant" of an antigen (or a fragment thereof) usually has an (amino acid) sequence that is at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% identical to a reference sequence. A "functional" sequence variant, in the context of an antigen / antigen fragment, means, for example, that the function of the epitope contained in the antigen (fragment) is not impaired or eliminated. In other words, a "functional" sequence variant of an antigen (fragment) is immunogenic, and preferably has substantially the same immunogenicity as the reference antigen (fragment). In some embodiments, for example, the amino acid sequence of one or more epitopes contained in a tumor antigen (fragment) is not mutated and is therefore identical to the (naturally occurring) reference epitope sequence.

[0016] It is understood that a person skilled in the art will usually select an antigen or a fragment thereof in consideration of the disease to be treated. Thus, the antigen or antigenic epitope is usually associated (or related) to the disease to be treated. A large number of antigens are known in the art in association with a particular disease. For example, to treat a tumor / cancer, a person skilled in the art will select a tumor antigen (or a fragment thereof) that is useful for a particular type of tumor / cancer. In some embodiments, a patient can be tested / screened for a particular antigen (e.g., by using an isolated sample) to identify whether the cancer / tumor expresses the particular antigen.

[0017] As used herein, a "tumor antigen" is an antigen produced by cancer / tumor cells. Tumor antigens include tumor-associated antigens and tumor-specific antigens. Tumor-associated (also called tumor-related) antigens (TAA) are antigens expressed by both cancer / tumor cells and normal cells. Tumor-specific antigens (TSA), in contrast, are antigens that are specifically expressed by cancer / tumor cells but not by normal cells. Examples of TSA include neo-antigens, which are not present in the body before cancer / tumor development, and therefore neo-antigens are "new" to the immune system. Neo-antigens often result from somatic mutations. Suitable epitopes of tumor antigens can be retrieved, for example, from cancer / tumor epitope databases as described in Vigneron et al. 2013, Cancer Immun. 13:15; URL: http: / / www.cancerimmunity.org / peptide / , or from the database "Tantigen" (TANTIGEN version 1.0, developed by the Bioinformatics Core Cancer, Vaccine Center, Dana-Farber Cancer Institute, Dec1, 2009; URL: http: / / cvc.dfci.harvard.edu / tadb / ).

[0018] The term "pharmaceutical composition" as used herein refers in particular to a preparation that is in a form that allows the biological activity of the active ingredient to be clearly effective and does not contain additional ingredients that are toxic to the subject to which the formulation is administered. In some embodiments, the (pharmaceutical) composition does not contain any further active ingredients (e.g., "active" with respect to cancer treatment) other than the active ingredients described herein below. As used herein, the term "vaccine" refers to a biological preparation that typically provides innate and / or adaptive immunity against a particular disease, preferably cancer. Thus, vaccines specifically support the innate and / or adaptive immune response of the immune system of the subject being treated. For example, the antigens or multi-antigenic domains described herein typically induce or support adaptive immune responses in the patient being treated, and the TLR peptide agonists described herein can induce or support innate immune responses.

[0019] As used herein, the term "disease" is intended to be generally synonymous with, and is used interchangeably with, the terms "disorder" and "condition" (in the context of medical conditions), in that all of these reflect an abnormal condition of the human or animal body or parts thereof that impairs normal functioning, is typically manifested by characteristic signs and symptoms, and causes a decrease in the lifespan or quality of life of the human or animal. As used herein, reference to "treatment" of a subject or patient is intended to include prevention, prophylaxis, attenuation, amelioration and cure. The terms "subject" or "patient" are used interchangeably herein to mean all mammals, including humans. Examples of subjects include humans, cows, dogs, cats, horses, goats, sheep, pigs, and rabbits. Preferably, the subject or patient is a human.

[0020] composition In a first aspect, the present invention provides a method for producing a composition comprising the steps of: (i) at least one tumor antigen, or a fragment or sequence variant thereof, At least one tumor antigen, or a fragment or sequence variant thereof, encoded in a 5' upstream open reading frame (uORF) within the 5' UTR of KRAS mRNA (KRAS-uORF1), TPX2 mRNA (TPX2-uORF1), or AURKA mRNA (AURKA-uORF2); (ii) a nucleic acid encoding the tumor antigen, or a fragment or sequence variant thereof; (iii) an antigen-presenting cell (APC) comprising (i) or (ii); or (iv) a T cell expressing either a T cell receptor or a CAR T cell receptor that targets the tumor antigen. A composition comprising:

[0021] The inventors have identified upstream open reading frames (ORFs), i.e., ORFs encoded in the 5'UTR (5' untranslated region) of tumor antigens, by analyzing selected Ribo-seq datasets from, for example, the RPFdb v.2.0 database (Wang et al., 2019, Nucleic Acids Res. 47, D230-D234). Thereby, the inventors have surprisingly identified peptides encoded in the 5' upstream open reading frame (uORF) in the 5'UTR of KRAS (Kirsten rat sarcoma viral oncogene homolog) mRNA, peptides encoded in the 5' upstream open reading frame (uORF) in the 5'UTR of TPX2 (targeting protein for Xklp2) mRNA, and peptides encoded in the 5' upstream open reading frame (uORF) in the 5'UTR of AURKA (Aurora A kinase) mRNA as tumor antigens. As demonstrated in the accompanying examples, such KRAS, TPX2 and AURKA transcripts were found to be strongly expressed in tumors, in contrast to normal healthy tissues. The inventors also found immunogenic epitopes within the KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2 peptides, and found that epitopes derived from the KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2 peptides are presented by human dendritic cells in the context of MHC class I and / or MHC class II. In summary, these findings support the role of the KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2 peptides as novel tumor-specific antigens useful in cancer immunotherapy.

[0022] Thus, the present invention provides compositions comprising at least one tumor antigen encoded in the 5' upstream open reading frame (uORF) in the 5'UTR of KRAS mRNA (KRAS-uORF1), TPX2 mRNA (TPX2-uORF1) or AURKA mRNA (AURKA-uORF2), or a fragment or sequence variant thereof. The tumor antigen is typically a peptide tumor antigen. These tumor antigens (i.e., at the peptide level) are also referred to herein as "KRAS-uORF1", "TPX2-uORF1" and "AURKA-uORF2", respectively. The skilled artisan will immediately distinguish from the respective context whether they refer to nucleotides or peptides.

[0023] In some embodiments, the composition comprises: - a tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5' UTR of KRAS mRNA, or a fragment or sequence variant thereof (KRAS-uORF1); - a tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5' UTR of TPX2 mRNA, or a fragment or sequence variant thereof (TPX2-uORF1), and / or - a tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5' UTR of AURKA mRNA, or a fragment or sequence variant thereof (AURKA-uORF2) Includes.

[0024] In some embodiments, the composition comprises a tumor antigen (KRAS-uORF1) encoded in the 5' upstream open reading frame (uORF) within the 5' UTR of KRAS mRNA and comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 1 or 2. The composition may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. The composition may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope).

[0025] In some embodiments, the composition comprises a tumor antigen (TPX2-uORF1) encoded in the 5' upstream open reading frame (uORF) within the 5'UTR of TPX2 mRNA and comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 3 or 4. The composition may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. The composition may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope).

[0026] In some embodiments, the composition comprises a tumor antigen (AURKA-uORF2) encoded in the 5' upstream open reading frame (uORF) in the 5'UTR of AURKA mRNA and comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 5. The composition may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). In addition to the above tumor antigens, the composition may further comprise at least one tumor antigen selected from the group consisting of CEACAM5 (or a fragment or sequence variant thereof as defined above), DUOXA2 (or a fragment or sequence variant thereof as defined above), and KRAS (or a fragment or sequence variant thereof as defined above). In contrast to the tumor antigens encoded in the 5' upstream open reading frame (uORF) in the 5'UTR as described above, the further antigens CEACAM5, DUOXA2 and KRAS as referred to herein are tumor antigens that are encoded in the ("classical") coding sequence (CDS) of the mRNA (i.e., not in the "untranslated" region / UTR).

[0027] In some embodiments, the composition comprises a fragment of CEACAM5 (carcinoembryonic antigen-associated cell adhesion molecule 5), which may comprise (or consist of) the amino acid sequence set forth in SEQ ID NO: 6. The composition may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids, or a sequence variant having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 6, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope).

[0028] In some embodiments, the composition comprises a fragment of CEACAM5, which may comprise (or consist of) the amino acid sequence set forth in SEQ ID NO: 7. The composition may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids, or a sequence variant having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 7, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope).

[0029] In some embodiments, the composition comprises a fragment of CEACAM5, which may comprise (or consist of) the amino acid sequence set forth in SEQ ID NO: 8. The composition may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids, or a sequence variant having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 8. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope).

[0030] In some embodiments, such fragments of CEACAM5 may be linked together (particularly as a fusion peptide / protein). Thus, the composition may comprise a (fusion) peptide comprising (or consisting of) the amino acids set forth in SEQ ID NO: 9. The composition may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 9, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope).

[0031] In some embodiments, the composition comprises a fragment of DUOXA2 (dual oxidase maturation factor 2), which may comprise (or consist of) the amino acid sequence set forth in SEQ ID NO: 10. The composition may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids, or a sequence variant having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope).

[0032] In some embodiments, the composition comprises a fragment of KRAS. Since KRAS mutations are common in cancer, the fragment of KRAS preferably comprises a mutation. The KRAS mutation may occur, for example, at G12, G13 or Q61 of KRAS. Thus, the fragment of KRAS may comprise positions G12, G13 and / or Q61 of KRAS, preferably with at least one of the amino acid residues at these positions being substituted. Preferably, the fragment of KRAS comprises a substitution at G12. Non-limiting examples of such substitutions include G12D, G12V, G12C, G12A and G12R. Preferably, the fragment of KRAS comprises position G12 of KRAS with a G12D or G12V substitution. Thus, the KRAS or fragment thereof is preferably KRAS-G12D or a fragment thereof, or KRAS-G12V or a fragment thereof (wherein the fragment comprises position G12 of KRAS and each of the substitutions).

[0033] In some embodiments, the composition comprises a fragment of KRAS, which may comprise (or consist of) the amino acid sequence set forth in SEQ ID NO: 11 or 12. The composition may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids, or a sequence variant having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 11, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. The composition may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 12. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope).

[0034] Preferably, the composition comprises different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the composition comprises (exactly or at least) two different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the composition comprises (exactly or at least) three different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the composition comprises (exactly or at least) four different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the composition comprises (exactly or at least) five different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the composition comprises all six different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. A multi-antigen vaccine (i) avoids the growth of antigen-deficient variants, (ii) targets different tumor cells within a heterogeneous tumor mass, and (iii) avoids tumor variability between patients.

[0035] Preferably, the composition comprises different tumor antigens selected from CEACAM5, DUOXA2, KRAS-G12D, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the composition comprises different tumor antigens selected from CEACAM5, DUOXA2, KRAS-G12V, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the composition comprises different tumor antigens selected from CEACAM5, DUOXA2, KRAS-G12D, KRAS-G12V, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof.

[0036] Preferably, the tumor antigen selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or a fragment or sequence variant thereof, comprises a CD4+ and / or CD8+ epitope.

[0037] In some embodiments, the composition according to the invention comprises at least one CD4 + Epitope and at least one CD8 + epitopes, which may be contained in the same or different tumor antigens (or fragments or variants thereof). h cells (CD4 + T cells) are involved in the licensing of DCs (dendritic cells) and the CTL (CD8 + T cells (cytotoxic T lymphocytes, cytotoxic T lymphocytes) play a central role in the antitumor immune response. + Epitope and at least one CD8 + The composition according to the invention containing the epitope is capable of inhibiting CTL and T h It provides an integrated immune response that allows for simultaneous priming of CD8 cells. + Epitope only or CD4 + This is preferable over immunization against epitopes alone.

[0038] Preferably, the composition comprises (i) different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof, and (ii) different CD4 + Epitopes and Different CD8 + Such compositions include polyepitopes. + CTL and CD4 + T h Induce cells to function synergistically against tumor cells to promote efficient antitumor immunity. h The cells are also involved in the maintenance of long-lasting cellular immunity monitored after vaccination. Such compositions are capable of generating polyclonal, multi-epitope immune responses as well as polyfunctional CD8 + and CD4 + It induces T cells and therefore effective anti-tumor activity.

[0039] In some embodiments, the composition comprises a tumor antigen comprising at least one amino acid sequence, wherein the amino acid sequence is - the amino acid sequence set forth in SEQ ID NO: 11 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 11, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - an amino acid sequence as set forth in SEQ ID NO: 12 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 12, - an amino acid sequence as set forth in SEQ ID NO: 1 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or an amino acid sequence as set forth in SEQ ID NO: 2 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 2, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - an amino acid sequence as set forth in SEQ ID NO: 3 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 3, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or an amino acid sequence as set forth in SEQ ID NO: 4 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 4, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - the amino acid sequence as set forth in SEQ ID NO: 5 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 5, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - the amino acid sequence as set forth in SEQ ID NO: 9 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9, and / or - an amino acid sequence as set forth in SEQ ID NO: 10, or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 10, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. It is.

[0040] In some embodiments, the composition comprises: - a tumor antigen comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 11 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 11, - a tumor antigen comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 12 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 12, - a tumor antigen comprising (or consisting of) an amino acid sequence as set forth in SEQ ID NO: 1 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or a tumor antigen comprising (or consisting of) an amino acid sequence as set forth in SEQ ID NO: 2 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 2 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 2, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - a tumor antigen comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 3 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 3, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or a tumor antigen comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 4 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 4, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - a tumor antigen comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 5 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5, - a tumor antigen comprising (or consisting of) an amino acid sequence as set forth in SEQ ID NO: 9 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9, and / or - a tumor antigen comprising (or consisting of) an amino acid sequence as set forth in SEQ ID NO: 10, or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10. Includes.

[0041] It will be appreciated that the tumor antigen (or a fragment or variant thereof) is preferably included in the composition as a peptide, as described above. Alternatively (or additionally), the composition may include a nucleic acid encoding the tumor antigen (or a fragment or variant thereof), as described above.

[0042] The nucleic acid preferably comprises a single-stranded, double-stranded or partially double-stranded nucleic acid, preferably selected from genomic DNA, cDNA, RNA, siRNA, antisense DNA, antisense RNA, ribozymes, complementary RNA / DNA sequences with or without expression elements, minigenes, gene fragments, control elements, promoters, and combinations thereof. Further preferred examples of nucleic acids (molecules) and / or polynucleotides include, for example, recombinant polynucleotides, vectors, oligonucleotides, RNA molecules, such as rRNA, mRNA, miRNA, siRNA, or tRNA, or DNA molecules as mentioned above. Thus, the nucleic acid (molecule) is a DNA molecule or an RNA molecule, preferably selected from genomic DNA, cDNA, siRNA, rRNA, mRNA, antisense DNA, antisense RNA, ribozymes, complementary RNA and / or DNA sequences, RNA and / or DNA sequences with or without expression elements, control elements, and / or promoters, vectors, and combinations thereof.

[0043] In some embodiments, the composition comprises: - a nucleic acid encoding KRAS-uORF1, preferably comprising the polynucleotide sequence set forth in SEQ ID NO: 37 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 37, - a nucleic acid encoding TPX2-uORF1, preferably comprising the polynucleotide sequence set forth in SEQ ID NO: 38 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 38, - a nucleic acid encoding AURKA-uORF2, preferably comprising the polynucleotide sequence set forth in SEQ ID NO: 39 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 39, - a nucleic acid encoding a fragment of CEACAM5, preferably comprising the polynucleotide sequence set forth in SEQ ID NO: 40 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 40, - a nucleic acid encoding a fragment of CEACAM5, preferably comprising the polynucleotide sequence set forth in SEQ ID NO: 41 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 41, - a nucleic acid encoding a fragment of CEACAM5, preferably comprising the polynucleotide sequence set forth in SEQ ID NO: 42 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 42, - a nucleic acid encoding a fragment of DUOXA2, preferably comprising a polynucleotide sequence as set forth in SEQ ID NO: 43, or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 43, - a nucleic acid encoding a fragment of KRAS-G12D, preferably comprising the polynucleotide sequence set forth in SEQ ID NO: 44 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 44, and / or - a nucleic acid encoding a fragment of KRAS-G12V, preferably comprising a polynucleotide sequence as set forth in SEQ ID NO: 45, or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 45; Includes.

[0044] The composition may comprise one or more nucleic acid molecules encoding different tumor antigens (or fragments or variants thereof) as described above, for example, two, three, four, five, or all six tumor antigens (or fragments or variants thereof) as described above. The different tumor antigens (or fragments or variants thereof) may be encoded by the same nucleic acid molecule (e.g., in a polycistronic manner) or may be encoded in different nucleic acid molecules, as described above.

[0045] Alternatively (or additionally), the composition may comprise an antigen presenting cell (APC) comprising (i) a tumor antigen (or a fragment or variant thereof) as described above, or (ii) a nucleic acid encoding the tumor antigen (or a fragment or variant thereof) as described above. In some embodiments, the antigen presenting cell (APC) may be a dendritic cell (DC). In some embodiments, the APCs may be loaded with a tumor antigen (or a fragment or variant thereof) as described above, such that the APCs are capable of presenting one or more epitopes of the tumor antigen (or a fragment or variant thereof). The composition may comprise different antigen-presenting cells that comprise (i) different tumor antigens (or fragments or variants thereof), or (ii) different nucleic acid molecules as described above. However, a single APC may comprise (i) different tumor antigens (or fragments or variants thereof), or (ii) different nucleic acid molecules as described above.

[0046] Alternatively (or additionally), the composition may comprise T cells expressing any of the T cell receptors or CAR T cell receptors that target a tumor antigen (or a fragment or variant thereof) as described above. In some embodiments, the composition may comprise different T cells expressing different T cell receptors or CAR T cell receptors, respectively, that target different tumor antigens (or fragments or variants thereof) as described above. Generally, the composition may be a pharmaceutical composition and / or a vaccine. In particular, such a composition is preferably a (pharmaceutical) composition that may comprise a pharma- ceutically acceptable carrier and / or vehicle, or any excipient, buffer, stabilizer or other material known to the person skilled in the art.

[0047] As further components, the (pharmaceutical) composition may in particular comprise a pharma- ceutically acceptable carrier and / or vehicle. In the context of the present invention, a pharma- ceutically acceptable carrier typically comprises a liquid or non-liquid base of the (pharmaceutical) composition. When the (pharmaceutical) composition is provided in liquid form, the carrier is typically pyrogen-free water, isotonic saline or a buffer (aqueous) solution, such as a phosphate, citrate or other buffer solution. In particular, for injection of the (pharmaceutical) composition, water or preferably a buffer, more preferably an aqueous buffer, may be used, containing a sodium salt, preferably at least 30 mM of a sodium salt, a calcium salt, preferably at least 0.05 mM of a calcium salt, and optionally a potassium salt, preferably at least 1 mM of a potassium salt. According to a preferred embodiment, the sodium salt, calcium salt and optionally the potassium salt may be present in the form of their halides, such as chlorides, iodides or bromides, in the form of their hydroxides, carbonates, bicarbonates or sulfates, etc. Examples of sodium salts include, but are not limited to, NaCl, NaI, NaBr, Na2CO3, NaHCO3, Na2SO4, examples of optional potassium salts include, for example, KCl, KI, KBr, K2CO3, KHCO3, K2SO4, examples of calcium salts include, for example, CaCl2, CaI2, CaBr2, CaCO3, CaSO4, Ca(OH)2. Furthermore, organic anions of the aforementioned cations may be contained in the buffer. According to a more preferred embodiment, the buffer suitable for injection purposes as defined above may contain a salt selected from sodium chloride (NaCl), calcium chloride (CaCl2) and optionally potassium chloride (KCl), and further anions may be present in addition to chloride. CaCl2 may also be replaced by another salt such as KCl. Typically, the salts in the injection buffer are present in concentrations of at least 30 mM sodium chloride (NaCl), at least 1 mM potassium chloride (KCl) and at least 0.05 mM calcium chloride (CaCl2).The injection buffer may be hypertonic, isotonic or hypotonic with respect to the particular reference medium, i.e. the buffer may have a higher, the same or a lower salt content with respect to the particular reference medium, preferably using the aforementioned salts in concentrations that do not lead to cell damage due to osmotic or other concentration effects. The reference medium may be, for example, a liquid present in an "in vivo" method, such as blood, lymph, cytoplasmic fluid or other body fluid, or a liquid that may be used as a reference medium in an "in vitro" method, such as a common buffer or liquid. Such common buffers or liquids are known to those skilled in the art. Physiological saline (0.9% NaCl) and lactated Ringer's solution are particularly preferred as liquid carriers. In some embodiments, the (pharmaceutical) composition further comprises arginine, such as L-arginine. In this context, the prescription of treatment when using the above (pharmaceutical) compositions, e.g., the determination of dosage, etc., typically falls within the competence of general practitioners and other physicians, and typically takes into account the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to physicians. Thus, the (pharmaceutical) composition typically comprises a therapeutically effective amount of the active component (tumor antigen). The (pharmaceutical) composition can be used as a general (pharmaceutical) composition or as a vaccine for humans, as well as for veterinary purposes, preferably for human medical purposes.

[0048] Examples of suitable adjuvants and / or immunomodulatory materials in the context of the present invention include MPL® (Corixa), aluminum-based minerals including aluminum compounds (commonly referred to as alum), ASO1-4, MF59, calcium phosphate, liposomes, iscoms, polyinosinic:polycytidylic acid (Poly IC) (including its stabilized form Poly ICLC (Hiltonol)), CpG oligodeoxynucleotides, granulocyte macrophage colony activator (GMC), and / or other immunomodulatory agents. These include knee stimulating factor (GM-CSF), lipopolysaccharide (LPS), montanide, polylactide coglycolide (PLG), flagellin, soap box saponin (QS21), aminoalkyl glucosamide compounds (e.g., RC529), bicomponent antimicrobial peptides with synthetic oligodeoxynucleotides (e.g., IC31), imiquimod, resiquimod, immunostimulatory sequence (ISS), monophosphoryl lipid A (MPLA), and fibroblast stimulatory lipopeptide (FSL1). Further materials, formulation processing techniques etc. which are useful in connection with or in connection with the preparation of compositions, in particular pharmaceutical compositions and vaccines, are known to those skilled in the art.

[0049] peptide In a further aspect, the present invention provides a method for producing a composition comprising the steps of: a) cell-penetrating peptides, b) a multi-antigenic domain comprising at least one tumor antigen, or a fragment or sequence variant thereof, A multi-antigenic domain, wherein the tumor antigen is encoded in a 5' upstream open reading frame (uORF) within the 5' UTR of KRAS mRNA (KRAS-uORF1), TPX2 mRNA (TPX2-uORF1), or AURKA mRNA (AURKA-uORF2), and c) TLR peptide agonists The present invention provides a peptide comprising:

[0050] In the peptides of the invention, components a) to c) are usually covalently linked by peptide bonds, and thus the peptides are either recombinant peptides (not occurring in nature) or "fusion" peptides (components a) to c) are "fused" to each other). Such peptides have the potential to: (i) stimulate polyepitopic cytotoxic T cell-mediated immunity; (ii) stimulate T h and (iii) simultaneously provide for the induction of immune cells and for the promotion of immune memory, thereby making the peptides according to the invention a potent vaccine with particularly improved antitumor activity.

[0051] Multiple antigenic domains The peptide of the present invention comprises multiple antigenic domains. As used herein, the term "multiple antigenic domain" refers to a domain, such as a peptide, that comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more) distinct antigens or fragments of at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more) distinct antigens. In some embodiments, the multiple antigenic domain comprises three or more different antigens or fragments thereof, in particular three, four, five, six, seven, eight, nine, ten or more different antigens or fragments thereof. Preferably, the "multiple antigenic domain" comprises at least two (e.g., 2, 3, 4, 5, 6, seven, eight, nine or more) distinct antigens (fragments), each fragment or antigen comprising at least one antigenic epitope. More preferably, the "multiple antigenic domain" comprises at least two to eight distinct antigens (fragments), each fragment / antigen comprising at least one antigenic epitope. Even more preferably, the "multiple antigenic domain" comprises (fragments of) five or six distinct antigens, each fragment comprising at least one antigenic epitope.

[0052] In particular, the (fragments of) different antigens are arranged consecutively in the multi-antigenic domain. In some embodiments, the (fragments of) different antigens are linked to each other, for example by a peptide spacer or linker (e.g., a GS linker). The spacer or linker is usually not a component a), i.e. a cell penetrating peptide, nor a component c), i.e. a TLR peptide agonist. In other embodiments, the (fragments of) different antigens are linked to each other directly, i.e., without a spacer or linker.

[0053] In some embodiments, each antigen or fragment thereof in the multiple antigenic domain comprises at least one CD4+ epitope and / or at least one CD8+ epitope, which may be contained in the same or different tumor antigens (or fragments or variants thereof). h cells (CD4 + T cells) are involved in the licensing of DCs (dendritic cells) and the CTL (CD8 + T cells (cytotoxic T lymphocytes, cytotoxic T lymphocytes) play a central role in the antitumor immune response. + Epitope and at least one CD8 + Multiple antigenic domains containing epitopes are capable of targeting CTL and T h It provides an integrated immune response that allows for simultaneous priming of CD8 cells. + Epitope only or CD4 + This is preferable over immunization against epitopes alone.

[0054] The peptide multi-antigenic domain comprises at least one tumor antigen encoded in the 5' upstream open reading frame (uORF) in the 5'UTR of KRAS mRNA (KRAS-uORF1), TPX2 mRNA (TPX2-uORF1) or AURKA mRNA (AURKA-uORF2), or a fragment or sequence variant thereof as defined above. The tumor antigen is typically a peptide tumor antigen. These tumor antigens (i.e., at the peptide level) are also referred to herein as "KRAS-uORF1", "TPX2-uORF1" and "AURKA-uORF2", respectively. The skilled person will immediately recognize from the respective context whether they refer to nucleotides and / or peptides.

[0055] In some embodiments, the multiple antigenic domain comprises: - a tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5' UTR of KRAS mRNA, or a fragment or sequence variant thereof (KRAS-uORF1) - a tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5' UTR of TPX2 mRNA, or a fragment or sequence variant thereof (TPX2-uORF1), and / or - a tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5' UTR of AURKA mRNA, or a fragment or sequence variant thereof (AURKA-uORF2) Includes.

[0056] In some embodiments, the multi-antigenic domain comprises a tumor antigen encoded in a 5' upstream open reading frame (uORF) within the 5' UTR of KRAS mRNA (KRAS-uORF1) and comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 1 or 2. The multi-antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. The multiple antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multiple antigenic domain preferably comprises the amino acid sequence according to SEQ ID NO: 1 or 2, or a sequence variant thereof.

[0057] In some embodiments, the multi-antigenic domain comprises a tumor antigen encoded in the 5' upstream open reading frame (uORF) in the 5'UTR of TPX2 mRNA (uORF-TPX2) and comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 3 or 4. The multi-antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. The multiple antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multiple antigenic domain preferably comprises the amino acid sequence according to SEQ ID NO: 3 or 4, or a sequence variant thereof.

[0058] In some embodiments, the multi-antigenic domain comprises a tumor antigen encoded in the 5' upstream open reading frame (uORF) in the 5' UTR of AURKA mRNA (AURKA-uORF2) and comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 5. The multi-antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multiple antigenic domain preferably comprises the amino acid sequence set forth in SEQ ID NO: 5, or a sequence variant thereof. In addition to the above tumor antigens, the multi-antigenic domain may further comprise at least one tumor antigen selected from the group consisting of CEACAM5 (or a fragment or sequence variant thereof as defined above), DUOXA2 (or a fragment or sequence variant thereof as defined above), and KRAS (or a fragment or sequence variant thereof as defined above).

[0059] In some embodiments, the multi-antigenic domain comprises (a fragment of) CEACAM5 (carcinoembryonic antigen-associated cell adhesion molecule 5), which may comprise (or consist of) the amino acid sequence set forth in SEQ ID NO: 6. The multi-antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids, or a sequence variant having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 6, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multi-antigenic domain preferably comprises the amino acid sequence set forth in SEQ ID NO: 6, or a sequence variant thereof.

[0060] In some embodiments, the multi-antigenic domain comprises (a fragment of) CEACAM5, which may comprise (or consist of) the amino acid sequence of SEQ ID NO: 7. The multi-antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids, or a sequence variant having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 7, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multi-antigenic domain preferably comprises the amino acid sequence set forth in SEQ ID NO: 7, or a sequence variant thereof.

[0061] In some embodiments, the multi-antigenic domain comprises (a fragment of) CEACAM5, which may comprise (or consist of) the amino acid sequence set forth in SEQ ID NO: 8. The multi-antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids, or a sequence variant having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 8, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multi-antigenic domain preferably comprises the amino acid sequence set forth in SEQ ID NO: 8, or a sequence variant thereof.

[0062] In some embodiments, such fragments of CEACAM5 may be linked together (particularly as a fusion peptide / protein). Thus, the multi-antigenic domain may comprise a (fusion) peptide comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 9. The multi-antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 9, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multi-antigenic domain preferably comprises the amino acid sequence set forth in SEQ ID NO: 9, or a sequence variant thereof.

[0063] In some embodiments, the multi-antigenic domain comprises (a fragment of) DUOXA2 (dual oxidase maturation factor 2), which may comprise (or consist of) the amino acid sequence set forth in SEQ ID NO: 10. The multi-antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids, or a sequence variant having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 10, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multi-antigenic domain preferably comprises the amino acid sequence set forth in SEQ ID NO: 10, or a sequence variant thereof.

[0064] In some embodiments, the multi-antigenic domain comprises (a fragment of) KRAS. Since KRAS mutations are common in cancer, (a fragment of) KRAS preferably comprises a mutation. KRAS mutations may occur, for example, at G12, G13 or Q61 of KRAS. Thus, the fragment of KRAS may comprise positions G12, G13 and / or Q61 of KRAS, preferably with at least one amino acid residue at these positions being substituted. Preferably, the fragment of KRAS comprises a substitution at G12. Non-limiting examples of such substitutions include G12D, G12V, G12C, G12A and G12R. Preferably, the fragment of KRAS comprises position G12 of KRAS with a G12D or G12V substitution. Thus, the KRAS or fragment thereof is preferably KRAS-G12D or a fragment thereof, or KRAS-G12V or a fragment thereof (wherein the fragment comprises position G12 of KRAS and the respective substitution).

[0065] In some embodiments, the multiple antigenic domain comprises a fragment of KRAS, which may comprise (or consist of) the amino acid sequence set forth in SEQ ID NO: 11 or 12. The multiple antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids, or a sequence variant having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 11, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. The multiple antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 12. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multiple antigenic domain preferably comprises the amino acid sequence according to SEQ ID NO: 11 or 12, or a sequence variant thereof.

[0066] Thus, multiple antigenic domains include - an amino acid sequence as set forth in SEQ ID NO: 9 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 9, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - an amino acid sequence as set forth in SEQ ID NO: 12 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 12, - the amino acid sequence set forth in SEQ ID NO: 11 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 11, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - an amino acid sequence as set forth in SEQ ID NO: 1 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or an amino acid sequence as set forth in SEQ ID NO: 2 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 2, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - an amino acid sequence as set forth in SEQ ID NO: 3 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 3, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or an amino acid sequence as set forth in SEQ ID NO: 4 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 4, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - the amino acid sequence as set forth in SEQ ID NO: 5, or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5, or - an amino acid sequence as set forth in SEQ ID NO: 10, or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 10, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. The amino acid sequence may comprise at least one amino acid sequence selected from the group consisting of:

[0067] Preferably, the multiple antigenic domain comprises different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the multiple antigenic domain comprises (exactly or at least) two different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the multiple antigenic domain comprises (exactly or at least) three different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the multiple antigenic domain comprises (exactly or at least) four different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the multiple antigenic domain comprises (exactly or at least) five different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the multiple antigenic domain comprises all six different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. Multi-antigen vaccines (i) avoid the growth of antigen-deficient variants, (ii) target different tumor cells within a heterogeneous tumor mass, and (iii) circumvent inter-patient tumor variability.

[0068] Preferably, the multiple antigenic domain comprises different tumor antigens selected from CEACAM5, DUOXA2, KRAS-G12D, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the composition comprises different tumor antigens selected from CEACAM5, DUOXA2, KRAS-G12V, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the composition comprises different tumor antigens selected from CEACAM5, DUOXA2, KRAS-G12D, KRAS-G12V, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof.

[0069] Preferably, the tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or sequence variants thereof, comprise CD4+ and / or CD8+ epitopes. Preferably, the multi-antigenic domain comprises (i) different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof, and (ii) different CD4 + Epitopes and Different CD8 + Such peptides containing multiple antigenic domains are useful for the synthesis of multi-epitope CD8 + CTL and CD4 + T h It induces cells to function synergistically against tumor cells, promoting efficient antitumor immunity. h These cells are also involved in the maintenance of long-lasting cellular immunity monitored after vaccination. Such peptides containing multiple antigenic domains have been shown to promote polyclonal, multi-epitope immune responses as well as polyfunctional CD8 + and CD4 + It induces T cells and therefore effective anti-tumor activity.

[0070] In some embodiments, the multiple antigenic domains preferably comprise, in the N-terminal to C-terminal direction: - CEACAM5 or a fragment or sequence variant thereof, - KRAS or a fragment or sequence variant thereof, - KRAS-uORF1, or a fragment or sequence variant thereof, - TPX2-uORF1, or a fragment or sequence variant thereof, - AURKA-uORF2, or a fragment or sequence variant thereof, and - DUOXA2 or a fragment or sequence variant thereof Includes.

[0071] Preferably, the multiple antigenic domains are arranged, preferably from N-terminus to C-terminus, as follows: - an amino acid sequence as set forth in SEQ ID NO: 9 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9, - an amino acid sequence as set forth in SEQ ID NO: 11 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 11, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and / or an amino acid sequence as set forth in SEQ ID NO: 12 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 12, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - an amino acid sequence as set forth in SEQ ID NO: 1 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or an amino acid sequence as set forth in SEQ ID NO: 2 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 2, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - an amino acid sequence as set forth in SEQ ID NO: 3 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 3, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or an amino acid sequence as set forth in SEQ ID NO: 4 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 4, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - the amino acid sequence as set forth in SEQ ID NO: 5 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5, and - an amino acid sequence as set forth in SEQ ID NO: 10, or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10. Includes.

[0072] More preferably, the multi-antigenic domain comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 13, or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 13. It is also more preferred that the multi-antigenic domain comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 14, or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 14, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0073] Cell-penetrating peptides Cell-penetrating peptides (CPPs) allow efficient delivery, i.e., trafficking and loading, of particularly multiple antigenic domains, to antigen-presenting cells (APCs), in particular to dendritic cells (DCs), and thus to the antigen-processing machinery of dendritic cells. The term "cell penetrating peptide" ("CPP") is generally used to denote short peptides that can transport various types of cargo molecules through the cell membrane and thus facilitate the cellular uptake of various molecular cargoes (from nano-sized particles to small chemical molecules and large fragments of DNA). "Cell internalization" of a cargo molecule linked to a cell penetrating peptide generally means the transport of the cargo molecule through the cell membrane and thus the cargo molecule enters the cell. Depending on the individual case, the cargo molecule can then be released into the cytoplasm, directed to intracellular organelles, or further presented on the cell surface. The cell penetrating ability or internalization of a cell penetrating peptide or peptides (including said cell penetrating peptides) according to the present invention can be confirmed by standard methods known to those skilled in the art, including flow cytometry or fluorescence microscopy of live and fixed cells, immunocytochemistry of cells transduced with said peptides, and Western blot.

[0074] Typically, cell penetrating peptides (CPPs) have a length of 8-50 residues. Cell penetrating peptides typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine, or has a sequence that contains an alternating pattern of polar / charged and non-polar hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively. Although cell penetrating peptides vary in size, amino acid sequence, and charge, CPPs all share a common feature, which is the ability to translocate cell membranes to facilitate the delivery of various molecular cargoes into the cytoplasm or organelles of cells. Cell penetrating peptides have found numerous applications in medicine as drug delivery agents in the treatment of various diseases, including cancer and viral inhibitors, and as contrast agents for cell labeling and imaging. Various CPPs which can be used as cell-penetrating peptides, i.e. as component a), in the peptides according to the invention are also disclosed in the review: Milletti, F., Cell-penetrating peptides: classes, origin, and current landscape. Drug Discov Today 17 (15-16): 850-60, 2012.

[0075] Preferably, the cell-penetrating peptide included in the peptide of the present invention has an amino acid sequence that includes or consists of the amino acid sequence set forth in SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23, or is a sequence variant thereof as described above. In some embodiments, the cell penetrating peptide according to the present invention has an amino acid sequence comprising or consisting of SEQ ID NO: 15, or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 15, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0076] In some embodiments, the cell penetrating peptide according to the present invention has an amino acid sequence comprising or consisting of SEQ ID NO:21, or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:21, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the cell penetrating peptide according to the present invention has an amino acid sequence comprising or consisting of SEQ ID NO:22, or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:22, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the cell penetrating peptide according to the present invention has an amino acid sequence comprising or consisting of SEQ ID NO:23, or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:23, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0077] These cell-penetrating peptides are derived from the "ZEBRA" protein of Epstein-Barr Virus (EBV). "ZEBRA" (also known as Zta, Z, EB1, or BZLF1) generally refers to the Epstein-Barr Virus (EBV) basic leucine zipper (bZIP) transcription activator. Without being bound by any theory, these cell-penetrating peptides are thought to act as a transcription factor (TFR) for CD8 + T cells and CD4 +It is believed to promote both MHC class I and II restricted presentation of cargo antigens to T cells. Thus, such CPPs can deliver multi-epitope peptides to dendritic cells (DCs) and subsequently promote CTL and Th cell activation and anti-tumor function. Thus, such CPPs can efficiently deliver peptides according to the invention to antigen presenting cells (APCs) leading to multi-epitope MHC class I and II restricted presentation.

[0078] Preferably, the cell penetrating peptide has an amino acid sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15, or a sequence variant thereof as described above. It will be understood by those skilled in the art that the primary amino acid sequence of the cell penetrating peptide may be further post-translationally modified, such as by glycosylation or phosphorylation, without departing from the invention. In certain embodiments, the cell penetrating peptide, in addition to its amino acid sequence as described above, comprises: (i) a nuclear localization signal (NLS), such signals being well known to those skilled in the art and described in Nair et al. (2003, Nucleic Acids Res. 31(1): 397-399); and / or (ii) targeting peptides, including tumor-homing peptides such as those described in Kapoor et al. (2012, PLoS ONE 7(4): e35187); It may further include. Preferably, the cell penetrating peptide is (directly) linked to the multiple antigenic domain to facilitate cellular internalization of the multiple antigenic domain.

[0079] TLR peptide agonists In the peptides according to the invention, the TLR peptide agonist allows for increased targeting of the vaccine to dendritic cells along with self-adjuvantity. The physical linkage of the TLR peptide agonist to at least one antigen or antigenic epitope in the CPP and the peptides according to the invention provides an enhanced immune response by co-stimulation of antigen presenting cells, in particular dendritic cells, which internalize, metabolize and present the antigen.

[0080] As used herein, a "TLR peptide agonist" is an agonist of a Toll-like receptor (TLR), i.e., it binds to and activates the TLR, in particular resulting in a biological response. Furthermore, a TLR peptide agonist is a peptide as defined above. Preferably, the TLR peptide agonist comprises 10-150 amino acids, more preferably 15-130 amino acids, even more preferably 20-120 amino acids, particularly preferably 25-110 amino acids, and most preferably 30-100 amino acids.

[0081] Toll-like receptors (TLRs) are transmembrane proteins characterized by an extracellular domain, a transmembrane domain, and a cytoplasmic domain. The extracellular domain, which contains leucine-rich repeats (LRRs) with a horseshoe-like shape, is involved in the recognition of common molecular patterns derived from diverse microorganisms. Toll-like receptors include TLRs 1-10. Compounds capable of activating TLR receptors and their modifications and derivatives have been well documented in the art. TLR1 can be activated by bacterial lipoproteins and their acetylated forms, and TLR2 can further be activated by gram-positive bacterial glycolipids, LPS, LPA, LTA, pili, outer membrane proteins, bacterial- or host-derived heat shock proteins, and mycobacterial lipoarabinomannan. TLR3 can be activated by dsRNA, particularly dsRNA of viral origin, or by a compound called poly(LC). TLR4 can be activated by gram-negative LPS, LTA, host or bacterial heat shock proteins, viral coat or envelope proteins, taxol or its derivatives, hyaluronan containing oligosaccharides, and fibronectin. TLR5 can be activated by bacterial flagella or flagellin. TLR6 can be activated by mycobacterial lipoproteins and group B streptococcal heat-labile soluble factor (GBS-F) or staphylococcal modulin. TLR7 can be activated by imidazoquinolines. TLR9 can be activated by unmethylated CpG DNA or chromatin-IgG complexes.

[0082] Preferably, the TLR peptide agonist comprised in the peptide according to the invention is an agonist of TLR1, 2, 4, 5, 6 and / or 10. TLRs are expressed either on the cell surface (TLR1, 2, 4, 5, 6 and 10) or on the membrane of intracellular organelles such as endosomes (TLR3, 4, 7, 8 and 9). The natural ligands of endosomal receptors have been shown to be nucleic acid-based molecules (except TLR4). Cell surface expressed TLR1, 2, 4, 5, 6 and 10 recognize extracellular microbial molecular patterns (Monie, TP, Bryant, CE, et al. 2009: Activating immunity: Lessons from the TLRs and NLRs. Trends Biochem. Sci. 34(11), 553-561). Although TLRs are expressed on several cell types, virtually all TLRs are expressed on DCs, enabling these specialized cells to sense all possible pathogens and danger signals.

[0083] However, TLR2, 4, and 5 are constitutively expressed on the surface of DCs. Thus, the TLR peptide agonist comprised in the peptide according to the invention is more preferably a TLR2, TLR4 and / or TLR5 peptide agonist. Even more preferably, the TLR peptide agonist is a TLR2 peptide agonist and / or a TLR4 peptide agonist. TLR2 can detect a wide variety of ligands derived from bacteria, viruses, parasites, and fungi, and interacts with a broad and structurally diverse range of ligands, including molecules expressed by microorganisms and fungi.

[0084] A preferred TLR2 peptide agonist is annexin II or an immunomodulatory fragment thereof (having TLR agonist functionality), as described in detail in WO2012 / 048190 and US Patent Application Publication No. 13 / 0331546, in particular a TLR2 peptide agonist comprising the amino acid sequence set forth in SEQ ID NO:7 of WO2012 / 048190, or a fragment or variant thereof.

[0085] The amino acid sequence set forth in SEQ ID NO: 16 or 24, or a sequence variant thereof (having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 16, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24, TLR2 peptide agonists comprising or consisting of component c) or a sequence variant thereof having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity are preferred as component c), i.e. as TLR peptide agonists comprised in a peptide. Particularly preferably, the TLR peptide agonist has the amino acid sequence according to SEQ ID NO: 16.

[0086] A variety of ligands interact with TLR4, including monophosphoryl lipid A from Salmonella minnesota R595 (MPLA), lipopolysaccharide (LPS), mannan (Candida albicans), glycoinositol phospholipids (Trypanosoma), viral envelope proteins (RSV and MMTV), and endogenous antigens including fibrinogen and heat shock proteins. Preferred TLR4 peptide agonists correspond to motifs that bind to TLR4, specifically (i) peptides that mimic the natural LPS ligand (RS01: Gln-Glu-Ile-Asn-Ser-Ser-Tyr and RS09: Ala-Pro-Pro-His-Ala-Leu-Ser) and (ii) fibronectin-derived peptides. The cellular glycoprotein fibronectin (FN) has multiple isoforms generated from a single gene by alternative splicing of three exons. One of these isoforms is the extra domain A (EDA) that interacts with TLR4. Thus, suitable TLR peptide agonists include fibronectin EDA domain or fragments or variants thereof. Such suitable fibronectin EDA domain or fragments or variants thereof are disclosed in EP1913954, EP2476440, US2009 / 0220532, and WO2011 / 101332.

[0087] In some embodiments, the peptide according to the invention comprises: a) a cell penetrating peptide having an amino acid sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23, or a sequence variant thereof having at least 70% sequence identity; b)- A fragment of CEACAM5 having a minimum length of 8 amino acids; - a fragment of DUOXA2 having a minimum length of 8 amino acids, - a fragment of KRAS having a minimum length of 8 amino acids, - KRAS-uORF1 or a fragment thereof having a minimum length of 8 amino acids, - TPX2-uORF1 or a fragment thereof having a minimum length of 8 amino acids, and / or AURKA-uORF2 or a fragment thereof with a minimum length of 8 amino acids and c) TLR peptide agonists that are TLR2 peptide agonists and / or TLR4 peptide agonists Includes.

[0088] The different components a), b) and c) of the peptide may be directly or indirectly linked (e.g. by a peptide bond). In other words, the two components may be directly adjacent or they may be linked by an additional component of the peptide, e.g. a peptide spacer or linker. Preferably, the peptide spacer consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, more preferably about 1, 2, 3, 4, or 5 amino acids. The amino acid sequence of the peptide spacer may be identical to the amino acid sequence of the N- or C-terminal flanking region of any of the components a), b), or c). Alternatively, the peptide spacer may consist of an amino acid sequence resulting from conservative amino acid substitutions of said naturally occurring flanking regions or a non-natural amino acid sequence, such as the sequence of a known cleavage site of a protease. In some embodiments, the peptide spacer does not contain any Cys(C) residues. In some embodiments, the linker sequence comprises at least 20%, more preferably at least 40%, even more preferably at least 50% of Gly or β-alanine residues. Suitable linker sequences can be readily selected and prepared by one of skill in the art. They can be composed of D and / or L amino acids.

[0089] Preferably, all three components a), b) and c) are linked via backbone / backbone bonds, thus resulting in a peptide backbone comprising a cell penetrating peptide backbone, a multiple antigenic domain backbone and a TLR peptide agonist backbone. In other words, the cell penetrating peptide backbone, the multiple antigenic domain backbone and the TLR peptide agonist backbone may together with further components, such as a linker or spacer, constitute the peptide backbone. Thus, the following arrangements of components a), b) and c) are preferred, said preferred arrangements being shown below from the N-terminus to the C-terminus of the peptide backbone, all three components a), b) and c) are linked via backbone / backbone bonds, and may be linked by a linker, spacer or another additional component: (α) component a) (cell penetrating peptide)-component b) (multiple antigenic domain)-component c) (TLR peptide agonist), (β) component c) (TLR peptide agonist)-component a) (cell penetrating peptide)-component b) (multiple antigenic domains); (γ) component a) (cell penetrating peptide)-component c) (TLR peptide agonist)-component b) (multiple antigenic domains); (δ) component c) (TLR peptide agonist)-component b) (multiple antigenic domain)-component a) (cell penetrating peptide); (ε) component b) (multiple antigenic domain)-component a) (cell penetrating peptide)-component c) (TLR peptide agonist), or (ζ) component b) (multiple antigenic domain)-component c) (TLR peptide agonist)-component a) (cell penetrating peptide).

[0090] Preferably, the multiple antigenic domains are located at the C-terminus of the cell penetrating peptide. More preferably, components a), b) and c) are arranged in the following order from the N-terminus to the C-terminus of the backbone of said peptide: (α) component a)-component b)-component c), or (β) Component c) - Component a) - Component b) where the components may be linked by further components, in particular by linkers or spacers.

[0091] Preferred exemplary peptides of the present invention include, preferably from N-terminus to C-terminus: a) a cell penetrating peptide having an amino acid sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15, or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 15; b) - an amino acid sequence as set forth in SEQ ID NO: 9 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 9, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - an amino acid sequence as set forth in SEQ ID NO: 11 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 11, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and / or an amino acid sequence as set forth in SEQ ID NO: 12 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 12, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - an amino acid sequence as set forth in SEQ ID NO: 1 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - an amino acid sequence as set forth in SEQ ID NO: 3 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3, - the amino acid sequence as set forth in SEQ ID NO: 5 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5, and - an amino acid sequence as set forth in SEQ ID NO: 10, or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10. and c) A TLR peptide agonist having an amino acid sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16, or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 16. Includes.

[0092] In some embodiments, the peptide of the present invention may comprise or consist of an amino acid sequence as set forth in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 46, or SEQ ID NO: 47, or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 46, or SEQ ID NO: 47. For example, the peptide of the present invention may comprise or consist of an amino acid sequence as set forth in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 46, or SEQ ID NO: 47.

[0093] Preferably, the peptide of the present invention may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18, or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18. For example, the peptide of the present invention may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18.

[0094] The present invention also provides a (pharmaceutical) composition comprising the peptide of the invention as described above. The (pharmaceutical) composition may comprise a pharma- ceutically acceptable carrier and / or vehicle, or any excipient, buffer, stabilizer or other material known to the skilled person. With regard to such further components, i.e. carrier, vehicle, excipient, buffer, stabilizer, adjuvant, etc., the detailed description of the composition according to the invention above applies accordingly to the (pharmaceutical) composition comprising the peptide of the invention. The (pharmaceutical) composition typically comprises a therapeutically effective amount of the active component (peptide). The (pharmaceutical) composition can be used as a general (pharmaceutical) composition or as a vaccine, for humans, and also for veterinary purposes, preferably for human medical purposes.

[0095] The present invention also provides a nucleic acid (molecule) comprising a polynucleotide encoding the peptide of the present invention as described above. The nucleic acid preferably comprises a single-stranded, double-stranded or partially double-stranded nucleic acid. Preferably, the nucleic acid (molecule) is selected from genomic DNA, cDNA, RNA, siRNA, mRNA, antisense DNA, antisense RNA, ribozyme, complementary RNA / DNA sequence. It may (or may not) comprise an expression element, a minigene, a gene fragment, a regulatory element, a promoter, and combinations thereof. Further preferred examples of nucleic acid (molecule) and / or polynucleotide include, for example, a recombinant polynucleotide, a vector, an oligonucleotide, an RNA molecule, such as rRNA, mRNA, miRNA, siRNA, or tRNA, or a DNA molecule. Thus, the nucleic acid (molecule) is preferably a DNA molecule or an RNA molecule, preferably selected from genomic DNA, cDNA, siRNA, rRNA, mRNA, antisense DNA, antisense RNA, ribozymes, complementary RNA and / or DNA sequences, RNA and / or DNA sequences with or without expression elements, regulatory elements, and / or promoters, vectors, and combinations thereof. In some embodiments, the nucleic acid (molecule) may be a vector, for example an expression vector for the expression of the peptide of the invention.

[0096] Vesicular stomatitis virus (VSV) In a further aspect, the present invention provides a recombinant vesicular stomatitis virus (VSV) encoding a multi-antigenic domain comprising at least one tumor antigen, or a fragment or sequence variant thereof, wherein the tumor antigen is encoded in a 5' upstream open reading frame (uORF) within the 5' UTR of KRAS mRNA (KRAS-uORF1), TPX2 mRNA (TPX2-uORF1) or AURKA mRNA (AURKA-uORF2). Vesicular stomatitis virus (VSV) belongs to the rhabdovirus (family Rhabdoviridae) and is a negative-stranded RNA virus. The genome of VSV is on a single molecule of negative-sense RNA molecule that codes for five major proteins: G protein (G), large protein (L), phosphoprotein (P), matrix protein (M) and nucleoprotein (N). In some embodiments, the vesicular stomatitis virus is vesicular stomatitis Indiana virus (VSIV) or vesicular stomatitis New Jersey virus (VSNJV).

[0097] Multiple antigenic domains The VSV of the present invention is a recombinant VSV, which comprises a multiple antigenic domain as defined above in the context of a peptide. This therefore refers to a domain, such as a peptide, that comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more) different antigens or fragments of at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more) different antigens. In some embodiments, the multiple antigenic domain comprises three or more different antigens or fragments thereof, in particular three, four, five, six, seven, eight, nine, ten or more different antigens or fragments thereof. Preferably, the "multiple antigenic domain" comprises at least two (e.g., 2, 3, 4, 5, 6, seven, eight, nine or more) different antigens (fragments), each fragment or antigen comprising at least one antigenic epitope. More preferably, the "multiple antigenic domain" comprises at least two to eight different antigens (fragments), each fragment / antigen comprising at least one antigenic epitope. Even more preferably, the "multiple antigenic domain" comprises (fragments of) five or six different antigens, each fragment comprising at least one antigenic epitope.

[0098] In particular, the (fragments of) different antigens are arranged consecutively in the multi-antigenic domain. In some embodiments, the (fragments of) different antigens are linked to each other, for example, by a peptide spacer or linker (e.g., a GS linker). In other embodiments, the (fragments of) different antigens are linked to each other directly, i.e., without a spacer or linker.

[0099] In some embodiments, each antigen or fragment thereof in the multiple antigenic domain comprises at least one CD4+ epitope and / or at least one CD8+ epitope, which may be contained in the same or different tumor antigens (or fragments or variants thereof). h cells (CD4 + T cells) are involved in the licensing of DCs (dendritic cells) and the CTL (CD8 + T cells (cytotoxic T lymphocytes, cytotoxic T lymphocytes) play a central role in the antitumor immune response. + Epitope and at least one CD8 + Multiple antigenic domains containing epitopes are capable of targeting CTL and T h It provides an integrated immune response that allows for simultaneous priming of CD8 cells. + Epitope only or CD4 + This is preferable over immunization against epitopes alone.

[0100] The multi-antigenic domain of VSV comprises at least one tumor antigen encoded in the 5' upstream open reading frame (uORF) in the 5'UTR of KRAS mRNA (KRAS-uORF1), TPX2 mRNA (TPX2-uORF1) or AURKA mRNA (AURKA-uORF2), or a fragment or sequence variant thereof as defined above. The tumor antigen is typically a peptide tumor antigen. These tumor antigens (i.e., at the peptide level) are also referred to herein as "KRAS-uORF1", "TPX2-uORF1" and "AURKA-uORF2", respectively. The skilled artisan will immediately recognize from the respective context whether they refer to nucleotides and / or peptides.

[0101] In some embodiments, the multiple antigenic domain comprises: - a tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5' UTR of KRAS mRNA, or a fragment or sequence variant thereof (KRAS-uORF1) - a tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5' UTR of TPX2 mRNA, or a fragment or sequence variant thereof (TPX2-uORF1), and / or - a tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5' UTR of AURKA mRNA, or a fragment or sequence variant thereof (AURKA-uORF2) Includes.

[0102] In some embodiments, the multi-antigenic domain comprises a tumor antigen encoded in the 5' upstream open reading frame (uORF) in the 5'UTR of KRAS mRNA (KRAS-uORF1) and comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 1 or 2. The multi-antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity. The functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multi-antigenic domain preferably comprises the amino acid sequence set forth in SEQ ID NO: 1 or 2, or a sequence variant thereof.

[0103] In some embodiments, the multi-antigenic domain comprises a tumor antigen (uORF-TPX2) encoded in the 5' upstream open reading frame (uORF) in the 5'UTR of TPX2 mRNA and comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 3 or 4. The multi-antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity. The functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multi-antigenic domain preferably comprises the amino acid sequence set forth in SEQ ID NO: 3 or 4, or a sequence variant thereof.

[0104] In some embodiments, the multi-antigenic domain comprises a tumor antigen encoded in the 5' upstream open reading frame (uORF) in the 5'UTR of AURKA mRNA (AURKA-uORF2) and comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 5. The multi-antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity. The functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multi-antigenic domain preferably comprises the amino acid sequence set forth in SEQ ID NO: 5, or a sequence variant thereof. In addition to the above tumor antigens, the multi-antigenic domain may further comprise at least one tumor antigen selected from the group consisting of CEACAM5 (or a fragment or sequence variant thereof as defined above), DUOXA2 (or a fragment or sequence variant thereof as defined above), and KRAS (or a fragment or sequence variant thereof as defined above).

[0105] In some embodiments, the multi-antigenic domain comprises (a fragment of) CEACAM5 (carcinoembryonic antigen-associated cell adhesion molecule 5), which may comprise (or consist of) the amino acid sequence set forth in SEQ ID NO: 6. The multi-antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids, or a sequence variant having at least 70% sequence identity. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multi-antigenic domain preferably comprises the amino acid sequence set forth in SEQ ID NO: 6, or a sequence variant thereof. In some embodiments, the multi-antigenic domain comprises (a fragment of) CEACAM5, which may comprise (or consist of) the amino acid sequence of SEQ ID NO: 7. The multi-antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids, or a sequence variant having at least 70% sequence identity. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multi-antigenic domain preferably comprises the amino acid sequence set forth in SEQ ID NO: 7, or a sequence variant thereof.

[0106] In some embodiments, the multi-antigenic domain comprises (a fragment of) CEACAM5, which may comprise (or consist of) the amino acid sequence set forth in SEQ ID NO: 8. The multi-antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids, or a sequence variant having at least 70% sequence identity. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multi-antigenic domain preferably comprises the amino acid sequence set forth in SEQ ID NO: 8, or a sequence variant thereof. In some embodiments, such fragments of CEACAM5 may be linked together (particularly as a fusion peptide / protein). Thus, the multi-antigenic domain may comprise a (fusion) peptide comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 9. The multi-antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids or a sequence variant having at least 70% sequence identity. The functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multi-antigenic domain preferably comprises the amino acid sequence set forth in SEQ ID NO: 9, or a sequence variant thereof.

[0107] In some embodiments, the multi-antigenic domain comprises (a fragment of) DUOXA2 (dual oxidase maturation factor 2), which may comprise (or consist of) the amino acid sequence set forth in SEQ ID NO: 10. The multi-antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids, or a sequence variant having at least 70% sequence identity. Functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multi-antigenic domain preferably comprises the amino acid sequence set forth in SEQ ID NO: 10, or a sequence variant thereof.

[0108] In some embodiments, the multi-antigenic domain comprises (a fragment of) KRAS. Since KRAS mutations are common in cancer, (a fragment of) KRAS preferably comprises a mutation. KRAS mutations may occur, for example, at G12, G13 or Q61 of KRAS. Thus, the fragment of KRAS may comprise positions G12, G13 and / or Q61 of KRAS, preferably with at least one amino acid residue at these positions being substituted. Preferably, the fragment of KRAS comprises a substitution at G12. Non-limiting examples of such substitutions include G12D, G12V, G12C, G12A and G12R. Preferably, the fragment of KRAS comprises position G12 of KRAS with a G12D or G12V substitution. Thus, the KRAS or fragment thereof is preferably KRAS-G12D or a fragment thereof, or KRAS-G12V or a fragment thereof (wherein the fragment comprises position G12 of KRAS and the respective substitution).

[0109] In some embodiments, the multi-antigenic domain comprises a fragment of KRAS, which may comprise (or consist of) the amino acid sequence set forth in SEQ ID NO: 11 or 12. The multi-antigenic domain may also comprise a fragment or sequence variant thereof as defined above, i.e. a fragment having a minimum length of 8 amino acids, or a sequence variant having at least 70% sequence identity. The functionality as a tumor antigen is preferably maintained in the fragment or sequence variant (e.g. in that the fragment or sequence variant comprises at least one epitope). Thus, the multi-antigenic domain preferably comprises the amino acid sequence set forth in SEQ ID NO: 11 or 12, or a sequence variant thereof.

[0110] Thus, multiple antigenic domains include - an amino acid sequence as set forth in SEQ ID NO: 9 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 9, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - an amino acid sequence as set forth in SEQ ID NO: 12 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 12, - the amino acid sequence set forth in SEQ ID NO: 11 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 11, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - an amino acid sequence as set forth in SEQ ID NO: 1 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or an amino acid sequence as set forth in SEQ ID NO: 2 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 2, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - an amino acid sequence as set forth in SEQ ID NO: 3 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 3, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or an amino acid sequence as set forth in SEQ ID NO: 4 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 4, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - the amino acid sequence as set forth in SEQ ID NO: 5, or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5, or - an amino acid sequence as set forth in SEQ ID NO: 10, or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 10, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. The amino acid sequence may comprise at least one amino acid sequence selected from the group consisting of:

[0111] Preferably, the multiple antigenic domain comprises different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the multiple antigenic domain comprises (exactly or at least) two different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the multiple antigenic domain comprises (exactly or at least) three different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the multiple antigenic domain comprises (exactly or at least) four different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the multiple antigenic domain comprises (exactly or at least) five different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the multiple antigenic domain comprises all six different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. Multi-antigen vaccines (i) avoid the growth of antigen-deficient variants, (ii) target different tumor cells within a heterogeneous tumor mass, and (iii) circumvent inter-patient tumor variability.

[0112] Preferably, the tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or sequence variants thereof, comprise CD4+ and / or CD8+ epitopes. Preferably, the multi-antigenic domain comprises (i) different tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof, and (ii) different CD4 + Epitopes and Different CD8 + Contains an epitope.

[0113] In some embodiments, the multiple antigenic domains preferably comprise, in the N-terminal to C-terminal direction: - CEACAM5 or a fragment or sequence variant thereof, - KRAS or a fragment or sequence variant thereof, - KRAS-uORF1, or a fragment or sequence variant thereof, - TPX2-uORF1, or a fragment or sequence variant thereof, - AURKA-uORF2, or a fragment or sequence variant thereof, and - DUOXA2 or a fragment or sequence variant thereof Includes.

[0114] Preferably, the multiple antigenic domains are arranged, preferably from N-terminus to C-terminus, as follows: - an amino acid sequence as set forth in SEQ ID NO: 9 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9, - an amino acid sequence as set forth in SEQ ID NO: 11 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 11, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and / or an amino acid sequence as set forth in SEQ ID NO: 12 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 12, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - an amino acid sequence as set forth in SEQ ID NO: 1 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or an amino acid sequence as set forth in SEQ ID NO: 2 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 2, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, - an amino acid sequence as set forth in SEQ ID NO: 3 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence as set forth in SEQ ID NO: 4 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4, - the amino acid sequence as set forth in SEQ ID NO: 5 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5, and - an amino acid sequence as set forth in SEQ ID NO: 10, or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10. Includes.

[0115] More preferably, the multiple antigenic domain comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 19 or 48, or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 19 or 48. For example, the multiple antigenic domain comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 19 or 48, preferably SEQ ID NO: 19.

[0116] VSV: Further Features In some embodiments, the vesicular stomatitis virus (VSV) is an oncolytic vesicular stomatitis virus (VSV). As used herein, "oncolytic" virus refers to a virus that preferentially infects and kills cancer cells. It is assumed that the infected cancer cells are destroyed by oncolysis, releasing new infectious virus particles or virions to target "remaining" cancer / tumor. The oncolytic activity of the recombinant rhabdovirus of the present invention can be tested in various assay systems known to those skilled in the art (an exemplary in vitro assay is described in Muik et al., Cancer Res., 74(13), 3567-78, 2014). It should be understood that oncolytic VSV can only infect and lyse certain types of cancer cells. Also, the oncolytic effect may vary depending on the type of cancer cells.

[0117] In some embodiments, the vesicular stomatitis virus (VSV) is capable of replication. The term "replicative" or "replicative virus" as used herein refers to a virus that contains all the information in its genome to enable it to replicate in a cell. For example, the replication capacity of the recombinant vesicular stomatitis virus of the present invention can be evaluated according to the method disclosed in Tani et al. JOURNAL OF VIROLOGY, Aug. 2007, p. 8601-8612; or Garbutt et al. JOURNAL OF VIROLOGY, May 2004, p. 5458-5465. Preferably, VSV can specifically replicate in cancer cells. Preferably, VSV replicates specifically in tumor cells that have lost the ability to mount and respond to antiviral innate immune responses (e.g., type I IFN signaling). It is assumed that viral replication in tumor cells leads to cell death, resulting in the release of tumor-associated antigens, local inflammation, and the induction of antitumor immunity. On the other hand, defective replication in "healthy cells" is preferred so that VSV can be rapidly cleared from normal tissues.

[0118] Preferably, in the genome of VSV, the gene encoding glycoprotein G is replaced by the gene encoding glycoprotein GP of Lymphocytic Choriomeningitis Virus (LCMV), which preferably comprises the amino acid sequence set forth in SEQ ID NO: 25 or a functional sequence variant thereof that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto. Thus, vesicular stomatitis virus (VSV), in particular VSV-GP (e.g. recombinant VSV with LCMV GP as disclosed in WO2010 / 040526) is preferred. Advantageous properties of VSV-GP include one or more of the following: highly potent and rapid killer (<8 hours), oncolytic virus, capable of systemic application, markedly reduced neurotropism with loss of neurotoxicity, grows cytolytically, potent activation of innate immunity, approximately 3 kb space for immunomodulatory cargo and antigens, recombinant with arenavirus glycoproteins from lymphocytic choriomeningitis virus (LCMV), favorable safety profile in terms of reduced neurotoxicity and less susceptibility to neutralizing antibody responses and complement destruction compared to wild-type VSV (VSV-G), replicates specifically in tumor cells that have lost the ability to mount and respond to antiviral innate immune responses (e.g., type I IFN signaling), and is rapidly cleared from normal tissues due to defective replication in "healthy cells". It is postulated that viral replication in tumor cells leads to cell death, resulting in the release of tumor-associated antigens, local inflammation and induction of antitumor immunity.

[0119] In some embodiments, the vesicular stomatitis virus (VSV) exhibits the following characteristics: - which encodes in its genome multiple antigenic domains as defined above, - which encodes in its genome the vesicular stomatitis virus nucleoprotein (N), the large protein (L), the phosphoprotein (P) and the matrix protein (M); - the gene encoding the glycoprotein G of the vesicular stomatitis virus has been replaced by the gene encoding the glycoprotein GP of the lymphocytic choriomeningitis virus (LCMV), and / or - The glycoprotein G of the vesicular stomatitis virus is replaced by the glycoprotein GP of LCMV.

[0120] Preferably, the vesicular stomatitis virus (VSV) exhibits the following characteristics: - which encodes in its genome a vesicular stomatitis virus nucleoprotein (N) comprising the amino acids set forth in SEQ ID NO: 26 or a sequence variant thereof having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, - which encodes in its genome a vesicular stomatitis virus phosphoprotein (P) comprising the amino acids set forth in SEQ ID NO: 27 or a sequence variant thereof having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, - which encodes in its genome a vesicular stomatitis virus large protein (L) comprising the amino acids set forth in SEQ ID NO: 28 or a sequence variant thereof having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, - which encodes in its genome a vesicular stomatitis virus matrix protein (M) comprising the amino acids set forth in SEQ ID NO: 29 or a sequence variant thereof having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, - which encodes in its genome multiple antigenic domains of vesicular stomatitis virus as defined above, - the gene encoding the glycoprotein G of the vesicular stomatitis virus has been replaced by the gene encoding the glycoprotein GP of the lymphocytic choriomeningitis virus (LCMV), and / or - The glycoprotein G is replaced by the glycoprotein GP of LCMV.

[0121] More preferably, - a phosphoprotein (P) comprising the amino acid sequence set forth in SEQ ID NO: 27, or a sequence variant thereof having at least 70% sequence identity, - a nucleoprotein (N) comprising the amino acid sequence set forth in SEQ ID NO: 26, or a sequence variant thereof having at least 70% sequence identity, - a matrix protein (M) comprising the amino acid sequence set forth in SEQ ID NO: 29, or a sequence variant thereof having at least 70% sequence identity, - a large protein (L) comprising the amino acid sequence set forth in SEQ ID NO: 28, or a sequence variant thereof having at least 70% sequence identity, - a glycoprotein (GP) comprising an amino acid sequence as set forth in SEQ ID NO: 25 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 25, and - a multi-antigenic domain comprising the amino acid sequence of SEQ ID NO: 19, or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 48; The vesicular stomatitis virus (VSV) according to any one of claims 43 to 85, which encodes in its genome.

[0122] Thus, particularly preferred is a vesicular stomatitis virus (VSV) comprising an RNA genome comprising (or consisting of) an RNA sequence as set forth in SEQ ID NO: 30, or a sequence variant thereof as defined above (e.g. a sequence variant having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity). In some embodiments, the vesicular stomatitis virus (VSV) comprises an RNA genome comprising (or consisting of) an RNA sequence corresponding to the cDNA sequence set forth in SEQ ID NO: 49, or a sequence variant thereof as defined above (e.g., a sequence variant having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0123] The present invention also provides a (pharmaceutical) composition comprising a VSV of the invention as described above. The (pharmaceutical) composition may further comprise a pharma- ceutically acceptable carrier and / or vehicle, or any excipient, buffer, stabilizer or other material known to a person skilled in the art. With regard to such further components, i.e. carriers, vehicles, excipients, buffers, stabilizers, adjuvants, etc., the detailed description of the composition according to the invention above applies accordingly to the (pharmaceutical) composition comprising a VSV of the invention. The (pharmaceutical) composition typically comprises a therapeutically effective amount of the active component (VSV). The (pharmaceutical) composition can be used as a general (pharmaceutical) composition or as a vaccine for humans, and also for veterinary purposes, preferably for human medical purposes.

[0124] Vaccines, kits and combinations In a further aspect, the present invention also provides a method for producing a composition comprising the steps of: (i) a composition according to the invention as described above, (ii) a peptide according to the invention as described above, or (iii) the vesicular stomatitis virus (VSV) according to the invention as described above Also provided is a vaccine comprising: In general, a vaccine induces, supports or enhances the immune response (innate and / or adaptive) of the immune system of the subject or patient being treated. For example, the antigens described herein, particularly the multiple antigenic domains, typically induce or support an adaptive immune response in the patient being treated. TLR peptide agonists of the peptides described herein can induce or support an innate immune response.

[0125] The vaccine may also comprise a pharma- ceutically acceptable carrier, adjuvant, and / or vehicle as defined above for the (pharmaceutical) composition. In the particular context of a vaccine, the choice of pharma- ceutically acceptable carrier is determined in principle by the manner in which the vaccine is to be administered. The vaccine can be administered, for example, systemically or locally, as described above. More preferably, the vaccine can be administered by intravenous, intratumoral, intradermal, subcutaneous, or intramuscular routes. For this reason, the vaccine is preferably formulated in liquid (or sometimes solid) form. The suitable amount of vaccine to be administered can be determined by routine experimentation with animal models. Such models include, but are not limited to, rabbits, sheep, mice, rats, dogs, and non-human primate models. Preferred unit dose forms for injection include sterile solutions of water, saline, or mixtures thereof. The pH of such solutions should be adjusted to about 7.4. Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid, and collagen matrices.

[0126] The vaccine may further contain one or more auxiliary substances to further enhance its immunogenicity. In some embodiments, a synergistic effect may be achieved between the composition containing the antigen, peptide and / or VSV as described above and the auxiliary substances that may be contained in the vaccine. Depending on the different types of auxiliary substances, different mechanisms may be considered in this respect. For example, compounds that allow the maturation of dendritic cells (DC), such as lipopolysaccharide or TNF-α, form a first class of suitable auxiliary substances. In general, any agent that affects the immune system in the manner of a "danger signal" (LPS, GP96, etc.) or a cytokine (e.g. GM-CSF) may be used as an auxiliary substance, which allows the immune response generated by the composition containing the antigen, peptide and / or VSV as described above to be strengthened and / or influenced in a targeted manner. Particularly preferred auxiliary substances are cytokines, e.g. monokines, lymphokines, interleukins or chemokines which further promote the innate immune response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors, e.g. hGH.

[0127] Preferably, the vaccine comprises: (i) a peptide according to the invention as described above, and (ii) the vesicular stomatitis virus (VSV) according to the invention as described above Includes. In a further aspect, the present invention also provides a method for producing a composition comprising the steps of: (i) a peptide according to the invention as described above, and (ii) the vesicular stomatitis virus (VSV) according to the invention as described above Also provided is a kit comprising: In a further aspect, the present invention provides a method for producing a composition comprising the steps of: (i) a peptide according to the invention as described above, and (ii) the vesicular stomatitis virus (VSV) according to the invention as described above Also provided is a combination comprising:

[0128] Generally, in the vaccines, kits and combinations of the present invention, the different components (i) and (ii) can be provided (a) in the same composition or in the same container, or (b) in a (spatially) separate manner (e.g., in different compositions or different containers). Preferably, in the vaccines, kits and combinations of the present invention, (i) peptide and (ii) VSV are provided in a separate manner (e.g., separate containers or separate compositions as described above). By providing the different components (i) and (ii) separately, it is possible to administer (i) peptide and (ii) VSV separately, for example, via different routes, in different compositions, and / or at different times / schedules. In particular, providing the different components (i) and (ii) separately allows the use of (i) peptide and (ii) VSV in a heterologous prime-boost regimen.

[0129] In the vaccine, kit or combination, the multiple antigenic domain encoded by (in the genome of) vesicular stomatitis virus (VSV) preferably comprises a (tumor) antigen, or a fragment or sequence variant thereof, also contained in the multiple antigenic domain of the peptide. It is also preferred that the multiple antigenic domain encoded by (in the genome of) vesicular stomatitis virus (VSV) comprises the amino acid sequence of each of the (tumor) antigens, or a fragment or sequence variant thereof, also contained in the multiple antigenic domain of the peptide. It is also preferred that the multiple antigenic domain of the peptide comprises an antigen, or a fragment thereof, contained in the multiple antigenic domain encoded in the genome of vesicular stomatitis virus (VSV). It is further preferred that the multiple antigenic domain of the peptide comprises the amino acid sequence of each of the (tumor) antigens, or a fragment or sequence variant thereof, contained in the multiple antigenic domain encoded in the genome of vesicular stomatitis virus (VSV).

[0130] This allows for an advantageous heterologous prime-boost regimen. The principle of the heterologous prime-boost approach is to make the immune system focus its response on a specific target antigen by avoiding immune responses against the antigen carrier or delivery system after successive administration of the same antigen carrier or delivery system as used in the allologous prime-boost regimen. In the heterologous prime-boost regimen, administration of a first immunogen primes cytotoxic T lymphocytes (CTLs) specific for the target antigen, but priming also occurs against the antigen carrier or delivery system. By administering an unrelated second antigen carrier or delivery system (e.g., a viral vector) during the "boost" phase, the immune system is confronted with a large number of new antigens. Since the second antigen carrier or delivery system also codes / delivers the target antigen targeted by the already existing primed cells, a strong memory response is elicited by the immune system, boosting the previously primed CTLs specific for the target antigen. Thus, the vaccines, kits or combinations of the present invention advantageously provide target antigens in different contexts, i.e., (i) contained in a peptide of the present invention and (ii) encoded by a VSV of the present invention, which allows for use in heterologous prime-boost regimes.

[0131] The corresponding antigen (tumor) antigen, or a fragment or sequence variant thereof, contained in the peptide multi-antigenic domain and the multi-antigenic domain encoded in VSV, may be any antigen (or a fragment or variant thereof) as described herein. Preferably, the corresponding antigen (tumor) antigen, or a fragment or sequence variant thereof, is selected from the group consisting of: - a tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5' UTR of KRAS mRNA, or a fragment or sequence variant thereof (KRAS-uORF1); - a tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5' UTR of TPX2 mRNA, or a fragment or sequence variant thereof (TPX2-uORF1); - a tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5' UTR of AURKA mRNA, or a fragment or sequence variant thereof (AURKA-uORF2); - CEACAM5 or a fragment or sequence variant thereof as defined above, - DUOXA2, or a fragment or sequence variant thereof as defined above, and - KRAS, or a fragment or sequence variant thereof as defined above.

[0132] Preferably, the KRAS (or a fragment or sequence variant thereof) is a mutant KRAS as described above. More preferably, the KRAS (or a fragment or sequence variant thereof) is KRAS-G12D (or a fragment or sequence variant thereof) or KRAS-G12V (or a fragment or sequence variant thereof). In some embodiments, the peptide multi-antigenic domain and the VSV-encoded multi-antigenic domain each comprise a tumor antigen encoded in a 5' upstream open reading frame (uORF) within the 5'UTR of KRAS mRNA, or a fragment or sequence variant thereof (KRAS-uORF1).

[0133] In some embodiments, the peptide multi-antigenic domain and the VSV-encoded multi-antigenic domain each comprise a tumor antigen encoded in a 5' upstream open reading frame (uORF) within the 5'UTR of TPX2 mRNA, or a fragment or sequence variant thereof (TPX2-uORF1). In some embodiments, the peptide multi-antigenic domain and the VSV-encoded multi-antigenic domain each comprise a tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5'UTR of AURKA mRNA, or a fragment or sequence variant thereof (AURKA-uORF2).

[0134] In some embodiments, the multiple antigenic domains of the peptide and the multiple antigenic domains encoded in VSV each comprise CEACAM5, or a fragment or sequence variant thereof as defined above. In some embodiments, the multiple antigenic domains of the peptide and the multiple antigenic domains encoded in VSV each comprise DUOXA2, or a fragment or sequence variant thereof as defined above.

[0135] In some embodiments, the multiple antigenic domains of the peptide and the multiple antigenic domains encoded in VSV each comprise KRAS, or a fragment or sequence variant thereof as defined above. Preferably, KRAS (or a fragment or sequence variant thereof) is a mutant KRAS as defined above. More preferably, KRAS (or a fragment or sequence variant thereof) is KRAS-G12D (or a fragment or sequence variant thereof) or KRAS-G12V (or a fragment or sequence variant thereof).

[0136] Preferably, the multiple antigenic domains of the peptide and the multiple antigenic domains encoded in VSV each comprise at least one amino acid sequence selected from the group consisting of: - the amino acid sequence set forth in SEQ ID NO: 9 or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence as set forth in SEQ ID NO: 12 or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence as set forth in SEQ ID NO: 11 or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 1 or 2, or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 3 or 4, or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence as set forth in SEQ ID NO: 5, or a sequence variant thereof having at least 70% sequence identity, or - the amino acid sequence as set forth in SEQ ID NO: 10, or a sequence variant thereof having at least 70% sequence identity.

[0137] More preferably, the multiple antigenic domains of the peptide and the multiple antigenic domains encoded in VSV each comprise different (i.e., two or more) corresponding tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or fragments or variants thereof. In some embodiments, the multiple antigenic domains of the peptide and the multiple antigenic domains encoded in VSV each comprise (exactly or at least) two corresponding tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or corresponding fragments or variants thereof. In some embodiments, the multiple antigenic domains of the peptide and the multiple antigenic domains encoded in VSV each comprise (exactly or at least) three corresponding tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1, and AURKA-uORF2, or corresponding fragments or variants thereof. In some embodiments, the multiple antigenic domains of the peptide and the multiple antigenic domains encoded in VSV each comprise (exactly or at least) four corresponding tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1, and AURKA-uORF2, or corresponding fragments or variants thereof. In some embodiments, the peptide multiple antigenic domain and the VSV encoded multiple antigenic domain each comprise (exactly or at least) five corresponding tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1, and AURKA-uORF2, or corresponding fragments or variants thereof. In some embodiments, the peptide multiple antigenic domain and the VSV encoded multiple antigenic domain each comprise all six different corresponding tumor antigens selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1, and AURKA-uORF2, or corresponding fragments or variants thereof.

[0138] Preferably, the multiple antigenic domain encoded in the genome of a vesicular stomatitis virus (VSV) comprises the amino acid sequence of each of the antigens included in the peptidic multiple antigenic domain, or a fragment or sequence variant thereof. In some embodiments, the multiple antigenic domain encoded in the genome of a vesicular stomatitis virus (VSV) comprises one or more additional amino acid sequences, preferably of antigens, or fragments or sequence variants thereof, not included in the peptidic multiple antigenic domain. In other embodiments, the multiple antigenic domain encoded in the genome of a vesicular stomatitis virus (VSV) consists of the amino acid sequence of each of the antigens included in the peptidic multiple antigenic domain, or a fragment or sequence variant thereof.

[0139] In some embodiments, the multiple antigenic domain of the peptide comprises the amino acid sequence of each of the antigens, or fragments thereof, of the multiple antigenic domain encoded in the genome of Vesicular Stomatitis Virus (VSV). In some embodiments, the multiple antigenic domain of the peptide comprises one or more additional amino acid sequences, preferably of antigens, or fragments or sequence variants thereof, that are not included in the multiple antigenic domain encoded in the genome of Vesicular Stomatitis Virus (VSV). In other embodiments, the multiple antigenic domain of the peptide consists of the amino acid sequence of each of the antigens, or fragments or sequence variants thereof, that are included in the multiple antigenic domain encoded in the genome of Vesicular Stomatitis Virus (VSV).

[0140] Preferably, the multiple antigenic domain encoded in the genome of vesicular stomatitis virus (VSV) may comprise both (i) KRAS-G12D, or a fragment or sequence variant thereof, and (ii) KRAS-G12V, or a fragment or sequence variant thereof, whereas the multiple antigenic domain of the peptide may comprise either (i) KRAS-G12D, or a fragment or sequence variant thereof, or (ii) KRAS-G12V, or a fragment or sequence variant thereof. Thus, a multiple antigenic domain encoded in the genome of vesicular stomatitis virus (VSV) can comprise both (i) the amino acid sequence set forth in SEQ ID NO: 11, or a sequence variant thereof having at least 70% sequence identity, and (ii) the amino acid sequence set forth in SEQ ID NO: 12, or a sequence variant thereof having at least 70% sequence identity, while a multiple antigenic domain of a peptide can comprise either (i) the amino acid sequence set forth in SEQ ID NO: 11, or a sequence variant thereof having at least 70% sequence identity, or (ii) the amino acid sequence set forth in SEQ ID NO: 12, or a sequence variant thereof having at least 70% sequence identity.

[0141] In some embodiments, the multiple antigenic domain of the peptide may comprise both (i) KRAS-G12D, or a fragment or sequence variant thereof, and (ii) KRAS-G12V, or a fragment or sequence variant thereof, while the multiple antigenic domain encoded in the genome of vesicular stomatitis virus (VSV) may comprise either (i) KRAS-G12D, or a fragment or sequence variant thereof, or (ii) KRAS-G12V, or a fragment or sequence variant thereof. Thus, a multiple antigenic domain of a peptide can comprise both (i) the amino acid sequence set forth in SEQ ID NO: 11, or a sequence variant thereof having at least 70% sequence identity, and (ii) the amino acid sequence set forth in SEQ ID NO: 12, or a sequence variant thereof having at least 70% sequence identity, whereas a multiple antigenic domain encoded in the genome of vesicular stomatitis virus (VSV) can comprise either (i) the amino acid sequence set forth in SEQ ID NO: 11, or a sequence variant thereof having at least 70% sequence identity, or (ii) the amino acid sequence set forth in SEQ ID NO: 12, or a sequence variant thereof having at least 70% sequence identity.

[0142] More preferably, in the vaccine, kit or combination of the invention, - the peptide multiple antigenic domain comprises an amino acid sequence as set forth in SEQ ID NO: 13 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 13, or an amino acid sequence as set forth in SEQ ID NO: 14 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 14, and - the multiple antigenic domain encoded in the genome of vesicular stomatitis virus (VSV) comprises the amino acid sequence set forth in SEQ ID NO: 19 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or the amino acid sequence set forth in SEQ ID NO: 48 or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 48, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0143] Even more preferably, in the vaccine, kit or combination of the invention - the peptide is selected from the group consisting of an amino acid sequence as set forth in SEQ ID NO: 17, or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 17, an amino acid sequence as set forth in SEQ ID NO: 18, or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 18, or an amino acid sequence set forth in SEQ ID NO: 47 or a sequence variant thereof having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 47; and - Vesicular stomatitis virus (VSV) comprises an RNA genome comprising the RNA sequence set forth in SEQ ID NO: 30, or a sequence variant thereof having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0144] In some embodiments of the vaccines, kits or combinations of the invention, the vesicular stomatitis virus (VSV) comprises an RNA genome comprising (or consisting of) an RNA sequence corresponding to the cDNA sequence set forth in SEQ ID NO: 49, or a sequence variant thereof as defined above (e.g., a sequence variant having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0145] The vaccine, kit or combination of the present invention further comprises: (iii) PD-1 / PD-L1 pathway inhibitors It is also preferred that the compound further comprises: Generally, the PD-1 / PD-L1 pathway inhibitor may be provided (a) in the same composition or in the same container as any one of components (i) and (ii) (peptide and VSV), or (b) in a (spatially) separate manner (e.g. in a different composition or in a different container), although in the vaccines, kits and combinations of the invention, the PD-1 / PD-L1 pathway inhibitor is preferably provided in a separate manner (separate from (i) the peptide and (ii) VSV), e.g. in a separate container or in a separate composition. As used herein, the term "inhibitor" includes lowering, reducing, blocking and inhibiting the PD-1 / PD-L1 pathway, including antagonists (and inverse agonists) of the PD-1 / PD-L1 pathway.

[0146] In general, the PD-1 / PD-L1 pathway is well known in the art and described, for example, in Han Y, Liu D, Li L. PD-1 / PD-L1 pathway: current researches in cancer. Am J Cancer Res. 2020 Mar 1;10(3):727-742. PD-1 (programmed cell death protein 1) and its ligand PD-L1 (programmed cell death ligand 1) are well known for their role in cancer immune escape. Non-limiting examples of inhibitors of the PD-1 / PD-L1 pathway include pembrolizumab (anti-PD-1 antibody), nivolumab (anti-PD-1 antibody), pidilizumab (anti-PD-1 antibody), cemiplimab (anti-PD-1 antibody), PDR-001 (anti-PD-1 antibody), atezolizumab (anti-PD-L1 antibody), avelumab (anti-PD-L1 antibody), durvalumab (anti-PD-L1 antibody), and PD1-1, PD1-2, and PD1-3 (anti-PD-1 antibodies) as described herein. Thus, the inhibitor of the PD-1 / PD-L1 pathway may be selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, cemiplimab, PDR-001, atezolizumab, avelumab, durvalumab, ezavenlimab, an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 32 (PD1-1), an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 34 (PD1-2), and an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and a light chain comprising the amino acid sequence of SEQ ID NO: 36 (PD1-3).

[0147] Additional PD-1 antagonists disclosed by Li et al. (supra) or known to be in clinical trials, such as AMP-224, MEDI0680 (AMP-514), BMS-936559, JS001-PD-1, SHR-1210, BMS-936559, TSR-042, JNJ-63723283, MEDI4736, MPDL3280A, can be used as an alternative to or in addition to the antagonists described above. As used herein, INN is meant to encompass all biosimilar antibodies that have the same or substantially the same amino acid sequence as the original antibody, including, but not limited to, biosimilar antibodies approved under 42 USC §262 subsection (k) in the United States and equivalent regulations in other jurisdictions.

[0148] The antibodies PD1-1, PD1-2 and PD1-3 are antibodies that comprise the following (full-length) heavy chain and (full-length) light chain sequences: - PD1-1: a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 32; - PD1-2: a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 34; - PD1-3: a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and a light chain comprising the amino acid sequence of SEQ ID NO: 36.

[0149] Preferably, the inhibitor of the PD-1 / PD-L1 pathway may be selected from the group consisting of ezabenlimab, an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 32 (PD1-1), an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 34 (PD1-2), and an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and a light chain comprising the amino acid sequence of SEQ ID NO: 36 (PD1-3).

[0150] Medical Treatment and Uses The composition according to the invention as described above, the peptide according to the invention as described above, the Vesicular Stomatitis Virus (VSV) according to the invention as described above, the kit according to the invention as described above, the vaccine according to the invention as described above, or the combination according to the invention as described above may be for use in medicine.Preferably, the composition according to the invention as described above, the peptide according to the invention as described above, the Vesicular Stomatitis Virus (VSV) according to the invention as described above, the kit according to the invention as described above, the vaccine according to the invention as described above, or the combination according to the invention as described above is for use in the treatment of cancer. Thus, the present invention also provides the use of a composition according to the present invention as described above, a peptide according to the present invention as described above, a vesicular stomatitis virus (VSV) according to the present invention as described above, a kit according to the present invention as described above, a vaccine according to the present invention as described above, or a combination according to the present invention as described above, for the manufacture of a medicament for the treatment of cancer.

[0151] In a further aspect, the present invention provides a method for ameliorating, treating or reducing (the risk of occurrence) cancer or for inducing or enhancing an anti-tumor response, comprising administering to a subject in need thereof an (effective amount of) a composition according to the present invention as described above, a peptide according to the present invention as described above, a Vesicular Stomatitis Virus (VSV) according to the present invention as described above, a kit according to the present invention as described above, a vaccine according to the present invention as described above, or a combination according to the present invention as described above. Preferably, the cancer (as mentioned in the above method and use) is a cancer of the gastrointestinal tract (GI). Non-limiting examples of GI cancer include anal cancer, appendiceal cancer, cholangiocarcinoma / bile duct cancer, particularly extrahepatic bile duct cancer, gastrointestinal carcinoid tumor, colorectal cancer, particularly colon cancer, rectal cancer and metastatic colorectal cancer, esophageal cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal stromal tumor (GIST), and pancreatic cancer, such as pancreatic ductal adenocarcinoma. Thus, the cancer is preferably selected from the group consisting of anal cancer, appendiceal cancer, cholangiocarcinoma / bile duct cancer, particularly extrahepatic bile duct cancer, gastrointestinal carcinoid tumor, colorectal cancer, particularly colon cancer, rectal cancer and metastatic colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor (GIST), and pancreatic cancer, such as pancreatic ductal adenocarcinoma. More preferably, the cancer is selected from the group consisting of colon cancer, rectal cancer, colorectal cancer, metastatic colorectal cancer, pancreatic cancer, and pancreatic ductal adenocarcinoma.

[0152] For the envisaged medical treatment, in particular for the treatment of cancer (e.g. as described above), the peptide of the present invention as described above is preferably combined with the vesicular stomatitis virus (VSV) of the present invention as described above. Thus, the peptide for the above-mentioned use can be administered in combination with the vesicular stomatitis virus (VSV) of the present invention as described above. Furthermore, the vesicular stomatitis virus (VSV) for the above-mentioned use can be administered in combination with the peptide of the present invention as described above. In such combined use, the multiple antigenic domains of the peptide and the multiple antigenic domains encoded in the VSV are preferably adapted to each other as described in detail above in the context of the vaccines, kits and combinations of the present invention comprising the peptide and VSV. In particular, the "combination" or "combined" use of a peptide as described herein and VSV generally means that treatment with a peptide as described herein is combined with treatment with a VSV as described herein. In such combined use of the peptide of the present invention as described above and VSV (i.e., for each vaccine, kit and combination of the present invention as described above), each of the peptide and the vesicular stomatitis virus (VSV) is generally administered at least once. Preferably, the peptide and the vesicular stomatitis virus (VSV) are administered sequentially (not simultaneously).

[0153] However, even if one component (peptide or VSV) is not administered, for example, on the same day as the other component, their treatment schedules are usually intertwined. Thereby, advantageous heterologous prime-boost regimes as described above can be achieved. In particular, one component (e.g., peptide) can be administered first (as "priming") and the other component (e.g., VSV) can be administered later, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or more days or weeks after "priming" (as "boost"). Thereby, the interval between priming and boosting is usually selected to find a strong immune response. Preferably, the peptide is administered prior to administration of vesicular stomatitis virus (VSV). Thus, in a heterologous prime-boost regimen, the peptide can function as the "priming" and VSV can function as the "boost."

[0154] In some embodiments, the peptide is administered at least twice, preferably before and after administration of VSV. Thus, the peptide and the vesicular stomatitis virus (VSV) are administered in the order KVK, where "K" refers to a (single) administration of the peptide and "V" refers to a (single) administration of the VSV. In some embodiments, the peptide is administered repeatedly. For example, the peptide and the vesicular stomatitis virus (VSV) can be administered in the order KVK, KVKK, KVKKK, or KVKKKK (where "K" refers to the peptide and "V" refers to VSV). In some embodiments, the treatment schedule includes a single administration of the vesicular stomatitis virus (VSV), i.e., VSV is administered only once.

[0155] In some embodiments, the peptide and VSV are administered 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, or 21 days apart from each other, preferably about 4 to about 24 days apart from each other, more preferably about 10 to about 18 days apart from each other, and even more preferably about 12 to about 16 days apart from each other. In some embodiments, the peptide is administered repeatedly, preferably once every 2 to 6 weeks, more preferably once every 3 to 5 weeks, and even more preferably once every 4 weeks. In some embodiments, the dose of peptide may be about 0.5 nmol to about 10 nmol. In some embodiments, the dose of VSV may be about 10 6 TCID 50 ~about 10 11 TCID 50 Preferably, the dose of VSV is about 10 7 TCID 50 ~about 10 9 TCID 50 , more preferably about 10 8 TCID 50 ~about 10 9 TCID 50 , or about 10 7 TCID 50 ~about 10 8 TCID 50 , more preferably about 1×10 7 TCID 50 ~Approx. 1×10 8 TCID 50 It may be.

[0156] Generally, the peptide and the vesicular stomatitis virus (VSV) can be administered via the same or different routes of administration. Preferably, the route of administration is selected from intravenous, subcutaneous, and intramuscular administration. Preferably, the peptide and the VSV are administered via different routes of administration. More preferably, the peptide is administered subcutaneously and the vesicular stomatitis virus (VSV) is administered intravenously or intratumorally, preferably intravenously.

[0157] The (combined) use of peptides and VSV (as well as in the vaccines, kits and combinations described herein) may further comprise the administration of an inhibitor of the PD-1 / PD-L1 pathway, for example an inhibitor of the PD-1 / PD-L1 pathway as described above. Thus, the inhibitor of the PD-1 / PD-L1 pathway may be selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, cemiplimab, PDR-001, atezolizumab, avelumab, durvalumab, ezavenlimab, an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 32 (PD1-1), an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 34 (PD1-2), and an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and a light chain comprising the amino acid sequence of SEQ ID NO: 36 (PD1-3).

[0158] The PD-1 / PD-L1 pathway inhibitor is administered simultaneously, sequentially, or alternating with the peptide or vesicular stomatitis virus (VSV). In some embodiments, the PD-1 / PD-L1 pathway inhibitor is administered on the same day and / or alternating with the peptide.

[0159] A brief description of the attached drawings follows below, which are intended to explain the invention in more detail, but are not intended to limit the subject matter of the invention in any way. [Brief description of the drawings]

[0160] [Figure 1]Figure 1 shows the expression of transcripts encoding KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2 measured by BaseScope on tissue arrays (HalioDX) and tumor whole sections (A) for Example 1. Probes corresponding to KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2-antigen sequences were used. The bar graph shows the quantification of the experiment by an automated scoring algorithm (HALO-Indica lab). Note that healthy tissue adjacent to CRC is characterized by substantially higher expression of the transcripts of interest than healthy colon tissue. (B) Comparison of RNA-Seq-based expression of KRAS, TPX2 and AURKA transcripts between healthy colon / pancreas and tumor adjacent colon / pancreas tissue (TCGA / GTEx). mRNA expression of KRAS, TPX2 and AURKA data were extracted from the following public databases: Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx). (C) Translation of KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2 tumor antigen ORFs in human pancreatic (PDAC), gastric (GC) and colorectal (CRC) tumors or tumor-adjacent healthy tissues. Translation was measured by RiboSeq. The number of in-frame reads across each ORF was normalized for ORF length (assuming translation is driven by the innermost possible start codon) and sample library size (i.e., the sum of all trimmed reads in the sample mapping to the human genome after removal of reads derived from rRNA and tRNA). Unpaired two-tailed t-tests were performed to assess statistical significance (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 2-1] For Example 2, (A) DUOXA2 and CEACAM5 transcript expression in various GI tract tumors and healthy tissues (TCGA / GTEx). (B) CEACAM5 protein expression measured by immunohistochemical staining on tissue arrays (HalioDX). Bar graphs (upper panel) show quantification of the experiment. [Figure 2-2]For Example 2, (C) Expression of DUOXA2 transcripts measured by RNAScope on tissue arrays (HalioDX). The bar graph (left panel) shows the quantification of the experiment. [Diagram 3] For Example 3, (A) Co-expression of mutant KRAS-G12D / V with CEACAM5 and DUOXA2 at the mRNA level (based on various RNASeq-based datasets including TCGA) in pancreatic (PDAC) and colorectal (CRC) cancers. (B) Immunogenicity of epitopes derived from KRAS-uORF1, TPX2-uORF1, AURKA-uORF2, KRAS-G12D / V, and DUOXA2. To stimulate specific T cell responses in healthy donor PBMCs, in vitro priming was performed using the indicated peptides (e.g., KLVVVGADGV) in the context of a given HLA allele (e.g., HLA-A*0201). Cultures enriched for peptide-specific T cells were then assayed on various tumor cell line targets pulsed with the respective peptides (specific responses). In parallel, the same tumor cell lines were pulsed with an irrelevant control peptide or not pulsed at all (non-specific background response). T cell activation was measured by IFNγ ELISpot. In panel (B), each spot corresponds to a different tumor cell line or moDC target. The ratio of specific response to non-specific background response is shown. A ratio above 1 indicates a specific T cell response. Negative controls (i.e. peptide epitopes that did not induce a specific T cell response) are included in the panel. [Figure 4]Figure 4: Presentation of ATP150- and ATP152-derived epitopes by human moDCs for Example 4. moDCs were incubated with either ATP150 or ATP152 to allow processing of the vaccine by endogenous antigen presentation mechanisms. Cross-presented antigens were then analyzed by mass spectrometry. In the panel, detected peptide epitopes were mapped against the ATP150 / ATP152 backbone to visualize the regions from which they were derived. Each line corresponds to one of ten PBMC donors. Note that the ten donors differ from each other in terms of their HLA haplotypes and are therefore expected to present different peptide epitopes. [Diagram 5] Immunogenicity after ATP150 / VSV-GP154 heterologous prime-boost vaccination for Example 5. IFNγ-producing CEACAM5 and DUOXA2 specific T cells by ELISpot. (A) Overview of prime-boost protocol. Mice [n=5] were primed (d0) and boosted (d14) with 10 nmol ATP150 (s.c.) or primed (d0) with 10 nmol ATP150 (s.c.) and boosted (d14) with 107 TCID50 VSV-GP154 (i.v.). (B) Splenocytes were harvested on day 21 and pulsed with overlapping 11mer peptides corresponding to the CEACAM5 and DUOXA2 regions incorporated in the vaccine. ***p≦0.001, ****p≦0.0001. [Figure 6] For Example 6, (A) tumor growth (mean ± SEM) and (B) survival of mice immunized with 1x105 TC-1 cells subcutaneously and either 2 nmol of peptide construct (multiple antigenic domain "Mad25") subcutaneously 7 days later or 1x107 TCID50 of VSV-GP-HPV intravenously 14 days after tumor implantation. Additional doses of K and V were administered as indicated by the dotted lines and tumor growth was monitored (n=7). [Figure 7]FIG. 7 shows the vaccination schedule (A) and detection of HPV-specific CD8+ T cells after heterologous prime-boost and anti-PD-1 in TC-1 tumor-bearing mice (B) for Example 7. Mice were bled on days 14, 21, 35, 42, and 56 and analyzed by flow cytometry with multimer staining to detect HPV-specific CD8+ T cells. [Figure 8] Figure 7 shows the efficacy of heterologous prime-boost vaccination and anti-PD-1 in TC-1 tumor-bearing mice for Example 7. Mean tumor volume and survival curves of C57BL / 6 mice (n=7 mice / group) implanted subcutaneously with TC-1 cells. Mice were vaccinated with peptide construct (multiple antigenic domain "Mad25") (K, 2 nmol / mouse, subcutaneous) and VSV-GP-HPV (V, 107 TCID50 / mouse, intravenous). Anti-PD-1 (200 μg) was administered intravenously. The number of tumor-free mice is shown. [Figure 9] Figure 8 shows the efficacy of heterologous prime-boost vaccination comparing three different doses of VSV-GP-HPV in tumor-bearing mice for Example 8. Tumor volumes (upper / left panel) and survival curves (lower / right panel) of C57BL / 6 mice (n=7 mice / group) subcutaneously implanted with TC-1 cells are shown. Mice were vaccinated at three different doses, 1x107, 3x107 or 5x107 TCID50, with either peptide construct (multiple antigenic domains "Mad25") (K) or VSV-GP-HPV (V) on days 7, 14 and 28 (Vac). Median survival times are shown on the graph (ms). *, p<0.05; **, p<0.01; ***, p<0.001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0161] Example 1 Tumor antigens encoded within the 5'UTR of KRAS mRNA (KRAS-uORF1), TPX2 mRNA (TPX2-uORF1), and AURKA mRNA (AURKA-uORF2) Novel tumor antigens encoded in the open reading frames (ORFs) of the 5'UTR of KRAS mRNA (KRAS-uORF1), TPX2 mRNA (TPX2-uORF1), and AURKA mRNA (AURKA-uORF2) have been identified. The tumor antigens (peptides) are also referred to herein as "KRAS-uORF1 peptide" or "KRAS-uORF1," "TPX2-uORF1 peptide" or "TPX2-uORF1," and "AURKA-uORF2 peptide" or "AURKA-uORF2," respectively. Each tumor antigen has the amino acid sequence set forth in SEQ ID NO: 1 (KRAS-uORF1 peptide), SEQ ID NO: 3 (TPX2-uORF1 peptide), and SEQ ID NO: 5 (AURKA-uORF2 peptide).

[0162] Because uORFs are often characterized by unusual, non-canonical translation initiation sites, and KRAS-uORF1, TPX2-uORF1, and AURKA-uORF2 do not contain a conventional initiation codon, we first tested whether they were translatable in human cancer cell lines. To this end, antigens were fused to the N-terminus of dTomato, and expression of the resulting fluorescent fusion proteins was confirmed by flow cytometry.

[0163] To investigate the tumor expression of KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2 at the RNA level, we used BaseScope detection of the relevant transcripts. For this purpose, we used probes corresponding only to the KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2 coding sequences themselves, which makes it possible to distinguish between mRNA transcripts of loci that code for the tumor antigen of interest and those that do not (for example, according to Ensembl, the AURKA locus encompasses 13 alternatively spliced ​​transcripts, and of these 13 transcripts, 7 code for the full-length AURKA kinase, whereas only 3 code for AURKA-uORF2).

[0164] In the first experiment, transcription, i.e. the production of relevant KRAS, TPX2, and AURKA mRNA transcripts, was evaluated in (i) pancreatic and colorectal tumors, (ii) "healthy" tissue adjacent to the tumor, and (iii) healthy tissue from non-diseased organs. For this purpose, staining was performed with LEICA BondMax©. Slides were assessed for RNA quality by using a positive control probe (housekeeping gene PPIB). After QC testing, slides were tested using BaseScope probes: Basescope® 2.5LS probe BA-Hs-Aurka-Tu-1-1zz-stC1 lot: 21175A (ACD#1084338-C1), Basescope® LS probe BA-Hs-KRAS-1zz-stC1 lot: 21175A (ACD#1084358) according to the manufacturer's instructions. Slides were counterstained with hematoxylin and mounted using VectaMount mounting medium. Entire slide images were scanned at 40x objective magnification using a Nanozoomer S360© Digital Pathology Scanning System (Hamamatsu). The number of cancer cells with one or more red signals (positive cancer cells) was scored as positive. The percentage of positive cancer cells was calculated using HALO software (Indica Lab) by a certified pathologist.

[0165] Since the KRAS-uORF1, TPX2-uORF1, and AURKA-uORF2 peptide tumor antigens lack any known domains, it seems unlikely that they would stably fold and accumulate in cells. Without being bound by any theory, it is assumed that they undergo rapid proteasomal degradation, which would be advantageous since it could lead to the production of antigenic epitopes. However, the predicted steady-state protein levels of these entities are very low, making them essentially impossible to detect with antibodies. Nevertheless, to assess their translation (i.e., their protein synthesis) in human pancreatic (PDAC), gastric (GC), and colorectal (CRC) tumors (or healthy tissue adjacent to the tumors), we employed the RiboSeq approach (Ingolia, NT, Ghaemmaghami, S., Newman, JRS, and Weissman, JS (2009). Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling. Science 324, 218-223). Briefly, at least 200 mg of fresh frozen tumor tissues and corresponding tumor-adjacent tissues from PDAC [n=12; only 5 of which had tumor-adjacent material], GC [n=5], and CRC [n=44] were purchased from a commercial provider (Indivumed GmbH, Hamburg) for RiboSeq library preparation, sequencing (NovaSeq6000, 200M+ reads / library), and bioinformatics analysis at a partner CRO (TB-Seq Inc, South San Francisco). After trimming adapters using Cutadapt, they were aligned against the human genome assembly GRCh38.p13 using STAR. Ribosome footprints were confirmed to match the expected length spectrum. 28-36mers were considered for further analysis.The quality of the dataset and proper 3-nt periodicity were verified by assessing the metagene read distribution around the start / stop codons. To quantify the translation of AURKA-uORF2, TPX2-uORF1, and KRAS-uORF1, the number of associated in-frame reads (10 7 The mean mean (multiplied by ) was normalized for ORF length (under the assumption that translation is driven by the innermost RiboSeq-assisted start codon) and sample library size (i.e., the sum of all trimmed mappable reads after removal of sequences derived from rRNA and tRNA) to allow comparison between samples.

[0166] The results of expression of transcripts encoding KRAS-uORF1, TPX2-uORF1, and AURKA-uORF2 (A), RNA-Seq-based comparison of expression of KRAS, TPX2, and AURKA transcripts between healthy colon / pancreas and tumor-adjacent colon / pancreas tissues (B), and translation of uORFs of interest in human pancreatic (PDAC), gastric (GC), and colorectal (CRC) tumors or tumor-adjacent healthy tissues (C) are shown in Figure 1 .

[0167] These data demonstrated strong expression of the relevant KRAS, TPX2, and AURKA transcripts in pancreatic and colorectal tumors (Figure 1A), whereas normal healthy tissues from colon, pancreas, and other organs showed only weak signals above background (Figure 1A). This strongly indicates that the encoded tumor antigens can be targeted in these cancers. The "healthy" tissues adjacent to the tumors showed intermediate transcriptional signals, unlike truly healthy tissues from non-diseased organs. This trend was also seen in the RNASeq-based TCGA / GTEx dataset (Figure 1B), pointing to transcriptional dysregulation spreading from the tumor site to the directly adjacent regions. Without being bound by any theory, it is therefore assumed that healthy tissues adjacent to tumors are not necessarily completely representative of truly healthy tissues.

[0168] FIG. 1C shows that KRAS-uORF1, TPX2-uORF1, and AURKA-uORF2 peptide tumor antigens are expressed in tumors, albeit to varying degrees, in various indications, including pancreatic, gastric, and colorectal cancer. In general, either low or no synthesis was observed in healthy tissues directly adjacent to the tumor (FIG. 1C). Without being bound by any theory, it is inferred that translation of KRAS-uORF1, TPX2-uORF1, and AURKA-uORF2 is not detectable in most healthy tissues adjacent to the tumor, despite the suggestion of expression of the corresponding mRNA transcripts from BaseScope (FIG. 1A) and RNASeq (FIG. 1B), due to tumor-selective upregulation of upstream ORF translation, which may be related to the process of tumorigenesis itself. Thus, for KRAS-uORF1, TPX2-uORF1, and AURKA-uORF2, cancer-specific translation appears to add an additional layer of tumor specificity on top of transcription that is largely cancer-specific.

[0169] Example 2 Expression profile of additional tumor antigens CEACAM5 and DUOXA2 To select further tumor antigens of interest, CEACAM5 and DUOXA2 mRNA expression data were extracted from the following public databases: Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx). CEACAM5 and DUOXA2 were selected based on their tumor-selective expression by public RNASeq (Figure 2A).

[0170] The expression profiles of CEACAM5 and DUOXA2 were then experimentally verified in human tumor and non-tumor GI tract tissues. To measure CEACAM5 expression, formalin-fixed paraffin-embedded (FFPE) tissue arrays were purchased from a commercial provider (Indivumed GmbH, Hamburg). After QC testing, CEACAM5 staining was performed by ImmunoHistoChemistry using a CEACAM5-specific antibody (DakoM707229-2). Slides were counterstained with hematoxylin and mounted using VectaMount mounting medium. The entire slide images were scanned at 40x objective magnification using a Nanozoomer S360© Digital Pathology Scanning System (Hamamatsu). The number of cancer cells with one or more red signals (positive cancer cells) was scored as positive. The percentage of positive cancer cells was calculated by a certified pathologist using HALO software (Indica Lab).

[0171] Furthermore, DUOXA2 expression was investigated in GI tract tissues. For this purpose, formalin-fixed paraffin-embedded (FFPE) tissue arrays were purchased from a commercial provider (Indivumed GmbH, Hamburg). Slides were assessed for RNA quality by using a positive control probe (housekeeping gene PPIB). After QC testing, expression of DUOXA2 transcripts was performed by using an RNAscope probe for DUOXA2 (Bio-Techne HD-RM-000619) according to the manufacturer's instructions. Slides were counterstained with hematoxylin and mounted using VectaMount mounting medium. The entire slide images were scanned at 40x objective magnification using a Nanozoomer S360© Digital Pathology Scanning System (Hamamatsu). The number of cancer cells with one or more red signals (positive cancer cells) was scored as positive. The percentage of positive cancer cells was calculated by a certified pathologist using HALO software (Indica Lab). The results are shown in Figure 2. Specifically, CEACAM5 was detected by immunohistochemistry in 80-100% of pancreatic and colorectal cancers, but not in completely healthy tissue or in healthy tissue adjacent to the tumor (Figure 2B). DUOXA2 expression was robustly detected in 37-74% of pancreatic and colorectal cancers, but expression in the corresponding healthy tissue was rare (e.g., only 1 of 16 pancreatic cancers) (Figure 2C).

[0172] Example 3 Immunogenicity of KRAS-uORF1, TPX2-uORF1, AURKA-uORF2, CEACAM5, DUOXA2 and KRAS-G12D / V antigens In addition to the tumor antigens of Examples 1 and 2 above, a further tumor antigen of interest was included in further analysis, namely KRAS, specifically KRAS-G12D / V. The oncogenic driver mutation KRAS-G12D / V is not generally found outside of tumors.

[0173] In the first step, we co-expressed mutant KRAS-G12D / V with CEACAM5 and DUOXA2 at the mRNA level in pancreatic cancer (PDAC) and colorectal cancer (CRC) (based on various RNASeq-based datasets including TCGA). Furthermore, to identify peptides that bind to specific HLA class I alleles, the NetMHCpan4.1 prediction algorithm was used to predict MHC class I epitopes of KRAS-uORF1, TPX2-uORF1, AURKA-uORF2, and DUOXA2. A subset of these peptides and donor PBMCs expressing the corresponding HLA alleles were then used for in vitro priming experiments to determine immunogenicity. For this purpose, dendritic cells were isolated from these PBMCs and, after pulsing with selected peptides, they were used to stimulate autologous CD8 T cells, whose responses were measured by IFNg ELISpot assay.

[0174] Next, the immunogenicity of KRAS-G12D / V was analyzed using peripheral blood mononuclear cells (PBMCs) from two independent healthy donors. For this purpose, CD8 + T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors. Monocytes were isolated by plastic adherence to CD8 +CD8+ T cells were isolated from depleted PBMCs and subsequently treated with cytokines for generation and maturation of dendritic cells (DCs, antigen presenting cells). Donors were selected by their expression of specific HLA alleles predicted for presentation of each peptide by DCs. CD8+ T cells were primed at least three times with peptide-loaded DCs before testing their specific immune response to the peptides. Priming cycles included co-culture of CD8+ T cells with peptide-loaded DCs for 7-10 days. After three priming cycles, CD8 T cells were plated in an ELISpot assay (Immunospot, Shaker Heights, OH44122) to assess their ability to secrete IFN-β after overnight stimulation with tumor cell lines or peptide-loaded DCs. Spots were counted using an ELISPOT reader.

[0175] The results are shown in Figure 3. As shown in Figure 3A, co-expression analysis of mutant KRAS-G12D / V with CEACAM5 and DUOXA2 at the mRNA level in pancreatic cancer (PDAC) and colorectal cancer (CRC) revealed that 59% of KRAS-G12D / V mutant PDAC / CRC tumors expressed both CEACAM5 and DUOXA2 transcripts at more than 5 TPM. Figure 3B shows the immunogenicity of epitopes derived from KRAS-uORF1, TPX2-uORF1, AURKA-uORF2, KRAS-G12D / V, and DUOXA2. All of the peptides tested were found to be immunogenic (Figure 3B), suggesting that cognate T cells are likely available for activation in cancer patients.

[0176] Example 4 Design and evaluation of peptides containing cell-penetrating peptides, multiple antigenic domains, and TLR agonists Next, peptides were designed that comprised (i) a cell penetrating peptide, (ii) multiple antigenic domains and (iii) a TLR agonist essentially as described in WO2016 / 146260. For this purpose, two different multiple antigenic domains were designed that comprised either (immunogenic) fragments of KRAS-uORF1, TPX2-uORF1, AURKA-uORF2, KRAS-G12D, CEACAM5 and DUOXA2 or (immunogenic) fragments of KRAS-uORF1, TPX2-uORF1, AURKA-uORF2, KRAS-G12V, CEACAM5 and DUOXA2.

[0177] The multiple antigenic domains of both constructs contain the following antigenic fragments of CEACAM5: CEACAM5-1 NRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVL [SEQ ID NO:6] CEACAM5-2 PDSSYLSGANLNLSCHSAS [SEQ ID NO: 7] CEACAM5-3 PQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSA [SEQ ID NO:8]

[0178] Based on these three different fragments, CEACAM5 fusion constructs of the following sequences were designed with multiple antigenic domains in both constructs: NRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSA [SEQ ID NO: 9] In addition, the following antigens or antigenic fragments were included in the multi-antigenic domain of both constructs: DUOXA2: TGVLSLFLGGAVVSLQYVRPSALRTLLDQSAKD [SEQ ID NO: 10] KRAS-uORF1: VAAARPVLPAPAISDWERARRRH [SEQ ID NO: 1] TPX2-uORF1: VGRKEGAARARVSLLPEFGTAEVHLPAPSAVRAARPGF [SEQ ID NO:3] AURKA-uORF2: GGDKGRLVGVAERQVPCRFLRP [SEQ ID NO:5]

[0179] The multi-antigenic domains of both constructs differ in that one contains a fragment of KRAS-G12D (SEQ ID NO: 11) and the other contains a fragment of KRAS-G12V (SEQ ID NO: 12). KRAS-G12D: TEYKLVVVGADGVGKSALTIQLIQ (SEQ ID NO:11) KRAS-G12V: TEYKLVVVGAVGVGKSALTIQLIQ (SEQ ID NO:12)

[0180] Two different peptide constructs are designated "ATP150" (containing KRAS-G12D) and "ATP152" (containing KRAS-G12V). They contain the following multiple antigenic domains: Multi-antigenic domain ATP150: NRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSATEYKLVVVGA DGVGKSALTIQLIQVAAARPVLPAPAISDWERARRRHVGRKEGAARARVSLLPEFGTAEVHLPAPSAVRAARPGFGGDKGRLVGVAERQVPCRFLRPTGVLSLFLGGAVVSLQYVRPSALRTLLDQSAKD [SEQ ID NO: 13] Multi-antigenic domain ATP152: NRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSATEYKLVVVGA VGVGKSALTIQLIQVAAARPVLPAPAISDWERARRRHVGRKEGAARARVSLLPEFGTAEVHLPAPSAVRAARPGFGGDKGRLVGVAERQVPCRFLRPTGVLSLFLGGAVVSLQYVRPSALRTLLDQSAKD [SEQ ID NO: 14]

[0181] As described above, the peptide constructs ATP150 and ATP152 contain, in addition to multiple antigenic domains, a cell-penetrating peptide. The cell-penetrating peptide contained in ATP150 and ATP152 has the following sequence: KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLK [SEQ ID NO: 15] In addition, the peptide constructs ATP150 and ATP152 further comprise a TLR agonist: STVHEILSKLSLEGDHSTPPSAYGSVKPYTNFDAE [SEQ ID NO: 16]

[0182] In summary, the peptide constructs ATP150 and ATP152 exhibit the following sequences: ATP150 MKRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKNRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSATEYKLV VVGADGVGKSALTIQLIQVAAARPVLPAPAISDWERARRRHVGRKEGAARARVSLLPEFGTAEVHLPAPSAVRAARPGFGGDKGRLVGVAERQVPCRFLRPTGVLSLFLGGAVVSLQYVRPSALRTLLDQSAKDSTVHEILSKLSLEGDHSTPPSAYGSVKPYTNFDAE [SEQ ID NO: 17] ATP152 MKRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKNRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSATEYKLV VVGAVGVGKSALTIQLIQVAAARPVLPAPAISDWERARRRHVGRKEGAARARVSLLPEFGTAEVHLPAPSAVRAARPGFGGDKGRLVGVAERQVPCRFLRPTGVLSLFLGGAVVSLQYVRPSALRTLLDQSAKDSTVHEILSKLSLEGDHSTPPSAYGSVKPYTNFDAE [SEQ ID NO:18]

[0183] Next, the immunogenicity of antigens / antigenic fragments contained in the multi-antigenic domains of ATP150 and ATP152 was tested in monocyte-derived human dendritic cells (moDCs). For this purpose, cells were extracted from a fresh human leukoapheresis product named Leukopak supplied by Tissue Solution (Scotland). HLA typing of 15 donors was performed by the Immunology and Transplant Unit and National Reference Laboratory for Histocompatibility (LNRH) of Geneva University Hospital. Peripheral blood mononuclear cells (PBMCs) from leukopak samples were prepared after density gradient centrifugation on Ficoll-Paque and monocytes were enriched by cryoaggregation. Residual T lymphocytes were removed using rosetting with sheep red blood cells and density gradient centrifugation on Ficoll-Paque. Monocytes were subsequently plated with complete RPMI medium supplemented with GM-CSF and IL-4 for 7 days. Cells were then concentrated and interferon alpha 2a was added along with 600 nM ATP150 or ATP152. Cells were incubated overnight at 37°C and 5% CO2. Cells were then detached, counted, and centrifuged. Supernatants were aspirated and cell pellets were frozen at -20°C prior to ligandome analysis.

[0184] The cell pellet was lysed in solubilization buffer and homogenized by pulse sonication. After debris removal and sterile filtration, HLA class I and class II molecules were isolated from the soluble fraction using standard immunoaffinity purification. HLA ligands were then eluted by acid elution, isolated by ultracentrifugation, desalted, and preconcentrated prior to LC-MS / MS analysis.

[0185] Peptide samples were separated by nanoflow high performance liquid chromatography (RSLCnano, Thermo Fisher Scientific) using a 50 μm×25 cm PepMap rapid separation liquid chromatography column (Thermo Fisher Scientific) and an acetonitrile gradient ranging from 2.4% to 32.0% over 90 min. Eluted peptides were analyzed on an online-coupled LTQ Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) using the highest velocity collision-induced dissociation (CID) fragmentation method for HLA class I or high-energy collision dissociation (HCD) fragmentation method for HLA class II. For label-free quantification (LFQ) of relative HLA ligand abundance between activated samples loaded with and without ATP150 (or ATP152), the injected peptide amount of the paired samples was normalized from the mean precursor ion intensity determined in the dose-finding run, and LC-MS / MS analysis was performed for five technical replicates for each sample.

[0186] Raw files were processed using Proteome Discoverer 1.4 (Thermo Fisher Scientific). The SEQUEST HT search engine (University of Washington) was used to search the human proteome contained in the Swiss-Prot database in combination with the construct ATP150 (or ATP152). After data processing, specific filters were applied. For class I, data were filtered with an FDR of 5%, a peptide length of 8-12 amino acids, and a search engine rank of 1. For class II, data were filtered with an FDR of 1%, a peptide length of 8-25 amino acids, and a search engine rank of 1. Protein guessing was disabled, which allowed multiple protein annotation of peptides. To determine HLA class I binding peptides, the NetMHCpan-3.0 algorithm was used with a percentile binding rank of less than 2% and the SYFPEITHI algorithm was used with a threshold of 50% of the maximum score.

[0187] The results are shown in Figure 4. These data show that monocyte-derived human dendritic cells (moDCs) incubated with ATP150 or ATP152 presented MHC class I and / or MHC class II epitopes from all incorporated antigens, further demonstrating that endogenous processing in professional antigen-presenting cells can drive meaningful antigen presentation to patient CD8 and CD4 T cells. CEACAM5 and DUOXA2 epitopes are presented by MHC class I in human GI tract tumors in vivo. Ligandome analysis studies confirm the precise delivery of ATP150 and ATP152 to human moDC cells, the translation of the multi-antigenic domains of ATP150 and ATP152, and the multi-epitope and multi-allelic processing and presentation of all antigens in the multi-antigenic domains of ATP150 and ATP152.

[0188] Example 5 CEACAM5- and DUOXA2-specific T cell responses to ATP150 / VSV-GP154 heterologous prime-boost Antigen-specific T cell responses were then evaluated in a heterologous prime-boost vaccination scheme combining ATP150 (described in Example 4 above) with a recombinant vesicular stomatitis virus (VSV) encoding the corresponding antigen / antigenic fragment as described below (referred to herein as "VSV-GP154").

[0189] The multiple antigenic domains encoded in VSV include the antigens / antigenic fragments set forth in SEQ ID NOs: 1, 3, and 5 to 12. The multiple antigenic domain of VSV-GP154 has the following amino acid sequence: MNRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSATEYKLVVVGADGVGKSALTIQ LIQTEYKLVVVGAVGVGKSALTIQLIQVAAARPVLPAPAISDWERARRRHVGRKEGAARARVSLLPEFGTAEVHLPAPSAVRAARPGFGGDKGRLVGVAERQVPCRFLRPTGVLSLFLGGAVVSLQYVRPSALRTLLDQSAKD [SEQ ID NO: 19] Specifically, VSV-GP154 has the sequence set forth in SEQ ID NO: 30 (the RNA sequence of VSV-GP154, non-inverted complement). The corresponding cDNA sequence is provided in SEQ ID NO: 49.

[0190] To test antigen-specific T cell responses, a heterologous prime-boost vaccination scheme was evaluated, peripheral immune responses were examined on day 21 (1 week after the VSV-GP154 boost) and CEACAM5- and DUOXA2-specific T cells were determined by enzyme-linked immunospot (ELISpot) in mice. For this purpose, C57BL / 6 mice (6 and 10 weeks old) were injected with two cycles of vaccination: initial subcutaneous vaccination with 10 nmol ATP150, subcutaneous administration of 10 nmol ATP150 on day 14 or 10 7 TCID 50 VSV-GP154 intravenously. Spleens were harvested on day 21. Spleen cells were isolated and evaluated in ELISpot assays. ELISpot assays were performed using the Mouse IFNγ ELISpot Diaclone Kit (Ref 862.031.015S) according to the manufacturer's instructions. Splenocytes were isolated using Ficoll Paque Plus, GE Healthcare, ref 171440.02, and 2 × 10 cells were cultured at 10 °C for 1 h. 5 pieces~10×10 6The cells were plated in a concentration gradient of 150. Cells were pulsed with ATP150 and overlapping 11mer peptides corresponding to the CEACAM5 and DUOXA2 regions incorporated into VSV-GP154. Spots were counted using an ELISPOT reader.

[0191] Figure 5 shows the prime-boost protocol (A) and results (B). Boosting with VSV-GP154 promotes a strong expansion of CEACAM5- and DUOXA2-specific IFNγ-producing T cells compared to allogeneic prime-boost.

[0192] Example 6 Heterologous prime-boost vaccination in mouse tumor models A mouse tumor model (TC-1 tumor model) was used to evaluate the treatment effects such as tumor volume and survival rate in a heterologous prime-boost vaccination regimen with peptide constructs and recombinant VSV. For this purpose, the multi-antigenic domains of the peptide constructs and the multi-antigenic domains of VSV had to be adapted to the model tumor. Although the multi-antigenic domains as described in Examples 4 and 5 above were designed for use in humans, the animal tumor model requires vaccination with antigens / antigenic fragments corresponding to the model tumor. For this purpose, the multiple antigenic domain described in Examples 4 and 5 above was replaced by "multiple antigenic domain 25" (Mad-25, SEQ ID NO: 20), which contains both the CD8 and CD4H-2b epitopes from E7HPV. Thus, the peptide construct of this example contains the multiple antigenic domain Mad25. In addition to wild-type E2, VSV-GP-HPV also encodes an attenuated E6 / E7 fusion construct (Cassetti et al., 2004, Vaccine 22(3-4): 520-527).

[0193] Briefly, TC-1 cells were provided by TCWu (Johns Hopkins University, Maryland, US) and cultured in complete RPMI 1640 containing 0.4 mg / ml geneticin. For tumor implantation, 1 × 10 5 TC-1 cells were subcutaneously injected. Seven days later, the mice were given a subcutaneous priming immunization with 2 nmol of the peptide construct (multiple antigenic domains "Mad25", "K" in FIG. 6) or 1×10 7 TCID 50 Fourteen days after tumor implantation, mice were intravenously immunized with 1 × 10 VSV-GP-HPV. 7 TCID 50 100 mg of VSV-GP-HPV was administered intravenously. Additional doses of peptide construct "K" and virus "V" were administered as indicated by the dotted lines in Figure 6A. To monitor tumor growth, tumor diameters were measured 2-3 times a week using calipers and calculated according to the formula: 0.4 x length x width. 2 Volumes were calculated using the ELISA kit (1000 KB) using the ELISA kit (1000 KB). Mice were sacrificed when tumor sizes reached sizes specified by the veterinary authorities of each institution or when tumors showed signs of ulceration.

[0194] The results are shown in Figure 6. Viral treatment ("V") alone resulted in tumor growth delay (Figure 6A). However, "priming" with peptide constructs followed by viral treatment ("boost") further led to complete remission in all tumors, even in large tumors (Figure 6B). This strongly indicates that priming with peptide (K) can induce tumor regression of sizable virally treated tumors 2 weeks after implantation. These data suggest that priming with peptide (K) lays the immunological foundation for robust tumor remission after viral (V) boost in the TC-1 tumor model.

[0195] Example 7 Immunogenicity and efficacy of heterologous prime-boost combined with anti-PD-1 in tumor-bearing mice To evaluate the immunogenicity and efficacy of heterologous prime-boost vaccination and combination treatment with checkpoint inhibitors (anti-PD-1), the TC-1 mouse tumor model was used essentially as described in Example 6 above. TC-1 cells were implanted as described in Example 6, and mice were administered peptide construct "K" (with multi-antigenic domain Mad25) and VSV-GP-HPV "V" as described in Example 6. Mice were also administered 200 μg of PD-1 antibody (clone RMP1-14, BioXcell, Lebanon, New Hampshire, US) intravenously on days 7, 15, 28, and 49 according to the schedule shown in Figure 7A. To evaluate circulating anti-HPVCD8 T cells, mice were bled on days 14, 21, 35, 42, and 56 and analyzed by flow cytometry with multimer staining to detect HPV-specific CD8 T cells.

[0196] The results are shown in Figures 7 and 8. As shown in Figure 7B, circulating levels of HPV-specific CD8 T cells were increased to a similar extent in the KVKK regimen, regardless of whether the mice received anti-PD-1 treatment or not. As shown in Figure 8, therapeutic vaccination starting on day 7 after tumor cell implantation mediated a strong anti-tumor effect in the TC-1 tumor model in both groups treated with KVKK. Increased survival was observed when mice received an additional anti-PD-1 injection at each vaccination, with 4 of 7 mice remaining tumor-free in the triple combination group (Figure 8B).

[0197] Example 8 Effect of different VSV doses in heterologous prime-boost vaccination To evaluate the efficacy of different doses of VSV-GP-HPV "V" in heterologous prime-boost vaccination, the TC-1 tumor model was used as described in Examples 6 and 7 above. For tumor implantation, mice were implanted with 1×10 5TC-1 cells were subcutaneously injected. Seven days later, mice received a subcutaneous priming immunization with 2 nmol of peptide construct ("K"). A VSV-GP-HPV boost was administered 14 days after tumor cell implantation at three different doses: 1×10 7 , 3×10 7 Or 5 x 10 7 TCID 50 Fourteen days later, the mice were administered 2 nmol of peptide construct ("K"). The results are shown in Figure 9. The previously observed antitumor effect is strongly indicated by the comparable transient remission in the three K / V / K groups, as observed by the large reduction in tumor volume after the first boost (VSV-GP-HPV injection). 7 TCID 50 Although the group shows a slightly increased survival rate, no major differences are observed among the three treatment groups representing different doses of VSV-GP-HPV.

[0198] Example 9 Effect of multiple immunogenic epitopes including fragments of KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2 To examine the effect of multiple immunogenic epitopes including fragments of KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2 in the peptide of the present invention (ATP150), a comparative construct ("ATP132") lacking fragments of KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2 was prepared.

[0199] The multi-antigenic domain of ATP132 has the following sequence: NRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSATEYKLVVVGADGVGKSALTIQLIQ [SEQ ID NO:50] In addition to the multiple antigenic domains, ATP132 comprises a cell penetrating peptide as set forth in SEQ ID NO:15 and a TLR agonist as set forth in SEQ ID NO:16. In summary, the peptide construct ATP132 exhibits the following sequence: MKRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKNRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIP QQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSATEYKLVVVGADGVGKSALTIQLIQSTVHEILSKLSLEGDHSTPPSAYGSVKPYTNFDAE [SEQ ID NO:51]

[0200] The immunogenicity of antigens / antigenic fragments contained in the multi-antigenic domains of ATP150 and ATP132 was compared in monocyte-derived human dendritic cells (moDCs). For this purpose, cells were extracted from fresh human leukoapheresis products named Leukopak supplied by Tissue Solution (Scotland). HLA typing of donors was performed by the Immunology and Transplant Unit and National Reference Laboratory for Histocompatibility (LNRH) of Geneva University Hospital. Peripheral blood mononuclear cells (PBMCs) from leukopak samples were prepared after density gradient centrifugation on Ficoll-Paque and monocytes were enriched by cryoaggregation. Residual T lymphocytes were removed using rosetting with sheep red blood cells and density gradient centrifugation on Ficoll-Paque. Monocytes were subsequently plated with complete RPMI medium supplemented with GM-CSF and IL-4 for 7 days. Cells were then concentrated and interferon alpha 2a was added along with 600 nM ATP150 or 600 nM ATP132. Cells were incubated overnight at 37°C and 5% CO2. Cells were then detached, counted, and centrifuged. Supernatants were aspirated and cell pellets were frozen at -20°C prior to ligandome analysis.

[0201] The cell pellet was lysed in solubilization buffer and homogenized by pulse sonication. After debris removal and sterile filtration, HLA class I and class II molecules were isolated from the soluble fraction using standard immunoaffinity purification. HLA ligands were then eluted by acid elution, isolated by ultracentrifugation, desalted, and preconcentrated prior to LC-MS / MS analysis. Peptide samples were separated by nanoflow high performance liquid chromatography (RSLCnano, Thermo Fisher Scientific) using a 50 μm×25 cm PepMap rapid separation liquid chromatography column (Thermo Fisher Scientific) and an acetonitrile gradient ranging from 2.4% to 32.0% over 90 min. Eluted peptides were analyzed on an online-coupled LTQ Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) using the highest velocity collision-induced dissociation (CID) fragmentation method for HLA class I or high-energy collision dissociation (HCD) fragmentation method for HLA class II. For label-free quantification (LFQ) of relative HLA ligand abundance between activated samples loaded with and without ATP150 or ATP132, the injected peptide amount of the paired samples was normalized from the mean precursor ion intensity determined in the dose-finding run, and LC-MS / MS analysis was performed for five technical replicates for each sample.

[0202] Raw files were processed using Proteome Discoverer 1.4 (Thermo Fisher Scientific). The SEQUEST HT search engine (University of Washington) was used to search the human proteome contained in the Swiss-Prot database in combination with constructs ATP150 or ATP132. After data processing, specific filters were applied. For class I, data were filtered with an FDR of 5%, peptide length of 8-12 amino acids, and search engine rank of 1. For class II, data were filtered with an FDR of 1%, peptide length of 8-25 amino acids, and search engine rank of 1. Protein guessing was disabled, which allowed multiple protein annotation of peptides. To determine HLA class I binding peptide affinity, the NetMHCpan-3.0 algorithm was used with a percentile binding rank of less than 2% and the SYFPEITHI algorithm with a threshold of 50% of the maximum score. The results are shown in Table 2 below.

[0203] [Table 2]

[0204] Table 2 shows the average number of peptide epitopes detected for MHC class I and MHC class II per donor. These data show that monocyte-derived human dendritic cells (moDCs) incubated with a peptide according to the invention (ATP150) containing fragments of KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2 presented more MHC class I and / or MHC class II epitopes from all incorporated antigens than moDCs incubated with a comparative peptide (ATP132) lacking the fragments of KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2. These data further demonstrate that a higher number of immunogenic fragments results in better endogenous processing in professional antigen-presenting cells that can drive meaningful presentation of antigens to the patient's CD8 and CD4 T cells. Table of Sequences and SEQ ID NOs:

[0205] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7]

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

Table 3-21

Claims

1. (i) at least one tumor antigen, or a fragment or sequence variant thereof, At least one tumor antigen, or a fragment or sequence variant thereof, wherein the tumor antigen is encoded in a 5' upstream open reading frame (uORF) within the 5' UTR of KRAS mRNA (KRAS-uORF1), TPX2 mRNA (TPX2-uORF1), or AURKA mRNA (AURKA-uORF2); (ii) a nucleic acid encoding the tumor antigen; (iii) an antigen-presenting cell (APC) containing (i) or (ii); or (iv) a T cell expressing either a T cell receptor or a CAR T cell receptor that targets the tumor antigen. A composition comprising:

2. 2. The composition of claim 1, further comprising at least one tumor antigen selected from the group consisting of CEACAM5, DUOXA2, and KRAS, or a fragment or sequence variant thereof.

3. The composition of claim 2, wherein the KRAS or a fragment thereof is KRAS-G12D or a fragment thereof, or KRAS-G12V or a fragment thereof.

4. 3. The composition of claim 2, wherein the tumor antigen selected from CEACAM5, DUOXA2, KRAS, KRAS-uORF1, TPX2-uORF1 and AURKA-uORF2, or a fragment or sequence variant thereof, comprises a CD4+ and / or CD8+ epitope.

5. The tumor antigen comprises at least one amino acid sequence, the amino acid sequence being - the amino acid sequence according to SEQ ID NO: 9 or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 11 or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 12 or a sequence variant thereof having at least 70% sequence identity, - an amino acid sequence according to SEQ ID NO: 1 or 2, or a sequence variant thereof having at least 70% sequence identity, - an amino acid sequence according to SEQ ID NO: 3 or 4, or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 5, or a sequence variant thereof having at least 70% sequence identity, or - the amino acid sequence according to SEQ ID NO: 10, or a sequence variant thereof having at least 70% sequence identity 2. The composition of claim 1 ,

6. a) a cell-penetrating peptide; b) a multi-antigenic domain comprising at least one tumor antigen, or a fragment or sequence variant thereof, A multi-antigenic domain, in which the tumor antigen is encoded in a 5' upstream open reading frame (uORF) within the 5' UTR of KRAS mRNA (KRAS-uORF1), TPX2 mRNA (TPX2-uORF1), or AURKA mRNA (AURKA-uORF2), and c) TLR peptide agonists A peptide comprising:

7. 7. The peptide of claim 6, wherein the multiple antigenic domain further comprises at least one tumor antigen selected from the group consisting of CEACAM5 or a fragment thereof, DUOXA2 or a fragment thereof, and KRAS or a fragment thereof, each fragment having a minimum length of 8 amino acids.

8. 7. The peptide of claim 6, wherein the multiple antigenic domain comprises three or more different antigens or fragments thereof having a minimum length of 8 amino acids, in particular 3, 4, 5, 6, 7, 8, 9, 10 or more different antigens or fragments thereof.

9. The multiple antigenic domains - a tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5'UTR of KRAS mRNA (KRAS-uORF1); - a tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5' UTR of TPX2 mRNA (TPX2-uORF1), and - A tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5' UTR of AURKA mRNA (AURKA-uORF2) The peptide of claim 6, comprising:

10. The multiple antigenic domains preferably comprise, in the N-terminal to C-terminal direction: CEACAM5 or a fragment thereof with a minimum length of 8 amino acids, KRAS or a fragment thereof with a minimum length of 8 amino acids, KRAS-uORF1, or a fragment thereof having a minimum length of 8 amino acids, TPX2-uORF1, or a fragment thereof having a minimum length of 8 amino acids, AURKA-uORF2, or a fragment thereof having a minimum length of 8 amino acids, and DUOXA2 or a fragment thereof with a minimum length of 8 amino acids The peptide of claim 6, comprising:

11. The peptide according to claim 7, wherein the KRAS or a fragment thereof is KRAS-G12D or a fragment thereof, or KRAS-G12V or a fragment thereof.

12. The peptide according to claim 7, wherein the fragment of CEACAM5 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 6 to 8, or a sequence variant thereof having at least 70% sequence identity, and preferably the multi-antigenic domain comprises an amino acid sequence as set forth in SEQ ID NO: 9, or a sequence variant thereof having at least 70% sequence identity.

13. The multiple antigenic domains - the amino acid sequence according to SEQ ID NO: 9 or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 12 or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 11 or a sequence variant thereof having at least 70% sequence identity, - an amino acid sequence according to SEQ ID NO: 1 or 2, or a sequence variant thereof having at least 70% sequence identity, - an amino acid sequence according to SEQ ID NO: 3 or 4, or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 5, or a sequence variant thereof having at least 70% sequence identity, or - the amino acid sequence according to SEQ ID NO: 10, or a sequence variant thereof having at least 70% sequence identity The peptide of claim 6, comprising at least one amino acid sequence selected from the group consisting of:

14. The multiple antigenic domains preferably comprise, in the N-terminal to C-terminal direction: - the amino acid sequence according to SEQ ID NO: 9 or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 11 or a sequence variant thereof having at least 70% sequence identity, and / or the amino acid sequence according to SEQ ID NO: 12 or a sequence variant thereof having at least 70% sequence identity, - an amino acid sequence according to SEQ ID NO: 1 or 2, or a sequence variant thereof having at least 70% sequence identity, - an amino acid sequence according to SEQ ID NO: 3 or 4, or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 5 or a sequence variant thereof having at least 70% sequence identity, and - the amino acid sequence according to SEQ ID NO: 10, or a sequence variant thereof having at least 70% sequence identity The peptide of claim 6, comprising:

15. 7. The peptide of claim 6, wherein the multiple antigenic domain comprises an amino acid sequence as set forth in SEQ ID NO: 13 or 14, or a sequence variant thereof having at least 70% sequence identity.

16. The peptide of claim 6, wherein the cell-penetrating peptide has an amino acid sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23, or a sequence variant thereof having at least 70% sequence identity.

17. The peptide of claim 6, wherein the cell-penetrating peptide has an amino acid sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15 or a sequence variant thereof having at least 70% sequence identity.

18. 7. The peptide of claim 6, wherein the TLR peptide agonist is a TLR2 peptide agonist and / or a TLR4 peptide agonist.

19. 7. The peptide of claim 6, wherein the TLR peptide agonist comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16 or 24, or a sequence variant thereof having at least 70% sequence identity.

20. a) the cell penetrating peptide has an amino acid sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23, or a sequence variant thereof having at least 70% sequence identity; b) the multiple antigenic domain comprises - a fragment of CEACAM5 having a minimum length of 8 amino acids, - a fragment of DUOXA2 having a minimum length of 8 amino acids, - a fragment of KRAS having a minimum length of 8 amino acids, KRAS-uORF1 or a fragment thereof with a minimum length of 8 amino acids, TPX2-uORF1, or a fragment thereof having a minimum length of 8 amino acids, and / or AURKA-uORF2, or a fragment thereof with a minimum length of 8 amino acids Including, c) the TLR peptide agonist is a TLR2 peptide agonist and / or a TLR4 peptide agonist; The peptide according to claim 6.

21. Preferably, in the N-terminal to C-terminal direction, a) a cell-penetrating peptide having an amino acid sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15, or a sequence variant thereof having at least 70% sequence identity; b) - the amino acid sequence set forth in SEQ ID NO: 9 or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 11 or a sequence variant thereof having at least 70% sequence identity, and / or the amino acid sequence according to SEQ ID NO: 12 or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 1 or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 3 or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 5 or a sequence variant thereof having at least 70% sequence identity, and - the amino acid sequence according to SEQ ID NO: 10, or a sequence variant thereof having at least 70% sequence identity and c) A TLR peptide agonist having an amino acid sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16, or a sequence variant thereof having at least 70% sequence identity. The peptide of claim 6, comprising:

22. From the N-terminus to the C-terminus, a) a cell-penetrating peptide having an amino acid sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15, or a sequence variant thereof having at least 90% sequence identity; b) - the amino acid sequence set forth in SEQ ID NO: 9, or a sequence variant thereof having at least 90% sequence identity; - the amino acid sequence according to SEQ ID NO: 11 or a sequence variant thereof having at least 90% sequence identity, and / or the amino acid sequence according to SEQ ID NO: 12 or a sequence variant thereof having at least 90% sequence identity, - the amino acid sequence according to SEQ ID NO: 1 or a sequence variant thereof having at least 90% sequence identity - the amino acid sequence according to SEQ ID NO: 3 or a sequence variant thereof having at least 90% sequence identity, - the amino acid sequence according to SEQ ID NO: 5 or a sequence variant thereof having at least 90% sequence identity, and - the amino acid sequence according to SEQ ID NO: 10, or a sequence variant thereof having at least 90% sequence identity and c) A TLR peptide agonist having an amino acid sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16, or a sequence variant thereof having at least 90% sequence identity. The peptide of claim 6, comprising:

23. 7. The peptide according to claim 6, comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 46 or SEQ ID NO: 47, preferably an amino acid sequence as set forth in SEQ ID NO: 17 or 18, or a sequence variant thereof having at least 70% sequence identity.

24. A recombinant vesicular stomatitis virus (VSV) encoding a multi-antigenic domain comprising at least one tumor antigen, or a fragment or sequence variant thereof, wherein the tumor antigen is encoded in a 5' upstream open reading frame (uORF) within the 5' UTR of KRAS mRNA (KRAS-uORF), TPX2 mRNA (TPX2-uORF), or AURKA mRNA (AURKA-uORF).

25. The vesicular stomatitis virus (VSV) of claim 24, wherein the multiple antigenic domain further comprises at least one tumor antigen selected from the group consisting of CEACAM5 or a fragment thereof, DUOXA2 or a fragment thereof, and KRAS or a fragment thereof, each fragment having a minimum length of 8 amino acids.

26. The vesicular stomatitis virus (VSV) of claim 24, wherein the multiple antigenic domain comprises three or more different antigens or fragments thereof having a minimum length of 8 amino acids, in particular 3, 4, 5, 6, 7, 8, 9, 10 or more different antigens or fragments thereof.

27. The multiple antigenic domains - a tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5'UTR of KRAS mRNA (KRAS-uORF1); - a tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5' UTR of TPX2 mRNA (TPX2-uORF1), and - A tumor antigen encoded in the 5' upstream open reading frame (uORF) within the 5' UTR of AURKA mRNA (AURKA-uORF2) The vesicular stomatitis virus (VSV) of claim 24, comprising:

28. The multiple antigenic domains preferably comprise, in the N-terminal to C-terminal direction: CEACAM5 or a fragment thereof with a minimum length of 8 amino acids, KRAS or a fragment thereof with a minimum length of 8 amino acids, KRAS-uORF1, or a fragment thereof having a minimum length of 8 amino acids, TPX2-uORF1, or a fragment thereof having a minimum length of 8 amino acids, AURKA-uORF2, or a fragment thereof having a minimum length of 8 amino acids, and DUOXA2 or a fragment thereof with a minimum length of 8 amino acids The vesicular stomatitis virus (VSV) of claim 24, comprising:

29. The vesicular stomatitis virus (VSV) of claim 25, wherein the KRAS or a fragment thereof is KRAS-G12D or a fragment thereof, or KRAS-G12V or a fragment thereof.

30. The vesicular stomatitis virus (VSV) of claim 25, wherein the fragment of CEACAM5 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 6 to 8, or a sequence variant thereof having at least 70% sequence identity, and preferably the multiple antigenic domain comprises an amino acid sequence set forth in SEQ ID NO: 9, or a sequence variant thereof having at least 70% sequence identity.

31. The multiple antigenic domains - the amino acid sequence according to SEQ ID NO: 9 or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 11 or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 12 or a sequence variant thereof having at least 70% sequence identity, - an amino acid sequence according to SEQ ID NO: 1 or 2, or a sequence variant thereof having at least 70% sequence identity, - an amino acid sequence according to SEQ ID NO: 3 or 4, or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 5, or a sequence variant thereof having at least 70% sequence identity, or - the amino acid sequence according to SEQ ID NO: 10, or a sequence variant thereof having at least 70% sequence identity The vesicular stomatitis virus (VSV) of claim 24, comprising at least one amino acid sequence selected from the group consisting of:

32. The multiple antigenic domains preferably comprise, in the N-terminal to C-terminal direction: - the amino acid sequence according to SEQ ID NO: 9 or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 11 or a sequence variant thereof having at least 70% sequence identity, and / or the amino acid sequence according to SEQ ID NO: 12 and / or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 1 or a sequence variant thereof having at least 70% sequence identity - the amino acid sequence according to SEQ ID NO: 3 or a sequence variant thereof having at least 70% sequence identity, - the amino acid sequence according to SEQ ID NO: 5 or a sequence variant thereof having at least 70% sequence identity, and - the amino acid sequence according to SEQ ID NO: 10, or a sequence variant thereof having at least 70% sequence identity The vesicular stomatitis virus (VSV) of claim 24, comprising:

33. The multiple antigenic domains are arranged in the N-terminal to C-terminal direction as follows: - the amino acid sequence according to SEQ ID NO: 9 or a sequence variant thereof having at least 90% sequence identity, - the amino acid sequence according to SEQ ID NO: 11 or a sequence variant thereof having at least 90% sequence identity, and / or the amino acid sequence according to SEQ ID NO: 12 and / or a sequence variant thereof having at least 90% sequence identity, - the amino acid sequence according to SEQ ID NO: 1 or a sequence variant thereof having at least 90% sequence identity - the amino acid sequence according to SEQ ID NO: 3 or a sequence variant thereof having at least 90% sequence identity, - the amino acid sequence according to SEQ ID NO: 5 or a sequence variant thereof having at least 90% sequence identity, and - the amino acid sequence according to SEQ ID NO: 10, or a sequence variant thereof having at least 90% sequence identity The vesicular stomatitis virus (VSV) of claim 24, comprising:

34. The vesicular stomatitis virus (VSV) of claim 24, wherein the multiple antigenic domain comprises an amino acid sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 48, preferably an amino acid sequence set forth in SEQ ID NO: 19, or a sequence variant thereof having at least 70% sequence identity.

35. The vesicular stomatitis virus (VSV) of claim 24, which is an oncolytic vesicular stomatitis virus (VSV).

36. The vesicular stomatitis virus (VSV) of claim 24, which is replication-competent.

37. 25. The vesicular stomatitis virus (VSV) of claim 24, wherein the gene encoding glycoprotein G is replaced by a gene encoding glycoprotein GP of Lymphocytic Choriomeningitis Virus (LCMV), wherein the glycoprotein GP of LCMV preferably comprises the amino acid sequence set forth in SEQ ID NO: 25, or a functional sequence variant thereof that is at least 80%, 85%, 90%, 95% identical thereto.

38. - encoding in its genome multiple antigenic domains as defined in claim 25, - encoding in its genome the nucleoprotein (N), the large protein (L), the phosphoprotein (P) and the matrix protein (M) of the vesicular stomatitis virus, - the gene encoding the glycoprotein G of the vesicular stomatitis virus has been replaced by the gene encoding the glycoprotein GP of the lymphocytic choriomeningitis virus (LCMV), and / or - the glycoprotein G of the vesicular stomatitis virus is replaced by the glycoprotein GP of LCMV, The vesicular stomatitis virus (VSV) of claim 24.

39. - encoding in its genome a vesicular stomatitis virus nucleoprotein (N) comprising the amino acid sequence set forth in SEQ ID NO: 26, or a sequence variant thereof having at least 80%, 85%, 90%, 92%, 94%, 96%, 98% sequence identity, - encoding in its genome a vesicular stomatitis virus phosphoprotein (P) comprising the amino acid sequence set forth in SEQ ID NO: 27 or a sequence variant thereof having at least 80%, 85%, 90%, 92%, 94%, 96%, 98% sequence identity, - encoding in its genome a vesicular stomatitis virus large protein (L) comprising the amino acid sequence set forth in SEQ ID NO: 28 or a sequence variant thereof having at least 80%, 85%, 90%, 92%, 94%, 96%, 98% sequence identity, - encoding in its genome a vesicular stomatitis virus matrix protein (M) comprising the amino acid sequence set forth in SEQ ID NO: 29, or a sequence variant thereof having at least 80%, 85%, 90%, 92%, 94%, 96%, 98% sequence identity, - encoding in its genome multiple antigenic domains of vesicular stomatitis virus as defined in claim 25, - the gene encoding the glycoprotein G of the vesicular stomatitis virus has been replaced by the gene encoding the glycoprotein GP of the lymphocytic choriomeningitis virus (LCMV), and / or - glycoprotein G is replaced by glycoprotein GP of LCMV, The vesicular stomatitis virus (VSV) of claim 24.

40. - a phosphorylated protein (P) comprising the amino acid sequence according to SEQ ID NO: 27, or a sequence variant thereof having at least 70% sequence identity, - a nucleoprotein (N) comprising the amino acid sequence according to SEQ ID NO: 26, or a sequence variant thereof having at least 70% sequence identity, - a matrix protein (M) comprising the amino acid sequence according to SEQ ID NO: 29, or a sequence variant thereof having at least 70% sequence identity, - a large protein (L) comprising the amino acid sequence according to SEQ ID NO: 28 or a sequence variant thereof having at least 70% sequence identity, - a glycoprotein (GP) comprising the amino acid sequence set forth in SEQ ID NO: 25, or a sequence variant thereof having at least 70% sequence identity, and - a multi-antigenic domain comprising the amino acid sequence according to SEQ ID NO: 19 or SEQ ID NO: 48, or preferably the amino acid sequence according to SEQ ID NO: 19, or a sequence variant thereof having at least 70% sequence identity; The vesicular stomatitis virus (VSV) of claim 24, which encodes in its genome:

41. The vesicular stomatitis virus (VSV) of claim 24, whose RNA genome comprises or consists of the RNA sequence set forth in SEQ ID NO: 30, or a sequence variant thereof having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

42. (i) the composition according to any one of claims 1 to 5, (ii) a peptide according to any one of claims 6 to 23, or (iii) The vesicular stomatitis virus (VSV) according to any one of claims 24 to 41. A vaccine comprising:

43. (i) a peptide according to claim 6, and (ii) The vesicular stomatitis virus (VSV) of claim 24. A vaccine comprising:

44. (i) a peptide according to claim 6, and (ii) The vesicular stomatitis virus (VSV) of claim 24. Kit including:

45. (i) a peptide according to claim 6, and (ii) The vesicular stomatitis virus (VSV) of claim 24. A combination including:

46. The vaccine of claim 43, the kit of claim 44, or the combination of claim 45, wherein the multiple antigenic domain encoded in the genome of vesicular stomatitis virus (VSV) comprises an antigen contained in the multiple antigenic domain of a peptide, or a fragment or sequence variant thereof.

47. 47. The vaccine, kit or combination of claim 46, wherein the multiple antigenic domain encoded in the genome of vesicular stomatitis virus (VSV) comprises the amino acid sequence of each of the antigens contained in the peptide multiple antigenic domain, or a fragment or sequence variant thereof.

48. The vaccine of claim 43, the kit of claim 44, or the combination of claim 45, wherein the multiple antigenic domain of the peptide comprises an antigen contained in a multiple antigenic domain encoded in the genome of vesicular stomatitis virus (VSV), or a fragment or sequence variant thereof.

49. 49. The vaccine, kit or combination of claim 48, wherein the peptide multiple antigenic domain comprises the amino acid sequence of each of the antigens of the multiple antigenic domain encoded in the genome of Vesicular Stomatitis Virus (VSV), or a fragment or sequence variant thereof.

50. - the multiple antigenic domain of the peptide comprises an amino acid sequence as set forth in SEQ ID NO: 13 or 14, or a sequence variant thereof having at least 70% sequence identity, - the multiple antigenic domain encoded in the genome of vesicular stomatitis virus (VSV) comprises the amino acid sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 48, preferably the amino acid sequence set forth in SEQ ID NO: 19, or a sequence variant thereof having at least 70% sequence identity; A vaccine, kit or combination according to any one of claims 43 to 45.

51. the peptide comprises an amino acid sequence as set forth in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 46 or SEQ ID NO: 47, preferably an amino acid sequence as set forth in SEQ ID NO: 17 or 18, or a sequence variant thereof having at least 70% sequence identity, - the vesicular stomatitis virus (VSV) comprises an RNA genome comprising an RNA sequence according to SEQ ID NO: 30, or a sequence variant thereof having at least 70% sequence identity; A vaccine, kit or combination according to any one of claims 43 to 45.

52. (iii) PD-1 / PD-L1 pathway inhibitor 46. ​​The vaccine, kit or combination of any one of claims 43 to 45, further comprising:

53. 53. The vaccine, kit or combination of claim 52, wherein the inhibitor of the PD-1 / PD-L1 pathway is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, cemiplimab, PDR-001, atezolizumab, avelumab, durvalumab, ezavenlimab, an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 32, an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 34, and an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and a light chain comprising the amino acid sequence of SEQ ID NO:

36.

54. 46. ​​A composition according to claim 1, a vaccine according to claim 43, a kit according to claim 44 or a combination according to claim 45 for use in medicine.

55. 46. ​​A composition according to claim 1, a vaccine according to claim 43, a kit according to claim 44, or a combination according to claim 45, for use in the treatment of cancer.

56. 56. The composition, vaccine, kit or combination of claim 55, wherein the cancer is a gastrointestinal (GI) cancer.

57. 56. The composition, vaccine, kit or combination of claim 55, wherein the cancer is selected from the group consisting of anal cancer, appendix cancer, cholangiocarcinoma / bile duct cancer, in particular extrahepatic bile duct cancer, gastrointestinal carcinoid tumors, colorectal cancer, in particular colon cancer, rectal cancer and metastatic colorectal cancer, esophageal cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal stromal tumors (GIST), and pancreatic cancer, e.g. pancreatic ductal adenocarcinoma.

58. 56. The composition, vaccine, kit, or combination of claim 55, wherein the cancer is selected from the group consisting of colon cancer, rectal cancer, colorectal cancer, metastatic colorectal cancer, pancreatic cancer, and pancreatic ductal adenocarcinoma.

59. 55. The vaccine, kit or combination of claim 54, wherein the peptide and the vesicular stomatitis virus (VSV) are each administered at least once, preferably the peptide is administered prior to administration of the vesicular stomatitis virus (VSV).

60. 55. The vaccine, kit or combination of claim 54, wherein the peptide is administered at least twice, preferably before and after administration of vesicular stomatitis virus (VSV).

61. 55. The vaccine, kit or combination of claim 54, wherein the peptide and vesicular stomatitis virus (VSV) are administered in the following order: K-V-K, K-V-K-K, K-V-K-K-K, or K-V-K-K-K-K.

62. 55. The vaccine, kit or combination of claim 54, wherein the treatment schedule comprises a single administration of vesicular stomatitis virus (VSV).

63. 55. The vaccine, kit or combination of claim 54, wherein the peptide and the vesicular stomatitis virus (VSV) are administered via the same or different routes, preferably intravenously, subcutaneously or intramuscularly.

64. 55. The vaccine, kit or combination of claim 54, wherein the peptide is administered subcutaneously and the vesicular stomatitis virus (VSV) is administered intravenously or intratumorally, preferably intravenously.

65. 55. The vaccine, kit or combination according to claim 54, further comprising the administration of an inhibitor of the PD-1 / PD-L1 pathway, preferably as defined in claim 53.

66. 66. The vaccine, kit or combination of claim 65, wherein the inhibitor of the PD-1 / PD-L1 pathway is administered simultaneously, sequentially or alternatingly with the peptide or the vesicular stomatitis virus (VSV).

67. Use of a composition described in claim 1, a peptide described in claim 6, a vesicular stomatitis virus (VSV) described in claim 24, a vaccine described in claim 43, a kit described in claim 44, or a combination described in claim 45 for the manufacture of a medicament for the treatment of cancer.

68. A tumor antigen comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:11, or a sequence variant thereof having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:

11. - a tumor antigen comprising or consisting of the amino acid sequence according to SEQ ID NO: 12 or a sequence variant thereof having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 12, - a tumor antigen comprising or consisting of the amino acid sequence according to SEQ ID NO: 1 or a sequence variant thereof having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1, - a tumor antigen comprising or consisting of the amino acid sequence according to SEQ ID NO: 3 or a sequence variant thereof having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 3, - a tumor antigen comprising or consisting of the amino acid sequence according to SEQ ID NO: 5 or a sequence variant thereof having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 5, - a tumor antigen comprising or consisting of an amino acid sequence according to SEQ ID NO: 9 or a sequence variant thereof having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 9, and - a tumor antigen comprising or consisting of the amino acid sequence according to SEQ ID NO: 10, or a sequence variant thereof having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:

10.

23. A pharmaceutical composition comprising:

69. A nucleic acid encoding a tumor antigen comprising or consisting of an amino acid sequence according to SEQ ID NO:11, or a sequence variant thereof having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:

11. - a nucleic acid encoding a tumor antigen comprising or consisting of an amino acid sequence according to SEQ ID NO: 12 or a sequence variant thereof having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 12, - a nucleic acid encoding a tumor antigen comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 or a sequence variant thereof having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1, - a nucleic acid encoding a tumor antigen comprising or consisting of the amino acid sequence according to SEQ ID NO: 3 or a sequence variant thereof having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 3, - a nucleic acid encoding a tumor antigen comprising or consisting of an amino acid sequence according to SEQ ID NO: 5 or a sequence variant thereof having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 5, - a nucleic acid encoding a tumor antigen comprising or consisting of an amino acid sequence according to SEQ ID NO: 9 or a sequence variant thereof having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 9, and - a nucleic acid encoding a tumor antigen comprising or consisting of the amino acid sequence according to SEQ ID NO: 10 or a sequence variant thereof having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 10 23. A pharmaceutical composition comprising:

70. A pharmaceutical composition comprising a peptide, the peptide being arranged in an N-terminal to C-terminal direction as follows: a) a cell-penetrating peptide having an amino acid sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15, or a sequence variant thereof having at least 90% sequence identity; b) - the amino acid sequence set forth in SEQ ID NO: 9 or a sequence variant thereof having at least 90% sequence identity; - the amino acid sequence according to SEQ ID NO: 11 or a sequence variant thereof having at least 90% sequence identity, and / or the amino acid sequence according to SEQ ID NO: 12 or a sequence variant thereof having at least 90% sequence identity, - the amino acid sequence according to SEQ ID NO: 1 or a sequence variant thereof having at least 90% sequence identity, - the amino acid sequence according to SEQ ID NO: 3 or a sequence variant thereof having at least 90% sequence identity, - the amino acid sequence according to SEQ ID NO: 5 or a sequence variant thereof having at least 90% sequence identity, and - the amino acid sequence according to SEQ ID NO: 10, or a sequence variant thereof having at least 90% sequence identity and c) A TLR peptide agonist having an amino acid sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16, or a sequence variant thereof having at least 90% sequence identity.

13. A pharmaceutical composition comprising:

71. The pharmaceutical composition described in claim 68, comprising a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17, or a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18, or a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 46, or a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:

47.

72. A pharmaceutical composition comprising a vesicular stomatitis virus, wherein the vesicular stomatitis virus is - a phosphorylated protein (P) comprising the amino acid sequence according to SEQ ID NO: 27, or a sequence variant thereof having at least 70% sequence identity, - a nucleoprotein (N) comprising the amino acid sequence according to SEQ ID NO: 26, or a sequence variant thereof having at least 70% sequence identity, - a matrix protein (M) comprising the amino acid sequence according to SEQ ID NO: 29, or a sequence variant thereof having at least 70% sequence identity, - a large protein (L) comprising the amino acid sequence according to SEQ ID NO: 28 or a sequence variant thereof having at least 70% sequence identity, - a glycoprotein (GP) comprising the amino acid sequence set forth in SEQ ID NO: 25, or a sequence variant thereof having at least 70% sequence identity, and - a multi-antigenic domain comprising the amino acid sequence according to SEQ ID NO: 19 or SEQ ID NO: 48, or a sequence variant thereof having at least 70% sequence identity; A pharmaceutical composition comprising a gene encoding the above gene in its genome.

73. The pharmaceutical composition of claim 72, wherein the RNA genome of vesicular stomatitis virus (VSV) comprises or consists of an RNA sequence set forth in SEQ ID NO: 30, or a sequence variant thereof having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

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