Antigenic peptides for the prevention and treatment of cancer - Patents.com

Abstract

The present invention relates to antigen-based immunotherapy, particularly cancer immunotherapy. In particular, the present invention provides antigenic peptides that are distinct from, but have amino acid similarity to, fragments of human tumor antigens. The present invention further provides immunogenic compounds, nanoparticles, cells, and pharmaceutical compositions comprising such antigenic peptides, as well as nucleic acids encoding such antigenic peptides.

Description

[Technical field]

[0001] The present invention relates to the field of cancer treatment, more particularly to immunotherapeutic methods, in particular the present invention provides various peptides that are useful in cancer immunotherapy. [Background technology]

[0002] Cancer is one of the leading causes of death worldwide. According to the World Health Organization (WHO), in 2012 alone, 14 million new cases and 8.2 million cancer-related deaths were reported worldwide, and the number of new cancer cases is expected to rise by about 70% within the next 20 years. So far, more than 60% of the world's total new annual cases occur in Africa, Asia, and Latin America. These regions also account for 70% of the world's cancer deaths. In men, the top five most common sites of cancer are lung, prostate, colorectal, stomach, and liver; while in women, they are breast, colorectal, lung, cervix, and stomach.

[0003] Cancer has long been managed using surgery, radiation therapy, cytotoxic chemotherapy, and endocrine manipulation, typically in combination with a sequence that best controls the disease. However, a major limitation to the true efficacy of these standard therapies is their imprecise specificity, which results in collateral damage of normal tissues incurred during treatment, low cure rates, and intrinsic drug resistance.

[0004] In the last few years, there has been an incredible increase in the development of cancer treatments, especially thanks to major advances in expression profiling of tumor and normal cells and recent research, and the first clinical results in immunotherapy or molecular targeted therapy have started to change our understanding of this disease.

[0005] Promising anti-cancer immunotherapies are now a reality, and evidence that the host immune system can recognize tumor antigens has led to the development of anti-cancer drugs that are now approved by regulatory agencies such as the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Various therapeutic approaches include, among others, adoptive transfer of ex vivo expanded tumor infiltrating lymphocytes (TILs), cancer cell vaccines, immune stimulatory cytokines and their variants, pattern recognition receptor (PRR) agonists, and immune-modulating monoclonal antibodies targeting tumor antigens or immune checkpoints (Galuzzi et al., Classification of current anticancer immunotherapies. Oncotarget. 2014 Dec. 30;5(24):12472-508).

[0006] Unfortunately, a significant proportion of patients may still exhibit intrinsic resistance to some of these immunotherapies, or may even acquire resistance during the course of treatment. For example, 3-year survival rates have been reported to be approximately 20% for the anti-CTLA-4 antibody ipilimumab in unresectable or metastatic melanoma (Snyder et al., Genetic basis for clinical response to CTLA-4 blockade in melanoma. N Engl J Med. 2014 Dec. 4;371(23):2189-2199; Schadendorf et al., Pooled Analysis of Long-Term Survival Data From Phase II and Phase III Trials of Ipilimumab in Unresectable or Metastatic Melanoma. J Clin Oncol. 2015 Jun. 10;33(17):1889-94), while 3-year survival rates for another checkpoint inhibitor, nivolumab, which targets PD-1, have been reported to be 44% in renal cell carcinoma (RCC) and 18% in non-small cell lung cancer (NSCLC) (Mc Dermott et al., Survival, Durable Response, and Long-Term Safety in Patients With Previously Treated Advanced Renal Cell Carcinoma Receiving Nivolumab. J Clin Oncol. 2015 June 20;33(18):2013~20; Gettinger et al., Overall Survival and Long-Term Safety of Nivolumab (Anti-Programmed Death 1 Antibody, BMS-936558, ONO-4538) in Patients With Previously Treated Advanced Non-Small-Cell Lung Cancer. J Clin Oncol. June 20, 2015;33(18):2004-12).Thus, underlying drug resistance remains a constant barrier to the efficacy of these immunotherapies, and it is clear that a different approach to cancer treatment is needed to break down this barrier.

[0007] The lack of response in many subjects treated with these immunotherapies may be related to a defective antitumor immune response, such as defects in antigen presentation by antigen-presenting cells (APCs) or antigen recognition by T cells. In other words, a positive response to immunotherapy correlates with the ability of the immune system to generate specific lymphocyte subsets capable of recognizing MHC class I-restricted antigens expressed by human cancer cells (Kvistborg et al., Human cancer regression antigens. Curr Opin Immunol. 2013 April;25(2):284-90). This hypothesis suggests that the response to adoptive transfer of tumor-infiltrating lymphocytes (TILs) is dependent on the expression of CD8 T cells infused into the patient. + This is strongly supported by data demonstrating a direct correlation with the number of T cells (Besser et al., Adoptive transfer of tumor-infiltrating lymphocytes in patients with metastatic melanoma: intent-to-treat analysis and efficacy after failure to prior immunotherapies. Clin Cancer Res. 2013 Sep 1;19(17):4792-800). Thus, a strong anti-tumor response will depend on the presentation of immunoreactive peptides and the presence of sufficient numbers of reactive cells "trained" to recognize these antigens.

[0008] Tumor antigen-based vaccination represents a unique approach to cancer therapy that has gained considerable interest because it can enlist the cooperation of the patient's own immune system to recognize, attack and destroy the tumor in a specific and durable manner. Indeed, tumor cells are known to express a large number of peptide antigens that are easily recognized by the immune system. Vaccines based on such antigens therefore offer great opportunities not only for improving the overall survival of patients, but also for monitoring the immune response and preparing GMP-grade products, thanks to the low toxicity and low molecular weight of the tumor antigens. Examples of tumor antigens include, among others, protein by-products transcribed from normally silent or overexpressed genes and from proteins expressed by oncoviruses (Kvistborg et al., Human cancer regression antigens. Curr Opin Immunol. 2013 April;25(2):284-90), as well as neo-antigens resulting from point mutations of cellular proteins. The latter are of particular interest as they have been shown to directly correlate with increased overall survival in patients treated with CTLA-4 inhibitors (Snyder et al., Genetic basis for clinical response to CTLA-4 blockade in melanoma. N Engl J Med. 2014 Dec. 4;371(23):2189-2199; Brown et al., Neo-antigens predicted by tumor genome meta-analysis correlate with increased patient survival. Genome Res. 2014 May;24(5):743-50).

[0009] Nevertheless, the number of human tumor antigens for which cancer vaccines can be developed is limited, especially antigens derived from mutated or modified self-proteins that may induce immune tolerance and / or undesirable autoimmune side effects.

[0010] Thus, there is a need in the art to identify alternative cancer therapeutics that can overcome the limitations encountered in this field.

[0011] The present invention has the object of fulfilling the above-mentioned need, which is achieved by means of the subject matter hereinafter and in particular by the provisions of the present invention and the appended claims. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] WO2013 / 135553A1 [Patent Document 2] EP2119726A1 [Patent Document 3] EP0372501 [Patent Document 4] EP0378881 [Patent Document 5] EP0427347 [Patent Document 6] WO93 / 17712 [Patent Document 7] WO98 / 58668 [Patent Document 8] WO00 / 56360 [Patent Document 9] WO00 / 61761 [Patent Document 10] WO96 / 26962 [Patent Document 11] WO01 / 18053 [Patent Document 12] WO2013 / 151672A2 [Patent Document 13] WO2013 / 101690A1 [Patent Document 14] WO2013 / 052523A [Patent Document 15] US2010 / 0113300 [Patent Document 16] US2013 / 0115191 [Patent Document 17] WO2004 / 033685A1 [Patent Document 18] WO2004 / 074322A1 [Patent Document 19] WO2012 / 056407A1 [Patent Document 20] WO2013 / 057586A1

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[0014] Invention items In particular, the present invention provides, inter alia: 1. An antigenic peptide comprising or consisting of an amino acid sequence shown in any one of SEQ ID NOs: 1 to 16 and 40 to 42. 2. An antigenic peptide comprising or consisting of an amino acid sequence shown in any one of SEQ ID NOs: 1 to 16 and 40 to 42, in which one or two amino acid residues may be optionally substituted, deleted, or added. 3. The antigenic peptide of item 2, wherein the core sequence is maintained. 4. An antigenic peptide according to any one of the preceding items, comprising or consisting of a microbiota variant of a human reference peptide according to any one of SEQ ID NOs: 17 to 31. 5. The antigenic peptide according to any one of the preceding items, consisting of an amino acid sequence shown in any one of SEQ ID NOs: 1 to 16 and 40 to 42. 6. The antigenic peptide according to any one of the preceding items, having a length of 8 to 15 amino acids or 8 to 11 amino acids, preferably 9 or 10 amino acids. 7. An antigenic peptide according to any one of the preceding items, comprising or consisting of the amino acid sequence set forth in SEQ ID NO:1. 8. The antigenic peptide of any one of the preceding items, comprising or consisting of the amino acid sequence set forth in SEQ ID NO:2. 9. The antigenic peptide of any one of the preceding items, comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3. 10. The antigenic peptide of any one of the preceding items, wherein the length of the antigenic peptide does not exceed 30 amino acids. 11. The antigenic peptide of any one of the preceding items, wherein the length of the antigenic peptide does not exceed 25 amino acids. 12. The antigenic peptide of any one of the preceding items, wherein the length of the antigenic peptide does not exceed 20 amino acids. 13. The antigenic peptide of any one of the preceding paragraphs, wherein the length of the antigenic peptide does not exceed 15 amino acids. 14. The antigenic peptide of any one of the preceding items, wherein the length of the antigenic peptide does not exceed 11 amino acids. 15. The antigenic peptide of any one of the preceding items, which is not a full-length (microbiota) protein. 16. An immunogenic compound comprising an antigenic peptide described in any one of the preceding items. 17. The immunogenic compound according to item 16, wherein the antigenic peptide is linked to a carrier molecule. 18. The immunogenic compound according to item 17, wherein the carrier molecule is a carrier protein or a carrier peptide. 19. An immunogenic compound according to any one of items 16 to 18, comprising or consisting of a polypeptide of formula (I): PepNt-CORE-PepCt (I) During the ceremony, - "PepNt" consists of a polypeptide having a length varying from 0 to 500 amino acid residues and is located at the N-terminus of the polypeptide of formula (I); - "CORE" consists of an antigenic peptide as defined in any one of items 1 to 15; and "PepCt" consists of a polypeptide having a length varying from 0 to 500 amino acid residues and is located at the C-terminus of the polypeptide of formula (I). 20. Nanoparticles loaded with, optionally with an adjuvant: - at least one of the antigenic peptides according to any one of items 1 to 15, or - at least one immunogenic compound according to any one of items 16 to 19. 21. Cells loaded with the antigenic peptide according to any one of items 1 to 15 or the immunogenic compound according to any one of items 16 to 19. 22. The cell according to item 38, wherein the cell is an antigen-presenting cell, preferably a dendritic cell. 23. A nucleic acid encoding an antigenic peptide according to any one of items 1 to 15, a polypeptide of formula (I) as defined in item 19, or an immunogenic compound according to any one of items 16 to 19, wherein the immunogenic compound is a peptide or a protein. 24. The nucleic acid according to item 23, which is a DNA or 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. 25. A host cell comprising the nucleic acid according to item 23 or 24. 26. The host cell according to item 25, wherein the nucleic acid is a vector. 27. The host cell according to item 25 or 26, which is a bacterial cell, preferably an enterobacterial cell. 28. A (cytotoxic and / or activating) T lymphocyte specific for an antigenic peptide according to any one of items 1 to 15. 29. An antibody that binds to the antigenic peptide according to any one of items 1 to 15. 30. A T cell receptor that binds to the antigenic peptide according to any one of items 1 to 15. 31. An antigenic peptide according to any one of items 1 to 15. - the immunogenic compound according to any one of items 16 to 19, - the nanoparticles according to item 20, - the cell according to item 21 or 22, - a nucleic acid according to item 23 or 24, - a host cell according to any one of items 25 to 27, - a T lymphocyte according to item 28, - the antibody according to item 29, or - a T cell receptor according to item 30, and optionally one or more pharma- ceutically acceptable excipients or carriers. 23. A pharmaceutical composition comprising: 32. (i) at least two distinct antigenic peptides according to any one of items 1 to 15; (ii) at least two distinct immunogenic compounds according to any one of items 16 to 19; (iii) at least two distinct nanoparticles according to item 20; (iv) at least two distinct nucleic acids according to item 21 or 22; or (v) at least two distinct cytotoxic T lymphocytes according to item 28; 32. The pharmaceutical composition according to item 31, comprising: 33. A pharmaceutical composition according to item 32, comprising at least three or four distinct components, preferably three or four distinct antigenic peptides, according to any one of (i) to (v). 34. At least three or four distinct active ingredients are - an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:1; - an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:2; and - an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:3. 35. The pharmaceutical composition according to any one of items 31 to 34, further comprising: - an antigenic peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 32; and an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 33, or an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 34; 34. The pharmaceutical composition according to item 33, 36. The pharmaceutical composition according to any one of items 31 to 35, further comprising a helper peptide, preferably a peptide comprising or consisting of an amino acid sequence according to SEQ ID NO: 39. 37. An antigenic peptide according to any one of items 1 to 15. - the immunogenic compound according to any one of items 16 to 19, - the nanoparticles according to item 20, - the cell according to item 21 or 22, - a nucleic acid according to item 23 or 24, - a host cell according to any one of items 25 to 27, - a T lymphocyte according to item 28, - the antibody according to item 29, - a T cell receptor according to item 30, or - The pharmaceutical composition according to any one of items 31 to 36. Including the kit. 38. The kit according to item 37, further comprising a package insert or instructions for use comprising instructions for preventing or treating cancer by using the antigenic peptide, immunogenic compound, nanoparticle, cell, nucleic acid, host cell, cytotoxic T lymphocyte, and / or pharmaceutical composition. 39. The kit according to item 37 or 38, comprising at least two distinct antigenic peptides according to any one of items 1 to 15. 40. A combination of at least two distinct antigenic peptides according to any one of items 1 to 15 for use in the prevention or treatment of cancer. 41. An antigenic peptide according to any one of items 1 to 15 for use as a medicament. 42. The antigenic peptide according to any one of items 1 to 15 for use in the prevention or treatment of cancer. 43. For use in medicine, in particular in the prevention and / or treatment of cancer, The antigenic peptide according to any one of items 1 to 15. The immunogenic compound according to any one of items 16 to 19, 21. The nanoparticles according to item 20, 23. The cell according to item 21 or 22. 25. The nucleic acid according to Item 23 or 24, 28. The host cell according to any one of Items 25 to 27, 29. The T lymphocyte according to item 28, 29. The antibody according to claim 29, 31. The T cell receptor according to item 30, The pharmaceutical composition according to any one of items 31 to 36, The kit according to any one of items 37 to 39, or The combination according to item 40. 44. A method for preventing and / or treating cancer, or initiating, enhancing or prolonging an anti-tumor response to cancer in a subject in need thereof, comprising administering to the subject: - the antigenic peptide according to any one of items 1 to 15, - the immunogenic compound according to any one of items 16 to 19, - the nanoparticles according to item 20, - the cell according to item 21 or 22, - a nucleic acid according to item 23 or 24, - a host cell according to any one of items 25 to 27, - a T lymphocyte according to item 28, - the antibody according to item 29, - a T cell receptor according to item 30, - the pharmaceutical composition according to any one of items 31 to 36, - a kit according to any one of items 37 to 39, or - The combination according to item 40. 45. A peptide-MHC (pMHC) multimer comprising an antigenic peptide according to any one of items 1 to 15.

[0015] definition Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context requires otherwise, the nomenclature and cell and tissue culture techniques used herein are those well known and commonly used in the art.

[0016] Such techniques are well described in the literature, such as Owen et al. (Kuby Immunology, 7th ed., 2013 - WH Freeman) and Sambrook et al. (Molecular cloning: A laboratory manual 4th ed., Cold Spring Harbor Laboratory Press - Cold Spring Harbor, NY, USA, 2012).

[0017] However, the following definitions apply more specifically with respect to the use of various terms throughout this specification.

[0018] The terms "peptide", "polypeptide", "protein" and variations of these terms refer to peptides, oligopeptides, polypeptides or proteins comprising at least two amino acids linked together, preferably by a normal peptide bond or alternatively, by a modified peptide bond, such as in the case of isosteric peptides. The term "(poly)peptide" refers to peptides and / or polypeptides. In particular, the terms "peptide", "polypeptide" and "protein" refer to a continuous chain of amino acids of any length linked together via peptide bonds (-NHCO-). Peptides, polypeptides and proteins can perform structural and / or functional roles in cells in vitro and / or in vivo. The terms "peptide", "polypeptide" and "protein" preferably encompass amino acid chains in the range of sizes from 2 amino acid residues to at least about 1000 amino acid residues. The term "peptide" preferably as used herein encompasses amino acid chains in the range of sizes less than about 30 amino acids, while the terms "polypeptide" and "protein" preferably encompass amino acid chains in the range of sizes at least 30 amino acids. The terms "polypeptide" and "protein" are used interchangeably herein. Preferably, the terms "peptide", "polypeptide", "protein" also include "peptidomimetics", defined as peptide analogues containing non-peptide structural elements, which peptides are capable of mimicking or antagonizing the biological action of the natural parent peptide. Peptidomimetics lack classical peptide characteristics, such as peptide bonds susceptible to enzymatic cleavage. In particular, a peptide, polypeptide or protein may contain, in addition to these amino acids, or may be composed of amino acids other than the 20 amino acids defined by the genetic code. In particular, a peptide, polypeptide or protein in the context of the present invention may equally be composed of amino acids modified by natural processes, such as post-translational maturation processes, or by chemical processes well known to those skilled in the art. Such modifications are well described in detail in the literature.These modifications can occur anywhere in a polypeptide, for example in the peptide backbone, in the amino acid chain, or even at the carboxy- or amino-terminus. In particular, a peptide or polypeptide can be branched after ubiquitination or can be cyclic, with or without branching. Modifications of this type can be the result of natural or synthetic post-translational processes well known to those skilled in the art. The terms "peptide", "polypeptide", and "protein" in the context of the present invention in particular also include modified peptides, polypeptides, and proteins. For example, peptide, polypeptide, or protein modifications may 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, seneloylation, sulfatation, arginylation, or ubiquitination, or other amino acid additions. Such modifications are well documented in the literature (Proteins Structure and Molecular Properties (1993) 2nd ed., T. E. Creighton, New York; Post-translational Covalent Modifications of Proteins (1983) Ed. B. C. Johnson, Academic Press, New York; Seifter et al. (1990) Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. 182: 626-646, and Rattan et al. (1992) Protein Synthesis: Post-translational Modifications and Aging, Ann NY Acad Sci, 663: 48-62).Thus, the terms "peptide", "polypeptide", and "protein" preferably include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, and the like.

[0019] Preferably, the (poly)peptide or protein is a "classical" (poly)peptide or protein, which is typically composed of amino acids selected from the 20 amino acids defined by the genetic code, linked to each other by conventional peptide bonds.

[0020] As is well known in the art, peptides, polypeptides, and proteins can be encoded by nucleic acids. The terms "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", "polynucleotide", "nucleotide sequence" are used interchangeably herein and refer to an exact sequence of natural nucleotides (e.g., A, T, G, C, and U) or synthetic nucleotides, i.e., a chain of at least two nucleotides. In particular, the terms "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", "polynucleotide", "nucleotide sequence" refer to DNA or RNA. Preferably, the nucleic acid comprises DNA or RNA, preferably single-stranded, double-stranded, or partially double-stranded, and is preferably selected from genomic DNA (gDNA), complementary DNA (cDNA), ribosomal DNA (rDNA), and transcription products of said DNA, such as RNA. Preferred examples of nucleic acids include ribosomal RNA (rRNA), messenger RNA (mRNA); antisense DNA, antisense RNA; complementary RNA and / or DNA sequences, ribozymes, (complementary) RNA / DNA sequences with or without expression elements, vectors; 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, or transfer RNA (tRNA), or DNA as described above. Thus, it is preferred that the nucleic acid (molecule) is a DNA or RNA molecule, preferably selected from gDNA; cDNA; rRNA; mRNA; antisense DNA; antisense RNA; complementary RNA and / or DNA sequences; RNA and / or DNA sequences with or without expression elements, control elements, and / or promoters; and combinations thereof. It is within the skill of the person skilled in the art to determine a nucleotide sequence capable of encoding a particular amino acid sequence.

[0021] The (poly)peptides and / or nucleic acids according to the invention may be prepared by any known method, including but not limited to any synthetic method, any recombinant method, any ex vivo production method, and the like, and any combination thereof. Such techniques are fully explained in the literature, such as those mentioned above.

[0022] As used herein, the term "antigenic peptide" refers to a peptide that tends to induce / elicit, increase, prolong or maintain an immune response in a subject to which it is administered. In particular, the antigenic peptide is a sequence variant of (a fragment / epitope of) a (human) tumor antigen. In other words, the antigenic peptide is preferably different from (a fragment / epitope of) a (human) tumor antigen, but it preferably has amino acid similarity with (a fragment / epitope of) a (human) tumor antigen. Importantly, the antigenic peptide shares the same core sequence with each of (a fragment / epitope of) a (human) tumor antigen. Preferably, the immune response induced / elicited, increased, prolonged or maintained by the antigenic peptide (also) targets each of (a fragment / epitope of) a (human) tumor antigen.

[0023] As used herein, the term "tumor antigen" includes tumor-specific antigens (TSA) and tumor-associated antigens (TAA). In general, the term "tumor antigen" or "tumor protein" herein refers to antigenic substances that are produced in tumor cells, and sometimes also in normal cells, and that can trigger an immune response in a subject upon administration. In humans, they are classified according to their expression patterns, functions, or genetic origins, and include, but are not limited to, overexpressed autoantigens (e.g., BIRC5); cancer-testis (CT) antigens (e.g., MAGE-1); mutant antigens also known as neoantigens (e.g., mutants derived from p53); tissue-specific differentiation antigens (e.g., melanoma antigen Melan A / MART-1); viral antigens expressed by oncoviruses (e.g., HPV, EBV); oncofetal antigens (e.g., alpha-fetoprotein AFP and carcinoembryonic antigen CEA); and universal antigens (telomerase).

[0024] As used herein, the term "core sequence" refers to amino acids in the middle of a sequence, e.g. in the middle of an antigenic peptide and / or (reference) epitope (also referred to as the "central amino acids" of the sequence). Thus, the core sequence consists of all amino acids except the two most N-terminal and the two most C-terminal. For example, in a 9 amino acid peptide (an antigenic peptide according to the invention, or (a fragment / epitope of) a (human) tumor antigen, respectively), the central 5 amino acids represent the core sequence, and changes can only occur in either the two most N-terminal or the two most C-terminal amino acid positions. Thus, a "shared core sequence" (or a "maintained" core sequence) usually means that mutations / differences are only allowed in the two most N-terminal and the two most C-terminal amino acids of the (reference) epitope / sequence.

[0025] As used herein, the term "microbiota" refers to the symbiotic microorganisms found in and on all multicellular organisms studied to date, from plants to animals. In particular, microbiota have been found to be important for the immunological, hormonal, and metabolic homeostasis of their hosts. Microbiota include bacteria, archaea, protists, fungi, and viruses. Thus, a "microbiota sequence variant" (or "microbiota variant") is a sequence variant of a (human) reference sequence (particularly an epitope / fragment of a human tumor antigen) present in a microbiota, such as bacteria (e.g., it may be contained in a microbiota protein, such as a bacterial protein). Preferably, the antigenic peptide of the present invention is a microbiota sequence variant (of a reference epitope / fragment of a human B-cell tumor antigen). Thus, the antigenic peptide is preferably present (e.g., contained) in at least one protein expressed by the human microbiota.

[0026] Anatomically, the microbiota is present on or in any of several tissues and biological fluids, including the skin, conjunctiva, mammary gland, vagina, placenta, semen, uterus, ovarian follicles, lungs, saliva, oral cavity (particularly oral mucosa), and gastrointestinal tract, particularly the intestine. In the context of the present invention, the microbiota sequence variant is preferably a sequence variant of the gastrointestinal tract microbiota (microorganisms living in the gastrointestinal tract), more preferably a sequence variant of the gut microbiota (microorganisms living in the gut). Thus, it is most preferred that the microbiota sequence variant is a (human) gut bacteria sequence variant (i.e., a sequence variant of a (human) gut-dwelling bacterium).

[0027] Although microbiota can be found in and on many multicellular organisms (from plants to animals, all multicellular organisms studied to date), the microbiota found in and on humans is preferred. Such microbiota is referred to herein as the "human microbiota" (the term human specifically refers to the location / residence of the microbiota). Within the context of the present invention, the microbiota sequence variant is a human microbiota sequence variant.

[0028] The term "immunogenic compound" refers to a compound comprising an antigenic peptide according to the present invention. An "immunogenic compound" is capable of inducing / eliciting, increasing, prolonging or maintaining an immune response against said antigenic peptide in a subject to which it is administered. In some embodiments, an immunogenic compound comprises at least one antigenic peptide or, alternatively, at least one compound comprising such an antigenic peptide linked to a protein, such as a carrier protein.

[0029] A "carrier protein" is a protein that is typically capable of transporting a cargo, such as an antigenic peptide according to the invention. For example, a carrier protein can transport its cargo across a membrane. In the context of the present invention, carrier proteins also include peptides or polypeptides that are capable of in particular eliciting an immune response against an antigenic peptide that is linked to it. Carrier proteins are known in the art.

[0030] Alternatively, such carrier peptides or polypeptides may be co-administered in the form of an immune adjuvant.

[0031] Preferably, the antigenic peptides as described herein are co-administered or may be linked, for example covalently or non-covalently, to proteins / peptides with immune adjuvant properties, such as stimulating CD4+ Th1 cells. The antigenic peptides as described herein preferably bind to MHC class I, but CD4+ helper epitopes may additionally be used to provide an efficient immune response. Th1 helper cells secrete interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin-2 (IL-2), and can sustain efficient DC activation and specific CTL activation by enhancing the expression of costimulatory signals in dendritic cells (DCs) and T cells (Galaine et al., Interest of Tumor-Specific CD4 T Helper 1 Cells for Therapeutic Anticancer Vaccine. Vaccines (Basel). 2015 Jun. 30;3(3):490-502).

[0032] For example, the adjuvant peptide / protein may preferably be different from the antigenic peptide according to the invention. Preferably, the adjuvant peptide / protein may be capable of recalling immune memory or may provide non-specific help or may be a specific helper peptide. Several helper peptides for providing non-specific T cell help such as tetanus helper peptides, keyhole limpet hemocyanin peptides or PADRE peptides have been described in the literature (Adotevi et al., Targeting antitumor CD4 helper T cells with universal tumor-reactive helper peptides derived from telomerase for cancer vaccine. Hum Vaccin Immunother. 2013 May;9(5):1073-7; Slingluff CL, The present and future of peptide vaccines for cancer: single or multiple, long or short, alone or in combination? Cancer J. 2011 Sep-Oct;17(5):343-50). Thus, tetanus helper peptide, keyhole limpet hemocyanin peptide and PADRE peptide are preferred examples of such adjuvant peptides / proteins. This peptide represents another example of a helper peptide (having immune adjuvant properties) that is preferred in the context of the present invention. Another preferred example is h-pAg T13L (Bhasin M, Singh H, Raghava GP (2003) MHCBN: a comprehensive database of MHC binding and non-binding peptides. Bioinformatics 19: 665-666).Further examples of preferred helper peptides include (e.g. WO2013 / 135553 A1 or Dosset M, Godet Y, Vauchy C, Beziaud L, Lone YC, Sedlik C, Liard C, Levionnois E, Clerc B, Sandoval F, Daguindau E, Wain-Hobson S, Tartour E, Langlade-Demoyen P, Borg C, Adotevi O: Universal cancer peptide-based therapeutic vaccine breaks tolerance against telomerase and eradicates established tumor. Clin Cancer Res. 2012 Nov 15;18(22):6284~95 doi: 10.1158 / 1078-0432.CCR-12-0896. Epub 2 Oct 2012) and BIRC5 peptides (e.g. as described in EP2119726A1, or Widenmeyer M, Griesemann H, Stevanovic S, Feyerabend S, Klein R, Attig S, Hennenlotter J, Wernet D, Kuprash DV, Sazykin AY, Pascolo S, Stenzl A, Gouttefangeas C, Rammensee HG: Promiscuous survivin peptide induces robust CD4+ T-cell responses in the majority of vaccinated cancer patients. Int J Cancer. 2012 Jul 1;131(1):140-9. doi: 10.1002 / ijc.26365 Epub 14 Sep 2011). The most preferred helper peptide is the UCP2 peptide (amino acid sequence: KSVWSKLQSIGIRQH; SEQ ID NO: 39, e.g., as described in WO2013 / 135553A1 or Dosset et al., Clin Cancer Res. 2012 Nov. 15; 18(22):6284-95).

[0033] As used herein, the term "immunogenic composition" refers to a composition that is capable of eliciting, inducing, increasing, prolonging or maintaining an immune response, particularly when it is administered to a mammal, and particularly when it is administered to a human individual. Preferably, the immunogenic composition further comprises one or more immune adjuvant substances.

[0034] By "pharmaceutical acceptable excipient or carrier" is meant herein a pharmaceutical grade compound that enhances the delivery, stability or bioavailability of an active agent, can be metabolized by the subject to which it is administered, and is non-toxic to the subject. Preferred excipients and carriers according to the present invention include any of the excipients or carriers commonly used in pharmaceuticals, such as water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it is preferred to include an isotonic agent, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable excipients or carriers may further include minor amounts of auxiliary substances, such as wetting or emulsifying agents, or preservatives.

[0035] By "vaccine" is meant herein a composition capable of stimulating the immune system of a living organism so as to confer protection from harmful antigens, either through prophylaxis or therapy. Prophylactic vaccines are preferred. Preferably, the vaccine or vaccine composition further comprises one or more immune adjuvant substances.

[0036] According to various aspects and embodiments of the invention described herein, a "subject" or "host" preferably refers to a mammal, most preferably a human. The subject may have cancer, may be suspected of having cancer, or may be at risk of developing cancer.

[0037] As used herein, the term "cancer" refers to a malignant neoplasm. In particular, the term "cancer" as used herein refers to any member of a class of diseases or disorders characterized by the uncontrolled division of cells and the ability of these cells to invade other tissues, either by direct growth into adjacent tissues through invasion, or by migration to distant sites by metastasis. Metastasis is defined as the stage where cancer cells are transported through the bloodstream or lymphatic system. The term encompasses, inter alia, esophageal, gastric, duodenal, small intestinal, appendix, large intestine, colon, rectal, colorectal, anal, pancreatic, liver, gallbladder, spleen, kidney, bladder, prostate, testicular, uterine, endometrial, ovarian, vaginal, vulvar, breast, lung, thyroid, thymus, brain, nervous system, glioma, oral, skin, blood, lymphoma, eye, bone, bone marrow, muscle, etc. In the context of the present invention, melanoma, head and neck, breast, colorectal, or kidney cancer (e.g., renal clear cell carcinoma) is preferred.

[0038] As used herein, the terms "preventing," "prevention," "prophylaxis," or "preventing" generally mean avoiding or minimizing the onset or occurrence of a disease or condition before its onset, while the terms "treating," "treatment," or "treating" encompasses reducing, ameliorating, or curing a disease or condition (or a symptom of a disease or condition) after its onset. The term "preventing" encompasses "reducing the likelihood of occurrence" or "reducing the likelihood of recurrence."

[0039] As used herein, an "effective amount" or "effective dose" is an amount that provides a desired effect. For therapeutic purposes, an effective amount is an amount sufficient to provide a beneficial or desired clinical result. A preferred effective amount for a given application can be readily determined by one of skill in the art, taking into account, for example, the subject's size, age, weight, the type of disease / disorder to be prevented or treated, and the amount of time since the disease / disorder began. In the context of the present invention, with respect to prevention or treatment, an effective amount of a composition is an amount that is sufficient to induce a humoral and / or cellular immune response directed against the disease / disorder.

[0040] 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 stated members, integers, or steps, but not the exclusion of any other unstated members, integers, or steps. The term "consist of" is a special embodiment of the term "comprise", in which any other unstated members, integers, or steps are excluded. In the context of the present invention, the term "comprise" encompasses the term "consist of". Thus, the term "comprising" encompasses "including" as well as "consisting", e.g., a composition "comprising" X may consist exclusively of X, or may include something additional, e.g., X+Y.

[0041] The terms "a," "an," and "the," and similar references used in the context of describing the present invention (particularly in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or otherwise clearly contradicted by context. Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into this specification 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.

[0042] The word "substantially" does not exclude "completely", e.g. a composition that is "substantially" free of Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of the invention.

[0043] The term "about" in reference to a number x means x±10%.

[0044] Additional definitions are provided throughout the specification.

[0045] The present invention may be understood more readily by reference to the following detailed description, including preferred embodiments of the invention, and the examples included therein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Although the present invention is described in detail below, it should be understood that the invention is not limited to the specific methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0047] In the following, elements of the present invention are described. Although these elements are listed with respect to specific embodiments, it should be understood that they can be combined in any number of ways to produce additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only those specifically described embodiments. The description should be understood to support and encompass embodiments that combine the specifically described embodiments with any of the disclosed and / or preferred elements. Furthermore, any permutation or combination of all described elements in this application should be considered to be disclosed by the description of this application, unless the context dictates otherwise.

[0048] Antigenic peptides according to the present invention In a first aspect, the present invention provides an antigenic peptide comprising or consisting of an amino acid sequence shown in any one of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 40 to 42.

[0049] The present invention also provides an antigenic peptide comprising or consisting of an amino acid sequence shown in any one of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 40 to 42, in which one or two amino acid residues may be optionally substituted, deleted, or added.

[0050] The inventors have identified a set of antigenic peptides that can be used to induce a specific immune response against tumor cells. The antigenic peptides are different from (fragments of) human tumor antigens, but have amino acid similarity to them, as shown in Table 1 below. In particular, the antigenic peptides according to the invention are contained in polypeptides and proteins produced by commensal bacteria from the human gut. The antigenic peptides according to the invention are therefore bacterial sequences, rather than human sequences. Without wishing to be bound by any particular theory, the inventors believe that the human immune repertoire contains T cell clones that are reactive to bacterial peptides (contained in proteins produced by commensal bacteria from the gut), which have amino acid similarity to fragments of human tumor antigens. In particular, the antigenic peptides according to the invention are able to induce a stronger immune response than the corresponding human peptides, since T cells capable of strictly recognizing human peptides are eliminated during maturation as they recognize self-antigens, which is not the case for the antigenic peptides according to the invention. This may explain why the antigenic peptides described herein can induce immune responses, especially T cell responses, when these peptides are administered to a (human) individual.Thus, without being bound by any theory, the inventors assume that proteins produced by commensal bacteria from the gut can "mimic" tumor antigens and can be used to trigger specific immune responses against tumor cells.These findings provide further evidence that commensal bacteria can contribute to tumor cell eradication.

[0051] Thus, the present invention relates to antigenic peptides that have amino acid similarity to tumor antigens. As used herein, the phrase "having amino acid similarity to a tumor antigen" refers in particular to sequence variants of fragments (epitopes) of human tumor antigens such as CDC20 or other exemplary human tumor antigens listed in Table 1 below.

[0052] A "sequence variant" typically shares at least 50% sequence identity with a reference sequence, such as a fragment of a (reference) tumor antigen, particularly over the entire length of the sequence. Preferably, a sequence variant shares at least 70% or 75%, preferably at least 80% or 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96% or 97%, particularly preferably at least 98% or 99% sequence identity with a reference sequence, such as a fragment of a (reference) tumor antigen. Sequence identity can be calculated as known in the art, in particular as described below. Preferably, a sequence variant preserves a particular function of the reference sequence, such as its function as a tumor epitope and / or its ability to induce or maintain an immune response. In particular, an amino acid sequence variant has an altered sequence in which one or more of the amino acids in the reference sequence are mutated, e.g. deleted or substituted, or one or more amino acids are inserted into the sequence of the reference amino acid sequence. For example, a variant sequence that is at least 90% identical has no more than 10 changes, ie, deletions, insertions, or substitutions, in any combination, per 100 amino acids of the reference sequence.

[0053] The method for comparing the identity (similarity) of two or more sequences is well known in the art.The percentage of identity of two sequences can be determined, for example, by using a mathematical algorithm.A preferred, but not limiting, example of the mathematical algorithm that can be used is the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877. Such algorithms are incorporated into the BLAST family of programs, e.g., the BLAST or NBLAST programs (see also Altschul et al., 1990, J. Mol. Biol. 215, 403-410 or Altschul et al., (1997), Nucleic Acids Res, 25:3389-3402), and FASTA (Pearson (1990), Methods Enzymol. 183, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci. USA 85, 2444-2448), which are accessible through the NCBI homepage at the worldwide website ncbi.nlm.nih.gov. Sequences that are identical to other sequences to a certain degree can be identified by these programs. In addition, programs available in the Wisconsin Sequence Analysis Package, Version 9.1 (Devereux et al., 1984, Nucleic Acids Res., pp. 387-395), such as the programs BESTFIT and GAP, can also be used to determine the % identity between two polynucleotides and the % identity between two (poly)peptide sequences. BESTFIT uses the "local homology" algorithm of Smith and Waterman (1981), J. Mol. Biol. 147, pp. 195-197, to find the best single region of similarity between two sequences.

[0054] In particular, the antigenic peptides according to the invention have a core sequence identical to that of an epitope (fragment) of a (human) reference tumor antigen, which core sequence further exhibits a high prevalence based on the frequency of proteins present in the human microbiome in which the core sequence is found.

[0055] The core sequence therefore represents a major feature of the antigenic peptides according to the invention. Thus, the inventors have identified core sequences of great interest with high prevalence, since they are present in some sequence variants of fragments of (see) tumor antigens and / or in some human microbiota proteins at high frequency in a significant part of the general human population. Preferably, the core sequence is maintained in the antigenic peptide comprising or consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 16 and 40 to 42, optionally with substitution, deletion or addition of one or two amino acid residues.

[0056] Preferably, an antigenic peptide according to the present invention comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 40 to 42. In some embodiments, an antigenic peptide consists of or consists essentially of an amino acid sequence according to any one of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 40 to 42.

[0057] In some embodiments, the antigenic peptide may be modified and / or may include non-peptide bonds (e.g., as described above). For example, the antigenic peptide may be modified at its N-terminus and / or its C-terminus (e.g., to be labeled or linked to a carrier or substrate). Such modifications will generally depend on the intended purpose and are well known in the art.

[0058] The antigenic peptides disclosed herein can be prepared using well-known techniques. For example, the peptides can be synthetically prepared by recombinant DNA technology or chemical synthesis. The peptides disclosed herein can be synthesized individually or as longer polypeptides comprising two or more peptides (e.g., two or more peptides, or a peptide and a non-peptide). The antigenic peptides can be isolated, i.e., purified to be substantially free of other naturally occurring host cell proteins and fragments thereof, e.g., at least about 70%, 80%, or 90% purified. Preferably, the antigenic peptides according to the present invention are isolated antigenic peptides.

[0059] In some embodiments, antigenic peptides according to the invention have the ability to bind to molecules of the human major histocompatibility complex (MHC), e.g., MHC class I (MHC I) molecules; or, in extended forms such as length variants, MHC class II (MHC II) molecules. Preferably, antigenic peptides according to the invention are capable of binding to MHC class I (major histocompatibility complex class I, MHC I) molecules.

[0060] MHC class I molecules present epitopes to killer T cells, also called cytotoxic T lymphocytes (CTLs). In addition to TCR (T cell receptor), CTLs express CD8 receptors. When the CD8 receptor of a CTL docks with an MHC class I molecule, if the TCR of the CTL matches an epitope in the MHC class I molecule, the CTL triggers the cell to undergo programmed cell death by apoptosis. This pathway is particularly useful for preventing and / or treating cancer, since cancer cells are directly attacked. In humans, there are three different genetic loci that code for MHC class I molecules (human MHC molecules are also named human leukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. Thus, MHC class I includes HLA-A, HLA-B, and HLA-C molecules in humans. HLA-A*01, HLA-A*02, HLA-A*24, and HLA-B*07 are examples of different MHC class I alleles that can be expressed from these loci. By way of example, an antigenic peptide according to the invention may bind to HLA-A*01, HLA-A*02, HLA-A*24, and HLA-B*07 molecules. In some embodiments, an antigenic peptide according to the invention binds to HLA-A*02. Typically, peptides (epitopes) having a length of 8-11 amino acids are presented by MHC I.

[0061] In general, the antigenic peptides according to the invention can be of any length. Preferably, the antigenic peptides according to the invention do not exceed 350 amino acids. For example, the maximum length of the antigenic peptides according to the invention can be 300 amino acids or 250 amino acids. More preferably, the maximum length of an antigenic peptide according to the present invention does not exceed 200 amino acids, for example 190 amino acids or less, 180 amino acids or less, 170 amino acids or less, 160 amino acids or less, 150 amino acids or less, 140 amino acids or less, 130 amino acids or less, 120 amino acids or less, 110 amino acids or less, 100 amino acids or less, 95 amino acids or less, 90 amino acids or less, 85 amino acids or less, 80 amino acids or less, 75 amino acids or less, 70 amino acids or less, 65 amino acids or less, 60 amino acids or less, 55 amino acids or less, 50 amino acids or less, 45 amino acids or less, 40 amino acids or less, 35 amino acids or less, 30 amino acids or less, 29 amino acids or less, 28 amino acids or less, 27 amino acids or less, 26 amino acids or less, 25 amino acids or less, 24 amino acids or less, 23 amino acids or less, 22 amino acids or less, 21 amino acids or less, 20 amino acids or less, 19 amino acids or less, 18 amino acids or less, 17 amino acids or less, 16 amino acids or less, 15 amino acids or less, 14 amino acids or less, or 13 amino acids or less. In particular, the length of the antigenic peptides according to the invention is preferably at most 30 or 25 amino acids, more preferably at most 20 or 15 amino acids, with smaller peptides of 8-15 or 8-11 amino acids in length (e.g., 8, 9, 10 or 11 amino acids in length) being even more preferred; peptides having a length of 9 or 10 amino acids being even more preferred.

[0062] In particular, the antigenic peptides are not the full-length proteins produced by the gut-derived commensal bacteria from which they are derived, in other words, the antigenic peptides of the present invention are preferably fragments of full-length proteins (produced by the human microbiota).

[0063] Similarly, a "fragment / epitope" of a (reference) tumor antigen, which typically serves as a reference sequence, preferably comprises 8 to 11 consecutive amino acids of the tumor antigen, preferably 9 or 10 amino acids. It is understood that a "fragment / epitope" of a (reference) tumor antigen is not the full-length tumor antigen (protein).

[0064] As used herein, a "fragment" (of a protein or nucleic acid (sequence)) preferably has a maximum length of 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the full-length (reference) protein / nucleic acid / sequence. In some embodiments, the length of a fragment does not exceed 50% of the length of the (full-length) (reference) protein / nucleic acid. In other embodiments, the length of a fragment of a (reference) protein / nucleic acid does not exceed 20% or 10% of the length of the (full-length) (reference) protein / nucleic acid.

[0065] More generally, the invention provides antigenic peptides comprising or consisting of a microbiota sequence variant of a fragment of a human tumor antigen. The human tumor antigen may be selected from the group consisting of CDC20, KIF2C, UBE2C, ANKRD30A, AURKA, CDH17, CEACAM5, MMP11, OR51E2, and TOP2A. The fragment / epitope of a human (reference) tumor antigen may be selected from the group consisting of SEQ ID NOs: 17-31 and 38. In some embodiments, the antigenic peptide comprises or consists of a microbiota variant of a human reference peptide according to any one of SEQ ID NOs: 17-31 and 38.

[0066] In some embodiments, the antigenic peptide induces T cell cross-reactivity against human epitopes of tumor antigens (see). T cell cross-reactivity is an immune system phenomenon defined as the recognition of two or more peptide-MHC complexes (pMHC) by the T cell receptor (TCR).

[0067] Epitope mimicry relates to the concept of sequence and structural similarity between foreign and self-antigens as a triggering mechanism to induce cross-reactive immune responses against self-antigens. Interestingly, such epitope mimicry offers a possible way to circumvent the repertoire restriction of human T cells by clonal deletion of T cells that recognize self-antigens.

[0068] In particular, antigens (i.e., antigenic peptides according to the present invention) that are distinct from self-antigens (e.g., human epitopes of tumor antigens) but share sequence similarity with self-antigens are expected to (i) still be recognized by T cell receptor cross-reactivity, and (ii) be recognized by T cells / TCRs that have not been eliminated during the T cell education process. Thus, such antigens can elicit strong immune responses that result in clonal expansion of T cells with potential cross-reactivity with the self-antigen.

[0069] T cell receptor cross-reactivity with epitopes of human (reference) tumor antigens can be measured by ELISPOT-IFNγ assay, as shown in the Examples section. Briefly, HLA-A2 transgenic mice (e.g., HHD DR1 mice expressing human HLA-A2 and HLA-DR1 MHC and lacking mouse H-2 class I and class II MHC, and / or HHD DR3 mice expressing human HLA-A2 and HLA-DR3 MHC) can be immunized with an antigenic peptide of the invention, or in a control group, with the corresponding human (reference) peptide, with a prime injection on day 0 (d0) and later, e.g., on d14, with a boost injection. Then, e.g., 7 days after the boost injection (i.e., on d21), the mice can be sacrificed and the ability of splenocytes to be stimulated in vitro with the antigenic peptide of the invention and secrete IFN-gamma, as assessed by ELISPOT.

[0070] Table 1 below provides an overview on the antigenic peptides according to the invention with their amino acid sequences and SEQ ID NOs. Table 1 also provides information on which tumor antigen each antigenic peptide according to the invention is associated with. SEQ ID NOs: 1 to 16 and SEQ ID NOs: 40 to 42 refer to HLA-A*02 antigenic peptides according to the invention.

[0071] [Table 1]

[0072] In some embodiments, an antigenic peptide according to the invention is a sequence variant of a fragment (human reference peptide) of the tumor antigen CDC20, such as "SLPDRILDA" (SEQ ID NO: 17). Preferably, an antigenic peptide according to the invention is a sequence variant of a fragment of the tumor antigen CDC20, such as an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0073] In some embodiments, the antigenic peptide according to the present invention is a sequence variant of a fragment of the tumor antigen KIF2C (human reference peptide), such as "AINPELLQL" (SEQ ID NO: 18). Preferably, the antigenic peptide according to the present invention is a sequence variant of a fragment of the tumor antigen KIF2C, such as an antigenic peptide comprising or consisting of the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NOs: 40 to 42.

[0074] In some embodiments, the antigenic peptide according to the present invention is a sequence variant of a fragment (human reference peptide) of the tumor antigen UBE2C, such as "ALYDVRTIL" (SEQ ID NO: 19), "ALYDVRTILL" (SEQ ID NO: 38), "ILLSIQSLL" (SEQ ID NO: 20), or "RLQQELMTL" (SEQ ID NO: 21). More preferably, the antigenic peptide according to the present invention is a sequence variant of the UBE2C fragment (human reference peptide) "ALYDVRTIL" (SEQ ID NO: 19), such as an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3-4. It is also more preferred that the antigenic peptide according to the present invention is a sequence variant of the UBE2C fragment (human reference peptide) "ILLSIQSLL" (SEQ ID NO: 20), such as an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 5. It is also more preferred that the antigenic peptide according to the present invention is a sequence variant of the UBE2C fragment (human reference peptide) "RLQQELMTL" (SEQ ID NO: 21), such as an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 6.

[0075] In some embodiments, the antigenic peptide according to the invention is a sequence variant of a fragment (human reference peptide) of the tumor antigen ANKRD30A, such as "AVYSEILSV" (SEQ ID NO: 22), "KILDTVHSC" (SEQ ID NO: 23), or "SLDQKLFQL" (SEQ ID NO: 24). More preferably, the antigenic peptide according to the invention is a sequence variant of the ANKRD30A fragment (human reference peptide) "AVYSEILSV" (SEQ ID NO: 22), such as an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 7. It is also more preferred that the antigenic peptide according to the invention is a sequence variant of the ANKRD30A fragment (human reference peptide) "KILDTVHSC" (SEQ ID NO: 23), such as an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8. It is also more preferred that the antigenic peptide according to the invention is a sequence variant of the ANKRD30A fragment (human reference peptide) "SLDQKLFQL" (SEQ ID NO: 24), such as an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 9.

[0076] In some embodiments, an antigenic peptide according to the invention is a sequence variant of a fragment (human reference peptide) of the tumor antigen AURKA, such as "YLILEYAPL" (SEQ ID NO: 25). Preferably, an antigenic peptide according to the invention is a sequence variant of a fragment of the tumor antigen AURKA, such as an antigenic peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10.

[0077] In some embodiments, an antigenic peptide according to the invention is a sequence variant of a fragment of the tumor antigen CDH17 (human reference peptide), such as "LVIGIILAV" (SEQ ID NO: 26). Preferably, an antigenic peptide according to the invention is a sequence variant of a fragment of the tumor antigen CDH17, such as an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11.

[0078] In some embodiments, an antigenic peptide according to the invention is a sequence variant of a fragment of the tumor antigen CEACAM5 (human reference peptide), such as "YLSGANLNL" (SEQ ID NO: 27). Preferably, an antigenic peptide according to the invention is a sequence variant of a fragment of the tumor antigen CEACAM5, such as an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 12.

[0079] In some embodiments, the antigenic peptide according to the invention is a sequence variant of a fragment (human reference peptide) of the tumor antigen MMP11, such as "KVWSDVTPL" (SEQ ID NO: 28). Preferably, the antigenic peptide according to the invention is a sequence variant of a fragment of the tumor antigen MMP11, such as an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 13.

[0080] In some embodiments, an antigenic peptide according to the invention is a sequence variant of a fragment of tumor antigen OR51E2 (human reference peptide), such as "AQIGIVAVV" (SEQ ID NO: 29). Preferably, an antigenic peptide according to the invention is a sequence variant of a fragment of tumor antigen OR51E2, such as an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14.

[0081] In some embodiments, the antigenic peptide according to the invention is a sequence variant of a fragment (human reference peptide) of the tumor antigen TOP2A, such as "ILNSTTIEI" (SEQ ID NO: 30) or "ALIFGQLLT" (SEQ ID NO: 31). More preferably, the antigenic peptide according to the invention is a sequence variant of the TOP2A fragment (human reference peptide) "ILNSTTIEI" (SEQ ID NO: 30), such as an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 15. It is also more preferred that the antigenic peptide according to the invention is a sequence variant of the TOP2A fragment (human reference peptide) "ALIFGQLLT" (SEQ ID NO: 31), such as an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 16.

[0082] Preferably, an antigenic peptide according to the present invention comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, and 3. In some embodiments, an antigenic peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, an antigenic peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, an antigenic peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3.

[0083] As shown in the examples herein, the specific antigenic peptides according to the invention allow for the generation of a strong immune response against themselves and, most importantly, against peptides contained in tumor antigens that share amino acid similarity with them, even if the human reference peptides contained in the tumor antigens may be tolerogenic.

[0084] Advantageously, the antigenic peptides according to the invention may be in the form of immunogenic compounds, particularly for use in the prevention or treatment of cancer.

[0085] Immunogenic compounds comprising antigenic peptides according to the invention In a further aspect, the present invention also provides an immunogenic compound comprising an antigenic peptide according to the invention as described above. In particular, the preferred embodiments of the antigenic peptide as described above also apply to the immunogenic compound according to the invention.

[0086] In general, the term "immunogenic compound" includes any kind of compound that comprises an antigenic peptide according to the invention. For example, an antigenic peptide according to the invention may be linked to a carrier molecule or may be comprised in a polypeptide or protein (which may be present "alone", i.e. not linked to any other compound, or the polypeptide or protein comprising the antigenic peptide may be linked to a carrier molecule).

[0087] The type of carrier molecule used to prepare the immunogenic compound of the present invention, such as an immunogenic compound comprising or consisting of a polypeptide of formula (I) linked to a carrier molecule, is well known as general knowledge to those skilled in the art. In particular, the function of the carrier molecule may be to provide cytokine help (or T cell help) to enhance the immune response against tumor antigens.

[0088] Preferably, the immunogenic compound according to the invention comprises an antigenic peptide and a carrier molecule, especially when the antigenic peptide (or a polypeptide or protein comprising the antigenic peptide) is linked to a carrier molecule. A preferred carrier molecule is a carrier protein or carrier peptide. According to a preferred embodiment, the antigenic peptide as defined above or the polypeptide / protein comprising said antigenic peptide is linked, for example by covalent or non-covalent bonds, to a carrier protein or carrier peptide. Alternatively, a carrier protein or carrier peptide as described herein may be co-administered (separately) in the form of an immune adjuvant (i.e. not as an "immunogenic compound" but as a co-administration / combination therapy as described herein below).

[0089] In some embodiments, the antigenic peptides described herein or polypeptides / proteins comprising antigenic peptides can be co-administered or linked, e.g., covalently or non-covalently, to proteins / peptides with immune adjuvant properties, such as stimulating CD4+ Th1 cells. The antigenic peptides described herein preferably bind to MHC class I, but CD4+ helper epitopes can additionally be used to provide an efficient immune response. Th1 helper cells secrete interferon-gamma (IFN-γ), tumor necrosis factor (TNF-α), and interleukin-2 (IL-2), and can sustain efficient DC activation and specific CTL activation by enhancing the expression of costimulatory signals on dendritic cells (DCs) and T cells (Galaine et al., Interest of Tumor-Specific CD4 T Helper 1 Cells for Therapeutic Anticancer Vaccine. Vaccines (Basel). 2015 Jun. 30;3(3):490-502).

[0090] For example, the adjuvant peptide / protein can be a non-tumor antigen that recalls immune memory or provides non-specific help, or it can be a specific tumor-derived helper peptide. Several helper peptides for providing non-specific T cell help have been described in the literature, such as tetanus helper peptide, keyhole limpet hemocyanin peptide, or PADRE peptide (Adotevi et al., Targeting antitumor CD4 helper T cells with universal tumor-reactive helper peptides derived from telomerase for cancer vaccine. Hum Vaccin Immunother. 2013 May;9(5):1073-7; Slingluff CL, The present and future of peptide vaccines for cancer: single or multiple, long or short, alone or in combination? Cancer J. 2011 Sep-Oct;17(5):343-50). Thus, tetanus helper peptide, keyhole limpet hemocyanin peptide, and PADRE peptide are examples of such adjuvant peptides / proteins. Furthermore, the adjuvant peptide / protein may be a specific tumor-derived helper peptide. The specific tumor-derived helper peptide is typically presented by MHC class II, in particular HLA-DR, HLA-DP, or HLA-DQ. The specific tumor-derived helper peptide may be a fragment of the sequence of a shared overexpressed tumor antigen, such as HER2, NY-ESO-1, hTERT, or IL13RA2. Such a fragment preferably has a length of at least 10 amino acids, more preferably at least 11 amino acids, even more preferably at least 12 amino acids, and most preferably at least 13 amino acids. In particular, a fragment of a shared overexpressed tumor antigen, such as HER2, NY-ESO-1, hTERT, having a length of 13 to 24 amino acids is preferred.Preferred fragments bind MHC class II and can therefore be identified, for example, using the MHC class II binding prediction tool of the IEDB (Immune Epitope Database and Analysis Resource; supported by contract from the National Institute of Allergy and Infectious Diseases, a component of the National Institutes of Health in the Department of Health and Human Services; URLs: http: / / www.iedb.org / ; http: / / tools.iedb.org / mhcii / ). Preferably, the adjuvant peptide / protein is the UCP2 peptide (amino acid sequence: KSVWSKLQSIGIRQH; SEQ ID NO: 39, e.g., as described in WO2013 / 135553A1 or Dosset et al., Clin Cancer Res. 2012 Nov. 15; 18(22):6284-95).

[0091] It is also preferred that the immunogenic compound according to the invention is a polypeptide or protein comprising an antigenic peptide according to the invention. Preferably, such a protein or polypeptide is a recombinant protein or polypeptide, such as a fusion protein. The term "recombinant" means that it does not occur in nature. In some embodiments, the antigenic peptide according to the invention can be part of a fusion protein, for example fused to the N-terminal amino acid of the HLA-DR antigen-associated invariant chain (Ii) or fused to an antibody (or fused into the sequence of an antibody), for example an antibody specific for dendritic cells.

[0092] Preferably, the immunogenic compound according to the invention comprises or consists of a polypeptide of formula (I): PepNt-CORE-PepCt (I) During the ceremony, - "PepNt" consists of a polypeptide having a length varying from 0 to 500 amino acid residues and is located at the N-terminus of the polypeptide of formula (I); - "CORE" consists of an antigenic peptide according to the invention as defined above; and "PepCt" consists of a polypeptide having a length varying from 0 to 500 amino acid residues and is located at the C-terminus of the polypeptide of formula (I).

[0093] For example, the immunogenic compound comprises or consists of a polypeptide of formula (Ia) or (Ib): PepNt-CORE (Ia); or CORE-PepCt (Ib) In the formula, "PepNt", "PepCt" and "CORE" are as defined above.

[0094] Preferably, the polypeptide of formula (I), (Ia) or (Ib) is a fusion peptide or protein, in particular a recombinant fusion peptide or protein.

[0095] A polypeptide or immunogenic compound as defined above may be, for example, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids , 36 amino acids, 37 amino acids, 38 amino acids, 39 amino acids, 40 amino acids, 41 amino acids, 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 ​​amino acids, 49 amino acids, 50 amino acids, 51 amino acids, 52 amino acids, 53 amino acids, 54 amino acids, 55 amino acids, 56 amino acids, 57 amino acids, 58 amino acids, 59 amino acids, 60 amino acids, 61 amino acids, 62 amino acids, 63 amino acids, 64 amino acids, 65 amino acids, 66 amino acids, 67? 68 amino acids, 69 amino acids, 70 amino acids, 71 amino acids, 72 amino acids, 73 amino acids, 74 amino acids, 75 amino acids, 76 amino acids, 77 amino acids, 78 amino acids, 79 amino acids, 80 amino acids, 81 amino acids, 82 amino acids, 83 amino acids, 84 amino acids, 85 amino acids, 86 amino acids, 87 amino acids, 88 amino acids, 89 amino acids, 90 amino acids, 91 amino acids, 92 amino acids, 93 amino acids, 94 amino acids, 95 amino acids, 96 amino acids, 97 amino acids, 98 amino acids, 99 Also preferred are those containing from 9 amino acids to 1000 amino acids, including 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1000 amino acids. Thus, the lengths of "PepNt" and "PepCt", if applicable, may be defined accordingly.

[0096] Thus, "PepNt" and "PepCt" as defined above include, for example, 0 amino acid residue, 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 11 amino acid residues, 12 amino acid residues, 13 amino acid residues, 14 amino acid residues, 15 amino acid residues, 16 amino acid residues, 17 amino acid residues, 18 amino acid residues, 19 amino acid residues, 20 amino acid residues, 21 amino acid residues, 22 amino acid residues, 23 amino acid residues, 24 amino acid residues, 25 amino acid residues, 26 amino acid residues, 27 amino acid residues, 28 amino acid residues, 29 amino acid residues, 30 amino acid residues, 31 amino acid residues, 32 amino acid residues, 33 amino acid residues, 34 amino acid residues, 35 amino acid residues, 36 amino acid residues, 37 amino acid residues, 38 amino acid residues, 39 amino acid residues, 40 amino acid residues, 41 amino acid residues, 42 amino acid residues, 43 amino acid residues, 44 amino acid residues, 45 amino acid residues, 46 amino acid residues, 47 amino acid residues, 48 ​​amino acid residues, 49 amino acid residues, 50 amino acid residues, 51 amino acid residues, 52 amino acid residues, 53 amino acid residues, 54 amino acid residues, 55 amino acid residues, 56 amino acid residues, 57 amino acid residues, 58 amino acid residues, 59 amino acid residues, 60 amino acid residues, 61 amino acid residues, 62 amino amino acid residue, 25 amino acid residue, 26 amino acid residue, 27 amino acid residue, 28 amino acid residue, 29 amino acid residue, 30 amino acid residue, 31 amino acid residue, 32 amino acid residue, 33 amino acid residue, 34 amino acid residue, 35 amino acid residue, 36 amino acid residue, 37 amino acid residue, 38 amino acid residue, 39 amino acid residue, 40 amino acid residue, 41 amino acid residue, 42 amino acid residue, 43 amino acid residue, 44 amino acid residue, 45 amino acid residue, 46 amino acid residue, 47 amino acid residue, 48 amino acid residue, 49 amino acid residue, 50 amino acid residue, 51 amino acid residue, 52 amino acid residue, 53 amino acid residue, 54 amino acid residue, 55 amino acid residue, 56 amino acid residue, 57 amino acid residue, 58 amino acid residue, 59 amino acid residue, 60 amino acid residue, 61 amino acid residue, 62 amino acid residue, 63 amino acid residue, 64 amino acid residue, 65 amino acid residue, 66 amino acid residue, 67 amino acid residue, 68 amino acid residue, 69 amino acid residue, 70 amino acid residue, 71 amino acid residue, 72 amino acid residue, 73 amino acid residue, 74 amino acid residue, 75 amino acid residue, 76 amino acid residue, 77 amino acid residue, 78 amino acid residue, 79 amino acid residue, 78 amino acid residue, 79 amino acid residue, 80 amino acid residue, 81 amino acid residue, 82 amino acid residue, 83 amino acid residue, 84 amino acid residue, 85 amino acid residue, 86 amino acid residue, 87 amino acid residue, 88 amino acid residue, 89 amino acid residue, 89 amino acid residue, 90 amino acid residue, 91 amino acid residue, 92 amino acid residue, 93 amino acid residue, 94 2 amino acid residues, 53 amino acid residues, 54 amino acid residues, 55 amino acid residues, 56 amino acid residues, 57 amino acid residues, 58 amino acid residues, 59 amino acid residues, 60 amino acid residues, 61 amino acid residues, 62 amino acid residues, 63 amino acid residues, 64 amino acid residues, 65 amino acid residues, 66 amino acid residues, 67 amino acid residues, 68 amino acid residues, 69 amino acid residues, 70 amino acid residues, 71 amino acid residues, 72 amino acid residues, 73 amino acid residues, 74 amino acid residues, 75 amino acid residues, 76 amino acid residues, 77 amino acid residues, 78 amino acid residues, 79 amino acid residues , 80 amino acid residues, 81 amino acid residues, 82 amino acid residues, 83 amino acid residues, 84 amino acid residues, 85 amino acid residues, 86 amino acid residues, 87 amino acid residues, 88 amino acid residues, 89 amino acid residues, 90 amino acid residues, 91 amino acid residues, 92 amino acid residues, 93 amino acid residues, 94 amino acid residues, 95 amino acid residues, 96 amino acid residues, 97 amino acid residues, 98 amino acid residues, 99 amino acid residues, 100 amino acid residues, 110 amino acid residues, 120 amino acid residues, 130 amino acid residues, 140 amino acid residues, 150 amino acid residues, 160 amino acid residues,It may contain from 0 to 500 amino acid residues, including 170 amino acid residues, 180 amino acid residues, 190 amino acid residues, 200 amino acid residues, 250 amino acid residues, 300 amino acid residues, 350 amino acid residues, 400 amino acid residues, 450 amino acid residues, and 500 amino acid residues.

[0097] Preferably, the antigenic peptide is linked to a carrier molecule, in particular a carrier protein, preferably by covalent or non-covalent bonds. The carrier molecule to which the peptide is optionally linked can be selected from a wide variety of known carriers. Examples of carrier molecules for vaccine purposes include proteins such as human or bovine serum albumin and keyhole limpet hemocyanin (KLH), as well as fatty acids. Other embodiments of carrier molecules to which the antigenic peptides of formula (I) can be covalently linked include bacterial toxins or toxoids, such as diphtheria, cholera, E. coli heat labile, or tetanus toxoids, N. meningitidis outer membrane proteins (European Patent Application EP 0372501), synthetic peptides (European Patent Applications EP 0378881 and EP 0427347), heat shock proteins (PCT Application WO 93 / 17712), pertussis proteins (PCT Application WO 98 / 58668), protein D from H. influenzae (PCT Application WO 00 / 56360), and toxins A or B from C. difficile (International Patent Application WO 00 / 61761).

[0098] Furthermore, in the polypeptides according to formula (I), (Ia) or (Ib), "PepNt" and / or "PepCt" may preferably correspond to such proteins / peptides having immune adjuvant properties, such as providing stimulation of CD4+ Th1 cells as described herein.

[0099] In some embodiments, the antigenic peptide (or polypeptide / protein comprising said antigenic peptide) according to the present invention is covalently linked to a carrier molecule through a linker moiety. For example, the linker substance may be selected from the group consisting of GMBS (N-[γ-maleimidobutyryl-oxy]succinimide ester), sulfo-GMBS (N-[γ-maleimidobutyryl-oxy]sulfosuccinimide ester), SMPB (succinimidyl 4-[p-maleimidophenyl]butyrate), and sulfo-SMPB (sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate).

[0100] Peptide-MHC (pMHC) multimers containing antigenic peptides In a further aspect, the present invention also provides peptide-MHC (pMHC) multimers comprising an antigenic peptide according to the invention.

[0101] As used herein, the term "peptide-MHC multimer" (pMHC) refers to a stable multimeric complex composed of major histocompatibility complex (MHC) protein subunits loaded with an antigenic peptide of the invention. In general, an "MHC multimer" is an oligomeric form of MHC molecule. The primary function of an MHC molecule is to bind an antigen. According to the invention, said antigen is an antigenic peptide according to the invention. Thus, a complex of MHC proteins "loaded" with an antigenic peptide of the invention typically means that the antigenic peptide of the invention is bound to one or more of the MHC proteins. "Peptide-MHC multimers" (pMHC) of the invention include, but are not limited to, peptide-MHC dimers, trimers, tetramers, pentamers, hexamers, heptamers, or octamers. MHC tetramers and pentamers are preferred. The term "major histocompatibility complex" (MHC) is a generic designation intended to encompass the histocompatibility antigen systems described in various species, including human leukocyte antigens (HLA). In humans, there are three major distinct genetic loci that code for MHC class I molecules: HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02, and HLA-A*11 are examples of the various MHC class I alleles that can be expressed from these loci.

[0102] In one embodiment of the present invention, the pMHC multimer is a peptide / MHC class I multimer. In another particular embodiment, the pMHC multimer is an HLA / peptide multimer corresponding to MHC class I. Thus, the pMHC multimer may be an HLA-peptide multimer selected from the group consisting of HLA-A-peptide multimers, HLA-B-peptide multimers, HLA-C-peptide multimers, HLA-E-peptide multimers, MICA-peptide multimers, and MICB-peptide multimers. Methods for obtaining pMHC multimers are known in the art and described, for example, in WO96 / 26962 and WO01 / 18053, which are incorporated herein by reference.

[0103] In addition to the MHC molecule and the antigenic peptide of the present invention, the pMHC may further contain components such as a multimerizing agent and / or a label (e.g., for visualization). Examples of labels include, but are not limited to, fluorescent labels, such as fluorescently labeled proteins, such as streptavidin. Fluorescent labels include allophycocyanin (APC), phycoerythrin (PE), R-phycoerythrin (R-PE), and fluorescein isothiocyanate (FITC). A preferred label is biotin.

[0104] In one embodiment of the invention, said pMHC multimers can be used to visualize T cell populations that are specific for MHC class I peptide complexes or HLA / peptide complexes corresponding to MHC class I as described herein above. For example, pMHC multimers can be multimers in which the heavy chains of the MHC are biotinylated, which allows their combination with streptavidin as tetramers. Such pMHC tetramers have an increased avidity for appropriate TCR carrier T lymphocytes and can therefore be used to visualize reactive populations by immunofluorescence. In another embodiment of the invention, said pMHC multimers can be used for the detection and / or isolation by screening (in flow cytometry or by immunomagnetic screening) of T cell populations that are specific for pMHC complexes as described herein above.

[0105] Antigenic peptide-specific T lymphocytes In a further aspect, the present invention also provides T lymphocytes specific for an antigenic peptide according to the invention, in particular cytotoxic T lymphocytes (CTLs) specific for an antigenic peptide according to the invention. The T lymphocytes, in particular CTLs, are preferably activated (cytotoxic) T lymphocytes specific for an antigenic peptide according to the invention. The "specificity" of the T lymphocytes is preferably understood as the T lymphocytes binding to the antigenic peptide and additionally binding to the tumor antigen corresponding to the antigenic peptide. In this respect, the T lymphocytes, in particular CTLs, cross-react with both the antigenic peptide and its tumor antigen counterpart, which typically shows a high level of sequence identity or similarity with the antigenic peptide.

[0106] The present invention further provides a method for producing (cytotoxic) T lymphocytes specific for an antigenic peptide according to the invention, in particular activated (cytotoxic) T lymphocytes specific for an antigenic peptide according to the invention, comprising a step of contacting T lymphocytes, in particular CTLs, in vitro with antigen-loaded human class I or II MHC molecules expressed on the surface of an antigen-presenting cell, or an artificial construct mimicking an antigen-presenting cell, wherein said antigen is an antigenic peptide according to the invention. Preferred antigen-presenting cells include dendritic cells. An artificial construct mimicking an antigen-presenting cell can be, by way of example, a peptide-MHC multimer according to the invention. The step of contacting T lymphocytes, in particular CTLs, with antigen-loaded human class I or II MHC molecules expressed on the surface of an antigen-presenting cell, or an artificial construct mimicking an antigen-presenting cell, can be carried out for a time sufficient to activate said T lymphocytes, in particular CTLs, in an antigen-specific manner. Preferably, the antigenic peptide is a preferred antigenic peptide as described above, such as an antigenic peptide comprising or consisting of an amino acid sequence shown in any one of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 40 to 42, more preferably any one of SEQ ID NOs: 1, 2, and 3.

[0107] The present invention further relates to an activated T cell produced by the method according to the invention, said T cell selectively recognizing cells expressing a polypeptide comprising an antigenic peptide according to the invention and / or a polypeptide comprising the respective human reference peptide. In one particular embodiment, said T cell recognizes cells expressing a polypeptide comprising an antigenic peptide according to the invention and a polypeptide comprising the respective human reference peptide, in particular tumor cells overexpressing the respective TAAs described herein.

[0108] (Activated) T cells directed against the antigenic peptides of the present invention are useful in therapy. In particular, the activated T cells produced by the above method selectively recognize cells that abnormally express a polypeptide comprising the amino acid sequence of SEQ ID NO: 17-31 and 38 (i.e., a tumor antigen), for example, a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 17, 18, 19, and 38. In a specific embodiment, the cells that abnormally express a polypeptide comprising the amino acid sequence of SEQ ID NO: 17-31 and 38 are tumor cells involved in the cancer to be treated.

[0109] Preferably, T lymphocytes according to the invention that are specific for an antigenic peptide of the invention may have (exhibit / express) memory markers, such memory markers being preferably memory markers of intestinal memory cells, such as CCR9, CXCR3, CD103, CX3CR1, and α4β7+.

[0110] T lymphocytes according to the invention that are specific for an antigenic peptide of the invention are preferably amplified more / more strongly after vaccination with an antigenic peptide of the invention (derived from a human microbiota sequence) compared to vaccination with peptides that are not derived from a microbiota sequence, such as human (reference) sequences and / or synthetic peptides (e.g. containing artificially introduced, mutations). In other words, vaccination of a subject with an antigenic peptide of the invention preferably increases the number of T lymphocytes according to the invention that are specific for said antigenic peptide of the invention more than vaccination with the respective human or synthetic peptide (not derived from the microbiota) associated with that same reference epitope.

[0111] T lymphocytes according to the invention that are specific for an antigenic peptide of the invention are preferably amplified more / stronger and / or faster after vaccination in a subject that has said peptide (expressed by the subject's microbiota) in the gut (e.g., the peptide can be found in a stool sample of the subject) compared to a subject that does not have said peptide (the peptide is not expressed by the subject's microbiota) in the gut, e.g., a subject in which the peptide is not detectable in a stool sample. In particular, subjects that have said peptide (the peptide is expressed by the subject's microbiota) in the gut may respond faster (faster T cell proliferation) and / or have T cells of the desired type Tc1.

[0112] Cells loaded with antigenic peptides or immunogenic compounds In a further aspect, the present invention also provides a cell loaded with an antigenic peptide according to the invention or an immunogenic compound comprising an antigenic peptide according to the invention as described above. In particular, the preferred embodiments of the antigenic peptide as described above also apply to such a cell according to the invention.

[0113] Preferred cells loaded with an antigenic peptide according to the invention or an immunogenic compound according to the invention are antigen-presenting cells (APCs), more preferably dendritic cells (DCs).

[0114] APCs are of particular interest, since their main function is to process antigens and present them on the cell surface to T cells of the immune system in order to elicit and modulate T cell responses in vivo. In the context of the present invention, it is preferred that APCs are loaded with the antigenic peptides and / or immunogenic compounds according to the invention. This can be done by exposing APCs to said antigenic peptides and / or immunogenic compounds in vitro (as described in Rizzo MM, Alaniz L, Mazzolini G. Ex vivo loading of autologous dendritic cells with tumor antigens. Methods Mol Biol. 2014;1139:41-4; Rolinski J, Hus I. Breaking immunotolerance of tumors: a new perspective for dendritic cell therapy. J Immunotoxicol. 2014 October;11(4):311-8).

[0115] A preferred APC according to the present invention is a dendritic cell (DC). It may be advantageous indeed to combine at least one antigenic peptide or immunogenic compound according to the present invention with DC, since they are the most abundant and potent APC and are reported to be frequently functionally defective in cancer patients. DC can be easily obtained by the skilled artisan, for example by direct isolation from peripheral blood or by derivation from peripheral blood cells such as CD14+ monocytes or CD34+ hematopoietic progenitor cells, either from a healthy compatible donor (i.e., DC are HLA-associated) or from the patient himself (i.e., DC are autologous), provided that they are functional (Figdor CG, de Vries IJ, Lesterhuis WJ, Melief CJ. Dendritic cell immunotherapy: mapping the way. Nat Med. 2004 May;10(5):475-80). DCs can indeed be distinguished from other cells of peripheral blood by their surface markers, such as S100, p55, CD83, and / or OX62, and therefore can be isolated and purified based on said markers using cell culture techniques well known in the art.

[0116] The present invention further relates to a method for preparing a cell loaded with an antigenic peptide according to the invention, comprising the step of contacting a cell, in particular an antigen-presenting cell, with (a sufficient amount of) an antigenic peptide, wherein the antigenic peptide is loaded onto class I or II MHC molecules expressed on the surface of the cell, in particular the antigen-presenting cell (or an artificial antigen-presenting cell).

[0117] Nucleic acid encoding an antigenic peptide and host cells containing the nucleic acid In a further aspect, the present invention also provides a nucleic acid encoding an antigenic peptide according to the present invention, a polypeptide of formula (I) as defined above, or an immunogenic compound according to the present invention, wherein the immunogenic compound is a peptide or a protein. In particular, the preferred embodiments of the antigenic peptide as described above also apply to such a nucleic acid according to the present invention. For example, an antigenic peptide according to the present invention comprising or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 40 to 42 is even more preferred. For example, an antigenic peptide according to the present invention comprising or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 1, 2, 3, 7, 11, 16 is even more preferred. For example, an antigenic peptide according to the present invention comprising or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 3 is even more preferred. Combinations thereof, i.e. nucleic acids encoding separate antigenic peptides according to the present invention are also preferred.

[0118] The nucleic acid preferably comprises a single-stranded, double-stranded or partially double-stranded nucleic acid, preferably selected from DNA, cDNA, PNA, RNA, or a combination thereof. Non-limiting examples of nucleic acids include gDNA, cDNA, RNA, antisense DNA, antisense RNA, complementary RNA / DNA sequences with or without expression elements, minigenes, gene fragments, control elements, promoters, and combinations thereof. Further examples of nucleic acids include recombinant polynucleotides, vectors, oligonucleotides, RNA molecules such as rRNA, mRNA, or tRNA, or DNA molecules, as described above. Thus, it is preferred that the nucleic acid (molecule) is a DNA molecule or an RNA molecule, preferably selected from gDNA; cDNA; rRNA; mRNA; antisense DNA; antisense RNA; 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.

[0119] The ability to deliver nucleic acids, such as ribonucleic acid (RNA), into cells, whether in vitro, in vivo, in situ, or ex vivo, such as to cause intracellular translation of the nucleic acid and production of the encoded peptide of interest, is of great interest in the fields of therapeutics, diagnostics, reagents, and for biological assays. Of particular importance is the delivery and function of non-integrated polynucleotides. Thus, nucleic acids that do not integrate into the host chromosome, such as mRNA, are preferred. In general, nucleic acids, such as mRNA, can be optimized for expression of the antigenic peptide of the invention, for example by methods known in the art, such as codon optimization. In addition, the nucleic acid can be modified, for example, to enhance its stability, extend its lifespan, and / or increase expression of the antigenic peptide of the invention. Thus, optimized or modified mRNA (mmRNA) encoding the antigenic peptide according to the invention is preferred. mmRNAs (e.g., as described in WO2013 / 151672A2, WO2013 / 101690A1, WO2013 / 052523A, which are incorporated herein by reference) are distinguished from wild-type mRNAs in their functional and / or structural design features for optimal delivery of mRNA and / or optimal expression of the antigenic peptides of the invention. In general, the nucleic acid can be delivered "naked" or associated with a carrier, e.g., a cationic carrier. Cationic carriers (positively charged) typically readily associate with negatively charged nucleic acids. The carrier can be of any type, including, for example, polymers, proteins, lipids, and nanoparticles. Cationic lipids and nanoparticles (particularly lipid nanoparticles, LNPs) are preferred for nucleic acid delivery. Thus, the present invention also provides a nucleic acid as described herein associated with a carrier (e.g., a lipid, particularly a cationic lipid or LNPs).

[0120] In some embodiments, the nucleic acid molecule may be a vector. As used in the context of the present invention, the term "vector" refers to a nucleic acid molecule, preferably an artificial nucleic acid molecule, i.e. a nucleic acid molecule that does not exist in nature. A vector in the context of the present invention is suitable for incorporating or possessing a desired nucleic acid sequence. Such a vector may be a storage vector, an expression vector, a cloning vector, a transfer vector, etc. A storage vector is a vector that allows for convenient storage of a nucleic acid molecule. Thus, a vector may comprise, for example, a sequence corresponding to a desired antigenic peptide according to the present invention. An expression vector may be used for the production of an expression product, such as an RNA, e.g. an mRNA, or a peptide, polypeptide, or protein. For example, an expression vector may comprise a sequence required for transcription of a sequence stretch of the vector, such as a promoter sequence. A cloning vector is typically a vector that contains a cloning site that may be used to incorporate a nucleic acid sequence into the vector. A cloning vector may be, for example, a plasmid vector or a bacteriophage vector. A transfer vector may be a vector, such as a viral vector, that is suitable for transferring a nucleic acid molecule into a cell or organism. A vector in the context of the present invention may be, for example, an RNA vector or a DNA vector. Preferably, the vector is a DNA molecule. For example, a vector in the context of the present application comprises a cloning site, a selection marker such as an antibiotic resistance factor, and sequences suitable for the propagation of the vector, such as an origin of replication. Preferably, the vector in the context of the present application is a plasmid vector. Preferably, the vector in the context of the present application is an expression vector. An expression vector is typically capable of expressing a coding sequence, in particular an antigenic peptide according to the present invention, a polypeptide of formula (I) as defined above, or an immunogenic compound according to the present invention. A preferred vector is a vector for expression in bacterial cells. More preferably, the vector is useful for expression in so-called "bacterial live vaccine vectors", in which live bacterial cells (bacteria or bacterial spores, such as endospores, exospores, or microbial cysts) can serve as a vaccine.A preferred example is described in da Silva et al., Live bacterial vaccine vectors: an overview; Braz J Microbiol. 2015 Mar. 4;45(4):1117-29.

[0121] Nucleic acids encoding antigenic peptides according to the invention may take the form of naked nucleic acid or nucleic acid cloned into a plasmid or viral vector (Tregoning and Kinnear, Using Plasmids as DNA Vaccines for Infectious Diseases. Microbiol Spectr. 2014 Dec;2(6). doi: 10.1128 / microbiolspec.PLAS-0028-2014), the latter being particularly preferred. Examples of suitable viral vectors according to the invention include, but are not limited to, retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, and pox viral vectors. It is within the skill of the art to clone a nucleic acid into a plasmid or viral vector using standard recombinant techniques in the art.

[0122] In a further aspect, the present invention also provides a host cell comprising a nucleic acid according to the invention, or in particular an expression vector as described above. Also preferred are host cells comprising combinations thereof, i.e. separate nucleic acids according to the invention, e.g. encoding separate antigenic peptides according to the invention.

[0123] Preferably, the nucleic acid contained in the host cell is preferably a vector. Preferably, the host cell is a bacterial cell. Such a host cell may preferably be used for the production of an antigenic peptide according to the invention or an immunogenic compound according to the invention. Furthermore, such a host cell may also be an active ingredient in a vaccine. Preferably, the host cell is a bacterial cell, more preferably an enterobacterial cell. The term "enterobacterial cell" refers to a bacterium living in the (human) intestine. Such a bacterial host cell may serve as a "live bacterial vaccine vector" in which live bacterial cells (e.g. bacteria or bacterial spores, e.g. endospores, exospores, or microbial cysts) may serve as a vaccine. A preferred example thereof is described in da Silva et al., Live bacterial vaccine vectors: an overview; Braz J Microbiol. 2015 Mar. 4; 45(4): 1117-29. Bacterial cells (e.g. bacteria or bacterial spores, e.g. endospores, exospores or microbial cysts), especially (whole) Enterobacteriaceae species, may be advantageous, since they have the potential to trigger a stronger immune response than the (poly)peptides or nucleic acids they contain. Alternatively, the bacterial cells according to the invention, especially Enterobacteriaceae, may take the form of probiotics, i.e. live Enterobacteriaceae, which can therefore be used as food additives due to the health benefits they may confer. They can be freeze-dried, for example, as granules, pills or capsules, or can be mixed directly with dairy products for consumption.

[0124] In some embodiments, the host cell may be an antigen-presenting cell or a dendritic cell, in particular as described above. In some embodiments, the antigen-presenting cell comprises an expression vector according to the invention as described above, in particular an expression vector capable of expressing said peptide comprising any one of SEQ ID NO:1 to SEQ ID NO:16 and SEQ ID NO:40 to 42, or a variant amino acid sequence.

[0125] The present invention further relates to a method for producing a peptide according to the invention, said method comprising the steps of culturing a host cell according to the invention and isolating the peptide from said host cell or its culture medium.

[0126] Nanoparticles containing antigenic peptides or immunogenic compounds In a further aspect, the present invention also provides nanoparticles comprising, in particular nanoparticles loaded with, optionally together with an adjuvant: at least one antigenic peptide according to the invention, or at least one immunogenic compound according to the invention.

[0127] In particular, the preferred embodiments of the antigenic peptides as described above also apply to such nanoparticles according to the invention.

[0128] Nanoparticles, particularly for use as vaccines, are known in the art and are described, for example, in Shao et al., Nanoparticle-based immunotherapy for cancer, ACS Nano 2015, 9(1):16-30; Zhao et al., Nanoparticle vaccines, Vaccine 2014, 32(3):327-37; and Gregory et al., Vaccine delivery using nanoparticles, Front Cell Infect Microbiol. 2013, 3:13, doi: 10.3389 / fcimb.2013.00013. eCollection 2013, Review. In particular, nanoparticles are used for the delivery of antigenic peptides (or immunogenic compounds / polypeptides / proteins / nucleic acids containing antigenic peptides) and may optionally also act as adjuvants. Antigenic peptides (immunogenic compounds / polypeptides / proteins / nucleic acids containing antigenic peptides) are typically either encapsulated within nanoparticles or linked / bound to (decorated on) the surface of nanoparticles ("coating"). Compared to conventional approaches, nanoparticles can protect payloads (antigens / adjuvants) from the surrounding biological environment, increase half-life, minimize systemic toxicity, facilitate delivery to APCs, or even directly trigger activation of TAA-specific T cells. Preferably, nanoparticles have a size (diameter) of 300 nm or less, more preferably 200 nm or less, and most preferably 100 nm or less. Such nanoparticles are adequately protected from phagocytic cell uptake, with high structural integrity in circulation and long circulation times, capable of accumulating at sites of tumor growth, and can penetrate deep into the tumor mass.

[0129] Examples of nanoparticles include polymeric nanoparticles such as poly(ethylene glycol) (PEG) and poly(D,L-lactic-coglycolic acid) (PLGA); inorganic nanoparticles such as gold nanoparticles, iron oxide beads, iron oxide zinc oxide nanoparticles, carbon nanotubes, and mesoporous silica nanoparticles; liposomes such as cationic liposomes; immunostimulating complexes (ISCOMs); virus-like particles (VLPs); and self-assembling proteins.

[0130] Polymeric nanoparticles are nanoparticles based on / containing polymers, such as poly(D,L-lactide-co-glycolide) (PLG), poly(D,L-lactic acid-glycolic acid) (PLGA), poly(γ-glutamic acid) (γ-PGA), poly(ethylene glycol) (PEG), and polystyrene. Polymeric nanoparticles can encapsulate or bind / conjugate with antigens (e.g., antigenic peptides, or (poly)peptides containing same). Polymeric nanoparticles can be used, for example, for delivery to certain cells, or sustain antigen release thanks to their slow biodegradation rate. For example, g-PGA nanoparticles can be used to encapsulate hydrophobic antigens. Polystyrene nanoparticles can be conjugated with various antigens, as they can be surface modified with various functional groups. Polymers such as poly(L-lactic acid) (PLA), PLGA, PEG, and natural polymers such as polysaccharides can also be used to synthesize hydrogel nanoparticles, which are a type of nano-sized hydrophilic three-dimensional polymer network. Nanogels have favorable properties including flexible mesh size, large surface area for multivalent conjugation, high water content, and high antigen loading capacity. Thus, preferred nanoparticles are nanogels such as chitosan nanogels. Preferred polymer nanoparticles are nanoparticles based on / including PEG and PLGA.

[0131] Inorganic nanoparticles are nanoparticles based on / comprising inorganic materials, examples of such nanoparticles include gold nanoparticles, iron oxide beads, iron oxide zinc oxide nanoparticles, carbon nanoparticles (e.g., carbon nanotubes), and mesoporous silica nanoparticles. Inorganic nanoparticles offer robust structure and controllable synthesis. For example, gold nanoparticles can be easily produced in various shapes such as spheres, rods, cubes, etc. Inorganic nanoparticles can be surface modified, for example with carbohydrates. Carbon nanoparticles offer good biocompatibility and can be produced, for example, as nanotubes or (mesoporous) spheres. For example, multiple copies of an antigenic peptide (or (poly)peptide comprising it) according to the invention can be conjugated onto carbon nanoparticles, for example carbon nanotubes. Mesoporous carbon nanoparticles are preferred for oral administration. Silica-based nanoparticles (SiNPs) are also preferred. SiNPs are biocompatible and show excellent properties in selective tumor targeting and vaccine delivery. The abundant silanol groups on the surface of SiNPs can be used for further modification to introduce additional functionalities such as cell recognition, absorption of specific biomolecules, improved interaction with cells, and enhanced cellular uptake. Mesoporous silica nanoparticles are particularly preferred.

[0132] Liposomes are typically formed from phospholipids such as 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP). In general, cationic liposomes are preferred. Liposomes are self-assembling and have a phospholipid bilayer shell and an aqueous core. Liposomes can be produced as unilamellar vesicles (with a single phospholipid bilayer) or multilamellar vesicles (with several concentric phospholipid shells separated by layers of water). Thus, antigens can be encapsulated within the core or between the different layers / shells. Preferred liposome systems are those approved for human use, such as Inflexal® V and Epaxal®.

[0133] Immunostimulating complexes (ISCOMs) are cage-like particles of about 40 nm (diameter), e.g., colloidal saponin-containing micelles made of the saponin adjuvant Quil-A, cholesterol, phospholipids, and (poly)peptide antigens (e.g., antigenic peptides or polypeptides containing same). These spherical particles can entrap antigens through non-polar interactions. Two types of ISCOMs have been described, both of which consist of cholesterol, phospholipids (typically either phosphatidylethanolamine or phosphatidylcholine), and saponin (such as Quil-A).

[0134] Virus-like particles (VLPs) are self-assembling nanoparticles formed by the self-assembly of biocompatible capsid proteins. Due to the naturally optimized nanoparticle size and repetitive structural regularity, VLPs can induce strong immune responses. VLPs can be derived from various viruses and have sizes ranging from 20 nm to 800 nm, typically ranging from 20-150 nm. VLPs can be engineered to express additional peptides or proteins, either by fusing these peptides / proteins with the particle or by expressing multiple antigens. Furthermore, antigens can be chemically linked to the viral surface to create bioconjugated VLPs.

[0135] Examples of self-assembling proteins include ferritin and major vault protein (MVP). Ferritin is a protein that can self-assemble into a roughly spherical 10 nm structure. 96 units of MVP can self-assemble into a barrel-shaped vault nanoparticle with a size of approximately 40 nm wide and 70 nm long. An antigen genetically fused with a minimal interaction domain can be packaged into the interior of the vault nanoparticle by a self-assembly process when mixed with MVP. Thus, an antigen (e.g., an antigenic peptide according to the present invention, or a polypeptide comprising the same) can be fused to a self-assembling protein or a fragment / domain thereof, such as the minimal interaction domain of MVP. Thus, the present invention also provides a fusion protein comprising a self-assembling protein (or a fragment / domain thereof) and an antigenic peptide according to the present invention.

[0136] In general, preferred examples of nanoparticles (NPs) include iron oxide beads, polystyrene microspheres, poly(γ-glutamic acid) (γ-PGA) NPs, iron oxide zinc oxide NPs, cationized gelatin NPs, Pluronic® stabilized poly(propylene sulfide) (PPS) NPs, PLGA NPs, (cationic) liposomes, (pH-responsive) polymeric micelles, PLGA, cancer cell membrane-coated PLGA, lipid-calcium phosphate (LCP) NPs, liposome-protamine-hyaluronic acid (LPH) NPs, polystyrene latex beads, magnetic beads, iron dextran particles, and quantum dot nanocrystals.

[0137] Preferably, the nanoparticles further comprise an adjuvant, such as a Toll-like receptor (TLR) agonist. Thereby, the antigenic peptide (immunogenic compound / polypeptide / protein / nucleic acid containing the antigenic peptide) can be delivered together with the adjuvant to an antigen-presenting cell (APC), such as a dendritic cell (DC). The adjuvant, like the antigenic peptide, can be preferably encapsulated by the nanoparticles or bound / conjugated to the surface of the nanoparticles.

[0138] A particularly preferred adjuvant is polyinosinic:polycytidylic acid (also called "poly I:C") and / or its derivative poly ICLC. Poly I:C is a mismatched double-stranded RNA in which one strand is a polymer of inosinic acid and the other is a polymer of cytidylic acid. Poly I:C is an immune stimulant known to interact with Toll-like receptor 3 (TLR3). Poly I:C is structurally similar to double-stranded RNA, the "natural" stimulant of TLR3. Poly I:C can therefore be considered a synthetic analogue of double-stranded RNA. Poly ICLC is a synthetic complex of carboxymethylcellulose, polyinosinic-polycytidylic acid double-stranded RNA, and poly-L-lysine. Like poly I:C, poly ICLC is also a ligand for TLR3. Poly I:C and poly ICLC typically stimulate the release of cytotoxic cytokines. A preferred example of poly ICLC is Hiltonol®.

[0139] Antibodies that bind to antigenic peptides In a further aspect, the present invention provides an antibody directed against (i.e. which binds to) an antigenic peptide according to the invention or to a complex of said peptide with MHC according to the invention.

[0140] As used herein, the term "antibody" encompasses various forms of antibodies, including, but not limited to, whole antibodies, antibody fragments (e.g., antigen-binding fragments), human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies, and genetically engineered antibodies (variants or mutant antibodies), so long as the characteristic properties according to the invention (i.e., binding to an antigenic peptide) are retained. In some embodiments, the antibody is a mammalian antibody, such as a mouse, rat, rabbit, goat, sheep, or human antibody. In some embodiments, the antibody is a monoclonal antibody. An antibody usually comprises (at least) three complementarity determining regions (CDRs) on the heavy chain and (at least) three CDRs on the light chain. In general, the complementarity determining regions (CDRs) are hypervariable regions present in the heavy chain variable domain and the light chain variable domain. Typically, the CDRs of an antibody's heavy chain and its associated light chain together form an antigen receptor. Usually, the three CDRs (CDR1, CDR2, and CDR3) are arranged non-contiguously within the variable domain. Since an antigen receptor is typically composed of two variable domains (on two different polypeptide chains, i.e., heavy and light chains; heavy chain variable region (VH) and light chain variable region (VL)), there are typically six CDRs for each antigen receptor (heavy chain: CDRH1, CDRH2, and CDRH3; light chain: CDRL1, CDRL2, and CDRL3). For example, a classical IgG antibody molecule usually has two antigen receptors and therefore contains 12 CDRs. The CDRs on the heavy and / or light chain may be separated by framework regions, framework regions (FR) being regions within the variable domains that are less "variable" than the CDRs. For example, a variable region (or each variable region, respectively) may be composed of four framework regions separated by three CDRs. In addition, an antibody may contain one or more constant regions. In some embodiments, an antibody comprises a FC region.

[0141] Antibodies can be obtained, for example, by immunizing a mammal (e.g., a mouse, a rat, a rabbit, a goat, a sheep, or a human) with an antigenic peptide of the invention (or an immunogenic compound or nanoparticle of the invention). Human antibodies can be isolated from a human (isolated) sample (e.g., a blood sample).

[0142] Therefore, the present invention also relates to a method of immunizing a non-human animal with an antigenic peptide (or an immunogenic compound or nanoparticle) of the present invention, comprising the steps of: - contacting (immunizing) a non-human animal with an antigenic peptide (or immunogenic compound or nanoparticle) of the invention, preferably an antigenic peptide according to any one of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 40 to 42 (or an immunogenic compound or nanoparticle comprising such an antigenic peptide).

[0143] As used herein, "immunizing" is understood to be of a non-therapeutic type, since it concerns the production of antibodies in said non-human animal.

[0144] Non-human animals are typically suitable for antibody production. Preferably, the non-human animal is a non-human mammal, more preferably an animal selected from goats, and rodents such as mice, rats, and rabbits.

[0145] The present invention also relates to a method for producing (polyclonal) antibodies that recognize an antigenic peptide of the present invention, comprising the steps of: - isolating (polyclonal) antibodies which recognize an antigenic peptide of the invention (or an immunogenic compound or nanoparticle), preferably from a non-human animal which has been in contact with (immunized with) an antigenic peptide according to any one of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 40 to 42 (or an immunogenic compound or nanoparticle comprising such an antigenic peptide).

[0146] The present invention also relates to a method for isolating cells that produce antibodies that recognize an antigenic peptide according to the present invention, comprising the steps of: - isolating cells producing said antibodies which recognize said antigenic peptide from a non-human animal which has previously been in contact with (immunized with) a JNK inhibitor of the invention, preferably an antigenic peptide according to any one of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 40 to 42 (or an immunogenic compound or nanoparticle comprising such an antigenic peptide), and optionally immortalizing said cells.

[0147] The present invention also relates to a method for producing (monoclonal) antibodies recognizing an antigenic peptide according to the invention, comprising the steps of: - isolating an antibody which recognizes an antigenic peptide of the present invention, more preferably an antigenic peptide according to any one of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 40 to 42, from a cell culture supernatant of cells producing said antibody, and optionally immortalizing the cells.

[0148] The skilled artisan will understand that the method of immunizing a non-human animal and the method of producing (polyclonal) antibodies as disclosed herein may be performed sequentially. Similarly, the method of immunizing a non-human animal, the method of isolating antibody-producing cells, and the method of producing (monoclonal) antibodies may be combined.

[0149] In a further aspect, the invention relates to an antibody producible (and / or produced) by the method according to the invention for producing polyclonal or monoclonal antibodies, which antibody recognises at least one antigenic peptide of the invention, preferably an antigenic peptide according to any one of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 40 to 42. In some embodiments, said antibody does not recognise (bind) to a lesser extent the corresponding human peptide.

[0150] The present invention also relates to a cell isolated by the above-specified method for isolating a cell that produces an antibody that recognizes an antigenic peptide according to the present invention, wherein the cell produces an antibody that preferably recognizes an antigenic peptide of the present invention, preferably an antigenic peptide according to any one of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 40 to 42.

[0151] Methods for testing (monoclonal and / or polyclonal) antibodies for their binding affinity are well known in the art. One possibility among others is to characterize the binding affinity of the antibodies by ELISA, by using an antigenic peptide of the invention as target peptide.

[0152] T cell receptor binding to antigenic peptide The invention further relates to T cell receptors (TCRs), in particular soluble TCRs (sTCRs) and cloned TCRs, which can be engineered into autologous or allogeneic T cells and methods for making these, as well as NK cells or other cells which bear or cross-react with said TCRs.

[0153] As used herein, a "T cell receptor" (TCR) is a protein complex found on the surface of T cells (T lymphocytes) that is responsible for recognizing antigenic peptides bound to major histocompatibility complex (MHC) molecules.

[0154] To obtain T cells having a T cell receptor that binds to an antigenic peptide of the invention, for example an (isolated) sample (for example a blood sample) from a mammal, for example a human, can be screened for binding to an antigenic peptide of the invention, such as a peptide according to any one of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 40 to 42. The antigenic peptide can then be used as a target peptide.

[0155] It is a further aspect of the present invention to provide a method for producing soluble T cell receptors (sTCRs) that recognize specific peptide-MHC complexes. Such soluble T cell receptors can be generated from specific T cell clones and their affinity can be increased by targeted mutagenesis of complementarity determining regions. For T cell receptor selection, phage display can be used (US2010 / 0113300, Liddy N et al. Monoclonal TCR-redirected tumor cell killing. Nat Med 2012 June; 18(6):980-987). For stabilization of the T-cell receptor during phage display and in the case of practical use as a drug, the alpha and beta chains can be linked, for example, by non-natural disulfide bonds, other covalent bonds (single-chain T-cell receptors) or by dimerization domains (Boulter JM et al., Stable, soluble T-cell receptor molecules for crystallization and therapeutics. Protein Eng 2003 September; 16(9):707-711; Card KF et al., A soluble single-chain T-cell receptor IL-2 fusion protein retains MHC-restricted peptide specificity and IL-2 bioactivity. Cancer Immunol Immunother 2004 April; 53(4):345-357; and Willcox BE et al., Production of soluble alphabeta T-cell receptor heterodimers suitable for biophysical analysis of ligand binding. Protein Sci 1999 November; 8(1):111-112). (11):2418-2423). T cell receptors can be linked to toxins, drugs, cytokines (see, e.g., US 2013 / 0115191), domains that recruit effector cells, such as anti-CD3 domains, and the like, to carry out specific functions on target cells.Furthermore, it can be expressed in T cells used for adoptive transfer. Further information can be found in WO2004 / 033685A1 and WO2004 / 074322A1. sTCR combinations are described in WO2012 / 056407A1. Further methods for production are disclosed in WO2013 / 057586A1.

[0156] Pharmaceutical Compositions In a further aspect, the present invention also provides a pharmaceutical composition comprising at least one of the following: - an antigenic peptide according to the invention as described herein, - an immunogenic compound according to the invention as described herein, - the nanoparticles according to the invention as described herein, - a cell according to the invention as described herein, - a nucleic acid according to the invention as described herein, - a host cell according to the invention as described herein, - a T lymphocyte according to the invention as described herein, - an antibody according to the invention as described herein, and / or - a T cell receptor according to the invention as described herein, and, optionally, one or more pharma- ceutically acceptable excipients or carriers.

[0157] In particular, the preferred embodiments of the antigenic peptides as described above also apply to such pharmaceutical compositions according to the invention.

[0158] Also preferred are pharmaceutical compositions comprising combinations thereof, i.e., separate antigenic peptides according to the invention. For example, a pharmaceutical composition comprising: - at least two distinct antigenic peptides according to the invention as described herein, - at least two distinct immunogenic compounds according to the invention as described herein, - at least two distinct nanoparticles according to the invention as described herein, - at least two separate cells according to the invention as described herein, - at least two distinct nucleic acids according to the invention as described herein, - at least two separate host cells according to the invention as described herein, - at least two distinct T lymphocytes according to the invention as described herein, - at least two distinct antibodies according to the invention as described herein, and / or - at least two distinct T cell receptors according to the invention as described herein may include.

[0159] Thus, a pharmaceutical composition may comprise at least "two separate components" (of a pharmaceutical composition according to the invention), preferably three, four or five separate components. Generally, as used herein, the phrase "separate components" refers to: (1) a first component, such as an antigenic peptide according to the invention described herein, an immunogenic compound according to the invention described herein, a nanoparticle according to the invention described herein, a cell according to the invention described herein, a nucleic acid according to the invention described herein, a host cell according to the invention described herein, a T lymphocyte according to the invention described herein, an antibody according to the invention described herein, and / or a T cell receptor according to the invention described herein; and (2) at least one other component (different from the first component; whereas in the case of more than two separate components, each component is different from each other component), such as a separate antigenic peptide according to the invention described herein, a separate immunogenic compound according to the invention described herein, a separate nanoparticle according to the invention described herein, a separate cell according to the invention described herein, a separate nucleic acid according to the invention described herein, a separate host cell according to the invention described herein, a separate T lymphocyte according to the invention described herein, a separate antibody according to the invention described herein, and / or a separate T cell receptor according to the invention described herein; or one or more human tumor antigens (fragments thereof), in any form ("naked", as an immunogenic compound described herein, as a nanoparticle described herein, as a (host) cell described herein, or as a nucleic acid described herein). The "separate components" are preferably active components (as described above) in the context of the disease (cancer) to be prevented and / or treated. In other words, each of the separate components may also be useful in the prevention and / or treatment of said cancer, especially when administered individually (and not in combination as described herein), although the combination (i.e., co-administration) typically enhances their prophylactic and / or therapeutic effects (e.g., immune response), preferably in a synergistic manner.

[0160] Preferably, the "distinct components" are of the same type (e.g. distinct antigenic peptides, distinct immunogenic compounds, distinct nanoparticles, distinct nucleic acids, distinct (host) cells, distinct T cell receptors, distinct antibodies, or distinct T lymphocytes) and differ from each other only in that they relate to the distinct antigenic peptides of the invention as described herein.

[0161] Preferably, the composition may comprise at least three, four, or five separate active ingredients, which are preferably of the same type, but differ (only) in that each of them relates to a distinct antigenic peptide.

[0162] More preferably, the composition may comprise at least three, four, or five distinct active ingredients, which are preferably of the same type but differ (only) in that each of them relates to a distinct antigenic peptide; - the first component relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen CDC20; - the (separate) second component relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen KIF2C; - the (separate) third component relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen UBE2C.

[0163] Preferably, such a composition comprises: - a (separate) fourth component related to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen BIRC5; and / or - a (separate) fifth component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen FOXM1; It may further include.

[0164] Table 2 below provides a list of additional peptides that may be useful in combination with the peptides of the invention. SEQ ID NOs: 32-34 also refer to HLA-A*02 antigenic peptides.

[0165] [Table 2]

[0166] It is understood that these additional antigenic peptides for combination with the antigenic peptides of the invention may be provided in the same form, i.e., as antigenic peptides, immunogenic compounds, nanoparticles, peptide-loaded cells, nucleic acids, host cells, T lymphocytes, antibodies, or T cell receptors, as described herein for the antigenic peptides of the invention.

[0167] Thus, the pharmaceutical composition preferably comprises: - a first component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 17; - a (separate) second component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 18; - a (separate) third component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 19; - optionally a (separate) fourth component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 35; and - optionally a (separate) fifth component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 36; Includes.

[0168] More preferably, the pharmaceutical composition preferably comprises: - a first component related to an antigenic peptide comprising or consisting of the amino acid set forth in SEQ ID NO:1; - a (separate) second component related to an antigenic peptide comprising or consisting of the amino acid set forth in SEQ ID NO:2; - a (separate) third component related to an antigenic peptide comprising or consisting of the amino acid set forth in SEQ ID NO: 3; - optionally a (separate) fourth component related to an antigenic peptide comprising or consisting of the amino acid set forth in SEQ ID NO: 32; and - optionally a (separate) fifth component related to an antigenic peptide comprising or consisting of the amino acid set forth in SEQ ID NO: 33; Includes.

[0169] Even more preferably, the pharmaceutical composition preferably comprises - an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:1; - an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:2; - an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:3; - optionally an antigenic peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 32; and - optionally an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 33 Includes.

[0170] The pharmaceutical composition may also contain, instead of the preferred antigenic peptide combinations described above, the respective immunogenic compound combinations of the invention, the respective nanoparticle combinations of the invention, the respective nucleic acid combinations of the invention, etc., as described above.

[0171] Preferably, the pharmaceutical composition further comprises one or more pharma- ceutically acceptable excipients or carriers.

[0172] The pharmaceutical composition of the present invention may be in any form suitable for the purpose of the present invention.For example, the composition may be in a form suitable for parenteral, enteral, or topical administration, such as a liquid suspension, a solid dosage form (granules, pills, capsules, or tablets), or a paste or gel.It is within the skill of the art to select the appropriate form of the composition for the intended purpose.

[0173] The composition according to the invention may further comprise other active substances, which may for example enhance the effect of the antigenic peptide or immunogenic compound, or the composition may not comprise any other active substances (i.e. other than the antigenic peptide according to the invention, the immunogenic compound according to the invention, the nanoparticles according to the invention, the cells according to the invention, the nucleic acid according to the invention, and / or the host cells according to the invention).

[0174] The pharmaceutical composition as defined herein is preferably an immunogenic composition, i.e. a composition capable of inducing, increasing, prolonging or maintaining an immune response. This can be achieved by an antigenic peptide according to the invention or by an immunogenic compound according to the invention contained in said composition. Preferably, the pharmaceutical composition further comprises one or more immune adjuvant substances. Pharmaceutical compositions, in particular immunogenic compositions, may also be referred to herein as "vaccine compositions".

[0175] Preferably, the pharmaceutical composition further comprises at least one immunostimulant, in particular to increase, enhance, prolong or maintain the immune response mediated by the antigenic peptide. Preferred immunostimulants according to the present invention include, but are not limited to, immune adjuvants, antigen presenting cells, and combinations thereof. Preferably, the immune stimulant is an immune adjuvant or an antigen presenting cell (APC).

[0176] Preferably, the immune stimulant is an immune adjuvant. Some immune adjuvants are capable of aiding and prolonging the interaction between antigen and immune system, while others are capable of recruiting and activating cells of innate immunity to induce adaptive responses. Adjuvants belonging to the former category include, but are not limited to, mineral compounds such as alum, aluminum hydroxide, aluminum phosphate, hydroxyapatite, and oil emulsions such as paraffin oil, starch oil, Freund's complete / incomplete adjuvant (FCA / FIA), saponin (e.g., from Quillaja, soybean, and Polygala senega plants). Adjuvants in the latter category include, but are not limited to, immune stimulating complexes (ISCOMs); cytokines (e.g., GM-CSF; interleukins such as IL-1, IL-2, IL6, IL8, or IL12; tumor necrosis factors (TNFs) such as TNFα or TNFβ; interferons IFNs such as IFNα, IFNβ, IFNγ, or IFNδ); ligands for toll-like receptors (TLRs) such as imiquimod, resiquimod, or MPL; exosomes such as exosomes derived from dendritic cells (DCs) or tumor cells; heat shock proteins (HSPs such as gp96, hsp90, hsp70, calreticulin, hsp110, hsp170), pathogen-associated molecular patterns (PAMPs), bacterial products such as polysaccharides (PLSs) such as trehalose dimycolate (TDM), muramyl dipeptide (MDP), and polysaccharide-K.

[0177] More preferably, the immune adjuvant is a protein / peptide with immune adjuvant properties, such as stimulating CD4+ Th1 cells, as described herein ("helper" peptides). This can be a non-tumor antigen that recalls immune memory or provides non-specific help, or it can be a specific tumor-derived helper peptide, such as tetanus helper peptide, keyhole limpet hemocyanin peptide, or PADRE peptide. Another example is a specific tumor-derived helper peptide that can be presented by MHC II, in particular HLA-DR, HLA-DP, or HLA-DQ, such as a fragment of a shared overexpressed tumor antigen, e.g., HER2, NY-ESO-1, hTERT, or IL13RA2. In some embodiments, the immune adjuvant may be HHD-DR3 peptide or h-pAg T13L (Bhasin M, Singh H, Raghava GP (2003) MHCBN: a comprehensive database of MHC binding and non-binding peptides. Bioinformatics 19: 665-666). Preferably, the helper peptide is UCP2 peptide (SEQ ID NO: 39).

[0178] Preferably, the pharmaceutical composition comprises at least two distinct antigenic peptides according to the invention and a helper peptide, preferably the UCP2 peptide (SEQ ID NO: 39).

[0179] Preferably, the pharmaceutical composition comprises a first antigenic peptide according to the present invention comprising or consisting of a sequence variant of a fragment of human tumor antigen CDC20, a second antigenic peptide according to the present invention comprising or consisting of a sequence variant of a fragment of human tumor antigen KIF2C, a third antigenic peptide according to the present invention comprising or consisting of a sequence variant of a fragment of human tumor antigen UBE2C, a fourth antigenic peptide according to the present invention comprising or consisting of a sequence variant of a fragment of human tumor antigen BIRC5, a fifth antigenic peptide according to the present invention comprising or consisting of a sequence variant of a fragment of human tumor antigen FOXM1, and a helper peptide.

[0180] More preferably, the pharmaceutical composition comprises a first antigenic peptide comprising or consisting of a sequence variant of the CDC20 fragment (human reference peptide) "SLPDRILDA" (SEQ ID NO: 17), for example, an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1; a second antigenic peptide comprising or consisting of a sequence variant of the KIF2C fragment (human reference peptide) "AINPELLQL" (SEQ ID NO: 18), for example, an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2; a third antigenic peptide comprising or consisting of a sequence variant of the UBE2C fragment (human reference peptide) "ALYDVRTIL" (SEQ ID NO: 19). a fourth antigenic peptide comprising or consisting of a sequence variant of the BIRC5 fragment (human reference peptide) "LTLGEFLKL" (SEQ ID NO: 35), for example, an antigenic peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 32; a fifth antigenic peptide comprising or consisting of a sequence variant of the FOXM1 fragment (human reference peptide) "LMDLSTTPL" (SEQ ID NO: 36), for example, an antigenic peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 33; and a helper peptide, preferably the UCP2 peptide (SEQ ID NO: 39).

[0181] Even more preferably, the pharmaceutical composition comprises five distinct antigenic peptides, wherein the antigenic peptides comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 32, and 33. In addition, the pharmaceutical composition may further comprise the UCP2 peptide set forth in SEQ ID NO:39.

[0182] In some embodiments, the pharmaceutical composition does not comprise additional antigenic peptides (in addition to the antigenic peptides as described above).

[0183] In some embodiments, the pharmaceutical composition comprises (as an immune adjuvant) polyinosinic:polycytidylic acid (also called "poly I:C") and / or its derivative poly ICLC. Poly I:C is a mismatched double-stranded RNA, one strand of which is a polymer of inosinic acid and the other of cytidylic acid. Poly I:C is an immune stimulant known to interact with Toll-like receptor 3 (TLR3). Poly I:C is structurally similar to double-stranded RNA, the "natural" stimulant of TLR3. Poly I:C can therefore be considered a synthetic analogue of double-stranded RNA. Poly ICLC is a synthetic complex of carboxymethylcellulose, polyinosinic-polycytidylic acid double-stranded RNA, and poly-L-lysine. Like poly I:C, poly ICLC is also a ligand for TLR3. Poly I:C and poly ICLC typically stimulate the release of cytotoxic cytokines. A preferred example of Poly ICLC is Hiltonol®.

[0184] More preferably, the pharmaceutical composition comprises Montanide, such as Montanide ISA 51 VG and / or Montanide ISA 720 VG. These adjuvants, when mixed with a water-based antigenic vehicle, become stable water-in-oil emulsions. Montanide ISA 51 VG is based on a blend of mannide monooleate surfactant and mineral oil, whereas Montanide ISA 720 VG uses a non-mineral oil (Aucouturier J, Dupuis L, Deville S, Ascarateil S, Ganne V. Montanide ISA 720 and 51: a new generation of water in oil emulsions as adjuvants for human vaccines. Expert Rev Vaccines. 2002 Jun;1(1):111-8; Ascarateil S, Puget A, Koziol ME. Safety data of Montanide ISA 51 VG and Montanide ISA 720 VG, two adjuvants dedicated to human therapeutic vaccines. Journal for Immunotherapy of Cancer. 2015;3(Suppl 2):P428. doi:10.1186 / 2051-1426-3-S2-P428).

[0185] In some embodiments, the pharmaceutical composition may further comprise at least one anti-cancer therapeutic agent. In another aspect, the present invention relates to a combination of the pharmaceutical composition of the present invention and at least one anti-cancer therapeutic agent. The pharmaceutical composition may comprise an anti-cancer therapeutic agent (as a mixed preparation); or the pharmaceutical composition of the present invention and the anti-cancer therapeutic agent may be provided in separate manner, for example as a kit of parts. Thus, the pharmaceutical composition of the present invention and the anti-cancer therapeutic agent may be provided for simultaneous, separate or sequential administration. In other words, the present invention proposes the combination of the pharmaceutical composition of the present invention and at least one anti-cancer therapeutic agent for simultaneous, separate or sequential administration.

[0186] Thus, said therapeutic agent is preferably capable of preventing and / or treating the same type of cancer as the cancer for which the antigenic peptide according to the invention is used. Preferably, the anti-cancer therapeutic agent is selected from antibodies, CAR-T cells, tumor cell lysates, chemotherapeutic agents, radiotherapeutic agents, immune checkpoint modulators, and combinations thereof.

[0187] Antibodies are particularly advantageous in cancer therapy, because they can either bind to specific antigens on the surface of cancer cells, thereby directing therapy to tumors (i.e., they are referred to as tumor-targeting antibodies), or block immune checkpoints that are dysregulated in cancer (i.e., they are referred to herein as immune-regulating antibodies). The purpose of the latter type of antibody is to inhibit cancer immune resistance, which can be prominently observed against T cells that are specific for tumor antigens. Indeed, as is well known in the art, under normal physiological conditions, immune checkpoints are important for maintaining self-tolerance (i.e., preventing autoimmunity) and protect tissues from damage when the immune system is responding to pathogenic infection. However, in cancer, immune checkpoint expression, as an important mechanism of immune resistance, can be dysregulated.This resistance has been observed notably in melanoma, ovarian cancer, lung cancer, glioblastoma, breast cancer, and pancreatic cancer with respect to the PD-L1 checkpoint (Konishi et al., B7-H1 expression on non-small cell lung cancer cells and its relationship with tumor-infiltrating lymphocytes and their PD-1 expression. Clin Cancer Res. 2004 Aug. 1;10(15):5094-100; Ghebeh et al., The B7-H1 (PD-L1) T lymphocyte-inhibitory molecule is expressed in breast cancer patients with infiltrating ductal carcinoma: correlation with important high-risk prognostic factors. Neoplasia. 2006 Mar. 8(3):190-8; Hino et al., Tumor cell expression of programmed cell death-1 ligand 1 is a prognostic factor for malignant melanoma. Cancer. 2010 Apr. 1;116(7):1757-66). Other examples of immune checkpoints include, but are not limited to, PD-L2, PD-1, CD80, CD86, CTLA-4, B7H3, B7H4, PVR, TIGIT, GAL9, LAG-3, GITR, CD137, TIM3, VISTA, VISTA-R (Pico de Coana et al., Checkpoint blockade for cancer therapy: revitalizing a suppressed immune system. Trends Mol Med. 2015 Aug;21(8):482-91; Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012 Mar. 22;12(4):252-64).

[0188] Antibodies are usually used for these purposes either in the form of naked monoclonal antibodies (i.e., unconjugated) or conjugated to another molecule, which may be toxic to cells or radioactive.

[0189] Examples of well-known monoclonal tumor-targeting antibodies used in cancer immunotherapy include, but are not limited to, alemtuzumab (chronic lymphocytic leukemia), bevacizumab (colorectal cancer, glioblastoma multiforme, cervical cancer, lung cancer, renal cancer), brentuximab / vedotin (lymphoma), blinatumomab (acute lymphoblastic leukemia), catumaxomab (malignant ascites in EPCAM+ cancers), cetuximab (head and neck cancer), and gliomas with ... (cancerous mellitus, colorectal cancer), denosumab (breast, prostate, and bone cancer), gemtuzumab / ozogamicin (acute myeloid leukemia), ibritumomab / tiuxetan (non-Hodgkin's lymphoma), panitumumab (colorectal cancer), pertuzumab (breast cancer), obinutuzumab (chronic lymphocytic leukemia), ofatumumab (chronic lymphocytic leukemia), ipilimumab (melanoma), ramucirumab (gastric Examples of immunomodulatory antibodies include, but are not limited to, ipilimumab (melanoma), nivolumab (melanoma, lung cancer), and pembrolizumab (melanoma) (both of which block the PDCD1-dependent immune checkpoint), as well as MPDL3280A, MEDI4736, MEDI0680, and MSB0010718C (all of which block the PD-L1-dependent immune checkpoint) (Sharma and Allison, The Journal of Clinical Oncology, 2011, 113(1):1311-1323, 2012). Future of immune checkpoint therapy. Science. 2015 / 04 / 3;348(6230):pp.56-61).

[0190] Other antibodies for cancer immunotherapy are described in Buque et al., Trial Watch: Immunomodulatory monoclonal antibodies for oncological indications. Oncoimmunology. 2015 Mar 2;4(4):e1008814. eCollection April 2015; Redman et al., Mechanisms of action of therapeutic antibodies for cancer. Mol Immunol. 2015 Oct;67(2 Pt A):28-45; Simpson and Caballero, Monoclonal antibodies for the therapy of cancer MC Proc. 2014;8(Suppl 4):O6, as well as the Antibody Society website (list of therapeutic monoclonal antibodies approved or under consideration in the European Union or the United States, available at the web link http: / / www.antibodysociety.org / news / approved_mabs.php).

[0191] Adoptive cellular immunotherapy with chimeric antigen receptor (CAR) T cells has transformed the treatment landscape for B-cell non-Hodgkin's lymphoma (NHL), especially aggressive B-cell lymphoma. As an example, CD19-targeted CAR T cells represent the new standard of care for patients with DLBCL who are resistant to at least two lines of therapy. Two CAR T cell products, axicabtagene ciloleucel (axi-cel) (KTE-019) (YESCARTA™) and tisagenlecleucel (CTL019) (KYMRIAH™), have received U.S. Food and Drug Administration approval for the treatment of refractory DLBCL after two lines of therapy. A third product, lisocabtagene maraleucel (liso-cel) (JCAR017), is currently being evaluated in clinical trials. Other CAR T cells include CD20-CAR-T cells.

[0192] Tumor cell lysates may also be combined with the antigenic peptides according to the invention. Tumor cells are in fact capable of priming an immune response by presenting endogenous peptide-MHC complexes in addition to the host's dendritic cells (DCs) capable of processing and presenting the antigens delivered by said lysates. Thereby, the range of antigens against which an immune response can be induced is increased. Tumor cell lysates can be easily obtained by treating tumor cells with heat shock and / or chemical treatment and may be autologous (i.e. isolated from the patient) or allogeneic (i.e. isolated from another subject).

[0193] Standard chemotherapy and radiotherapy agents need not be described further herein, as they have been extensively described in the literature, in particular by Baskar et al. (Baskar et al., Cancer and radiation therapy: current advances and future directions. Int J Med Sci. 2012;9(3):193-9), Paci et al. (Paci et al., Review of therapeutic drug monitoring of anticancer drugs part 1--cytotoxics. Eur J Cancer. 2014 August;50(12):2010-9), and Widmer et al. (Widmer et al., Review of therapeutic drug monitoring of anticancer drugs part two--targeted therapies. Eur J Cancer. 2014 August;50(12):2020-36). A list of such drugs and agents is also available on the cancer.gov website (http: / / www.cancer.gov / about-cancer / treatment / drugs).

[0194] Preferably, the immune checkpoint modulating agent for combination with the antigenic peptides defined herein is an activator or inhibitor of one or more checkpoint molecules selected from CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CD40, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, CEACAM1, GARP, PS, CSF1R, CD94 / NKG2A, TDO, GITR, TNFR, and / or FasR / DcR3; or an activator or inhibitor of one or more ligands thereof.

[0195] More preferably, the immune checkpoint modulator is an activator of a (co)stimulatory checkpoint molecule or an inhibitor of an inhibitory checkpoint molecule, or a combination thereof. Thus, the immune checkpoint modulator is more preferably (i) an activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR, and / or ICOS, or (ii) an inhibitor of A2AR, B7-H3, B7-H4, BTLA, CD40, CTLA-4, IDO, KIR, LAG3, PD-1, PDL-1, PD-L2, TIM-3, VISTA, CEACAM1, GARP, PS, CSF1R, CD94 / NKG2A, TDO, TNFR, and / or FasR / DcR3.

[0196] Even more preferably, the immune checkpoint modulator is an inhibitor of an inhibitory checkpoint molecule (but preferably not an inhibitor of a stimulatory checkpoint molecule).Thus, the immune checkpoint modulator is even more preferably an inhibitor of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, PDL-1, PD-L2, TIM-3, VISTA, CEACAM1, GARP, PS, CSF1R, CD94 / NKG2A, TDO, TNFR, and / or DcR3, or a ligand thereof.

[0197] Preferably, checkpoint modulators for combination with the antigenic peptides defined herein may be selected from known modulators of the CTLA-4 or PD-1 pathways. More preferably, the immune checkpoint modulator is an inhibitor of CTLA-4, PD-L1, PD-L2, or PD-1; even more preferably, an inhibitor of the PD-1 pathway.

[0198] It is within the skill of the art to select an appropriate immunological anti-cancer therapeutic agent for the purpose of the present invention. For example, if one wishes to prevent or treat melanoma, one can preferably use lysates from melanoma cells and / or the antibody ipilimumab, together with an appropriate antigenic peptide. An appropriate antigenic peptide can be selected by (i) selecting an appropriate tumor antigen for a particular type of cancer, as known in the art, and (ii) selecting an appropriate antigenic peptide according to the present invention for that selected tumor antigen, as described above, for example in Table 1.

[0199] Anti-cancer therapeutic agents can also be administered in combination with the compositions of the present invention, either simultaneously, separately or sequentially. If the composition and the therapeutic agent are administered in a separate or sequential manner, they may be administered in different pharmaceutical forms.

[0200] Therefore, in another aspect, the present invention relates to a composition of the present invention and at least one anti-cancer therapeutic agent as described above as a combined preparation for simultaneous, separate or sequential administration. In other words, the present invention proposes the combined use of a composition of the present invention and at least one anti-cancer therapeutic agent as described above for simultaneous, separate or sequential administration.

[0201] Kit of parts In a further aspect, the present invention also provides a kit-of-parts (also referred to herein as a "kit") comprising at least one of the following: - an antigenic peptide according to the invention as described herein, - an immunogenic compound according to the invention as described herein, - the nanoparticles according to the invention as described herein, - a cell according to the invention as described herein, - a nucleic acid according to the invention as described herein, - a host cell according to the invention as described herein, - a T lymphocyte according to the invention as described herein, an antibody according to the invention as described herein, - a T cell receptor according to the invention as described herein, and / or - a pharmaceutical composition according to the invention as described herein.

[0202] In particular, the preferred embodiments of the antigenic peptides as described above also apply to such kits according to the invention.

[0203] Also preferred are combinations thereof, i.e. kits comprising separate antigenic peptides according to the invention. In particular, a kit-of-parts according to the invention may comprise more than one of the above components, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 separate components. For example, a kit-of-parts according to the invention may comprise at least 2 (for example 2, 3, 4, 5, 6, 7, 8, 9 or 10) different immunogenic compounds, at least 2 (for example 2, 3, 4, 5, 6, 7, 8, 9 or 10) different antigenic peptides, at least 2 (for example 2, 3, 4, 5, 6, 7, 8, 9 or 10) different nanoparticles, at least 2 (for example 2, 3, 4, 5, 6, 7, 8, 9 or 10) different peptide ... The kit may comprise at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) different cells, at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) different nucleic acids, at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) different host cells, at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) different pharmaceutical compositions, and the like. Preferably, such different components comprised in the kit-of-parts as described above differ in terms of antigenic peptides according to the invention, e.g., one component related to a first antigenic peptide and one component related to a second antigenic peptide (different from the first antigenic peptide). For example, the kit may comprise at least two separate immunogenic compounds according to the invention. For example, the kit may comprise at least two distinct antigenic peptides according to the present invention.For example, the kit may comprise at least two distinct nanoparticles according to the present invention.For example, the kit may comprise at least two distinct nucleic acids according to the present invention.For example, the kit may comprise at least two distinct cytotoxic T lymphocytes according to the present invention.

[0204] A preferred combination of components, such as an antigenic peptide, according to the present invention, contained in the kit corresponds to a preferred combination of components, such as an antigenic peptide, according to the present invention, contained in the above-mentioned pharmaceutical composition.

[0205] Thus, the present invention provides a kit comprising at least two, preferably three, more preferably four or five distinct antigenic peptides as described above (or immunogenic compounds, nanoparticles, nucleic acids, cells, etc. as described above that differ with respect to the antigenic peptides), and optionally a helper peptide, such as the UCP2 peptide of SEQ ID NO: 39 and / or an adjuvant, such as MONTANIDE ISA 51.

[0206] Preferably, the kit comprises separate antigenic peptides (in peptide form or in any other form as described above), including: (i) at least one antigenic peptide of the invention, e.g., as shown in Table 1; and (ii) at least one additional antigenic peptide as described above, e.g., as shown in Table 2 above.

[0207] In some embodiments, the kit comprises: - a first component relating to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen CDC20; - a (separate) second component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen KIF2C; - a (separate) third component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen UBE2C; - optionally a (separate) fourth component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen BIRC5; and - optionally a (separate) fifth component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen FOXM1. Includes.

[0208] Preferably, the kit comprises: - a first component related to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 17; - a (separate) second component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 18; - a (separate) third component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 19; - optionally a (separate) fourth component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 35; and - optionally a (separate) fifth component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 36; Includes.

[0209] More preferably, the kit comprises: - a first component related to an antigenic peptide comprising or consisting of the amino acid set forth in SEQ ID NO:1; - a (separate) second component related to an antigenic peptide comprising or consisting of the amino acid set forth in SEQ ID NO:2; - a (separate) third component related to an antigenic peptide comprising or consisting of the amino acid set forth in SEQ ID NO: 3; - optionally a (separate) fourth component related to an antigenic peptide comprising or consisting of the amino acid set forth in SEQ ID NO: 32; and - optionally a (separate) fifth component related to an antigenic peptide comprising or consisting of the amino acid set forth in SEQ ID NO: 33; Includes.

[0210] The kits may also contain, instead of the preferred antigenic peptide combinations described above, respective combinations of immunogenic compounds of the invention, respective combinations of nanoparticles of the invention, respective combinations of nucleic acids of the invention, etc., as described above.

[0211] Preferably, the kit further comprises a UCP2 helper peptide of SEQ ID NO:39.

[0212] In some embodiments, in addition to any of the components as described above, the kit further comprises an anti-cancer therapeutic agent as described above.

[0213] Thus, said therapeutic agent is preferably capable of preventing and / or treating the same type of cancer as the cancer for which the antigenic peptide according to the invention is used. Preferably, the anti-cancer therapeutic agent is selected from an antibody as described above, a CAR-T cell as described above, a tumor cell lysate as described above, a chemotherapeutic agent as described above, a radiotherapeutic agent as described above, an immune checkpoint modulator as described above, and combinations thereof.

[0214] The various components of the kit-of-parts may be packaged in one or more containers. The above components may be provided in lyophilized or dried form or dissolved in a suitable buffer. The kit may also include additional reagents, such as preservatives, growth media, and / or buffers for storage and / or reconstitution of the above-mentioned components, washing solutions, and the like.

[0215] The separate antigenic peptides (or immunogenic compounds as described above, nanoparticles, nucleic acids, cells, etc., which differ with respect to the antigenic peptides) may be contained in the same or separate containers. For example, the kit may contain a (single) container containing a first antigenic peptide as described herein and a second antigenic peptide as described herein. The (single) container may additionally also contain a helper peptide, such as UCP2. Optionally, the first and second antigenic peptides (and optionally the helper peptide) contained in a (single) container may be formulated together, for example, in water for injection and / or dimethyl sulfoxide (DMSO). Additionally, the kit may contain a further container (different from the container containing the antigenic peptides) containing an adjuvant, for example MONTANIDE ISA 51.

[0216] Optionally, the kit may also include a vial of water for injection and / or a vial adapter. Sterile needles may also be included, for example for vaccinating a patient after obtaining the emulsion. The kit may also include one or more syringes.

[0217] In addition, the kit of parts according to the present invention may optionally contain instructions for use. Thus, it is preferred that the kit comprises a package insert or instructions for use that include instructions for preventing or treating cancer by using the immunogenic compound according to the present invention, the antigenic peptide according to the present invention, the nanoparticle according to the present invention, the cell according to the present invention, the nucleic acid according to the present invention, the host cell according to the present invention, the pharmaceutical composition according to the present invention, etc., as described above.

[0218] Furthermore, the present invention also provides a vaccination kit for treating, preventing and / or stabilizing cancer comprising a pharmaceutical composition as described herein or a vaccine as described herein and instructions for the use of said pharmaceutical composition or said vaccine in the prevention and / or treatment of cancer.

[0219] Medical Treatment and Uses As mentioned above, the antigenic peptides of the invention (in their many different forms as described above) may be particularly useful for prophylactic or therapeutic purposes (as pharmaceuticals), especially for triggering a specific immune response to a particular tumor antigen / protein, e.g., for the prevention or treatment of cancer, e.g., in a patient in need thereof.

[0220] With that in mind, the present invention provides: For use in medicine, in particular in the prevention and / or treatment of proliferative diseases, preferably in the prevention and / or treatment of cancer. - an antigenic peptide according to the invention as described herein, - an immunogenic compound according to the invention as described herein, - the nanoparticles according to the invention as described herein, - a cell according to the invention as described herein, - a nucleic acid according to the invention as described herein, - a host cell according to the invention as described herein, - a T lymphocyte according to the invention as described herein, - an antibody according to the invention as described herein, and / or - a T cell receptor according to the invention as described herein, a pharmaceutical composition according to the invention as described herein, or - A kit according to the invention as described herein.

[0221] Furthermore, the present invention also provides a method for preventing (reducing the incidence of) and / or treating cancer, or initiating, enhancing or prolonging an anti-tumor response in a subject (in need thereof), comprising administering to the subject (an effective amount of): - an antigenic peptide according to the invention as described herein, - an immunogenic compound according to the invention as described herein, - the nanoparticles according to the invention as described herein, - a cell according to the invention as described herein, - a nucleic acid according to the invention as described herein, - a host cell according to the invention as described herein, - a T lymphocyte according to the invention as described herein, - an antibody according to the invention as described herein, and / or - a T cell receptor according to the invention as described herein, a pharmaceutical composition according to the invention as described herein, or - A kit according to the invention as described herein.

[0222] In particular, preferred embodiments of the antigenic peptides as described above also apply to the use according to the invention in the prevention and / or treatment of cancer, non-limiting examples of which include colorectal cancer, lung cancer, prostate cancer, and / or breast cancer.

[0223] Furthermore, the present invention relates to C8 + Provided is a method for inducing or enhancing a cytotoxic T cell dependent immune response in a subject against one or more epitopes, said method comprising administering to said subject any one of the following: - an antigenic peptide according to the invention as described herein, - an immunogenic compound according to the invention as described herein, - the nanoparticles according to the invention as described herein, - a cell according to the invention as described herein, - a nucleic acid according to the invention as described herein, - a host cell according to the invention as described herein, - a T lymphocyte according to the invention as described herein, - an antibody according to the invention as described herein, and / or - a T cell receptor according to the invention as described herein, a pharmaceutical composition according to the invention as described herein, or - A kit according to the invention as described herein.

[0224] CD8 + The immune response dependent response can be determined by evaluating the inflammatory response, pro-inflammatory cytokine response, including an increase in the expression of one or more of IFN-γ, TNF-α, and IL-2 mRNA or protein compared to the levels before administration of the compound of the present invention. It can also be measured by an increase in the frequency or absolute number of antigen-specific T cells after administration of the compound of the present invention, as measured by HLA-peptide multimer staining, ELISPOT assay, and delayed type hypersensitivity test. It can also be measured indirectly by an increase in antigen-specific serum antibodies that are dependent on antigen-specific helper T cells.

[0225] The present invention also provides a method for inducing or enhancing an immune response in a subject to one or more antigens or antigenic epitopes restricted by multiple MHC class I molecules, said method comprising administering to said subject any one of the following: - an antigenic peptide according to the invention as described herein, - an immunogenic compound according to the invention as described herein, - the nanoparticles according to the invention as described herein, - a cell according to the invention as described herein, - a nucleic acid according to the invention as described herein, - a host cell according to the invention as described herein, - a T lymphocyte according to the invention as described herein, - an antibody according to the invention as described herein, and / or - a T cell receptor according to the invention as described herein, a pharmaceutical composition according to the invention as described herein, or - A kit according to the invention as described herein.

[0226] A method for inducing or enhancing an immune response in a subject against multiple epitopes described herein restricted by multiple MHC class I molecules can be determined by evaluating the increase in the expression of one or more of cytokine responses, such as IFN-γ, TNF-α, and IL-2 mRNA or protein, after in vitro stimulation of T cells with individual peptides that bind to distinct MHC class I molecules on antigen-presenting cells, compared to the levels before administration of the compound of the present invention. Restriction to MHC class I molecules can also be confirmed by using antigen-presenting cells expressing MHC class I molecules or by using MHC class I molecule blocking antibodies. It can also be measured by an increase in the frequency or absolute number of antigen-specific T cells after administration of the compound of the present invention, measured by HLA-peptide multimer staining using multimers assembled with MHC class I molecules.

[0227] The present invention relates more particularly to a composition as defined above for use as a vaccine for immunotherapy. Furthermore, the following may be used as vaccines, in particular for (cancer) immunotherapy: - an antigenic peptide according to the invention as described herein, - an immunogenic compound according to the invention as described herein, - the nanoparticles according to the invention as described herein, - a cell according to the invention as described herein, - a nucleic acid according to the invention as described herein, - a host cell according to the invention as described herein, - a T lymphocyte according to the invention as described herein, - an antibody according to the invention as described herein, and / or - a T cell receptor according to the invention as described herein, a pharmaceutical composition according to the invention as described herein, or - A kit according to the invention as described herein.

[0228] As used in the context of the present invention, the term "vaccine" refers to a (biological) preparation that provides natural and / or adaptive immunity, typically against a particular disease, preferably cancer. Thus, a vaccine specifically supports the natural and / or adaptive immune response of the immune system of the subject to be treated. For example, the antigenic peptide according to the present invention typically provides or supports an adaptive immune response in the patient to be treated.

[0229] In the context of the present invention, the vaccine (composition) is capable of inducing a specific immune response against tumor antigens and is therefore preferably used for the prevention or treatment of cancer.

[0230] Thus, in a preferred embodiment, the present invention relates to a composition as defined above for use in the prevention and / or treatment of cancer in a subject in need thereof. More preferably, the present invention relates to the use of a composition of the present invention for the manufacture of a medicament for preventing or treating cancer in a subject in need thereof. In other words, the present invention relates to a method for preventing or treating cancer in a subject in need thereof, comprising administering to said subject an effective amount of a composition of the present invention.

[0231] Preferably, the cancers that can be prevented and / or treated by are related to tumor antigens (references) of the antigenic peptides described herein: - an antigenic peptide according to the invention as described herein, - an immunogenic compound according to the invention as described herein, - the nanoparticles according to the invention as described herein, - a cell according to the invention as described herein, - a nucleic acid according to the invention as described herein, - a host cell according to the invention as described herein, - a T lymphocyte according to the invention as described herein, - an antibody according to the invention as described herein, and / or - a T cell receptor according to the invention as described herein, a pharmaceutical composition according to the invention as described herein, or - A kit according to the invention as described herein. That is, a suitable antigenic peptide can be selected by (i) selecting a suitable tumor antigen for a particular type of cancer known in the art, and (ii) selecting a suitable antigenic peptide according to the invention for that selected tumor antigen, as described above, for example in Table 1 (and optionally in addition, in Table 2). The skilled artisan will readily appreciate that the antigenic peptide of the present invention can be selected based on the nature of the cancer to be prevented or treated and / or the human genes / human tumor antigens involved in said cancer.

[0232] Particularly preferred for the prevention or treatment of cancer are peptides according to the invention selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 16 and SEQ ID NO: 40 to SEQ ID NO: 42, alone or in combination. More preferred are peptides selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 16 and SEQ ID NO: 40 to SEQ ID NO: 42 (see Table 1), alone or in combination, optionally in combination with at least one peptide selected from the group consisting of SEQ ID NO: 32 to SEQ ID NO: 34 (see Table 2), and their use in the immunotherapy of colorectal cancer, lung cancer, prostate cancer and / or breast cancer.

[0233] Therefore, another aspect of the present invention relates to the use of at least one peptide according to any one of SEQ ID NOs: 1 to 16 and 40 to 42 for the treatment, preferably in combination, of a proliferative disease selected from the group of colorectal cancer, lung cancer, prostate cancer and breast cancer.

[0234] Therefore, another aspect of the present invention relates to the use of a peptide according to the invention for the treatment, preferably in combination, of a proliferative disease, in particular of cancer, for example selected from the group of colorectal cancer, lung cancer, prostate cancer and breast cancer.

[0235] As mentioned above, in the context of the pharmaceutical compositions and kits of the present invention, combinations of the above items are preferred, i.e. separate antigenic peptides according to the present invention are preferably used for the prevention and / or treatment of cancer.

[0236] Preferably, more than one of the above components may be used in the prevention and / or treatment of cancer. For example, at least two different antigenic peptides, at least two different immunogenic compounds, at least two different nanoparticles, at least two different cells, at least two different nucleic acids, at least two different host cells, at least two different pharmaceutical compositions, etc. may be used in the prevention and / or treatment of cancer. Preferably, such different components used in the prevention and / or treatment of cancer differ in terms of the antigenic peptide according to the present invention, e.g., one component is related to a first antigenic peptide and one component is related to a second antigenic peptide (different from the first antigenic peptide), e.g., in the context of a pharmaceutical composition or kit.

[0237] Thus, the present invention also provides a combination of at least two distinct antigenic peptides according to the invention for use in the prevention or treatment of cancer.

[0238] For the prevention or treatment of cancers such as colorectal, lung, prostate and / or breast cancer, combinations of antigenic peptides as described above (in the context of a pharmaceutical composition or kit) are preferred.

[0239] In some embodiments, the following may be administered (in combination, i.e., simultaneously or sequentially) for the prevention and / or treatment of cancer: - a first component relating to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen CDC20; - a (separate) second component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen KIF2C; - a (separate) third component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen UBE2C; - optionally a (separate) fourth component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen BIRC5; and - optionally a (separate) fifth component related to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen FOXM1.

[0240] Preferably, the following may be administered (in combination, i.e. simultaneously or sequentially) for the prevention and / or treatment of cancer: - a first component related to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 17; - a (separate) second component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 18; - a (separate) third component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 19; - optionally a (separate) fourth component associated with an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 35; and - optionally a (separate) fifth component related to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 36.

[0241] More preferably, the following may be administered (in combination, i.e. simultaneously or sequentially) for the prevention and / or treatment of cancer: - a first component related to an antigenic peptide comprising or consisting of the amino acid set forth in SEQ ID NO:1; - a (separate) second component related to an antigenic peptide comprising or consisting of the amino acid set forth in SEQ ID NO:2; - a (separate) third component related to an antigenic peptide comprising or consisting of the amino acid set forth in SEQ ID NO: 3; - optionally a (separate) fourth component related to an antigenic peptide comprising or consisting of the amino acid set forth in SEQ ID NO: 32; and - optionally a (separate) fifth component related to an antigenic peptide comprising or consisting of the amino acid set forth in SEQ ID NO: 33.

[0242] Methods of administration are well known to those skilled in the art. With regard to the composition of the present invention, it can be administered to a subject directly to the affected organ (i.e., local administration) or systemically (i.e., enteral or parenteral administration), or it can be further applied ex vivo to cells or human cell lines derived from the subject, which are then administered to the subject, or it can be further used in vitro to select a subpopulation of immune cells derived from the subject, which subpopulation is then re-administered to said subject. Enteral administration includes oral and rectal administration, as well as administration via a gastric feeding tube, a duodenal feeding tube, or a gastrostomy, while parenteral administration includes, among others, subcutaneous, intravenous, intramuscular, intraarterial, intradermal, intraosseous, intracerebral, and intrathecal injection. The method of administration often depends on the antigenic peptides and / or immunogenic compounds present in the composition, as well as the type of cancer to be treated and other active substances that may be contained in the composition. For example, administration is preferably intramuscular or intradermal injection when the immunogenic compound is a nucleic acid as defined above, and oral / intranasal administration is particularly preferred when said nucleic acid is cloned into a viral vector. Alternatively, administration is preferably intramuscular, intradermal or oral administration when the antigenic peptide and / or immunogenic compound is a (poly)peptide as defined above or when it is loaded in / onto a nanoparticle as described herein. Still alternatively, administration is preferably oral administration when the antigenic peptide and / or immunogenic compound is delivered in the form of an intestinal bacterium as defined above, particularly when the intestinal bacterium is in the form of a probiotic.

[0243] The antigenic peptides, immunogenic compounds, and nucleic acids according to the present invention can be further encapsulated to facilitate their administration to a subject in need thereof. For example, they can be encapsulated into peptide nanocarriers (preferred when the immunogenic compound is a nucleic acid or (poly)peptide), virosomes (preferred when the immunogenic compound is a nucleic acid or (poly)peptide), or lipid-based carrier systems such as liposome-polycation-DNA complexes (preferred when the immunogen is a nucleic acid or (poly)peptide) (Trovato M, De Berardinis P. Novel antigen delivery systems. World J Virol. 2015 Aug 12;4(3):156-68; Saade F, Petrovsky N. Technologies for enhanced efficacy of DNA vaccines. Expert Rev Vaccines. 2012 Feb;11(2):189-209; Li et al., Peptide Vaccine: Progress and Challenges. Vaccines (Basel). 2014 Jul 2;2(3):515-36).

[0244] The composition may also be administered more than once to achieve the desired effect. In a preferred embodiment, the composition is administered repeatedly, at least twice, preferably more than twice. This can be done weekly, biweekly, monthly, yearly, or even for extended periods of time, such as years after the first administration, to ensure that the subject is adequately immunized.

[0245] The present invention further relates to a method of killing or reducing the number of target cells in a patient in which the target cells aberrantly express a polypeptide comprising an antigenic peptide according to the invention or a corresponding human reference peptide, the method comprising administering to the patient an effective number of T cells as produced by the present invention.

[0246] The present invention further relates to the use of any peptide as described, a nucleic acid according to the invention, an expression vector according to the invention, a cell according to the invention, an activated T lymphocyte, a T cell receptor or an antibody or other peptide and / or peptide-MHC binding molecule according to the invention as a medicament or in the manufacture of a medicament. Preferably, the medicament is active against cancer.

[0247] In some embodiments, the medicament is for a cell therapy, a vaccine, or a soluble TCR or antibody based protein.

[0248] In the following, a brief description of the attached figures is given. The figures are intended to illustrate the invention in more detail. However, they are not intended to limit the subject matter of the invention in any way. [Brief description of the drawings]

[0249] [Figure 1] FIG. 10 shows in vitro affinity for antigenic peptide CDC20-B1 (ENT204_B1) compared to the corresponding human CDC20 epitope CDC20-H1 (ENT204-H) for Example 1. [Diagram 2] FIG. 10 shows in vitro affinity for antigenic peptide KIF2C-B1 (ENT207_B1) compared to the corresponding human KIF2C epitope KIF2C-H1 (ENT207-H) for Example 1. [Diagram 3] With reference to Example 1, FIG. 1 shows the in vitro affinity for the antigenic peptide UBE2C-B1 (ENT168_B1) compared to the corresponding human UBE2C epitope UBE2C-H1 (ENT168-H) and another human UBE2C epitope UBE2C-H11 (ENT168-HL). [Figure 4] FIG. 10 shows in vitro affinity for antigenic peptide ANKRD30A-B1 (ENT169_B1) compared to the corresponding human ANKRD30A epitope ANKRD30A-H1 (ENT169-H) for Example 1. [Diagram 5]FIG. 10 shows in vitro affinity for antigenic peptide CDH17-B1 (ENT176_B1) compared to the corresponding human CDH17 epitope CDH17-H1 (ENT176-H) with reference to Example 1. [Figure 6] FIG. 10 shows in vitro affinity for antigenic peptide TOP2A-B2 (ENT205_B1) compared to the corresponding human TOP2A epitope TOP2A-H2 (ENT205-H) for Example 1. [Figure 7] With reference to Example 2, ELISPOT results for HHD DR1 HLA-A2 transgenic mice vaccinated with antigenic peptide CDC20-B1 (ENT204_B1) and cross-reactivity with the human counterpart peptide CDC20-H1 (ENT204-H) as indicated in the figure. Data provided as number of spots per 1×106 total T cells. [Figure 8] With reference to Example 2, ELISPOT results for HHD DR1 HLA-A2 transgenic mice vaccinated with antigenic peptide KIF2C-B1 (ENT207_B1) and cross-reactivity with the human counterpart peptide KIF2C-H1 (ENT207-H) as indicated in the figure. Data provided as number of spots per 1×106 total T cells. [Figure 9] For Example 2, ELISPOT results for HHD DR1 HLA-A2 transgenic mice vaccinated with antigenic peptide UBE2C-B1 (ENT168_B1) and cross-reactivity with the human counterpart peptide UBE2C-H1 (ENT168-H) and another human UBE2C epitope UBE2C-H11 (ENT168-HL) as indicated in the figure. Data provided as number of spots per 1×106 total T cells. [Figure 10]With reference to Example 2, ELISPOT results for HHD DR1 HLA-A2 transgenic mice vaccinated with antigenic peptide ANKRD30A-B1 (ENT169_B1) and cross-reactivity with the human counterpart peptide ANKRD30A-H1 (ENT169-H) as indicated in the figure. Data provided as number of spots per 1×106 total T cells. [Figure 11] With reference to Example 2, ELISPOT results for HHD DR1 HLA-A2 transgenic mice vaccinated with antigenic peptide CDH17-B1 (ENT176_B1) and cross-reactivity with the human counterpart peptide CDH17-H1 (ENT176-H) as indicated in the figure. Data provided as number of spots per 1×106 total T cells. [Figure 12] With reference to Example 2, ELISPOT results for HHD DR1 HLA-A2 transgenic mice vaccinated with antigenic peptide TOP2A-B2 (ENT205_B1) and cross-reactivity with the human counterpart peptide TOP2A-H2 (ENT205-H) as indicated in the figure. Data provided as number of spots per 1×106 total T cells. [Figure 13] Referring to Example 3, FIG. 1 shows detection of CDC20-B1, KIF2C-B1, and UBE2C-B1 peptide-specific CD8+ T cells detected in peripheral blood from healthy donors (HLA-A2 positive). [Figure 14] FIG. 10 shows in vitro affinity for antigenic peptide KIF2C-B11 (ENT207_B2) compared to the corresponding human KIF2C epitope KIF2C-H1 (ENT207-H) for Example 1. [Figure 15] FIG. 10 shows in vitro affinity for antigenic peptide KIF2C-B12 (ENT207_B3) compared to the corresponding human KIF2C epitope KIF2C-H1 (ENT207-H) with reference to Example 1. [Figure 16]FIG. 10 shows in vitro affinity for antigenic peptide KIF2C-B13 (ENT207_B4) compared to the corresponding human KIF2C epitope KIF2C-H1 (ENT207-H), with reference to Example 1. [Figure 17] FIG. 10 shows in vitro affinity for antigenic peptide UBE2C-B11 (ENT168_B2) compared to the corresponding human UBE2C epitope UBE2C-H1 (ENT168-H), with reference to Example 1. [Figure 18] With reference to Example 3, this figure shows the cytotoxicity ability of CDC20-B1, KIF2C-B1, and UBE2C-B1 peptide-specific human T cell clones expanded in vitro by stimulation with microbiota-derived peptides. CDC20-B1, KI2C-B1, and UBE2C-B1 peptide-specific T cells have the ability to kill T2 cells loaded with bacteria or human peptides. EXAMPLES

[0250] In the following, specific examples are presented to illustrate various embodiments and aspects of the present invention. However, the present invention should not be limited in scope by the specific embodiments described herein. The following preparations and examples are presented to enable those skilled in the art to more clearly understand and practice the present invention. However, the present invention is not limited in scope by the exemplified embodiments, which are intended only as illustrations of a single aspect of the present invention, and methods that are functionally equivalent are within the scope of the present invention. Indeed, various modifications of the present invention in addition to those described herein will be readily apparent to those skilled in the art from the foregoing description, the accompanying figures, and the following examples. All such modifications are within the scope of the appended claims.

[0251] Example 1 Antigenic peptides have excellent affinity for the HLA-A*0201 allele Next, the binding affinity of various selected antigenic peptides and corresponding fragments of human tumor antigens (human reference peptides) to the HLA-A*0201 allele was confirmed in vitro. That is, the antigenic peptide of the sequence of SEQ ID NO:1 ("SLPDRILTV"; also referred to herein as CDC20-B1) was compared with the corresponding reference human peptide derived from CDC20 ("SLPDRILDA"; SEQ ID NO:17, also referred to herein as CDC20-H1). Furthermore, the antigenic peptide of the sequence of SEQ ID NO:2 ("ALNPELLAL"; also referred to herein as KIF2C-B1) was compared with the corresponding reference human peptide derived from KIF2C ("AINPELLQL"; SEQ ID NO:18, also referred to herein as KIF2C-H1). Furthermore, the antigenic peptide of the sequence of SEQ ID NO:3 ("FLAFVPLQL"; also referred to herein as UBE2C-B1) was compared with the corresponding reference human peptides derived from UBE2C ("ALYDVRTIL"; SEQ ID NO:19, also referred to herein as UBE2C-H1; and "ALYDVRTILL", SEQ ID NO:38, also referred to herein as UBE2C-H11). Furthermore, the antigenic peptide of the sequence of SEQ ID NO:4 ("YLAFVPLAL"; also referred to herein as UBE2C-B11) was compared with the corresponding reference human peptides derived from UBE2C ("ALYDVRTIL"; SEQ ID NO:19, also referred to herein as UBE2C-H1). Furthermore, the antigenic peptide of the sequence of SEQ ID NO:5 ("SLLSIQSYV"; also referred to herein as UBE2C-B2) was compared with the corresponding reference human peptide derived from UBE2C ("ILLSIQSLL", SEQ ID NO:20, also referred to herein as UBE2C-H2). Furthermore, the antigenic peptide of the sequence of SEQ ID NO:6 ("YLQQELMNL"; also referred to herein as UBE2C-B3) was compared with the corresponding reference human peptide derived from UBE2C ("RLQQELMTL", SEQ ID NO:21, also referred to herein as UBE2C-H3).Furthermore, the antigenic peptide of the sequence of SEQ ID NO: 7 ("ALYSEILTV"; also referred to herein as ANKRD30A-B1) was compared with the corresponding reference human peptide derived from ANKRD30A ("AVYSEILSV", SEQ ID NO: 22, also referred to herein as ANKRD30A-H1). Furthermore, the antigenic peptide of the sequence of SEQ ID NO: 8 ("LILDTVHSL"; also referred to herein as ANKRD30A-B2) was compared with the corresponding reference human peptide derived from ANKRD30A ("KILDTVHSC", SEQ ID NO: 23, also referred to herein as ANKRD30A-H2). Furthermore, the antigenic peptide of the sequence of SEQ ID NO: 9 ("TLDQKLFMV"; also referred to herein as ANKRD30A-B3) was compared with the corresponding reference human peptide derived from ANKRD30A ("SLDQKLFQL", SEQ ID NO: 24, also referred to herein as ANKRD30A-H3). Furthermore, the antigenic peptide of the sequence of SEQ ID NO: 10 ("YLILEYATV"; also referred to herein as AURKA-B1) was compared with the corresponding reference human peptide derived from AURKA ("YLILEYAPL", SEQ ID NO: 25, also referred to herein as AURKA-H1). Furthermore, the antigenic peptide of the sequence of SEQ ID NO: 11 ("KIIGIILAV"; also referred to herein as CDH17-B1) was compared with the corresponding reference human peptide derived from CDH17 ("LVIGIILAV", SEQ ID NO: 26, also referred to herein as CDH17-H1). Furthermore, the antigenic peptide of the sequence of SEQ ID NO: 12 ("YLSGANLFV"; also referred to herein as CEACAM5-B1) was compared with the corresponding reference human peptide derived from CEACAM5 ("YLSGANLNL", SEQ ID NO: 27, also referred to herein as CEACAM5-H1). Additionally, the antigenic peptide of sequence SEQ ID NO: 13 ("IVWSDVTYV"; also referred to herein as MMP11-B1) was compared to the corresponding reference human peptide derived from MMP11 ("KVWSDVTPL", SEQ ID NO: 28, also referred to herein as MMP11-H1).Additionally, the antigenic peptide of sequence SEQ ID NO: 14 ("AVIGIVAAV"; also referred to herein as OR51E2-B1) was compared to the corresponding reference human peptide derived from OR51E2 ("AQIGIVAVV", SEQ ID NO: 29, also referred to herein as OR51E2-H1). Additionally, the antigenic peptide of sequence SEQ ID NO: 16 ("ALIFGQLLL"; also referred to herein as TOP2A-B2) was compared to the corresponding reference human peptide derived from TOP2A ("ALIFGQLLT", SEQ ID NO: 31, also referred to herein as TOP2A-H2).

[0252] A. Materials and Methods A1. Measurement of peptide affinity for T2 cell line The experimental protocol is similar to that established for peptides presented by HLA-A*0201 (Tourdot et al., A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides: implication in the identification of cryptic tumor epitopes. Eur J Immunol. December 2000; 30(12):3411-21). Peptide affinity measurements are achieved using human neoplastic cells T2, which express HLA-A*0201 molecules but are TAP1 / 2 negative and unable to present endogenous peptides.

[0253] T2 cells (5 × 10 per well 4 Cells) are incubated with decreasing concentrations of peptides from 100 μM to 0.1 μM (4 points: 100 μM, 10 μM, 1 μM, 0.1 μM) in serum-free medium (TexMacs) supplemented with 100 ng / μl β2-microglobulin for 16 h at 37° C. Cells are then washed twice and labeled with PE-conjugated anti-HLA-A2 antibody (clone REA517, Miltenyi).

[0254] Analysis is accomplished by FACS (Macsquant Analyzer 10 or Macsquant Analyzer 16 Miltenyi).

[0255] For each peptide concentration, the geometric mean of the label associated with the peptide of interest is subtracted from the background noise and reported as a percentage of the geometric mean of HLA-A*0202 labeling obtained for the reference peptide HIV pol 589-597 at a concentration of 100 μM. Relative affinity is then determined as follows: Relative affinity = concentration of each peptide that induces 20% of HLA-A*0201 expression / concentration of reference peptide that induces 20% of HLA-A*0201 expression

[0256] A2. Solubilization of peptides Each peptide is solubilized by considering the amino acid composition. For peptides that do not contain any cysteine, methionine or tryptophan, DMSO can be added up to 10% of the total volume. Other peptides are resuspended in water or NH4OH.

[0257] B. Results The mean relative fluorescence intensity values ​​of T2 cells obtained for various concentrations of each peptide (data are normalized to the mean fluorescence of the HIV peptide, i.e., a value of 100 is equal to the highest binding observed with the HIV peptide) are shown in Table 3 below.

[0258] [Table 3]

[0259] Table 4 below summarizes the concentration and in vitro binding affinity required to induce 20% of HLA-A2 expression for each test peptide (*concentration of peptide inducing 20% ​​of HLA-A2 expression normalized to HIV-pol performed in the same experiment).

[0260] [Table 4]

[0261] In addition, Figures 1 to 6 and Figures 14 to 17 show the results of comparing selected examples, namely, antigenic peptide CDC20-B1 with the corresponding human CDC20 fragment CDC20-H1 (Figure 1), antigenic peptide KIF2C-B1 with the corresponding human KIF2C fragment KIF2C-H1 (Figure 2), antigenic peptide KIF2C-B11 with the corresponding human KIF2C fragment KIF2C-H1 (Figure 14), antigenic peptide KIF2C-B12 with the corresponding human KIF2C fragment KIF2C-H1 (Figure 15), antigenic peptide KIF2C-B13 with the corresponding human KIF2C fragment KIF2C-H1 (Figure 16), antigenic peptide KIF2C-B15 with the corresponding human KIF2C fragment KIF2C-H1 (Figure 17), and antigenic peptide KIF2C-B16 with the corresponding human KIF2C fragment KIF2C-H1 (Figure 18). The results are shown below: peptide UBE2C-B1 compared with the corresponding human UBE2C fragment UBE2C-H1 and another human UBE2C epitope UBE2C-H11 (Figure 3); antigenic peptide UBE2C-B11 compared with the corresponding human UBE2C fragment UBE2C-H1 (Figure 17); antigenic peptide ANKRD30A-B1 compared with the corresponding human ANKRD30A fragment ANKRD30A-H1 (Figure 4); antigenic peptide CDH17-B1 compared with the corresponding human CDH17 fragment CDH17-H1 (Figure 5); and antigenic peptide TOP2A-B2 compared with the corresponding human TOP2A fragment TOP2A-H2 (Figure 6).

[0262] In summary, the results show that the antigenic peptides according to the invention show at least similar binding affinity to HLA-A*0201 as the corresponding human tumor antigen fragments. In most cases, the binding affinity observed for the antigenic peptides according to the invention was stronger than that of the corresponding human epitopes. Without intending to be bound by any theory, it is assumed that such strong binding affinity of the antigenic peptides according to the invention reflects their ability to generate an immune response (i.e., their immunogenicity).

[0263] Example 2 Immunogenicity of UBE2C-B1 (ENT_168-B1), ANKRD30A-B1 (ENT_169-B1), CDH17-B1 (ENT_176-B1), CDC20-B1 (ENT_204-B1), TOP2A-B2 (ENT_205-B1), and KIF2C-B1 (ENT_207-B1) in HLA-A2 transgenic mice and cross-reactivity with the corresponding human peptides A. Materials and Methods A.1 Mouse model Briefly, HLA-A2 HHD-DR1 humanized mice (C57BL / 6JB2mtm1UncIAb- / -Tg(HLA-DRA,HLA- DRB1*0101)#GjhTg(HLA-A / H2-D / B2M)1Bpe) were randomly assigned (based on the sex and age of the mice) to experimental groups and each group was immunized with a specific vaccination peptide (vacc-pAg) combined with a common helper peptide (h-pAg UCP2; sequence: KSVWSKLQSIGIRQH; SEQ ID NO: 39) (as outlined in Table 6 below).

[0264] [Table 5]

[0265] The peptides were supplied as follows: · vacc-pAg: UBE2C-B1, ANKRD30A-B1, CDH17-B1, CDC20-B1, TOP2A-B2, and KIF2C-B1 were all made and supplied at 4 mM concentration; h-pAg: UCP2 was resuspended in pure distilled water at a concentration of 10 mg / mL.

[0266] The injected peptide formulations (emulsions) were prepared fresh for each group on each injection day. Mixtures for 10 animals were prepared using 2 mL Luer-lock syringes (4606701V, B BRAUN) and Combifix® adapters for Luer-lock female-female connections (B. Braun, 5206634): 500 μL of peptide mixture in syringe 1 was emulsified with 500 μL of Montanide ISA 51 VG (Seppic) contained in syringe 2. The emulsification process was carried out by first pushing the peptide mixture contained in syringe 1 into syringe 2 (containing Montanide) at a very slow speed. The transfer of the mixture from one syringe to another was then carried out for 6 cycles (one cycle corresponds to one pass from one syringe to another) at a very slow speed and then for 1 minute at the highest possible speed. Each emulsion was prepared in excess to compensate for dead volume during injection.

[0267] Animals were immunized with a prime injection on day 0 (d0) and a boost injection on d14. Each mouse was injected in the loose skin covering the animal's neck with 100 μL of an oily emulsion containing: 30 nmol of vacc-pAg and 30 μg or 100 μg of UCP2 helper peptide in 50 μL of the final solvent Montanide ISA 51 VG (Seppic) added in a 1:1 (v:v) ratio (50 μL per mouse).

[0268] A.2 Analysis Seven days after the boost injection (i.e., on d21), the animals were euthanized and the spleens were harvested. Splenocytes were prepared by mechanical disruption of the organ, followed by 70 μm filtering and red blood cell lysis.

[0269] The cell suspension was further used for ELISPOT-IFNγ assay (Table 5). Cells were cultured in 200 μL of complete T cell medium. Experimental conditions (in duplicate) were as follows: 2 × 10 cells per well when cultured in the presence of various pAgs (10 μM) or medium alone. 5 total cells; and 2 × 10 when cultured in the presence of positive controls PMA / ionomycin (PMA: Sigma P8139: 0, 1 μM final; ionomycin: Sigma I0634: 1 μM final). 4 Total cells. Cultures were assessed for their ability to secrete IFNγ (Mouse IFNγ ELISpotPLUS kit, Mabtech 3321-4APT-10) according to the manufacturer's instructions (approximately 12-48 h incubation time before performing the assay). Peptides used for restimulation are listed in Table 7.

[0270] [Table 6]

[0271] Spots were counted with an iSpot Fluorospot Reader System (AID). Data plotting and statistical analysis were performed with Prism-9 software (GraphPad Software Inc.).

[0272] B. Results All mice were 8-15 weeks old at the start of the experiment. Both males and females were used in the study. Animals were housed in groups of a maximum of six per cage. At the time of sacrifice, splenic T-cell populations were analyzed by flow cytometry and showed that the majority belonged to the CD4+ T-cell subset.

[0273] After plating and incubation with the appropriate stimuli, IFNγ-producing cells were visualized and counted. Data are from 1×10 6Results were presented as number of spots per total T cells. Individual means (obtained from duplicates) were then used to plot group means. Statistical analysis for comparisons (with irrelevant peptide conditions) was performed using unpaired non-parametric tests (Mann-Whitney) ( ** :p<0.01; * : p<0.05).

[0274] Overall, vaccination with antigenic peptides according to the present invention (CDC20-B1, KIF2C-B1, UBE2C-B1, ANKRD30A-B1, CDH17-B1, and TOP2A-B2) induced significant T cell responses in HHD DR1 mice in ELISPOT-IFNγ assays (Figures 7 to 12).

[0275] The results (Figure 7) show that immunization of HHD-DR1 mice with CDC20-B1 makes it possible to induce T cells that can react strongly after challenge with either CDC20-B1 or the human counterpart peptide CDC20-H1. Thus, CDC20-B1 is strongly immunogenic and can drive an effective immune response against the corresponding human peptide.

[0276] The results (Figure 8) show that immunization of HHD-DR1 mice with KIF2C-B1 allows the induction of T cells that can react strongly after challenge with either KIF2C-B1 or the human counterpart peptide KIF2C-H1. Thus, KIF2C-B1 is highly immunogenic and can drive an effective immune response against the corresponding human peptide.

[0277] The results (Figure 9) show that immunization of HHD-DR1 mice with UBE2C-B1 allows the induction of T cells capable of reacting strongly after challenge with either UBE2C-B1 or the human counterpart peptide UBE2C-H1, but not with another human UBE2C epitope UBE2C-H11. Thus, UBE2C-B1 is strongly immunogenic and capable of driving an effective immune response to the corresponding human peptide, but not to another human UBE2C epitope UBE2C-H11.

[0278] The results (Figure 10) show that immunization of HHD-DR1 mice with ANKRD30A-B1 allows the induction of T cells that can react strongly after challenge with either ANKRD30A-B1 or the human counterpart peptide ANKRD30A-H1. Thus, ANKRD30A-B1 is highly immunogenic and can drive an effective immune response against the corresponding human peptide.

[0279] The results (Figure 11) show that immunization of HHD-DR1 mice with CDH17-B1 allows the induction of T cells that can react strongly after challenge with either CDH17-B1 or the human counterpart peptide CDH17-H1. Thus, CDH17-B1 is highly immunogenic and can drive an effective immune response against the corresponding human peptide.

[0280] The results (Figure 12) show that immunization of HHD-DR1 mice with TOP2A-B2 allows the induction of T cells that can react strongly after challenge with either TOP2A-B2 or the human counterpart peptide TOP2A-H2. Thus, TOP2A-B2 is highly immunogenic and can drive an effective immune response against the corresponding human peptide.

[0281] In summary, these immunogenicity studies described in Example 2, performed in HHD DR1 mice, showed that the six antigenic peptides of the present invention, CDC20-B1, KIF2C-B1, UBE2C-B1, ANKRD30A-B1, CDH17-B1, and TOP2A-B2, induced strong immune responses. Cross-reactivity of T cells generated against CDC20-H1, KIF2C-B2, UBE2C-B1, ANKRD30A-B1, CDH17-B1, and TOP2A-B2 to the corresponding human peptides was demonstrated in HHD DR1 mice.

[0282] These results therefore provide experimental evidence that antigen-based immunotherapy can enhance T cell responses in vivo, and that the antigenic peptides according to the invention are particularly efficient for that purpose.

[0283] Example 3 Ex vivo cytotoxic effects of UBE2C-B1 (ENT_168-B1), CDC20-B1 (ENT_204-B1), and KIF2C-B1 (ENT_207-B1)-specific CD8 human T cells Several researchers support the idea of ​​the existence of a repertoire of specific T cells against microbial peptides, and although the number of microbial-specific T cells against peptides is expected to be small, it is expected to be sufficient to be reactivated by vaccine exposure.

[0284] To identify and functionally characterize circulating UBE2C-B1 (ENT_168-B1), CDC20-B1 (ENT_204-B1), and KIF2C-B1 (ENT_207-B1)-specific T cells in humans, an in vitro expansion protocol has been developed to detect T cells specific for each antigenic peptide and examine their cytotoxic capacity.

[0285] 3.1 Identification of antigenic peptide-specific CD8 T cells in humans In vitro amplification methods and specific pMHC multimers have been used for the identification of UBE2C-B1 (ENT_168-B1), CDC20-B1 (ENT_204-B1), and KIF2C-B1 (ENT_207-B1) specific T cells. pMHC multimers were generated for all bacterial peptides and their respective human counterparts. PBMCs from several HLA-A*02 healthy donors (up to 13 donors) were collected, enriched after CD137 and CD8 selection, and subjected to multiple rounds of in vitro amplification on T2 cells loaded with EO4010 peptide to increase the number of specific T cell clones. Detection of OMP peptide-specific CD8 T cells using cytometric analysis with fluorescent multimers was performed on the enriched CD8 T cell population.

[0286] Figure 13 illustrates the results obtained with one HLA-A2 healthy donor, for which cell proliferation allows the detection of UBE2C-B1 (ENT_168-B1)-specific cells (7,58%), CDC20-B1 (ENT_204-B1)-specific cells (7,46%) and KIF2C-B1 (ENT_207-B1)-specific cells (0,63%).

[0287] In conclusion, these results demonstrate the presence of CD8 T cells in the blood of healthy HLA-A2 donors capable of recognizing microbiota-derived peptides.

[0288] 3.2 Antigenic peptide-specific CD8 T cytotoxic function Using the CD8+ T cells expanded as above, cytotoxicity assays were performed in the presence of different ratios of target and effector cells to evaluate their cytotoxic capacity using flow cytometry readout. Target cells were T2 cell lines loaded with bacterial peptides or human counterpart peptides. As negative controls, unloaded T2 cells and T2 cells loaded with irrelevant peptides were used. As shown in Figure 18, in vitro expanded antigenic peptide-specific human T cells have the ability to kill T2 cells loaded with all bacterial peptides UBE2C-B1 (ENT_168-B1), CDC20-B1 (ENT_204-B1), and KIF2C-B1 (ENT_207-B1). More importantly, in vitro expanded UBE2C-B1 (ENT_168-B1), CDC20-B1 (ENT_204-B1), and KIF2C-B1 (ENT_207-B1)-specific human T cell clones were able to kill T2 cells loaded with human UBEC2, CDC20, and KIF2C.

[0289] Overall, these results demonstrate the presence of T cell clones in healthy volunteers that can recognize microbial peptides and kill targets bearing microbial peptides and human counterparts. These data are particularly encouraging because T cell clones were obtained in healthy donors, and therefore it can be expected that specific T cell clones can be efficiently expanded in patients exposed to immunization with the antigenic peptides of the invention.

Claims

1. An antigenic peptide comprising or consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 16 and 40 to 42.

2. The antigenic peptide of claim 1, consisting of an amino acid sequence shown in any one of SEQ ID NOs: 1 to 16 and 40 to 42.

3. The antigenic peptide of claim 1, having a length of 9 or 10 amino acids.

4. 2. The antigenic peptide of claim 1, comprising or consisting of the amino acid sequence set forth in SEQ ID NO:

1.

5. 2. The antigenic peptide of claim 1, comprising or consisting of the amino acid sequence set forth in SEQ ID NO:

2.

6. 2. The antigenic peptide of claim 1, comprising or consisting of the amino acid sequence set forth in SEQ ID NO:

3.

7. An immunogenic compound comprising an antigenic peptide according to any one of claims 1 to 6.

8. A nucleic acid encoding an antigenic peptide according to any one of claims 1 to 6 or an immunogenic compound according to claim 7, wherein the immunogenic compound is a peptide or a protein.

9. - an antigenic peptide according to any one of claims 1 to 6, - an immunogenic compound according to claim 7, or - a nucleic acid according to claim 8, 10. A pharmaceutical composition comprising:

10. The pharmaceutical composition of claim 9, further comprising one or more pharmaceutically acceptable excipients or carriers.

11. (i) at least two distinct antigenic peptides according to any one of claims 1 to 6; (ii) at least two distinct immunogenic compounds according to claim 7; or (iii) at least two distinct nucleic acids according to claim 8 10. The pharmaceutical composition of claim 9, comprising:

12. The separate components are - an antigenic peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1; - an antigenic peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2; and an antigenic peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3 12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition is associated with

13. - an antigenic peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 32; and an antigenic peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 33, or an antigenic peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 34; 10. The pharmaceutical composition of claim 9, further comprising:

14. 10. The pharmaceutical composition of claim 9, further comprising a helper peptide.

15. The pharmaceutical composition of claim 9, further comprising a helper peptide comprising or consisting of an amino acid sequence according to SEQ ID NO:

39.

16. For use in medicine, in particular in the prevention and / or treatment of cancer, The antigenic peptide according to any one of claims 1 to 6. The immunogenic compound according to claim 7, A nucleic acid according to claim 8, or 10. The pharmaceutical composition of claim 9.

17. A peptide-MHC (pMHC) multimer comprising an antigenic peptide according to any one of claims 1 to 6.

18. A cytotoxic T lymphocyte (CTL) specific to an antigenic peptide described in any one of claims 1 to 6.

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