Antigen peptide for preventing and treating cancer

Abstract

The present invention relates to antigen-based immunotherapy, in particular cancer immunotherapy. In particular, the present invention provides an antigen peptide which is different from a fragment of a human tumor antigen but has amino acid similarity to the fragment of the human tumor antigen. The invention also provides immunogenic compounds, nanoparticles, cells and pharmaceutical compositions comprising such antigen peptides and nucleic acids encoding such antigen peptides.

Description

Technical Field

[0001] The present invention relates to the field of cancer therapy, and more specifically, to immunotherapy. In particular, the present invention provides various peptides that can be used for cancer immunotherapy. Background Art

[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 increase by about 70% in the next two decades. So far, more than 60% of the total number of new cases each year worldwide occurs in Africa, Asia, and Central and South America. These regions also account for 70% of the world's cancer deaths. In men, the five most common sites of cancer are lung, prostate, colorectal, stomach, and liver; while in women, these sites are breast, colorectal, lung, cervix, and stomach.

[0003] Cancer has long been managed with surgery, radiotherapy, cytotoxic chemotherapy, and endocrine modulation, often used in sequential combinations to achieve optimal disease control. However, the true efficacy of these standard therapies is primarily limited by their imprecise specificity, which results in collateral damage to normal tissues during treatment, low cure rates, and intrinsic drug resistance.

[0004] In recent years, the development of cancer therapies has made great leaps forward due to major advances in the expression profiles of tumor and normal cells, and recent studies and initial clinical results of immunotherapy or molecular targeted therapies are also beginning to change our understanding of this disease.

[0005] Promising anticancer immunotherapies are now a reality, and evidence that the host immune system can recognize tumor antigens has led to the development of anticancer drugs that have been 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, immunostimulatory cytokines and their variants, pattern recognition receptor (PRR) agonists, and immunomodulatory 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 considerable number of patients still show intrinsic resistance to some of these immunotherapies, or even acquire resistance during treatment. For example, the three-year survival rate of patients with unresectable or metastatic melanoma treated with the anti-CTLA-4 antibody ipilimumab is reported to be approximately 20% (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 the three-year survival rate of nivolumab, another checkpoint inhibitor targeting PD-1, was reported to be 44% for renal cell carcinoma (RCC) and 18% for non-small cell lung cancer (NSCLC) (McDermott et al., Survival, Durable Response, and Long-Term Safety in Patients With Previously Treated Advanced Renal Cell Carcinoma Receiving Nivolumab. J Clin Oncol. 2015 Jun 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. 2015 Jun 20;33(18):2004-12). Therefore, fundamental drug resistance presents an inherent barrier to the efficacy of these immunotherapies. Therefore, a different approach to cancer therapy is clearly needed to break this barrier.

[0007] The lack of response in many subjects treated with these immunotherapies may be related to defects in the antitumor immune response (e.g., defects in antigen presentation by antigen presenting cells (APCs) or defects in antigen recognition by T cells). In other words, a positive response to immunotherapy is associated with the ability of the immune system to develop specific lymphocyte subsets that can recognize MHC class I-restricted antigens expressed by human cancer cells (Kvistborg et al., Human cancer regression antigens. Curr Opin Immunol. 2013 Apr;25(2):284-90). This hypothesis is strongly supported by data showing that responses to adoptive transfer of tumor infiltrating lymphocytes (TILs) are associated with CD8 + The number of T cells is directly related (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). Therefore, an effective 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] Vaccination based on tumor antigens represents a unique approach to cancer therapy that has attracted great interest because it can use the patient's own immune system to recognize, attack and destroy tumors in a specific and lasting manner. It is known that tumor cells do express a large number of peptide antigens that are easily recognized by the immune system. Therefore, vaccines based on such antigens offer great opportunities not only to improve the overall survival of patients, but also to monitor immune responses and prepare GMP-grade products thanks to the low toxicity and low molecular weight of tumor antigens. Examples of tumor antigens include, in particular, byproducts of proteins transcribed from normally silent genes or overexpressed genes, byproducts of proteins expressed by tumor viruses (Kvistborg et al., Human cancer regression antigens. Curr Opin Immunol. 2013 April; 25(2): 284-90), and new antigens generated by point mutations of cellular proteins. The latter are of particular interest as they have been shown to be directly associated 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] However, there are a limited number of human tumor antigens that can be used to develop cancer vaccines. In particular, antigens derived from mutated or modified self-proteins may induce immune tolerance and / or undesirable autoimmune side effects.

[0010] Therefore, there is a need in the art to identify alternative cancer treatment methods that overcome the limitations encountered in the field.

[0011] The object of the present invention is to meet the above needs. This object is achieved by the subject matter described below, in particular by the provisions of the present invention and the appended claims. Summary of the invention

[0012] In particular, the present invention particularly provides the following provisions:

[0013] 1. An antigenic peptide comprising or consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1-16 and SEQ ID NOs: 40-42.

[0014] 2. An antigenic peptide comprising or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 1-16 and SEQ ID NOs: 40-42, wherein, optionally, one or two amino acid residues may be replaced, deleted or added.

[0015] 3. The antigenic peptide according to clause 2, wherein the core sequence is retained.

[0016] 4. The antigenic peptide according to any of the preceding clauses, wherein the antigenic peptide comprises or consists of a microbiota variant of the human reference peptide of any of SEQ ID NOs: 17-31.

[0017] 5. The antigenic peptide according to any one of the preceding clauses, wherein the antigenic peptide consists of the amino acid sequence shown in any one of SEQ ID NOs: 1-16 and SEQ ID NOs: 40-42.

[0018] 6. The antigenic peptide according to any one of the preceding clauses, wherein the antigenic peptide is 8 to 15 amino acids or 8 to 11 amino acids in length, preferably the antigenic peptide is 9 or 10 amino acids in length.

[0019] 7. The antigenic peptide according to any one of the preceding clauses, wherein the antigenic peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0020] 8. The antigenic peptide according to any one of the preceding clauses, wherein the antigenic peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 2.

[0021] 9. The antigenic peptide according to any one of the preceding clauses, wherein the antigenic peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 3.

[0022] 10. The antigenic peptide according to any of the preceding clauses, wherein the antigenic peptide is no more than 30 amino acids in length.

[0023] 11. An antigenic peptide according to any one of the preceding clauses, wherein the antigenic peptide is no more than 25 amino acids in length.

[0024] 12. An antigenic peptide according to any one of the preceding clauses, wherein the antigenic peptide is no more than 20 amino acids in length.

[0025] 13. An antigenic peptide according to any of the preceding clauses, wherein the antigenic peptide is no more than 15 amino acids in length.

[0026] 14. An antigenic peptide according to any of the preceding clauses, wherein the antigenic peptide is no more than 11 amino acids in length.

[0027] 15. An antigenic peptide according to any of the preceding clauses, wherein the antigenic peptide is not a full-length (microbiota) protein.

[0028] 16. An immunogenic compound comprising an antigenic peptide according to any one of the preceding clauses.

[0029] 17. The immunogenic compound according to clause 16, wherein the antigenic peptide is linked to a carrier molecule.

[0030] 18. The immunogenic compound according to clause 17, wherein the carrier molecule is a carrier protein or a carrier peptide.

[0031] 19. An immunogenic compound according to any one of clauses 16 to 18, comprising or consisting of a polypeptide of formula (I)

[0032] PepNt-CORE-PepCt(I)

[0033] in:

[0034] - "PepNt" consists of a polypeptide having a length of 0 to 500 amino acid residues, located at the N-terminus of the polypeptide of formula (I);

[0035] - CORE consists of an antigenic peptide as defined in any of clauses 1 to 15; and

[0036] - "PepCt" consists of a polypeptide having a length of 0 to 500 amino acid residues, located at the C-terminus of the polypeptide of formula (I).

[0037] 20. Nanoparticles loaded with

[0038] - at least one antigenic peptide according to any one of clauses 1 to 15, or

[0039] - at least one immunogenic compound according to any one of clauses 16 to 19;

[0040] and, optionally, loaded with an adjuvant.

[0041] 21. A cell loaded with an antigenic peptide according to any one of clauses 1-15 or an immunogenic compound according to any one of clauses 16-19.

[0042] 22. The cell according to clause 38, wherein the cell is an antigen presenting cell, preferably a dendritic cell.

[0043] 23. A nucleic acid encoding an antigenic peptide according to any one of clauses 1 to 15, a polypeptide of formula (I) as defined in clause 19, or an immunogenic compound according to any one of clauses 16 to 19, wherein the immunogenic compound is a peptide or a protein.

[0044] 24. A nucleic acid according to clause 23, wherein the nucleic acid is a DNA molecule or an RNA molecule; preferably selected from genomic DNA; cDNA; siRNA; rRNA; mRNA; antisense DNA; antisense RNA; ribozymes; complementary RNA and / or DNA sequences; RNA and / or DNA sequences with or without expression elements, regulatory elements and / or promoters; vectors; and combinations thereof.

[0045] 25. A host cell comprising a nucleic acid according to clause 23 or 24.

[0046] 26. A host cell according to clause 25, wherein the nucleic acid is a vector.

[0047] 27. A host cell according to clause 25 or 26, wherein the host cell is a bacterial cell, preferably an enteric bacterial cell.

[0048] 28. (Cytotoxic and / or activated) T lymphocytes specific for an antigenic peptide according to any one of clauses 1-15.

[0049] 29. An antibody that binds to an antigenic peptide according to any one of clauses 1-15.

[0050] 30. A T cell receptor which binds to an antigenic peptide according to any one of clauses 1 to 15.

[0051] 31. A pharmaceutical composition comprising:

[0052] - an antigenic peptide according to any one of clauses 1 to 15,

[0053] - an immunogenic compound according to any one of clauses 16 to 19,

[0054] - nanoparticles according to item 20,

[0055] - a cell according to item 21 or 22,

[0056] - a nucleic acid according to item 23 or 24,

[0057] - a host cell according to any one of clauses 25 to 27,

[0058] - T lymphocytes according to item 28,

[0059] - an antibody according to clause 29, or

[0060] - a T-cell receptor according to item 30,

[0061] and, optionally, one or more pharmaceutically acceptable excipients or carriers.

[0062] 32. A pharmaceutical composition according to clause 31, wherein the composition comprises

[0063] (i) at least two different antigenic peptides according to any one of clauses 1 to 15;

[0064] (ii) at least two different immunogenic compounds according to any one of clauses 16 to 19;

[0065] (iii) at least two different nanoparticles according to clause 20;

[0066] (iv) at least two different nucleic acids according to clause 21 or 22; or

[0067] (v) at least two different cytotoxic T lymphocytes according to clause 28.

[0068] 33. A pharmaceutical composition according to item 32, comprising at least three or four different components according to any one of (i) to (v), preferably three or four different antigenic peptides.

[0069] 34. A pharmaceutical composition according to clause 33, wherein the at least three or four different active ingredients are related to

[0070] - an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0071] - an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2; and

[0072] - an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3.

[0073] 35. The pharmaceutical composition according to any one of clauses 31 to 34, wherein the pharmaceutical composition further comprises - an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 32; and

[0074] - 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.

[0075] 36. A pharmaceutical composition according to any of clauses 31 to 35, further comprising an auxiliary peptide, preferably a peptide comprising or consisting of the amino acid sequence according to SEQ ID NO: 39.

[0076] 37. A kit comprising

[0077] - an antigenic peptide according to any one of clauses 1 to 15,

[0078] - an immunogenic compound according to any one of clauses 16 to 19,

[0079] - nanoparticles according to item 20,

[0080] - a cell according to item 21 or 22,

[0081] - a nucleic acid according to item 23 or 24,

[0082] - a host cell according to any one of clauses 25 to 27,

[0083] - T lymphocytes according to item 28,

[0084] - the antibody according to clause 29,

[0085] - a T cell receptor according to clause 30, or

[0086] - A pharmaceutical composition according to any of clauses 31-36.

[0087] 38. The kit according to clause 37, further comprising a package insert or instructions providing instructions on how to use the antigenic peptide, immunogenic compound, nanoparticle, cell, nucleic acid, host cell, cytotoxic T lymphocyte and / or pharmaceutical composition to prevent or treat cancer.

[0088] 39. A kit according to clause 37 or 38, wherein the kit comprises at least two different antigenic peptides according to any one of clauses 1-15.

[0089] 40. A combination of at least two different antigenic peptides according to any one of clauses 1 to 15 for use in preventing or treating cancer.

[0090] 41. An antigenic peptide according to any one of clauses 1 to 15 for use as a medicament.

[0091] 42. An antigenic peptide according to any one of clauses 1 to 15, for use in preventing or treating cancer.

[0092] 43. An antigenic peptide according to any one of clauses 1 to 15,

[0093] An immunogenic compound according to any one of clauses 16 to 19,

[0094] The nanoparticle according to clause 20,

[0095] The cell according to item 21 or 22,

[0096] The nucleic acid according to clause 23 or 24,

[0097] A host cell according to any one of clauses 25 to 27,

[0098] The T lymphocyte according to clause 28,

[0099] The antibody according to clause 29,

[0100] The T cell receptor according to clause 30,

[0101] The pharmaceutical composition according to any one of clauses 31 to 36,

[0102] A kit according to any one of clauses 37 to 39, or

[0103] A combination according to clause 40,

[0104] For medical use, in particular for the prevention and / or treatment of cancer.

[0105] 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

[0106] - an antigenic peptide according to any one of clauses 1 to 15,

[0107] - an immunogenic compound according to any one of clauses 16 to 19,

[0108] - nanoparticles according to item 20,

[0109] - a cell according to item 21 or 22,

[0110] - a nucleic acid according to item 23 or 24,

[0111] - a host cell according to any one of clauses 25 to 27,

[0112] - T lymphocytes according to item 28,

[0113] - the antibody according to clause 29,

[0114] - a T-cell receptor according to item 30,

[0115] - a pharmaceutical composition according to any one of clauses 31 to 36,

[0116] - a kit according to any of clauses 37 to 39, or

[0117] - Combination according to Article 40.

[0118] 45. A peptide-MHC (pMHC) multimer comprising an antigenic peptide according to any one of clauses 1-15.

[0119] definition

[0120] Unless otherwise defined herein, the scientific and technical terms used in this application should have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, the nomenclature used herein and cell and tissue culture techniques are well known and commonly used in the art.

[0121] Such techniques are fully explained in the literature, for example Owen et al. (Kuby Immunology, 7th edition, 2013 - WH Freeman) and Sambrook et al. (Molecular cloning: A laboratory manual, 4th edition, Cold Spring Harbor Laboratory Press - Cold Spring Harbor, NY, USA, 2012).

[0122] However, for the use of various terms in this specification, the following definitions apply more particularly.

[0123] The terms "peptide", "polypeptide", "protein" and variants thereof refer to peptides, oligopeptides, polypeptides or proteins comprising at least two amino acids, which are preferably linked to each other by conventional peptide bonds, or alternatively by modified peptide bonds, 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 by peptide bonds (-NHCO-). Peptides, polypeptides and proteins can play a structural and / or functional role in vitro and / or in vivo cells. The terms "peptide", "polypeptide" and "protein" preferably include amino acid chains ranging in size from 2 to at least about 1000 amino acid residues. The term "peptide" preferably includes amino acid chains less than about 30 amino acids in size herein, while the terms "polypeptide" and "protein" preferably include amino acid chains of at least 30 amino acids in size. The terms "polypeptide" and "protein" are used interchangeably herein. Preferably, the terms "peptide", "polypeptide", "protein" also include "peptoids", which are defined as peptide analogs containing non-peptide structural elements, which can simulate or antagonize the biological effects of natural parent peptides. Peptoids lack classical peptide features, such as enzymatically cleavable peptide bonds. In particular, peptides, polypeptides or proteins may contain amino acids other than the 20 amino acids defined by the genetic code, or may be composed of amino acids other than the 20 amino acids defined by the genetic code. In particular, peptides, polypeptides or proteins in the context of the present invention may also be composed of amino acids modified by natural processes (such as post-translational maturation processes) or chemical processes, which are well known to those skilled in the art. Such modifications are described in detail in the literature. These modifications may occur at any position of the polypeptide: in the peptide backbone, in the amino acid chain, or even at the carboxyl or amino terminus. In particular, peptides or polypeptides may be branched after ubiquitination, or may be cyclic with or without branches. This type of modification may be the result of natural or synthetic post-translational processes known to those skilled in the art. The terms "peptide", "polypeptide", "protein" in the context of the present invention also include modified peptides, polypeptides and proteins. For example, peptide, polypeptide or protein modifications can include acetylation, acylation, ADP-ribosylation, amidation, covalent immobilization of nucleotides or nucleotide derivatives, covalent immobilization of lipids or lipid derivatives, covalent immobilization of phosphatidylinositols, 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, sulfation, amino acid addition (e.g., arginylation) or ubiquitination.Such modifications are fully described in detail in the literature (Proteins Structure and Molecular Properties (1993) 2nd edition, E.C. Creighton, New York; Post-translational Covalent Modifications of Proteins (1983) ed. BC 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). Therefore, the terms "peptide", "polypeptide" and "protein" preferably include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, etc.

[0124] Preferably, the (poly)peptide or protein is a "classical" (poly)peptide or protein, wherein a "classical" (poly)peptide or protein is usually composed of amino acids selected from the 20 amino acids defined by the genetic code, which are linked to each other by normal peptide bonds.

[0125] 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 the precise continuity of natural nucleotides (e.g., A, T, G, C and U) or synthetic nucleotides, i.e., forming 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. Nucleic acids preferably include single-stranded, double-stranded or partially double-stranded DNA or RNA, preferably selected from genomic DNA (gDNA), complementary DNA (cDNA), ribosomal DNA (rDNA) and the transcription product of the 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; mini-genes, gene fragments, regulatory elements, promoters and combinations thereof. Examples of further preferred nucleic acids (molecules) and / or polynucleotides include, for example, recombinant polynucleotides, vectors, oligonucleotides, RNA molecules (e.g., rRNA, mRNA, or transfer RNA (tRNA)), or DNA molecules as described above. Thus, preferred nucleic acids (molecules) are DNA molecules or RNA molecules; 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, regulatory elements and / or promoters; vectors; and combinations thereof. A person skilled in the art is able to determine a nucleotide sequence that can encode a specific amino acid sequence.

[0126] The (poly)peptide and / or nucleic acid of the present invention can be prepared by any method known in the art, including but not limited to any synthetic method, any recombinant method, any in vitro generation method, etc., and any combination thereof. These technologies have been fully explained in the above-mentioned documents.

[0127] As used herein, the term "antigenic peptide" refers to a peptide that is easy to induce / trigger, increase, prolong or maintain an immune response in a subject to which the peptide 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 preferably has amino acid similarity with (a fragment / epitope of) a (human) tumor antigen. Importantly, the antigenic peptide shares the same core sequence with the corresponding (fragment / epitope) of the (human) tumor antigen. Preferably, the antigenic peptide induces / trigger, increases, prolongs or maintains an immune response that (also) targets the corresponding (fragment / epitope) of the (human) tumor antigen.

[0128] As used herein, the term "tumor antigen" includes tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). In general, the term "tumor antigen" or "tumor protein" refers to an antigenic substance in this article, which is produced in tumor cells and sometimes in normal cells, and can induce an immune response after administration in a subject. In humans, these antigens have been classified according to their expression pattern, function or genetic origin, including but not limited to overexpressed self-antigens (e.g., BIRC5); cancer testis (CT) antigens (e.g., MAGE-1); mutant antigens, also called new antigens (e.g., mutants from p53); tissue-specific differentiation antigens (e.g., melanoma antigen Melan A / MART-1); viral antigens expressed by tumor viruses (e.g., HPV, EBV); carcinoembryonic antigens (e.g., alpha-fetoprotein AFP and carcinoembryonic antigen CEA); and universal antigens (telomerase).

[0129] As used herein, the term "core sequence" refers to the amino acid in the middle of the sequence (also referred to as the "central amino acid" of the sequence), such as the amino acid in the middle of an antigenic peptide and / or (reference) epitope. Therefore, the core sequence is composed of all amino acids except the two most N-terminal amino acids and the two most C-terminal amino acids. For example, in a nine-amino acid peptide (such as the corresponding (fragment / epitope) of the antigenic peptide of the present invention or (human) tumor antigen), five middle amino acids represent the core sequence, and changes can only occur in any one of the two N-terminal and two C-terminal amino acid positions. Therefore, a "shared core sequence" (or "retained" core sequence) generally means that only mutations / differences are allowed to occur in the two most N-terminal amino acids and the two most C-terminal amino acids of a (reference) epitope / sequence.

[0130] As used herein, the term "microbiota" refers to the symbiotic microorganisms found in all multicellular organisms (from plants to animals) studied so far. In particular, it was found that the microbiota is essential for the immune homeostasis, hormone homeostasis and metabolic homeostasis of its host. The microbiota includes bacteria, archaea, protists, fungi and viruses. Therefore, 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) that is present in the microbiota, for example, in bacteria (for example, it may be contained in a microbiota protein, for example, in 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). Therefore, the antigenic peptide is preferably present in (for example, contained in) at least one protein expressed by the human microbiota.

[0131] Anatomically, microbiota are present on or in any of a number of tissues and biological fluids, including the skin, conjunctiva, breast, vagina, placenta, semen, uterus, follicles, lungs, saliva, oral cavity (particularly oral mucosa) and gastrointestinal tract, particularly the intestinal tract. In the context of the present invention, the microbiota sequence variants are preferably sequence variants of gastrointestinal microbiota (microorganisms residing in the gastrointestinal tract), more preferably sequence variants of intestinal microbiota (microorganisms residing in the intestinal tract). Therefore, most preferably, the microbiota sequence variants are (human) intestinal bacterial sequence variants (i.e., sequence variants of bacteria residing in the (human) intestinal tract).

[0132] Although microbiota can be found in and on many multicellular organisms (all multicellular organisms studied so far, from plants to animals), microbiota found in and on humans are preferred. Such microbiota are referred to herein as "human microbiota" (wherein the term human refers in particular to the location / residence of the microbiota). In the context of the present invention, microbiota sequence variants are human microbiota sequence variants.

[0133] The term "immunogenic compound" refers to a compound comprising an antigenic peptide of the present invention. An "immunogenic compound" is capable of inducing / eliciting, increasing, prolonging or maintaining an immune response to the antigenic peptide in a subject to which the compound is administered. In some embodiments, the immunogenic compound comprises at least one antigenic peptide, or alternatively comprises at least one compound comprising such an antigenic peptide, wherein the antigenic peptide or compound is connected to a protein (e.g., a carrier protein).

[0134] A "carrier protein" is generally a protein capable of transporting cargo (e.g., an antigenic peptide of the present invention). For example, a carrier protein can transport its cargo across a membrane. In the context of the present invention, a carrier protein particularly (also) comprises a peptide or polypeptide capable of eliciting an immune response to the antigenic peptide to which it is attached. Carrier proteins are known in the art.

[0135] Alternatively, such carrier peptides or polypeptides may be co-administered in the form of an immunoadjuvant.

[0136] Preferably, the antigenic peptides as described herein can be co-administered or linked to proteins / peptides having immunoadjuvant properties (e.g., providing stimulation of CD4+Th1 cells), e.g., by covalent or non-covalent bonds. Although the antigenic peptides as described herein are preferably bound to MHC class I, CD4+ helper epitopes can also be used to provide an effective immune response. Th1 helper cells can maintain effective dendritic cell (DC) activation and specific CTL activation by secreting interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2) and enhancing the expression of costimulatory signals on DC and T cells (Galaine et al., Interest of Tumor-Specific CD4 T Helper 1 Cells for Therapeutic Anticancer Vaccine. Vaccines (Basel). June 30, 2015; 3 (3): 490-502).

[0137] For example, the adjuvant peptide / protein may preferably be different from the antigenic peptide of the present invention. Preferably, the adjuvant peptide / protein is capable of evoked immune memory or provides nonspecific assistance, or may be a specific helper peptide. Several helper peptides for providing nonspecific T cell assistance are described in the literature, such as tetanus helper peptide, keyhole limpet hemocyanin peptide or PADRE peptide (Adotévi et al., Targeting antitumor CD4 helper T cells with universal tumor-reactive helper peptides derived from telomerase for cancer vaccine. Hum Vaccin Immunother. May 2013; 9(5): 1073-7, Slingluff CL, The present and future of peptide vaccines for cancer: single or multiple, long or short, alone or incombination. Cancer J. September-October 2011; 17(5): 343-50). Therefore, tetanus helper peptide, keyhole limpet hemocyanin peptide and PADRE peptide are preferred examples of such adjuvant peptides / proteins. This peptide represents another example of an auxiliary peptide (having immunoadjuvant properties) that is preferred in the context of the present invention. Another preferred example is h-pAgT13L (Bhasin M, Singh H, Raghava GP (2003) MHCBN: a comprehensive database of MHC binding and non-binding peptides. Bioinformatics 19: 665-666).Other examples of preferred auxiliary peptides include UCP2 peptides (e.g., as described in WO 2013 / 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, Adotévi 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 2012 Oct 2) and BIRC5 peptides (eg, as described in: EP2119726 A1 or Widenmeyer M, Griesemann H,. 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 JCancer. 2012 Jul 1;131(1):140-9. doi:10.1002 / ijc.26365. Epub 2011 Sep 14). 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).

[0138] As used herein, the term "immunogenic composition" refers to a composition that can trigger, induce, increase, prolong or maintain an immune response, especially when it is administered to a mammal, especially when it is administered to a human individual, it can trigger, induce, increase, prolong or maintain an immune response. Preferably, the immunogenic composition also contains one or more immune adjuvant substances.

[0139] "Pharmaceutically acceptable excipient or carrier" refers herein to a pharmaceutical grade compound that improves the delivery, stability or bioavailability of an active agent, and can be metabolized by a subject to which it is administered, and is non-toxic to a subject to which it is administered. Preferred excipients and carriers of the present invention include any excipient or carrier commonly used in medicines, such as water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof. In many cases, it will be preferred to include an isotonic agent in the composition, such as a sugar, a polyol (e.g., mannitol, sorbitol) or sodium chloride. Pharmaceutically acceptable excipients or carriers may also include a small amount of auxiliary substances, such as a wetting agent or an emulsifier or a preservative.

[0140] "Vaccine" herein refers to a composition capable of stimulating the immune system of a living organism, thereby providing protection against harmful antigens by prevention or by therapy. Preventive vaccines are preferred. Preferably, the vaccine or vaccine composition also comprises one or more immunoadjuvant substances.

[0141] According to various aspects and embodiments of the invention described herein, "subject" or "host" preferably refers to a mammal, most preferably a human. The subject may have cancer, be suspected of having cancer, or be at risk of having cancer.

[0142] As used herein, the term "cancer" refers to a malignant tumor. In particular, the term "cancer" refers to any member of a class of diseases or illnesses in this article, characterized in that cells divide uncontrollably, and these cells can directly grow into adjacent tissues by invasion or be implanted into distal sites by transfer to invade other tissues. Transfer is defined as the stage in which cancer cells are transported by blood flow or lymphatic system. It especially includes esophageal cancer, gastric cancer, duodenal cancer, small intestine cancer, appendix cancer, large intestine cancer, colon cancer, rectal cancer, colorectal cancer, anal cancer, pancreatic cancer, liver cancer, gallbladder cancer, spleen cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, endometrial cancer, ovarian cancer, vaginal cancer, vulvar cancer, breast cancer, lung cancer, thyroid cancer, thymus cancer, brain cancer, nervous system cancer, glioma, oral cancer, skin cancer, blood cancer, lymphoma, eye cancer, bone cancer, bone marrow cancer, muscle cancer etc. In the context of the present invention, melanoma, head and neck cancer, breast cancer, colorectal cancer or renal cancer (eg clear cell renal cell carcinoma) are preferred.

[0143] As used herein, the terms "preventing," "prevention," "prophylaxis," or "prevent" generally refer to avoiding or minimizing the onset or occurrence of a disease or condition before it occurs, while the terms "treating," "treatment," or "treat" include reducing, ameliorating, or curing a disease or condition (or symptoms of a disease or condition) after it has occurred. The term "prevention" includes "reducing the likelihood of occurrence" or "reducing the likelihood of recurrence."

[0144] 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 produce a beneficial or desired clinical result. A skilled artisan can readily determine the preferred effective amount for a given application, for example, taking into account the subject's size, age, weight, type of disease / disorder to be prevented or treated, and the amount of time since the onset of the disease / disorder. In the context of the present invention, an effective amount of a composition, with respect to prevention or treatment, is an amount sufficient to induce a humoral immune response and / or a cell-mediated immune response to the disease / disorder.

[0145] Throughout this specification and the claims that follow, unless the context requires otherwise, the term "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of stated members, integers or steps, but not the exclusion of any other unstated members, integers or steps. The term "consisting of is a specific embodiment of the term "comprising", in which any other unstated members, integers or steps are excluded. In the context of the present invention, the term "comprising" encompasses the term "consisting of". Thus, the term "comprising" encompasses "including" as well as "consisting of", for example, a composition "comprising" X may consist of X alone or may include additional things, for example, X+Y.

[0146] In the context of describing the present invention (especially in the context of the claims), the terms "a", "an", "the", and similar references should be interpreted as covering both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of numerical ranges herein is intended merely to serve as a shorthand method of referring individually to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.

[0147] The word "substantially" does not exclude "completely", for example, a composition "substantially free of" Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of the present invention.

[0148] The term "about" in connection with a numerical value x means x ± 10%.

[0149] Additional definitions are provided throughout the specification.

[0150] The present invention can be understood more readily by reference to the following detailed description, including preferred embodiments of the invention and the Examples included therein. Specific implementation plan

[0151] Although the present invention is described in detail below, it will be understood that the present invention is not limited to the specific methods, protocols and reagents as described herein, as these may vary. It will also be understood that the terms used herein are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art.

[0152] Hereinafter, the elements of the present invention will be described. These elements are listed together with specific embodiments, but it should be understood that they can be combined in any way and in any number to produce additional embodiments. The various described embodiments and preferred embodiments should not be construed as limiting the present invention to only the embodiments clearly described. This specification should be understood to support and encompass the embodiments that combine the embodiments clearly described with any number of disclosed elements and / or preferred elements. In addition, unless the context otherwise indicates, any arrangement and combination of all described elements in this application should be considered to be disclosed by the specification of the application.

[0153] Antigenic peptides of the present invention

[0154] 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: 1-16 and SEQ ID NOs: 40-42.

[0155] The present invention also provides an antigenic peptide comprising or consisting of an amino acid sequence shown in any one of SEQ ID NOs: 1-16 and SEQ ID NOs: 40-42, wherein, optionally, one or two amino acid residues may be replaced, deleted or added.

[0156] The inventors have identified a group of antigenic peptides that can be used to induce a specific immune response against tumor cells. These antigenic peptides are different from (fragments of) human tumor antigens, but have amino acid similarity, as shown in Table 1 below. In particular, the antigenic peptides of the present invention are contained in polypeptides and proteins produced by symbiotic bacteria from the human intestinal tract. Therefore, the antigenic peptides of the present invention are not human sequences, but bacterial 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 symbiotic bacteria from the intestinal tract), and these bacterial peptides have amino acid similarity with fragments of human tumor antigens. In particular, the antigenic peptides of the present invention can induce a stronger immune response than the corresponding human peptides, because the T cells that can strictly recognize human peptides have been exhausted in the maturation process because of recognition of self-antigens, which is not the case for the antigenic peptides of the present invention. This can explain why when the antigenic peptides described herein are applied to (human) individuals, these peptides can induce immune responses, especially T cell responses. Therefore, without being bound by any theory, the inventors hypothesize that proteins produced by commensal bacteria from the intestine can "mimic" tumor antigens and can be used to trigger a specific immune response against tumor cells. These findings further prove that commensal bacteria can contribute to the eradication of tumor cells.

[0157] Therefore, the present invention relates to antigenic peptides having amino acid similarity with tumor antigens. As used herein, the expression "having amino acid similarity with tumor antigens" refers in particular to sequence variants of fragments (epitopes) of (reference) human tumor antigens (e.g., CDC20 or other exemplary human tumor antigens described in Table 1 below).

[0158] A "sequence variant" generally has at least 50% sequence identity with a reference sequence (e.g. a fragment of a (reference) tumor antigen), in particular over the total length of the sequence. Preferably, a sequence variant has at least 70% or 75%, preferably at least 80% or 85%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 96% or 97%, and particularly preferably at least 98% or 99% sequence identity with a reference sequence (e.g. a fragment of a (reference) tumor antigen). Sequence identity can be calculated in a manner known in the art, in particular as described below. Preferably, the sequence variant retains a specific function of the reference sequence, such as its function as a tumor epitope and / or its ability to elicit or maintain an immune response. In particular, an amino acid sequence variant has an altered sequence in which one or more 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, for every 100 amino acids of the reference sequence, a variant sequence that is at least 90% identical has no more than 10 changes, i.e. any combination of deletions, insertions or substitutions.

[0159] Methods for comparing the identity (similarity) of two or more sequences are well known in the art. The percentage of two sequences that are identical can, for example, be determined using a mathematical algorithm. A preferred but non-limiting example of a mathematical algorithm that can be used is the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877. This algorithm is integrated in the BLAST family of programs, such as 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, accessible via the World Wide Web site NCBI homepage ncbi.nlm.nih.gov) and FASTA (Pearson (1990), Methods Enzymol. 183, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci. USA 85, 2444-2448). Sequences that are identical to other sequences to a certain extent can be identified by these programs. In addition, the programs in the Wisconsin Sequence Analysis Software Package, Version 9.1 (Devereux et al., 1984, Nucleic Acids Res., 387-395), such as BESTFIT and GAP programs, can also be used to determine the percentage identity between two polynucleotides and the percentage identity between two (multiple) peptide sequences. BESTFIT uses the "local homology" algorithm of Smith and Waterman (1981), J. Mol. Biol. 147, 195-197, to find the best single similarity region between two sequences.

[0160] In particular, the core sequence of the antigenic peptides of the present invention is identical to the core sequence of the epitope (fragment) sequence of the (human) reference tumor antigen. In addition, the core sequence shows high prevalence based on the frequency of proteins present in the human microbiome in which the core sequence is located.

[0161] Therefore, the core sequence is the main feature of the antigenic peptides of the present invention. Therefore, the inventors have identified core sequences that are highly interesting and have high prevalence, because these sequences are present in several sequence variants of (reference) tumor antigen fragments and / or in several human microbiota proteins that appear frequently in a considerable proportion of the general population. Preferably, in an antigenic peptide comprising or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 1-16 and SEQ ID NOs: 40-42, wherein optionally, one or two amino acid residues can be replaced, deleted or added, the core sequence is still retained.

[0162] Preferably, the antigenic peptide of 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, the 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.

[0163] In some embodiments, the antigenic peptide may be modified and / or may include a non-peptide bond (e.g., as described above). For example, the antigenic peptide may be modified at its N-terminus and / or C-terminus (e.g., labeled or connected to a carrier or substrate). Such modifications generally depend on the intended purpose and are well known in the art.

[0164] The antigenic peptides disclosed herein can be prepared using known techniques. For example, peptides can be prepared by synthesis through 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 peptides and non-peptides). 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 of the present invention are isolated antigenic peptides.

[0165] In some embodiments, the antigenic peptides of the present invention have the ability to bind to human major histocompatibility complex (MHC) molecules (e.g., MHC class I (MHC I) molecules); or - in an extended form, such as a length variant - the ability to bind to MHC class II (MHC II) molecules. Preferably, the antigenic peptides of the present invention can bind to MHC class I (major histocompatibility complex class I, MHC I) molecules.

[0166] MHC class I molecules present epitopes to killer T cells (also known as cytotoxic T lymphocytes, CTL). In addition to TCR (T cell receptor), CTL also expresses CD8 receptors. When the CD8 receptor of CTL docks to MHC class I molecules, if the TCR of CTL is suitable for the epitope within the MHC class I molecule, the CTL triggers the cell to undergo programmed cell death by apoptosis. This approach is particularly useful in preventing and / or treating cancer because cancer cells are directly attacked. In humans, there are three different gene sites encoding MHC class I molecules (human MHC molecules are also called human leukocyte antigens (HLA)): HLA-A, HLA-B and HLA-C. Therefore, 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 sites. For example, the antigenic peptides of the present invention can bind to HLA-A*01, HLA-A*02, HLA-A*24, and HLA-B*07 molecules. In some embodiments, the antigenic peptides of the present invention bind to HLA-A*02. Typically, peptides (epitopes) of 8-11 amino acids in length are presented by MHC I.

[0167] In general, the antigenic peptide of the present invention can be any length. Preferably, the length of the antigenic peptide of the present invention does not exceed 350 amino acids. For example, the maximum length of the antigenic peptide of the present invention can be 300 or 250 amino acids. More preferably, the maximum length of the antigenic peptide of the present invention does not exceed 200 amino acids, for example, does not exceed 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14 or 13 amino acids. In particular, the length of the antigenic peptide of the present invention is preferably up to 30 or 25 amino acids, more preferably up to 20 or 15 amino acids, wherein even more preferably a smaller peptide of 8 to 15 amino acids or 8-11 amino acids (e.g., 8, 9, 10 or 11 amino acids) in length; and still more preferably a peptide of 9 or 10 amino acids in length. In particular, the antigenic peptide is not a full-length protein produced by the symbiotic bacteria from the intestinal tract from which the antigenic peptide is derived. In other words, the antigenic peptide of the present invention is preferably a fragment of a full-length protein (produced by the human microbiota).

[0168] Similarly, a "fragment / epitope" of a (reference) tumor antigen, which is usually used as a reference sequence, preferably comprises 8 to 11 consecutive amino acids, preferably 9 or 10 amino acids, of the tumor antigen. It should be understood that a "fragment / epitope" of a (reference) tumor antigen is not a full-length tumor antigen (protein).

[0169] 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.

[0170] More generally, the present invention provides an antigenic peptide comprising or consisting of a microbiota sequence variant of a human tumor antigen fragment. The human tumor antigen may be selected from CDC20, KIF2C, UBE2C, ANKRD30A, AURKA, CDH17, CEACAM5, MMP11, OR51E2 and TOP2A. The fragment / epitope of a human (reference) tumor antigen may be selected from any one 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.

[0171] In some embodiments, the antigenic peptide induces T cell cross-reactivity against a human epitope of a (reference) tumor antigen.T cell cross-reactivity is a phenomenon of the immune system defined as the recognition of two or more peptide-MHC complexes (pMHC) by a T cell receptor (TCR).

[0172] Epitope mimicry involves the concept of sequence and structural similarities between foreign and self-antigens as a triggering mechanism for eliciting cross-reactive immune responses against self-antigens. Interestingly, such epitope mimicry provides a possible avenue to circumvent the restriction of the human T-cell repertoire due to the exhaustion of T-cell clones recognizing self-antigens.

[0173] In particular, antigens (i.e., antigenic peptides of the present invention) are different from self-antigens (e.g., human epitopes of tumor antigens), but have sequence similarity with self-antigens, (i) can still be recognized due to the cross-reactivity of T cell receptors, and (ii) such antigens are expected to be recognized by T cells / TCRs that are not exhausted during the T cell education process. Therefore, such antigens are able to trigger a strong immune response, resulting in the expansion of T cell clones with potential cross-reactivity with self-antigens.

[0174] The cross-reactivity of T cell receptors with human (reference) tumor antigen epitopes can be measured by ELISPOT-IFNγ assay, as shown in the Examples section. Briefly, HLA-A2 transgenic mice (e.g., HHDDR1 mice expressing human HLA-A2 and HLA-DR1 MHC and lacking murine 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 a primary injection on day 0 (d0), followed by a booster injection with the antigenic peptides of the present invention, for example, on day 14, or a booster injection with the corresponding human (reference) peptide in a control group. Thereafter, for example, seven days after the booster injection (i.e., on day 21), the mice can be sacrificed and spleen cells stimulated in vitro with the antigenic peptides of the present invention to assess their ability to secrete IFN-γ, as assessed by ELISPOT.

[0175] Table 1 below provides an overview of the antigenic peptides of the present invention and their amino acid sequences and SEQ ID NOs. Table 1 also provides information on the tumor antigens (also referred to herein as "human reference peptides") to which each antigenic peptide of the present invention relates. SEQ ID NOs: 1 to 16 and SEQ ID NOs: 40-42 refer to the HLA-A*02 antigenic peptides of the present invention.

[0176] Table 1. HLA-A*02 antigen peptides of the present invention.

[0177]

[0178]

[0179] In some embodiments, the antigenic peptide of the present invention is a sequence variant of a fragment of the tumor antigen CDC20 (human reference peptide), such as "SLPDRILDA" (SEQ ID NO: 17). Preferably, the antigenic peptide of the present 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.

[0180] In some embodiments, the antigenic peptide of 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 of 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 sequence shown in SEQ ID NO: 2 and SEQ ID NO: 40-42.

[0181] In some embodiments, the antigenic peptide of the present invention is a sequence variant of a fragment of the tumor antigen UBE2C (human reference peptide), such as "ALYDVRTIL" (SEQ ID NO: 19), "ALYDVRTILL" (SEQ ID NO: 38), "ILLSIQSLL" (SEQID NO: 20) or "RLQQELMTL" (SEQ ID NO: 21). More preferably, the antigenic peptide of 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. Still more preferably, the antigenic peptide of 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. Still more preferably, the antigenic peptide of the present invention is a sequence variant of the UBE2C fragment (human reference peptide) "RLQQELMTL" (SEQ ID NO: 21), for example, an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 6.

[0182] In some embodiments, the antigenic peptide of the present invention is a sequence variant of a fragment of the tumor antigen ANKRD30A (human reference peptide), such as "AVYSEILSV" (SEQ ID NO: 22), "KILDTVHSC" (SEQ ID NO: 23) or "SLDQKLFQL" (SEQ ID NO: 24). More preferably, the antigenic peptide of the present 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. Still more preferably, the antigenic peptide of the present 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. Still more preferably, the antigenic peptide of the present 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.

[0183] In some embodiments, the antigenic peptide of the present invention is a sequence variant of a tumor antigen AURKA (human reference peptide) fragment, such as "YLILEYAPL" (SEQ ID NO: 25). Preferably, the antigenic peptide of the present invention is a sequence variant of a tumor antigen AURKA fragment, such as an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 10.

[0184] In some embodiments, the antigenic peptide of the present invention is a sequence variant of a fragment of the tumor antigen CDH17 (human reference peptide), such as "LVIGIILAV" (SEQ ID NO: 26). Preferably, the antigenic peptide of the present 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.

[0185] In some embodiments, the antigenic peptide of the present invention is a sequence variant of a tumor antigen CEACAM5 (human reference peptide) fragment, such as "YLSGANLNL" (SEQ ID NO: 27). Preferably, the antigenic peptide of the present invention is a sequence variant of a tumor antigen CEACAM5 fragment, such as an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 12.

[0186] In some embodiments, the antigenic peptide of the present invention is a sequence variant of a tumor antigen MMP11 fragment (human reference peptide), such as "KVWSDVTPL" (SEQ ID NO: 28). Preferably, the antigenic peptide of the present invention is a sequence variant of a tumor antigen MMP11 fragment, such as an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 13.

[0187] In some embodiments, the antigenic peptide of the present invention is a sequence variant of the tumor antigen OR51E2 fragment (human reference peptide), such as "AQIGIVAVV" (SEQ ID NO: 29). Preferably, the antigenic peptide of the present invention is a sequence variant of the tumor antigen OR51E2 fragment, such as an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14.

[0188] In some embodiments, the antigenic peptide of the present invention is a sequence variant of the tumor antigen TOP2A fragment (human reference peptide), such as "ILNSTTIEI" (SEQ ID NO: 30) or "ALIFGQLLT" (SEQ ID NO: 31). More preferably, the antigenic peptide of the present 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. Still more preferably, the antigenic peptide of the present 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.

[0189] Preferably, the antigenic peptide of the present invention comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 1, 2 and 3. In some embodiments, the antigenic peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the antigenic peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the antigenic peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 3.

[0190] As shown in the examples herein, the specific antigenic peptides of the present invention allow for the induction of a strong immune response against themselves and, most importantly, they also allow for the induction of a strong immune response against peptides with amino acid similarities thereto (which are contained in tumor antigens), even though the human reference peptides contained in the tumor antigens may be tolerogenic.

[0191] Advantageously, the antigenic peptides of the present invention may be in the form of immunogenic compounds, particularly for use in the prevention or treatment of cancer.

[0192] Immunogenic compounds comprising the antigenic peptides of the present invention

[0193] In another aspect, the present invention also provides an immunogenic compound comprising the antigenic peptide of the present invention as described above. In particular, the preferred embodiments of the antigenic peptide as described above are also applicable to the immunogenic compound of the present invention.

[0194] Generally speaking, the term "immunogenic compound" includes all types of compounds comprising the antigenic peptide of the present invention. For example, the antigenic peptide of the present invention can be connected to a carrier molecule, or the antigenic peptide of the present invention can be contained in a polypeptide or protein (the polypeptide or protein can appear "alone", i.e., not connected to any other compound, or the polypeptide or protein comprising the antigenic peptide can be connected to a carrier molecule).

[0195] Those skilled in the art are familiar with the types of carrier molecules used to produce the immunogenic compounds of the present invention (e.g., an immunogenic compound comprising or consisting of a polypeptide of formula (I) linked to a carrier molecule). In particular, the function of the carrier molecule may be to provide cytokine assistance (or T cell assistance) to enhance the immune response against tumor antigens.

[0196] Preferably, the immunogenic compound of the present invention comprises an antigenic peptide and a carrier molecule, particularly wherein the antigenic peptide (or a polypeptide or protein comprising the antigenic peptide) is connected to a carrier molecule. A preferred carrier molecule is a carrier protein or a carrier peptide. According to a preferred embodiment, the antigenic peptide as defined above or the polypeptide / protein comprising the antigenic peptide is, for example, connected to a carrier protein or a carrier peptide by a covalent bond or a non-covalent bond. Alternatively, for example, such a carrier protein or a carrier peptide can be co-administered (separately) in the form of an immunoadjuvant (i.e., not as an "immunogenic compound", but as co-administration / combination therapy, as described herein below).

[0197] In some embodiments, the antigenic peptide as described herein or the polypeptide / protein comprising the antigenic peptide can be co-administered with a protein / peptide having immunoadjuvant properties (e.g., providing stimulation of CD4+Th1 cells), or, for example, connected to the protein / peptide having immunoadjuvant properties by a covalent bond or a non-covalent bond. Although the antigenic peptide as described herein is preferably bound to MHC class I, the CD4+ helper epitope can also be used to provide an effective immune response. Th1 helper cells can maintain effective dendritic cell (DC) activation and specific CTL activation by secreting interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2) and enhancing the expression of costimulatory signals on DC and T cells (Galaine et al., Interest of Tumor-Specific CD4 T Helper 1 Cells for Therapeutic Anticancer Vaccine. Vaccines (Basel). 2015 June 30; 3(3): 490-502).

[0198] For example, the adjuvant peptide / protein can be a non-tumor antigen that evokes immune memory or provides non-specific assistance, or can be a specific tumor-derived helper peptide. Several helper peptides for providing non-specific T cell assistance are described in the literature, such as tetanus helper peptide, keyhole limpet hemocyanin peptide or PADRE peptide (Adotévi et al., Targeting antitumor CD4 helper T cells with universal tumor-reactive helper peptides derived from telomerase for cancer vaccine. Hum Vaccin Immunother. May 2013; 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. September-October 2011; 17(5): 343-50). Therefore, tetanus helper peptide, keyhole limpet hemocyanin peptide and PADRE peptide are examples of such adjuvant peptides / proteins. In addition, the adjuvant peptide / protein can be a specific tumor-derived helper peptide. Specific tumor-derived helper peptides are usually presented by MHC class II, in particular by HLA-DR, HLA-DP or HLA-DQ. Specific tumor-derived helper peptides can be fragments of common overexpressed tumor antigen (e.g., HER2, NY-ESO-1, hTERT or IL13RA2) sequences. Such fragments are preferably at least 10 amino acids in length, 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 common overexpressed tumor antigen (e.g., HER2, NY-ESO-1, hTERT) fragment with a length of 13 to 24 amino acids is preferred. Preferred fragments bind to MHC class II and can therefore be identified using, for example, the IEDB (Immune Epitope Database and Analysis Resource; contract support provided by the National Institute of Allergy and Infectious Diseases, Health and Human Services Division, National Institutes of Health; URL: http: / / www.iedb.org / ; http: / / tools.iedb.org / mhcii / ) MHC class II binding prediction tool.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).

[0199] It is also preferred that the immunogenic compound of the present invention is a polypeptide or protein comprising the antigenic peptide of the present invention. Preferably, this protein or polypeptide is a recombinant protein or polypeptide, such as a fusion protein. The term "recombinant" means that it does not exist in nature. In some embodiments, the antigenic peptide of the present invention can be a part of a fusion protein, such as fusion with the N-terminal amino acids of the HLA-DR antigen-related invariant chain (Ii), or fused with an antibody (e.g., an antibody specific for dendritic cells) (or fused to an antibody sequence).

[0200] Preferably, the immunogenic compound of the present invention comprises or consists of a polypeptide of formula (I)

[0201] PepNt-CORE-PepCt (I)

[0202] in:

[0203] - "PepNt" consists of a polypeptide having a length of 0 to 500 amino acid residues, located at the N-terminus of the polypeptide of formula (I);

[0204] - "CORE" consists of the antigenic peptide of the invention as defined above; and

[0205] - "PepCt" consists of a polypeptide having a length of 0 to 500 amino acid residues, located at the C-terminus of the polypeptide of formula (I).

[0206] For example, the immunogenic compound may comprise or consist of a polypeptide of formula (Ia) or (Ib):

[0207] PepNt-CORE (Ia); or

[0208] CORE-PepCt (Ib)

[0209] wherein "PepNt" and "PepCt" and "CORE" are as defined above.

[0210] Preferably, the polypeptide of formula (I), (Ia) or (Ib) is a fusion peptide or fusion protein, in particular a recombinant fusion peptide or protein.

[0211] It is also preferred that the polypeptide or immunogenic compound as defined above comprises 9 to 1000 amino acids; including 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 , 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 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” may be defined accordingly, if applicable.

[0212] 43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108 , 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450 and 500 amino acid residues.

[0213] Preferably, the antigenic peptide is linked to a carrier molecule, in particular to a carrier protein, preferably by covalent or non-covalent bonds. The carrier molecule to which the peptide is optionally bound can be selected from a variety of known carriers. Examples of carrier molecules for vaccine purposes include proteins such as human serum albumin or bovine serum albumin and keyhole limpet hemocyanin (KLH) and fatty acids. Other embodiments of carrier molecules that can be covalently linked to the antigenic peptide of formula (I) include bacterial toxins or toxoids (e.g., diphtheria, cholera, E. coli heat-labile or tetanus toxoids), Neisseria meningitidis (N. meningitidis) outer membrane proteins (European Patent Application No. EP0372501), synthetic peptides (European Patent Application No. EP0378881 and EP0427347), heat shock proteins (PCT Application No. WO93 / 17712), pertussis proteins (PCT Application No. WO98 / 58668), protein D from Haemophilus influenzae (H. influenzae) (PCT Application No. WO00 / 56360) and toxin A or B from Clostridium difficile (C. difficile) (International Patent Application No. WO00 / 61761).

[0214] Furthermore, in the polypeptides according to formula (I), (Ia) or (Ib), "PepNt" and / or "PepCt" may preferably correspond to a protein / peptide having immunoadjuvant properties (eg providing stimulation of CD4+ Th1 cells as described herein).

[0215] In some embodiments, the antigenic peptide of the present invention (or the polypeptide / protein comprising the antigenic peptide) is covalently bound to the carrier molecule via a linker moiety. For example, the linker can be selected from GMBS (N-[γ-maleimidobutyryl-oxy] succinimide ester), sulfo-GMBS (N-[γ-maleimidobutyryloxy] sulfosuccinimide ester), SMPB (succinimidyl 4-[p-maleimidophenyl] butyrate) and sulfo-SMPB (sulfosuccinimidyl 4-[p-maleimidophenyl] butyrate).

[0216] Peptide-MHC (pMHC) multimers containing antigenic peptides

[0217] In another aspect, the present invention also provides a peptide-MHC (pMHC) multimer comprising the antigenic peptide of the present invention.

[0218] As used herein, the term "peptide-MHC multimer" (pMHC) refers to a stable multimeric complex consisting of major histocompatibility complex (MHC) protein subunits loaded with antigenic peptides of the present invention. In general, "MHC multimers" are oligomeric forms of MHC molecules. The main function of MHC molecules is to bind to antigens. According to the present invention, the antigen is an antigenic peptide of the present invention. Therefore, the complex of MHC proteins "loaded with" the antigenic peptide of the present invention generally means that the antigenic peptide of the present invention is bound to one or more MHC proteins. The "peptide-MHC multimer" (pMHC) of the present invention includes, but is 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 name intended to cover tissue compatibility antigen systems described in different species, including human leukocyte antigens (HLA). In humans, there are three major different gene loci encoding MHC class I molecules: HLA-A, HLA-B and HLA-C. HLA-A*01, HLA-A*02, and HLA-A*11 are examples of different MHC class I alleles that can be expressed from these loci.

[0219] In one embodiment of the invention, the pMHC multimer is a peptide / MHC class I multimer. In another specific 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 pHMC multimers are known in the art and are described, for example, in WO96 / 26962 and WO01 / 18053, which are incorporated herein by reference.

[0220] In addition to the MHC molecules and antigenic peptides of the present invention, pMHC may also contain other components, such as polymerizing agents and / or labels (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.

[0221] In one embodiment of the invention, the pMHC polymer can be used to visualize a T cell population that is specific to an MHC class I peptide complex as described above or an HLA / peptide complex corresponding to an MHC class I. For example, a pMHC polymer can be a polymer in which the MHC heavy chain is biotinylated, which allows for combination with streptavidin as a tetramer. Such pMHC tetramers have increased affinity for suitable TCR carrier T lymphocytes and can therefore be used to visualize reactive populations by immunofluorescence. In another embodiment of the invention, the pMHC polymer can be used to detect and / or separate a T cell population that is specific to a pMHC complex as described above by screening (in flow cytometry or by immunomagnetic screening).

[0222] Antigen peptide-specific T lymphocytes

[0223] On the other hand, the present invention also provides T lymphocytes specific to the antigenic peptides of the present invention, particularly cytotoxic T lymphocytes (CTLs) specific to the antigenic peptides of the present invention. T lymphocytes, particularly CTLs, are preferably activated (cytotoxic) T lymphocytes specific to the antigenic peptides of the present invention. The "specificity" of T lymphocytes is preferably understood as T lymphocytes binding to the antigenic peptides, and in addition binding to the tumor antigens corresponding to the antigenic peptides. In this regard, T lymphocytes, particularly CTLs, cross-react with the antigenic peptides and their tumor antigen counterparts, and generally show a high level of sequence identity or similarity with the antigenic peptides.

[0224] The present invention also provides a method for producing (cytotoxic) T lymphocytes specific for the antigenic peptides of the present invention (particularly activated (cytotoxic) T lymphocytes specific for the antigenic peptides of the present invention), the method comprising contacting T lymphocytes, particularly CTLs, with antigen-loaded human class I or class II MHC molecules expressed on the surface of an antigen presenting cell or an artificial construct simulating an antigen presenting cell in vitro, wherein the antigen is an antigenic peptide of the present invention. Preferred antigen presenting cells include dendritic cells. The artificial construct simulating an antigen presenting cell can be, for example, a peptide-MHC multimer of the present invention. The step of contacting T lymphocytes, particularly CTLs, with antigen-loaded human class I or class II MHC molecules expressed on the surface of an antigen presenting cell or an artificial construct simulating an antigen presenting cell can be carried out for a period of time sufficient to activate the T lymphocytes, particularly CTLs, in an antigen-specific manner. Preferably, the antigenic peptide is the preferred antigenic peptide as described above, for example, an antigenic peptide comprising or consisting of the 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).

[0225] The present invention also relates to activated T cells produced by the method of the present invention, wherein the T cells selectively recognize cells expressing polypeptides comprising the antigenic peptides of the present invention and / or polypeptides comprising the corresponding human reference peptides. In a specific embodiment, the T cells recognize cells expressing polypeptides comprising the antigenic peptides of the present invention and polypeptides comprising the corresponding human reference peptides, especially tumor cells overexpressing the corresponding TAA as described herein.

[0226] (Activated) T cells against the antigenic peptides of the present invention can be used for therapy. In particular, the activated T cells generated by the above method selectively recognize cells that abnormally express polypeptides comprising the amino acid sequences of SEQ ID NOs: 17-31 and 38 (i.e., tumor antigens), such as polypeptides comprising the amino acid sequences shown in any one of SEQ ID NOs: 17, 18, 19 and 38. In a specific embodiment, cells that abnormally express polypeptides comprising the amino acid sequences of SEQ ID NOs: 17-31 and 38 are tumor cells involved in the cancer to be treated.

[0227] Preferably, the T lymphocytes of the present invention specific to the antigenic peptides of the present invention may have (show / express) memory markers. Such memory markers are preferably memory markers of intestinal memory cells, such as CCR9, CXCR3, CD103, CX3CR1 and α4β7+.

[0228] After vaccination with the antigenic peptides of the invention (derived from human microbiota sequences), the T lymphocytes of the invention specific for the antigenic peptides of the invention are preferably more / strongly amplified compared to vaccination with peptides not derived from microbiota sequences, such as human (reference) sequences and / or synthetic peptides (e.g., including mutations, e.g., artificially introduced mutations). In other words, vaccination of a subject with the antigenic peptides of the invention preferably increases the number of T lymphocytes of the invention specific for said antigenic peptides of the invention compared to vaccination with corresponding human or synthetic peptides (not derived from microbiota) associated with the same reference epitope.

[0229] After vaccination, in a subject having the peptide (expressed by the subject's microbiota) in the intestine (e.g., the peptide can be found in the subject's stool sample, compared to a subject not having the peptide (not expressed by the subject's microbiota) in the intestine (e.g., a subject in whose stool sample the peptide cannot be detected), the T lymphocytes of the present invention specific for the antigenic peptide of the present invention are preferably more / stronger and / or more rapidly expanded. In particular, a subject having the peptide (expressed by the subject's microbiota) in the intestine may react faster (T cells expand faster) and / or have T cells from the desired type Tc1.

[0230] Cells loaded with antigenic peptides or immunogenic compounds

[0231] In another aspect, the present invention also provides a cell loaded with the antigenic peptide of the present invention or an immunogenic compound comprising the antigenic peptide of the present invention as described above. In particular, the preferred embodiments of the antigenic peptide as described above are also applicable to the cell of the present invention.

[0232] Preferred cells loaded with the antigenic peptide of the present invention or the immunogenic compound of the present invention are antigen presenting cells (APCs), more preferably dendritic cells (DCs).

[0233] APCs are particularly interesting because their main function is to process antigens and present them to T cells of the immune system on the cell surface, thereby initiating and regulating T cell responses in vivo. In the context of the present invention, it is preferred that APCs are loaded with antigenic peptides and / or immunogenic compounds of the present invention. This can be achieved by exposing APCs to the 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).

[0234] Preferred APCs of the present invention are dendritic cells (DCs). Combining at least one antigenic peptide or immunogenic compound of the present invention with DCs can indeed be advantageous, because DCs are the most effective APCs, and are reported to often have functional defects in cancer patients. Those skilled in the art can easily obtain DCs from healthy compatible donors (i.e., DCs are HLA-related) or from the patient himself (provided that DCs are functional (i.e., DCs are autologous)), for example, by direct separation from peripheral blood, or from peripheral blood cells (e.g., CD14+ monocytes or CD34+ hematopoietic precursors) (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 (e.g., S100, p55, CD83 and / or OX62), and therefore can be separated and purified based on the markers using cell culture techniques known in the art.

[0235] The present invention also relates to a method for preparing a cell loaded with the antigenic peptide of the present invention, wherein the antigenic peptide is loaded onto a class I or class II MHC molecule expressed on the surface of a cell (particularly an antigen presenting cell (or an artificial antigen presenting cell)), and the method comprises the step of contacting (a sufficient amount of) the antigenic peptide with the cell (particularly an antigen presenting cell).

[0236] Nucleic acid encoding antigenic peptide and host cell containing nucleic acid

[0237] On the other hand, the present invention also provides a nucleic acid encoding an antigenic peptide of the present invention, a polypeptide of formula (I) as defined above, or an immunogenic compound of the present invention, wherein the immunogenic compound is a peptide or a protein. In particular, the preferred embodiments of the above-mentioned antigenic peptides are also applicable to such nucleic acids of the present invention. For example, even more preferably, an antigenic peptide of the present invention comprising or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 1-16 and SEQ ID NOs: 40-42. For example, more preferably, an antigenic peptide of 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. For example, still more preferably, an antigenic peptide of the present invention comprising or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 1-3. Also preferred are combinations thereof, i.e., nucleic acids encoding different antigenic peptides of the present invention.

[0238] 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, regulatory elements, promoters and combinations thereof. Further examples of nucleic acids include recombinant polynucleotides, vectors, oligonucleotides, RNA molecules (e.g., rRNA, mRNA or tRNA) or the above-mentioned DNA molecules. Therefore, the nucleic acid (molecule) is preferably 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, regulatory elements and / or promoters; vectors; and combinations thereof.

[0239] In the field of treatment, diagnosis, reagents and bioassays, it is of great interest that nucleic acids (e.g., ribonucleic acids (RNA)) can be delivered into cells (whether in vitro, in vivo, in situ or ex vivo) to cause intracellular translation of nucleic acids and produce the encoded peptides of interest. The delivery and function of non-integrative polynucleotides are particularly important. Therefore, nucleic acids such as mRNA that are not integrated into the host chromosome are preferred. Generally, nucleic acids (e.g., mRNA) can be optimized to express the antigenic peptides of the present invention, for example, by methods known in the art, such as codon optimization. In addition, nucleic acids can be modified, for example, to enhance their stability, extend their lifespan and / or increase the expression of the antigenic peptides of the present invention. Therefore, optimized or modified mRNA (mmRNA) encoding the antigenic peptides of the present invention is preferred. mmRNA differs from wild-type mRNA in its functional and / or structural design features for optimal delivery of mRNA and / or for optimal expression of the antigenic peptides of the present invention (e.g., as described in WO 2013 / 151672A2, WO 2013 / 101690 A1, WO2013 / 052523A, which are incorporated herein by reference). Typically, nucleic acids can be delivered "naked" or associated with a carrier (e.g., a cationic carrier). Cationic carriers (positively charged) are often easily associated with negatively charged nucleic acids. The carrier can be any of any type including, for example, polymers, proteins, lipids, and nanoparticles. Cationic lipids and nanoparticles (particularly lipid nanoparticles, LNPs) are preferably used for nucleic acid delivery. Therefore, the present invention also provides nucleic acids as described herein associated with a carrier (e.g., a lipid, particularly a cationic lipid or LNP).

[0240] In some embodiments, the nucleic acid molecule can be a vector. The term "vector" used in the context of the present invention refers to a nucleic acid molecule, preferably an artificial nucleic acid molecule, i.e., a nucleic acid molecule that does not exist in nature. The vector in the context of the present invention is suitable for incorporating or accommodating a desired nucleic acid sequence. Such vectors can be storage vectors, expression vectors, cloning vectors, transfer vectors, etc. Storage vectors are vectors that allow convenient storage of nucleic acid molecules. Therefore, the vector can contain a sequence corresponding to, for example, a desired antigenic peptide of the present invention. An expression vector can be used to produce an expression product, such as RNA (e.g., mRNA) or a peptide, polypeptide, or protein. For example, an expression vector can contain a sequence required for transcription of a vector sequence segment, such as a promoter sequence. A cloning vector is typically a vector containing a cloning site, which can be used to incorporate a nucleic acid sequence into a vector. A cloning vector can be, for example, a plasmid vector or a phage vector. A transfer vector can be a vector suitable for transferring a nucleic acid molecule into a cell or an organism, such as a viral vector. The vector in the context of the present invention can be, for example, an RNA vector or a DNA vector. Preferably, the vector is a DNA molecule. For example, a vector in the sense of the present application comprises a cloning site, a selection marker (e.g., an antibiotic resistance factor) and a sequence suitable for vector proliferation (e.g., 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 generally capable of expressing a coding sequence, in particular an antigenic peptide of the present invention, a polypeptide of formula (I) as defined above, or an immunogenic compound of the present invention. A preferred vector is a vector for expression in a bacterial cell. More preferably, the vector can be used for expression in a so-called "live bacterial vaccine vector", in which live bacterial cells (e.g., bacteria or bacterial spores, e.g., endospores, exospores, or microbial cysts) can be used as a vaccine. A preferred example thereof is described in da Silva et al., Live bacteria vaccine vectors: an overview; Braz J Microbiol. March 4, 2015; 45(4): 1117-29.

[0241] The nucleic acid encoding the antigenic peptide of the present invention can be in the form of naked nucleic acid, or it can be cloned into a plasmid or a viral vector (Tregoning and Kinnear, Using Plasmids as DNA Vaccines for InfectiousDiseases.Microbiol Spectr.2014 December; 2(6).doi:10.1128 / microbiolspec.PLAS-0028-2014), the latter being particularly preferred. Examples of suitable viral vectors of the present invention include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses (AAV), herpes viruses, and poxvirus vectors. Those skilled in the art can use standard recombinant techniques in the art to clone nucleic acids into plasmids or viral vectors.

[0242] On the other hand, the present invention also provides a host cell comprising the nucleic acid of the present invention (particularly the above-mentioned expression vector). Also preferred are combinations thereof, i.e., host cells comprising different nucleic acids of the present invention (e.g., encoding different antigenic peptides of the present invention).

[0243] 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 can be preferably used to produce the antigenic peptide of the present invention or the immunogenic compound of the present invention. In addition, such a host cell can also be an active component in a vaccine. Preferably, the host cell is a bacterial cell, more preferably an intestinal bacterial cell. The term "intestinal bacterial cell" refers to bacteria residing in the (human) intestine. Such a bacterial host cell can be used as a "live bacterial vaccine carrier", wherein live bacterial cells (e.g., bacteria or bacterial spores, e.g., endospores, exospores or microbial cysts) can be used as vaccines. Its preferred example is described in da Silva et al., Live bacteria vaccine vectors: an overview; Braz J Microbiol. March 4, 2015; 45 (4): 1117-29. Bacterial cells (e.g., bacteria or bacterial spores, e.g., endospores, exospores or microbial cysts), in particular (whole) intestinal bacterial species, can be advantageous because they have the potential to induce a greater immune response than the (poly)peptides or nucleic acids they contain. Alternatively, the bacterial cells (particularly enteric bacteria) of the present invention can be in the form of probiotics (i.e. live enteric bacteria) and can be used as food additives because they can provide health benefits. These bacterial cells can be, for example, freeze-dried into granules, pills or capsules, or directly mixed with dairy products for consumption.

[0244] In some embodiments, the host cell may be an antigen presenting cell and a dendritic cell, in particular as described above. In some embodiments, the antigen presenting cell comprises the above-described expression vector of the present invention, in particular an expression vector capable of expressing or expressing the peptide containing the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 16 and SEQ ID NO: 40 to 42 or variants.

[0245] The present invention also relates to a method for producing the peptide of the present invention, the method comprising culturing the host cell of the present invention, and isolating the peptide from the host cell or its culture medium.

[0246] Nanoparticles containing antigenic peptides or immunogenic compounds

[0247] In another aspect, the present invention also provides a nanoparticle comprising, in particular loaded with,

[0248] - at least one antigenic peptide according to the invention, or

[0249] - at least one immunogenic compound according to the invention;

[0250] and, optionally, loaded with an adjuvant.

[0251] In particular, the preferred embodiments of the antigenic peptide described above also apply to the nanoparticles of the present invention.

[0252] Nanoparticles, in particular nanoparticles used 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.e Collection 2013, Review. In particular, nanoparticles are used to deliver antigenic peptides (or immunogenic compounds / polypeptides / proteins / nucleic acids comprising antigenic peptides), and can also optionally serve as adjuvants. Antigenic peptides (immunogenic compounds / polypeptides / proteins / nucleic acids comprising antigenic peptides) are typically encapsulated in nanoparticles or connected / bound to (decorated to) the surface ("coating") of nanoparticles. Compared with traditional methods, nanoparticles can protect the payload (antigen / adjuvant) from the surrounding biological environment, increase half-life, minimize systemic toxicity, promote delivery to APC, or even directly trigger the activation of TAA-specific T cells. Preferably, the size (diameter) of the nanoparticle does not exceed 300nm, more preferably does not exceed 200nm, and most preferably does not exceed 100nm. Such nanoparticles fully avoid being taken up by phagocytes, have high structural integrity and long circulation time in the circulation, can accumulate at the site of tumor growth, and can penetrate deep into the tumor mass.

[0253] Examples of nanoparticles include polymer nanoparticles, such as poly(ethylene glycol) (PEG) and poly(D,L-lactic-co-glycolic 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; immunostimulatory complexes (ISCOMs); virus-like particles (VLPs); and self-assembling proteins.

[0254] Polymer nanoparticles are nanoparticles based on / comprising polymers, such as poly(D,L-lactide-co-glycolide) (PLG), poly(D,L-lactic acid-co-glycolic acid) (PLGA), poly(γ-glutamic acid) (γ-PGA), poly(ethylene glycol) (PEG) and polystyrene. Polymer nanoparticles can encapsulate (entrap) antigens (e.g., antigenic peptides or (poly)peptides comprising them) or bind / couple to antigens (e.g., antigenic peptides or (poly)peptides comprising them). Polymer nanoparticles can be used for delivery, such as delivery to certain cells, or to maintain antigen release by their slow biodegradation rate. For example, g-PGA nanoparticles can be used to encapsulate hydrophobic antigens. Polystyrene nanoparticles can be coupled to a variety of antigens because they can be surface-modified with various functional groups. Polymers such as poly(L-lactic acid) (PLA), PLGA, PEG and natural polymers (e.g., polysaccharides) can also be used to synthesize hydrogel nanoparticles, which are nanoscale hydrophilic three-dimensional polymer networks. Nanogels have good properties, including flexible mesh size, large surface area for multivalent coupling, high water content and high antigen loading capacity. Therefore, preferred nanoparticles are nanogels, such as chitosan nanogels. Preferred polymer nanoparticles are nanoparticles based on / comprising PEG and PLGA.

[0255] Inorganic nanoparticles are nanoparticles based on / containing inorganic substances, and 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 provide rigid structures and controlled synthesis. For example, gold nanoparticles can be easily made into different shapes, such as spheres, rods, cubes. Inorganic nanoparticles can be surface-modified, such as surface-modified with carbohydrates. Carbon nanoparticles provide good biocompatibility and can be made into, for example, nanotubes or (mesoporous) spheres. For example, multiple copies of the antigenic peptide of the present invention (or (poly)peptides comprising it) can be coupled to carbon nanoparticles (e.g., carbon nanotubes). For oral administration, mesoporous carbon nanoparticles are preferred. Silica-based nanoparticles (SiNPs) are also preferred. SiNPs have biocompatibility and show excellent properties in terms of selective tumor targeting and vaccine delivery. The abundant silanol groups on the SiNP surface can be used for further modification to introduce additional functions, such as cell recognition, absorption of specific biomolecules, improved interaction with cells, and enhanced cell uptake. Mesoporous silica nanoparticles are particularly preferred.

[0256] Liposomes are usually formed from phospholipids, such as 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP). In general, cationic liposomes are preferred. Liposomes are self-assembled, having a phospholipid bilayer shell and an aqueous core. Liposomes can be generated as unilamellar vesicles (having a single phospholipid bilayer) or multilamellar vesicles (having several concentric phospholipid shells separated by aqueous layers). Thus, antigens can be encapsulated in the core or between different layers / shells. Preferred liposome systems are those approved for human use, such as V and

[0257] Immunostimulatory complexes (ISCOMs) are cage-like particles of about 40 nm (diameter), micelles containing colloidal saponins, such as made from the saponin adjuvant Quil-A, cholesterol, phospholipids and (poly)peptide antigens (e.g. antigenic peptides or polypeptides containing them). These spherical particles can trap antigens through non-polar interactions. Two types of ISCOMs have been described, both of which consist of cholesterol, phospholipids (usually phosphatidylethanolamine or phosphatidylcholine) and saponins (e.g. Quil-A).

[0258] Virus-like particles (VLPs) are self-assembled nanoparticles formed by the self-assembly of biocompatible capsid proteins. Due to the naturally optimized nanoparticle size and repeated structural order, VLPs can induce a potent immune response. VLPs can be derived from various viruses and range in size from 20nm to 800nm, usually in the range of 20-150nm. VLPs can be engineered to express additional peptides or proteins by fusing these peptides / proteins into the particles or by expressing multiple antigens. In addition, antigens can be chemically coupled to the surface of the virus to produce bioconjugated VLPs.

[0259] Examples of self-assembling proteins include ferritin and major vault protein (MVP). Ferritin is a protein that can self-assemble into a nearly spherical 10 nm structure. 96 MVP units can self-assemble into barrel-shaped vault nanoparticles with a size of about 40 nm wide and 70 nm long. After the antigen fused to the minimal interaction domain gene is mixed with MVP, it can be packaged in the vault nanoparticles through the self-assembly process. Therefore, the antigen (e.g., the antigenic peptide of the present invention or a polypeptide comprising it) can be fused with a self-assembling protein or its fragment / domain (e.g., the minimal interaction domain of MVP). Therefore, the present invention also provides a fusion protein comprising a self-assembling protein (or its fragment / domain) and an antigenic peptide of the present invention.

[0260] 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) polymer 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.

[0261] Preferably, the nanoparticle also comprises an adjuvant, such as a toll-like receptor (TLR) agonist. Therefore, the antigenic peptide (immunogenic compound / polypeptide / protein / nucleic acid comprising the antigenic peptide) can be delivered together with the adjuvant, such as delivered to antigen presenting cells (APC), such as dendritic cells (DC). The adjuvant can be encapsulated by the nanoparticle or combined / coupled to the surface of the nanoparticle, preferably similar to the antigenic peptide.

[0262] Particularly preferred adjuvants are polyinosinic acid:polycytidylic acid (also known as "poly I:C") and / or its derivative poly-ICLC. Poly I:C is a mismatched double-stranded RNA in which one strand is an inosinic acid polymer and the other strand is a cytidylic acid polymer. Poly I:C is an immunostimulatory agent known to interact with toll-like receptor 3 (TLR3). Poly I:C is structurally similar to double-stranded RNA, which is a "natural" stimulator of TLR3. Therefore, poly I:C can be considered a synthetic analog of double-stranded RNA. Poly-ICLC is a synthetic complex of carboxymethylcellulose, polyinosinic acid-polycytidylic acid and poly-L-lysine double-stranded RNA. Similar to poly I:C, poly-ICLC is also a ligand for TLR3. Poly I:C and poly-ICLC typically stimulate the release of cytotoxic cytokines. Preferred examples of poly-ICLC are

[0263] Antibodies that bind to antigenic peptides

[0264] In another aspect, the present invention provides antibodies directed against (ie, binding to) an antigenic peptide of the present invention or a complex of the peptide of the present invention with MHC.

[0265] As used herein, the term "antibody" encompasses various forms of antibodies, including but not limited to complete antibodies, antibody fragments (e.g., antigen binding fragments), human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies, and genetically engineered antibodies (variants or mutant antibodies), as long as the characteristic properties of the present invention (i.e., binding to antigenic peptides) 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. Antibodies generally comprise (at least) three complementary determining regions (CDRs) on the heavy chain and (at least) three CDRs on the light chain. In general, complementary determining regions (CDRs) are hypervariable regions present in the heavy chain variable domain and the light chain variable domain. Typically, the heavy chain of an antibody and the CDRs of the light chain connected together form an antigen receptor. Typically, three CDRs (CDR1, CDR2, and CDR3) are arranged non-continuously in the variable domain. Since antigen receptors are usually composed of two variable domains (on two different polypeptide chains, i.e., heavy chain and light chain: heavy chain variable region (VH) and light chain variable region (VL)), each antigen receptor usually has six CDRs (heavy chain: CDRH1, CDRH2 and CDRH3; light chain: CDRL1, CDRL2 and CDRL3). For example, a classic IgG antibody molecule usually has two antigen receptors and therefore contains twelve CDRs. The CDRs on the heavy chain and / or light chain can be separated by a framework region, wherein the framework region (FR) is a region in the variable domain that is less "variable" than the CDR. For example, the variable region (or each variable region, respectively) can be composed of four framework regions, separated by three CDRs, and the antibody can contain one or more constant regions. In some embodiments, the antibody comprises an Fc region.

[0266] For example, antibodies can be obtained by immunizing mammals (e.g., mice, rats, rabbits, goats, sheep or humans) with antigenic peptides of the present invention (or immunogenic compounds or nanoparticles of the present invention). Human antibodies can be isolated from (isolated) samples (e.g., blood samples) of humans.

[0267] Therefore, the present invention also relates to a method for immunizing a non-human animal with the antigenic peptide (or immunogenic compound or nanoparticle) of the present invention, the method comprising the following steps:

[0268] - contacting (immunizing) a non-human animal with the antigenic peptide (or immunogenic compound or nanoparticle) of the present invention,

[0269] 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) is used.

[0270] As used herein, "immunization" is to be understood as being non-therapeutic in nature, as it involves the production of antibodies in the non-human animal.

[0271] Non-human animals are generally suitable for producing antibodies. Preferably, the non-human animal is a non-human mammal, more preferably an animal selected from goats and rodents (eg, mice, rats and rabbits).

[0272] The present invention also relates to a method for producing (polyclonal) antibodies recognizing the antigenic peptides of the present invention, said method comprising the following steps:

[0273] - isolating (polyclonal) antibodies recognizing the antigenic peptide from a non-human animal that has been previously contacted (immunized) with the antigenic peptide (or immunogenic compound or nanoparticle) of the present invention,

[0274] Preferably, the contacting (immunization) is performed using 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).

[0275] The present invention also relates to a method for isolating cells that produce antibodies that recognize the antigenic peptides of the present invention, the method comprising the following steps:

[0276] - isolating cells from a non-human animal that has been previously contacted (immunized) with a JNK inhibitor of the present invention, said cells producing said antibodies that recognize said antigenic peptide,

[0277] Preferably, the contacting (immunization) is performed using 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

[0278] The cells are optionally immortalized.

[0279] The present invention also relates to a method for producing (monoclonal) antibodies recognizing the antigenic peptides of the present invention, said method comprising the following steps:

[0280] - isolating an antibody that recognizes the antigenic peptide of the present invention from a cell culture supernatant of cells producing the antibody,

[0281] More preferably, an antibody that recognizes an antigenic peptide according to any one of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 40 to 42,

[0282] The cells are optionally immortalized.

[0283] Those skilled in the art will appreciate that the methods of immunizing non-human animals disclosed herein and methods of producing (polyclonal) antibodies can be performed sequentially. Similarly, methods of immunizing non-human animals, methods of separating cells producing antibodies, and methods of producing (monoclonal) antibodies can be combined.

[0284] In another aspect, the present invention relates to an antibody that can be produced (and / or has been produced) by the method for producing polyclonal antibodies or monoclonal antibodies of the present invention, wherein the antibody recognizes at least one antigenic peptide of the present invention, preferably an antigenic peptide according to any one of SEQ ID NOs: 1-16 and SEQ ID NOs: 40-42. In some embodiments, the antibody does not recognize (bind to) the corresponding human peptide to a lesser extent.

[0285] The present invention also relates to cells isolated according to the above-mentioned method for isolating cells, which produce antibodies that recognize the antigenic peptides of the present invention, wherein the cells produce antibodies that preferably recognize the antigenic peptides of the present invention, preferably the antigenic peptides according to any one of SEQ ID NOs: 1-16 and SEQ ID NOs: 40-42.

[0286] Methods for testing the binding affinity of (monoclonal and / or polyclonal) antibodies are well known in the art. One possibility is to characterize the binding affinity of the antibody by an ELISA method using the antigenic peptides of the invention as target peptides.

[0287] T cell receptors that bind to antigenic peptides

[0288] The invention also relates to T cell receptors (TCRs), particularly soluble TCRs (sTCRs) and cloned TCRs, which can be engineered into autologous or allogeneic T cells, and methods of making these, as well as NK cells or other cells bearing or cross-reacting with said TCRs.

[0289] As used herein, a "T cell receptor" (TCR) is a protein complex on the surface of T cells (T lymphocytes) that is responsible for recognizing antigenic peptides bound to major histocompatibility complex (MHC) molecules.

[0290] In order to obtain T cells having T cell receptors that bind to the antigenic peptides of the present invention, for example, (isolated) samples (e.g., blood samples) of mammals (e.g., humans) can be screened to screen whether they bind to the antigenic peptides of the present invention (e.g., peptides according to any one of SEQ ID NOs: 1-16 and SEQ ID NOs: 40-42). Thus, the antigenic peptides can be used as target peptides.

[0291] Another aspect of the present invention is to provide a method for producing a soluble T cell receptor (sTCR) that recognizes a specific peptide-MHC complex. Such soluble T cell receptors can be produced by specific T cell clones, and their affinity can be increased by mutagenesis for the complementary determining region. For the purpose of selecting T cell receptors, 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 T cell receptors during phage display and in the case of actual use as drugs, the α and β chains can be linked, for example, by non-native disulfide bonds, other covalent bonds (single-chain T cell receptors) or by a dimerization domain (see Boulter JM et al., Stable, soluble T-cell receptor molecules for crystallization and therapeutics. Protein Eng 2003 Sep;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 Apr;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 Nov;8(11):2418-2423). T cell receptors can be connected to toxins, drugs, cytokines (see, e.g., US2013 / 0115191), domains for recruiting effector cells (e.g., anti-CD3 domains), etc., to perform specific functions on target cells. In addition, it can be expressed in T cells for adoptive transfer. More information can be found in WO 2004 / 033685A1 and WO 2004 / 074322A1. Combinations of sTCRs are described in WO 2012 / 056407A1. Additional production methods are disclosed in WO 2013 / 057586A1.

[0292] Pharmaceutical composition

[0293] In another aspect, the present invention also provides a pharmaceutical composition comprising at least one of the following:

[0294] - an antigenic peptide of the invention as described herein,

[0295] - an immunogenic compound of the invention as described herein,

[0296] - a nanoparticle of the invention as described herein,

[0297] - a cell of the invention as described herein,

[0298] - a nucleic acid of the invention as described herein,

[0299] - a host cell of the invention as described herein,

[0300] - a T-lymphocyte according to the invention as described herein,

[0301] - an antibody of the invention as described herein, and / or

[0302] - a T-cell receptor of the invention as described herein,

[0303] and, optionally, one or more pharmaceutically acceptable excipients or carriers.

[0304] In particular, the preferred embodiments of the antigenic peptide described above are also applicable to the pharmaceutical composition of the present invention.

[0305] Also preferred are combinations thereof, i.e. pharmaceutical compositions comprising different antigenic peptides of the present invention. For example, a pharmaceutical composition may comprise

[0306] - at least two different antigenic peptides of the invention as described herein,

[0307] - at least two different immunogenic compounds of the invention as described herein,

[0308] - at least two different nanoparticles of the invention as described herein,

[0309] - at least two different cells of the invention as described herein,

[0310] - at least two different nucleic acids of the invention as described herein,

[0311] - at least two different host cells of the invention as described herein,

[0312] - at least two different T-lymphocytes of the invention as described herein,

[0313] - at least two different antibodies of the invention as described herein, and / or

[0314] - at least two different T-cell receptors of the invention as described herein.

[0315] Therefore, the pharmaceutical composition may comprise at least "two different components", preferably three, four or five different components (of the pharmaceutical composition of the present invention). Generally speaking, as used herein, the expression "different components" refers to

[0316] (1) a first component, e.g., an antigenic peptide of the invention as described herein, an immunogenic compound of the invention as described herein, a nanoparticle of the invention as described herein, a cell of the invention as described herein, a nucleic acid of the invention as described herein, a host cell of the invention as described herein, a T lymphocyte of the invention as described herein, an antibody of the invention as described herein, and / or a T cell receptor of the invention as described herein; and

[0317] (2) at least one other component (which is different from the first component; and in the case of more than two different components, each component is different from the other), for example, different antigenic peptides of the present invention as described herein, different immunogenic compounds of the present invention as described herein, different nanoparticles of the present invention as described herein, different cells of the present invention as described herein, different nucleic acids of the present invention as described herein, different host cells of the present invention as described herein, different T lymphocytes of the present invention as described herein, different antibodies of the present invention as described herein and / or different T cell receptors of the present invention as described herein; or one or more human tumor antigens (fragments) in any form ("naked", as an immunogenic compound as described herein, a nanoparticle as described herein, a (host) cell as described herein or a nucleic acid as described herein).

[0318] The "different 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 different components may also be used to prevent and / or treat said cancer, particularly if administered alone (rather than in combination as described herein) - although the combination (i.e. combined administration) will generally enhance its preventive and / or therapeutic effect (e.g. immune response), preferably in a synergistic manner.

[0319] Preferably, the "different components" are of the same type (e.g., different antigenic peptides, different immunogenic compounds, different nanoparticles, different nucleic acids, different (host) cells, different T-cell receptors, different antibodies or different T-lymphocytes) and differ from each other only in that they involve different antigenic peptides of the invention as described herein.

[0320] Preferably, the composition may comprise at least three, four or five different active ingredients, which are preferably of the same type but differ (only) in that they each relate to a different antigenic peptide.

[0321] More preferably, the composition may comprise at least three, four or five different active ingredients, which are preferably of the same type but differ (only) in that they each relate to a different antigenic peptide, wherein

[0322] - the first component relates to antigenic peptides comprising or consisting of (microbiota) sequence variants of fragments of the human tumor antigen CDC20;

[0323] - a (different) second component relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen KIF2C; and

[0324] - The (different) third component relates to antigenic peptides comprising or consisting of (microbiota) sequence variants of fragments of the human tumor antigen UBE2C.

[0325] Preferably, this composition may also comprise

[0326] - a (different) fourth component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen BIRC5; and / or

[0327] - A (different) fifth component, which relates to antigenic peptides comprising or consisting of (microbiota) sequence variants of fragments of the human tumor antigen FOXM1.

[0328] Table 2 below provides a list of additional peptides that can be used in combination with the peptides of the present invention (described above). SEQ ID NOs: 32 to 34 also refer to HLA-A*02 antigen peptides.

[0329] Table 2. Additional HLA-A*02 antigenic peptides that can be used in combination with the antigenic peptides of the present invention.

[0330]

[0331] It will be appreciated 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).

[0332] Therefore, the pharmaceutical composition preferably comprises

[0333] - a first component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 17;

[0334] - a (different) second component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 18;

[0335] - a (different) third component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 19;

[0336] - optionally, a (different) fourth component relating to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 35; and

[0337] - Optionally, a (different) fifth component relating to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 36.

[0338] More preferably, the pharmaceutical composition preferably comprises

[0339] - A first component, which involves an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0340] - a (different) second component, which relates to an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0341] - a (different) third component, which relates to an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3;

[0342] - optionally, a (different) fourth component, which relates to an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 32, and

[0343] - Optionally, a (different) fifth component which relates to an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 33.

[0344] Even more preferably, the pharmaceutical composition preferably comprises

[0345] - an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0346] - an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0347] - an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3;

[0348] -optionally, an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 32, and

[0349] - Optionally, an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 33.

[0350] It will be appreciated that the pharmaceutical composition 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.

[0351] Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients or carriers.

[0352] The pharmaceutical composition of the present invention can be any form suitable for the purpose of the present invention. For example, the composition can be a form suitable for parenteral, enteral or topical administration, such as a liquid suspension, solid dosage form (granules, pills, capsules or tablets) or a paste or gel. Those skilled in the art can select the appropriate composition form for the intended purpose.

[0353] The composition of the present invention may also include other active agents, such as active agents that can enhance the effect of antigenic peptides or immunogenic compounds. Alternatively, the composition may not include any other active agent (i.e., except antigenic peptides of the present invention, immunogenic compounds of the present invention, nanoparticles of the present invention, cells of the present invention, nucleic acids of the present invention and / or host cells of the present invention).

[0354] The pharmaceutical composition 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 the antigenic peptide of the present invention or the immunogenic compound of the present invention contained in the composition. Preferably, the pharmaceutical composition also comprises one or more immunoadjuvant substances. Pharmaceutical compositions, in particular immunogenic compositions, may also be referred to as "vaccine compositions" in this specification.

[0355] Preferably, the pharmaceutical composition further comprises at least one immunostimulant, particularly in order to increase, enhance, prolong or maintain the immune response mediated by the antigenic peptide. Preferred immunostimulants of the present invention include but are not limited to immune adjuvants, antigen presenting cells and combinations thereof. Preferably, the immunostimulant is an immune adjuvant or an antigen presenting cell (APC).

[0356] Preferably, the immunostimulant is an immunoadjuvant. Some immunoadjuvants can promote and prolong the time of interaction between antigen and immune system, while other immunoadjuvants can recruit and activate innate immune cells, thereby inducing adaptive response. Adjuvants belonging to the former class include but are not limited to mineral compounds, such as alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide; and oil-based emulsions, such as paraffin oil, starch oil, Freund's complete / incomplete adjuvant (FCA / FIA), saponin (e.g., from plant soap bark tree (Quillaja), soybean, Polygala senega (Polygala senega)). Adjuvants belonging to the latter category include, but are not limited to, immunostimulatory complexes (ISCOMs), such as cytokines (e.g., GM-CSF; interleukins, such as IL-1, IL-2, IL6, IL8, or IL12; tumor necrosis factor (TNF), such as TNFα or TNFβ; interferon IFN, such as IFNα, IFNβ, IFNγ, or IFNδ, etc.); ligands of toll-like receptors (TLRs), such as imiquimod, resiquimod, or MPL; exosomes, such as exosomes derived from dendritic cells (DCs) or tumor cells; bacterial products, such as heat shock proteins (HSPs, such as gp96, hsp90, hsp70, calreticulin, hsp110, hsp170), pathogen-associated molecular patterns (PAMPs), trehalose dimycolate (TDM), muramyl dipeptide (MDP); polysaccharides (PLS), such as polysaccharide-K.

[0357] More preferably, the immunoadjuvant is a protein / peptide having immunoadjuvant properties (e.g., providing stimulation of CD4+Th1 cells), as described herein ("helper" peptide). This can be a non-tumor antigen that evokes immune memory or provides nonspecific help, or it can be a helper peptide of specific tumor origin, such as a tetanus helper peptide, a keyhole limpet hemocyanin peptide, or a PADRE peptide. Another example is a helper peptide of specific tumor origin that can be presented by MHC II (particularly by HLA-DR, HLA-DP, or HLA-DQ), such as a fragment of a shared overexpressed tumor antigen, such as HER2, NY-ESO-1, hTERT, or IL13RA2. In some embodiments, the immunoadjuvant can be a HHD-DR3 peptide or h-pAg T13L (Bhasin M, Singh H, Raghava GP (2003) MHCBN: a comprehensive database of MHC binding and non-bindingpeptides. Bioinformatics 19: 665-666). Preferably, the helper peptide is the UCP2 peptide (SEQ ID NO: 39).

[0358] Preferably, the pharmaceutical composition comprises at least two different antigenic peptides of the present invention and one helper peptide, preferably the UCP2 peptide (SEQ ID NO: 39).

[0359] Preferably, the pharmaceutical composition comprises the first antigenic peptide of the present invention, which comprises or consists of a sequence variant of a fragment of the human tumor antigen CDC20; the second antigenic peptide of the present invention, which comprises or consists of a sequence variant of a fragment of the human tumor antigen KIF2C; the third antigenic peptide of the present invention, which comprises or consists of a sequence variant of a fragment of the human tumor antigen UBE2C; the fourth antigenic peptide of the present invention, which comprises or consists of a sequence variant of a fragment of the human tumor antigen BIRC5; the fifth antigenic peptide of the present invention, which comprises or consists of a sequence variant of a fragment of the human tumor antigen FOXM1; and an auxiliary peptide.

[0360] 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), for example, an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3; 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 shown in SEQ ID NO: 40. An antigenic peptide comprising or consisting of the amino acid sequence shown in 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 shown in SEQ ID NO:33; and an auxiliary peptide, preferably a UCP2 peptide (SEQ ID NO:39).

[0361] Even more preferably, the pharmaceutical composition comprises five different antigenic peptides, wherein the antigenic peptides comprise or consist of the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 32 and 33. In addition, the pharmaceutical composition may further comprise the UCP2 peptide shown in SEQ ID NO:39.

[0362] In some embodiments, the pharmaceutical composition does not comprise additional antigenic peptides (other than those described above).

[0363] In some embodiments, the pharmaceutical composition comprises polyinosinic acid:polycytidylic acid (also known as "poly I:C") and / or its derivative poly-ICLC (as an immunoadjuvant). Poly I:C is a mismatched double-stranded RNA in which one strand is an inosinic acid polymer and the other strand is a cytidylic acid polymer. Poly I:C is an immunostimulator known to interact with toll-like receptor 3 (TLR3). Poly I:C is structurally similar to double-stranded RNA, which is a "natural" stimulator of TLR3. Therefore, poly I:C can be regarded as a synthetic analog of double-stranded RNA. Poly-ICLC is a synthetic complex of carboxymethyl cellulose, polyinosinic acid-polycytidylic acid and poly-L-lysine double-stranded RNA. Similar to poly I:C, poly-ICLC is also a ligand for TLR3. Poly I:C and poly-ICLC typically stimulate the release of cytotoxic cytokines. Preferred examples of poly-ICLC are

[0364] More preferably, the pharmaceutical composition comprises Montanide, such as Montanide ISA 51VG and / or Montanide ISA 720VG. When these adjuvants are mixed with a water-based antigen medium, a stable water-in-oil emulsion is formed. Montanide ISA 51VG is based on a mixture of mannitol monooleate surfactant and mineral oil, while Montanide ISA 720VG 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 M-E. Safety data of Montanide ISA 51VG and Montanide ISA 720VG, 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).

[0365] In some embodiments, the pharmaceutical composition may also include at least one anticancer therapeutic agent. On the other hand, the present invention relates to a combination of a pharmaceutical composition of the present invention and at least one anticancer therapeutic agent. The pharmaceutical composition may include an anticancer therapeutic agent (as a combined preparation); or the pharmaceutical composition of the present invention and the anticancer therapeutic agent may be provided in a separate manner, for example as a kit of parts. Therefore, the pharmaceutical composition of the present invention and the anticancer therapeutic agent may be administered simultaneously, separately or sequentially. In other words, the present invention proposes the combined use of the pharmaceutical composition of the present invention and at least one anticancer therapeutic agent for simultaneous, separate or sequential administration.

[0366] Therefore, the therapeutic agent is preferably capable of preventing and / or treating the same cancer type as the antigenic peptide of the present invention. Preferably, the anti-cancer therapeutic agent is selected from antibodies, CAR-T cells, tumor cell lysates, chemotherapeutic agents, radiotherapeutic agents, immune checkpoint regulators and combinations thereof.

[0367] Antibodies are particularly advantageous in cancer therapy because they can bind to specific antigens on the surface of cancer cells, thereby directing therapy to the tumor (i.e., these are referred to as tumor-targeting antibodies), or block immune checkpoints that are dysregulated in cancer (i.e., these are referred to herein as immunomodulatory antibodies). The purpose of the latter class of antibodies is to inhibit cancer immune resistance, which is particularly observed to be directed against T cells specific for tumor antigens. In fact, as is well known in the art, under normal physiological conditions, immune checkpoints are essential for maintaining self-tolerance (i.e., preventing autoimmunity) and protecting tissues from damage when the immune system responds to pathogen infection. However, in cancer, immune checkpoint expression may be dysregulated, which is an important mechanism of immune resistance. Resistance to the PD-L1 checkpoint has been observed in particular in melanoma, ovarian cancer, lung cancer, glioblastoma, breast cancer, and pancreatic cancer (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 formant 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 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 cancerimmunotherapy. Nat Rev Cancer. 2012 Mar 22;12(4):252-64).

[0368] Antibodies are typically used for these purposes in the form of naked monoclonal antibodies (ie, unconjugated) or conjugated to other molecules, which may be toxic to cells or radioactive.

[0369] Examples of well-known monoclonal tumor-targeting antibodies used for cancer immunotherapy include, but are not limited to, alemtuzumab (chronic lymphocytic leukemia), bevacizumab (colorectal cancer, glioblastoma multiforme, cervical cancer, lung cancer, kidney cancer), brentuximab / vedotin (lymphoma), blinatumumab (acute lymphocytic leukemia), catumaxomab (malignant ascites in EPCAM+ cancers), cetuximab (cetoxan) (cancer), and scleroderma (splenic leukemia). tuximab (head and neck cancer, colorectal cancer), denosumab (breast cancer, prostate cancer, and bone cancer), gemtuzumab / ozogamicin (acute myeloid leukemia), ibritumomab / tiuxetan (non-Hodgkin lymphoma), panitumumab (colorectal cancer), pertuzumab (breast cancer), obinutuzumab (chronic lymphocytic leukemia), disease), ofatumumab (chronic lymphocytic leukemia), ipilimumab (melanoma), ramucirumab (gastric and gastroesophageal cancers), rituximab (chronic lymphocytic leukemia and non-Hodgkin lymphoma), siltuximab (multicentric Catsleman disease), tositumomab (non-Hodgkin lymphoma), and trastuzumab (breast cancer, gastric cancer and gastroesophageal cancer); while examples of immunomodulatory antibodies include, but are not limited to: ipilimumab (melanoma), which blocks the CTLA4-dependent immune checkpoint; nivolumab (melanoma, lung cancer) and prembrolizumab (melanoma), both of which block the PDCD1-dependent immune checkpoint; and MPDL3280A, MEDI4736, MEDI0680, and MSB0010718C, all of which block the PD-L1-dependent immune checkpoint (Sharma and Allison, The future of immune checkpoint therapy. Science. 2015 Apr 3;348(6230):56-61).

[0370] Other descriptions of antibodies used for cancer immunotherapy are found in Buqué et al., Trial Watch: Immunomodulatory monoclonal antibodies for oncological indications. Oncoimmunology. 2015 Mar 2;4(4):e1008814. eCollection 2015 Apr; Redman et al., Mechanisms of action of therapeutic antibodies for cancer. Mol Immunol. 2015 Oct;67(2Pt A):28-45; Simpson and Caballero, Monoclonal antibodies for the therapy of cancer MC Proc. 2014;8(Suppl 4):O6 and on the Antibody Society website (a list of therapeutic monoclonal antibodies approved or under review in the European Union or the United States is available at the following web link: http: / / www.antibodysociety.org / news / approved_mabs.php).

[0371] Adoptive cellular immunotherapy using chimeric antigen receptor (CAR) T cells has changed the treatment landscape for B-cell non-Hodgkin lymphoma (NHL), especially for aggressive B-cell lymphomas. For example, CD19-targeted CAR T cells represent a new standard of care for patients with refractory DLBCL who have previously received at least two lines of therapy. Two CAR T-cell products, axicabtagene ciloleucel (axi-cel) (KTE-019) and YESCARTA TM ) and tisagenlecleucel (CTL019) (KYMRIAH TM ) has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of refractory DLBCL after two lines of therapy. The third product, lisocabtagene maraleucel (liso-cel) (JCAR017), is currently being evaluated in clinical trials. Other CAR T cells include CD20-CAR-T cells.

[0372] Tumor cell lysate can also be combined with antigenic peptides of the present invention. Tumor cells can indeed initiate (prime) immune response by presenting endogenous peptide-MHC complexes and by host's dendritic cells (DC), and dendritic cells can process and present the antigen delivered by the lysate. Thus the antigen range of inducible immune response can be increased. Tumor cells can be processed by heat shock and / or chemical treatment, thereby easily obtaining tumor cell lysate, and can be autologous (i.e., separated from the patient) or allogeneic (i.e., separated from another experimenter).

[0373] Standard chemotherapeutic drugs and radiotherapeutic agents need not be described further here, as they have been described in detail 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).

[0374] Preferably, the immune checkpoint regulator combined with the antigenic peptide defined herein is an activator or inhibitor of one or more immune checkpoint molecules selected from the group consisting of 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 of its ligands.

[0375] More preferably, immune checkpoint regulator is an activator of (co) stimulatory checkpoint molecule or an inhibitor of inhibitory checkpoint molecule or a combination thereof. Therefore, immune checkpoint regulator is more preferably an activator of (i) CD27, CD28, CD40, CD122, CD137, OX40, GITR and / or ICOS or an inhibitor of (ii) 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.

[0376] Even more preferably, the immune checkpoint regulator is an inhibitor of an inhibitory checkpoint molecule (but preferably not an inhibitor of a stimulatory checkpoint molecule). Therefore, the immune checkpoint regulator 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 its ligand.

[0377] Preferably, the checkpoint regulator used in combination with the antigenic peptide defined herein can be selected from known regulators of the CTLA-4 pathway or the PD-1 pathway. More preferably, the immune checkpoint regulator is an inhibitor of CTLA-4, PD-L1, PD-L2 or PD-1; even more preferably, an inhibitor of the PD-1 pathway.

[0378] Those of ordinary skill in the art can select suitable immunological anticancer therapeutic agents for the purposes of the present invention. For example, if it is desired to prevent or treat melanoma, lysates from melanoma cells and / or the antibody ipilimumab, as well as suitable antigenic peptides, may be preferably used. Suitable antigenic peptides may be selected by (i) selecting suitable tumor antigens known in the art for a certain type of cancer and (ii) selecting suitable antigenic peptides of the present invention for the selected tumor antigen, as described above, for example, as described in Table 1.

[0379] Anticancer therapeutic agents may also be administered in combination with the compositions of the invention, either simultaneously, separately or sequentially. If the composition and therapeutic agent are administered separately or sequentially, they may be administered in different pharmaceutical forms.

[0380] Therefore, in another aspect, the present invention relates to a composition of the present invention and at least one anticancer 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 anticancer therapeutic agent as described above, for simultaneous, separate or sequential administration.

[0381] Complete kit

[0382] In another aspect, the present invention also provides a kit of parts (also referred to herein as a "kit") comprising at least one of the following:

[0383] - an antigenic peptide of the invention as described herein,

[0384] - an immunogenic compound of the invention as described herein,

[0385] - a nanoparticle of the invention as described herein,

[0386] - a cell of the invention as described herein,

[0387] - a nucleic acid of the invention as described herein,

[0388] - a host cell of the invention as described herein,

[0389] - a T-lymphocyte according to the invention as described herein,

[0390] - an antibody of the invention as described herein,

[0391] - a T cell receptor of the invention as described herein, and / or

[0392] -A pharmaceutical composition according to the invention as described herein.

[0393] In particular, the preferred embodiments of the antigenic peptide described above are also applicable to the kit of the present invention.

[0394] Combinations thereof, ie kits comprising different antigenic peptides of the invention are also preferred. In particular, the kit of parts of the invention may comprise more than one of the above components, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 different components. For example, the kit of parts of the present invention may comprise at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) different immunogenic compounds, at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) different antigenic peptides, at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) different nanoparticles, 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, etc. Preferably, such different components contained in the above kit of parts differ in terms of the antigenic peptides of the present invention, for example, one component relates to a first antigenic peptide and one component relates to a second antigenic peptide (different from the first antigenic peptide). For example, the kit may include at least two different immunogenic compounds of the invention. For example, the kit may include at least two different antigenic peptides of the invention. For example, the kit may include at least two different nanoparticles of the invention. For example, the kit may include at least two different nucleic acids of the invention. For example, the kit may include at least two different cytotoxic T lymphocytes of the invention.

[0395] The preferred combinations of the components (eg, antigenic peptides) of the present invention contained in the kit correspond to the preferred combinations of the components (eg, antigenic peptides) of the present invention contained in the above-mentioned pharmaceutical composition.

[0396] Therefore, the present invention provides a kit comprising at least two, preferably three, more preferably four or five different antigenic peptides as described herein (or the above-mentioned immunogenic compounds, nanoparticles, nucleic acids, cells, etc., which differ in antigenic peptides), and optionally comprising an auxiliary peptide (e.g., the UCP2 peptide of SEQ ID NO: 39) and / or an adjuvant (e.g., MONTANIDE ISA 51).

[0397] Preferably, the kit comprises different antigenic peptides (in the form of peptides or in any other form as described above), including (i) at least one antigenic peptide of the present invention, for example as shown in Table 1; and (ii) at least one additional antigenic peptide as described above, for example as shown in Table 2 above.

[0398] In some embodiments, the kit comprises

[0399] - a first component, which relates to antigenic peptides comprising or consisting of (microbiota) sequence variants of fragments of the human tumor antigen CDC20;

[0400] - a (different) second component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen KIF2C;

[0401] - a (different) third component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen UBE2C;

[0402] - Optionally, a (different) fourth component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen BIRC5; and

[0403] - Optionally, a (different) fifth component relating to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen FOXM1.

[0404] Preferably, the kit comprises

[0405] - a first component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 17;

[0406] - a (different) second component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 18;

[0407] - a (different) third component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 19;

[0408] - optionally, a (different) fourth component relating to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 35; and

[0409] - Optionally, a (different) fifth component relating to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 36.

[0410] More preferably, the kit comprises

[0411] - A first component, which involves an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0412] - a (different) second component, which relates to an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0413] - a (different) third component, which relates to an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3;

[0414] - optionally, a (different) fourth component, which relates to an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 32, and

[0415] - Optionally, a (different) fifth component which relates to an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 33.

[0416] It should be understood that the kit 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.

[0417] Preferably, the kit further comprises the UCP2 helper peptide of SEQ ID NO:39.

[0418] In some embodiments, the kit comprises, in addition to any of the components described above, an anti-cancer therapeutic agent as described above.

[0419] Therefore, the therapeutic agent is preferably capable of preventing and / or treating the same cancer type as the antigen peptide of the present invention. Preferably, the anticancer therapeutic agent is selected from the above-mentioned antibodies, the above-mentioned CAR-T cells, the above-mentioned tumor cell lysates, the above-mentioned chemotherapeutic agents, the above-mentioned radiotherapeutic agents, the above-mentioned immune checkpoint regulators and combinations thereof.

[0420] The individual components of the kit may be packaged in one or more containers. The components may be provided in a lyophilized or dried form or dissolved in a suitable buffer. The kit may also contain additional reagents, including, for example, preservatives, growth media and / or buffers, washing solutions, etc., for storing and / or redissolving the components.

[0421] Different antigenic peptides (or the above-mentioned immunogenic compounds, nanoparticles, nucleic acids, cells, etc., which are different in terms of antigenic peptides) can be contained in the same or different containers. For example, the test kit can include a (single) container, which contains a first antigenic peptide as described herein and a second antigenic peptide as described herein. The (single) container can also additionally include an auxiliary peptide, such as UCP2. Optionally, the first antigenic peptide and the second antigenic peptide (and optionally, the auxiliary peptide) contained in the (single) container can be formulated together, for example, formulated in water for injection and / or dimethyl sulfoxide (DMSO). In addition, the test kit can also include another container (different from the container containing the antigenic peptide), which contains an adjuvant, such as MONTANIDE ISA 51.

[0422] Optionally, the kit may also include a vial of water for injection and / or a vial adapter. A sterile needle may also be included, for example for vaccinating a patient after obtaining the emulsion. The kit may also include one or more syringes.

[0423] In addition, the kit of parts of the present invention may optionally contain instructions for use. Therefore, preferably, the kit comprises a package insert or instructions, wherein there is guidance for preventing or treating cancer by using the immunogenic compound of the present invention, the antigenic peptide of the present invention, the nanoparticle of the present invention, the cell of the present invention, the nucleic acid of the present invention, the host cell of the present invention, the pharmaceutical composition of the present invention, etc., as described above.

[0424] In addition, the present invention also provides a vaccination kit for treating, preventing and / or stabilizing cancer, which comprises the pharmaceutical composition as described herein or the vaccine as described herein and instructions for use of the pharmaceutical composition or the vaccine in preventing and / or treating cancer.

[0425] Medical treatments and uses

[0426] As described above, the antigenic peptides of the present invention (in the various different forms described above) can be used in particular for preventive or therapeutic purposes (as drugs), in particular for triggering a specific immune response against a specific tumor antigen / protein, such as for the prevention or treatment of cancer, for example in patients in need thereof.

[0427] In view of this, the present invention provides

[0428] - an antigenic peptide of the invention as described herein,

[0429] - an immunogenic compound of the invention as described herein,

[0430] - a nanoparticle of the invention as described herein,

[0431] - a cell of the invention as described herein,

[0432] - a nucleic acid of the invention as described herein,

[0433] - a host cell of the invention as described herein,

[0434] - a T-lymphocyte according to the invention as described herein,

[0435] - an antibody of the invention as described herein, and / or

[0436] - a T-cell receptor of the invention as described herein,

[0437] - a pharmaceutical composition of the invention as described herein, or

[0438] - a kit of the invention as described herein,

[0439] Use in medicine, in particular for preventing and / or treating proliferative diseases, preferably for preventing and / or treating cancer.

[0440] In addition, the present invention also provides a method for preventing (reducing the occurrence of) and / or treating cancer or initiating, enhancing or prolonging the anti-tumor response of a subject (in need thereof), the method comprising administering to the subject (an effective amount of)

[0441] - an antigenic peptide of the invention as described herein,

[0442] - an immunogenic compound of the invention as described herein,

[0443] - a nanoparticle of the invention as described herein,

[0444] - a cell of the invention as described herein,

[0445] - a nucleic acid of the invention as described herein,

[0446] - a host cell of the invention as described herein,

[0447] - a T-lymphocyte according to the invention as described herein,

[0448] - an antibody of the invention as described herein, and / or

[0449] - a T-cell receptor of the invention as described herein,

[0450] - a pharmaceutical composition of the invention as described herein, or

[0451] - A kit of parts according to the invention as described herein.

[0452] In particular, the preferred embodiments of the above-mentioned antigenic peptides are also applicable to the use of the present invention in preventing and / or treating cancer. Non-limiting examples of cancer include colorectal cancer, lung cancer, prostate cancer and / or breast cancer.

[0453] In addition, the present invention provides a method for inducing or improving CD8-dependent + A method for inducing an immune response of cytotoxic T cells against one or more epitopes, wherein the method comprises administering to the subject any of:

[0454] - an antigenic peptide of the invention as described herein,

[0455] - an immunogenic compound of the invention as described herein,

[0456] - a nanoparticle of the invention as described herein,

[0457] - a cell of the invention as described herein,

[0458] - a nucleic acid of the invention as described herein,

[0459] - a host cell of the invention as described herein,

[0460] - a T-lymphocyte according to the invention as described herein,

[0461] - an antibody of the invention as described herein, and / or

[0462] - a T-cell receptor of the invention as described herein,

[0463] - a pharmaceutical composition of the invention as described herein, or

[0464] - A kit of parts according to the invention as described herein.

[0465] CD8-dependent + The immune response of the reaction includes an increase in the expression of one or more of IFN-γ, TNF-α and IL-2 mRNA or protein relative to the level before the administration of the compound of the present invention. It can also be measured by the increase in the frequency or absolute number of antigen-specific T cells after the administration of the compound of the present invention, measured by HLA-peptide polymer staining, ELISPOT determination and delayed hypersensitivity test. It can also be indirectly measured by the increase in antigen-specific serum antibodies that depend on antigen-specific T helper cells.

[0466] The present invention also provides a method for inducing or improving an immune response against one or more antigens or antigenic epitopes in a subject, wherein the immune response is restricted by a plurality of MHC class I molecules, wherein the method comprises administering to the subject any of the following:

[0467] - an antigenic peptide of the invention as described herein,

[0468] - an immunogenic compound of the invention as described herein,

[0469] - a nanoparticle of the invention as described herein,

[0470] - a cell of the invention as described herein,

[0471] - a nucleic acid of the invention as described herein,

[0472] - a host cell of the invention as described herein,

[0473] - a T-lymphocyte according to the invention as described herein,

[0474] - an antibody of the invention as described herein, and / or

[0475] - a T-cell receptor of the invention as described herein,

[0476] - a pharmaceutical composition of the invention as described herein, or

[0477] - A kit of parts according to the invention as described herein.

[0478] The method of inducing or improving an immune response to multiple epitopes as described herein in a subject can be determined by evaluating a cytokine response, which is restricted by multiple MHC class I molecules, the method comprising stimulating T cells in vitro with a single peptide bound to a discrete MHC class I molecule on an antigen presenting cell, and increasing the expression of one or more of the mRNA or protein of IFN-γ, TNF-α and IL-2 compared to the level before administering the compound of the invention. The restriction of MHC class I molecules can also be verified by using antigen presenting cells expressing MHC class I molecules or by using MHC class I blocking antibodies. It can also be measured by the increase in the frequency or absolute number of antigen-specific T cells after administering the compound of the invention, measured by HLA-peptide multimer staining using multimers assembled with MHC class I molecules.

[0479] The invention more particularly relates to a composition as defined above for use as a vaccine for immunotherapy.

[0480] - an antigenic peptide of the invention as described herein,

[0481] - an immunogenic compound of the invention as described herein,

[0482] - a nanoparticle of the invention as described herein,

[0483] - a cell of the invention as described herein,

[0484] - a nucleic acid of the invention as described herein,

[0485] - a host cell of the invention as described herein,

[0486] - a T-lymphocyte according to the invention as described herein,

[0487] - an antibody of the invention as described herein, and / or

[0488] - a T-cell receptor of the invention as described herein,

[0489] - a pharmaceutical composition of the invention as described herein, or

[0490] - a kit of the invention as described herein

[0491] Can be used as a vaccine, in particular for (cancer) immunotherapy.

[0492] In the context of the present invention, the term "vaccine" refers to a (biological) preparation that provides innate and / or adaptive immunity, usually against a specific disease, preferably cancer. Therefore, a vaccine particularly supports the innate and / or adaptive immune response of the immune system of the subject to be treated. For example, the antigenic peptides of the present invention generally cause or support an adaptive immune response of the patient to be treated.

[0493] In the context of the present invention, the vaccine (composition) can induce a specific immune response against a tumor antigen and is therefore preferably used to prevent or treat cancer.

[0494] Therefore, in a preferred embodiment, the present invention relates to a composition as defined above for use in preventing and / or treating cancer in a subject in need thereof. More preferably, the present invention relates to the use of a composition of the present invention in the preparation 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, the method comprising administering an effective amount of a composition of the present invention to the subject.

[0495] Preferably, the cancer to be prevented and / or treated by any of the following

[0496] - an antigenic peptide of the invention as described herein,

[0497] - an immunogenic compound of the invention as described herein,

[0498] - a nanoparticle of the invention as described herein,

[0499] - a cell of the invention as described herein,

[0500] - a nucleic acid of the invention as described herein,

[0501] - a host cell of the invention as described herein,

[0502] - a T-lymphocyte according to the invention as described herein,

[0503] - an antibody of the invention as described herein, and / or

[0504] - a T-cell receptor of the invention as described herein,

[0505] - a pharmaceutical composition of the invention as described herein, or

[0506] - a kit of the invention as described herein,

[0507] (reference) tumor antigens related to antigenic peptides as described herein. That is, suitable antigenic peptides can be selected by (i) selecting suitable tumor antigens known in the art for a certain type of cancer and (ii) selecting suitable antigenic peptides of the present invention for the selected tumor antigen, as described above, for example, as described in Table 1 (and optionally, in addition to Table 2). Those skilled in the art will readily appreciate that antigenic peptides 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 the cancer.

[0508] Particularly preferred for use in the prevention or treatment of cancer are peptides of the present invention (alone or in combination) 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. More preferred are peptides (alone or in combination) 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), 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 immunotherapy of colorectal cancer, lung cancer, prostate cancer and / or breast cancer.

[0509] Therefore, another aspect of the invention relates to the use of at least one peptide according to any one of SEQ ID NO: 1 to 16 and SEQ ID NO: 40 to SEQ ID NO: 42 for the treatment (preferably in combination) of a proliferative disease selected from colorectal cancer, lung cancer, prostate cancer and breast cancer.

[0510] Therefore, another aspect of the invention relates to the use of the peptides of the invention for the treatment, preferably in combination, of a proliferative disease, in particular a cancer, for example selected from the group consisting of colorectal cancer, lung cancer, prostate cancer and breast cancer.

[0511] As described above, in the context of the pharmaceutical compositions and kits of the present invention, combinations of the above clauses are preferred, i.e., different antigenic peptides of the present invention are preferably used for the prevention and / or treatment of cancer. Preferably, more than one of the above components can be used for 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. can be used for the prevention and / or treatment of cancer. Preferably, such different components for the prevention and / or treatment of cancer are different in terms of the antigenic peptides of the present invention, for example, one component relates to a first antigenic peptide and one component relates to a second antigenic peptide (different from the first antigenic peptide), as described above, for example in the context of a pharmaceutical composition or a kit.

[0512] Therefore, the present invention also provides a combination of at least two different antigenic peptides of the present invention for use in preventing or treating cancer.

[0513] For the prevention or treatment of cancer, such as colorectal cancer, lung cancer, prostate cancer and / or breast cancer, combinations of the above-mentioned antigenic peptides (in the context of a pharmaceutical composition or kit) are preferred.

[0514] In some embodiments,

[0515] - a first component, which relates to antigenic peptides comprising or consisting of (microbiota) sequence variants of fragments of the human tumor antigen CDC20;

[0516] - a (different) second component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen KIF2C;

[0517] - a (different) third component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen UBE2C;

[0518] - Optionally, a (different) fourth component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen BIRC5; and

[0519] - Optionally, a (different) fifth component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of a fragment of the human tumor antigen FOXM1

[0520] The administration may be (in combination, ie simultaneously or sequentially) for the prevention and / or treatment of cancer.

[0521] Preferably,

[0522] - a first component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 17;

[0523] - a (different) second component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 18;

[0524] - a (different) third component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 19;

[0525] - optionally, a (different) fourth component relating to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 35; and

[0526] - Optionally, a (different) fifth component, which relates to an antigenic peptide comprising or consisting of a (microbiota) sequence variant of SEQ ID NO: 36

[0527] (combined, i.e., simultaneous or sequential) administration for the prevention and / or treatment of cancer.

[0528] More preferably,

[0529] - A first component, which involves an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0530] - a (different) second component, which relates to an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0531] - a (different) third component, which relates to an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3;

[0532] - optionally, a (different) fourth component, which relates to an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 32, and

[0533] - Optionally, a (different) fifth component, which relates to an antigenic peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 33

[0534] (combined, i.e., simultaneous or sequential) administration for the prevention and / or treatment of cancer.

[0535] The method of administration is well known to those skilled in the art. About the composition of the present invention, it can be directly administered to the subject, administered to the affected organ (i.e., local administration) or systemically administered (i.e., enteral or parenteral administration), or even can be applied to the cell or human cell line derived from the subject in vitro, and then administered to the subject, or even can be used in vitro for selecting the immune cell subpopulation derived from the subject, and then re-administered to the subject. Enteral administration includes oral administration and rectal administration, and administration via a gastric feeding tube, a duodenal feeding tube or a gastrostomy, and parenteral administration especially includes subcutaneous injection, intravenous injection, intramuscular injection, intraarterial injection, intradermal injection, intraosseous injection, intracerebral injection and intrathecal injection. The method of administration will generally depend on the antigenic peptides and / or immunogenic compounds present in the composition, the cancer type to be treated, and other activating agents that may be contained in the composition. For example, if the immunogenic compound is a nucleic acid as defined above, it is preferably intramuscular injection or intradermal injection to administer, and if the nucleic acid is cloned into a viral vector, oral / nasal administration is particularly preferred. Alternatively, if the antigenic peptide and / or immunogenic compound is a (poly)peptide as defined above, or if it is loaded in / on a nanoparticle as described herein, administration is preferably intramuscular, intradermal or oral administration. And, still alternatively, if the antigenic peptide and / or immunogenic compound is delivered in the form of an enteric bacterium as defined above, especially if the enteric bacterium is in the form of a probiotic, administration is preferably oral administration.

[0536] The antigenic peptides, immunogenic compounds and nucleic acids of the present invention may be further packaged so as to facilitate their administration to a subject in need thereof. For example, these can be encapsulated into peptide nanocarriers (preferred if the immunogenic compound is a nucleic acid or (poly)peptide), into virosomes (preferred if the immunogenic compound is a nucleic acid or (poly)peptide), or into lipid-based carrier systems such as liposome-polycation-DNA complexes (preferred if 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).

[0537] The composition can also be administered multiple times 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 over a long period of time, such as weekly, every other week, monthly, annually, or even several years after the first administration to ensure that the subject is properly immunized.

[0538] The present invention also relates to a method for killing or reducing the number of target cells in a patient, wherein the target cells abnormally express a polypeptide comprising the antigenic peptide of the present invention or the corresponding human reference peptide, the method comprising administering an effective number of T cells produced by the present invention to the patient.

[0539] The present invention also relates to the use of any of the peptides, nucleic acids of the present invention, expression vectors of the present invention, cells of the present invention, activated T lymphocytes, T cell receptors or antibodies or other peptide binding molecules and / or peptide-MHC binding molecules of the present invention as a medicament or for the preparation of a medicament. Preferably, the medicament has activity against cancer.

[0540] In some embodiments, the medicament is for cell therapy, vaccine, or soluble TCR or antibody based protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0541] A brief description of the accompanying drawings will be given below. The accompanying drawings are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the present invention in any way.

[0542] Figure 1 : Shows the in vitro affinity of the antigenic peptide CDC20-B1 (ENT204_B1) in Example 1 compared with the corresponding human CDC20 epitope CDC20-H1 (ENT204-H).

[0543] Figure 2 : Shows the in vitro affinity of the antigenic peptide KIF2C-B1 (ENT207_B1) compared with the corresponding human KIF2C epitope KIF2C-H1 (ENT207-H) in Example 1.

[0544] Figure 3 : Shows the in vitro affinity of the antigenic peptide UBE2C-B1 (ENT168_B1) in Example 1 compared with the corresponding human UBE2C epitope UBE2C-H1 (ENT168-H) and another human UBE2C epitope UBE2C-H11 (ENT168-HL).

[0545] Figure 4: Shows the in vitro affinity of the antigenic peptide ANKRD30A-B1 (ENT169_B1) in Example 1 compared to the corresponding human ANKRD30A epitope ANKRD30A-H1 (ENT169-H).

[0546] Figure 5 : Shows the in vitro affinity of the antigenic peptide CDH17-B1 (ENT176_B1) in Example 1 compared with the corresponding human CDH17 epitope CDH17-H1 (ENT176-H).

[0547] Figure 6 : Shows the in vitro affinity of the antigenic peptide TOP2A-B2 (ENT205_B1) in Example 1 compared with the corresponding human TOP2A epitope TOP2A-H2 (ENT205-H).

[0548] Figure 7 : Shows the ELISPOT results of HHDDR1 HLA-A2 transgenic mice vaccinated with the antigen peptide CDC20-B1 (ENT204_B1) in Example 2 (as shown), and the cross-reaction with the human corresponding peptide CDC20-H1 (ENT204-H). Data are expressed as 1×10 6 The number of total T cells is provided.

[0549] Figure 8 : Shows the ELISPOT results of HHDDR1 HLA-A2 transgenic mice vaccinated with the antigen peptide KIF2C-B1 (ENT207_B1) in Example 2 (as shown), and the cross-reaction with the human corresponding peptide KIF2C-H1 (ENT207-H). Data are expressed as 1×10 6 The number of total T cells is provided.

[0550] Fig. 9 : Shows the ELISPOT results of HHDDR1 HLA-A2 transgenic mice vaccinated with the antigen peptide UBE2C-B1 (ENT168_B1) in Example 2 (as shown), as well as cross-reactions with the human corresponding peptide UBE2C-H1 (ENT168-H) and another human UBE2C epitope UBE2C-H11 (ENT168-HL). Data are expressed as 1×10 6 The number of total T cells is provided.

[0551] Fig.10: Shows the ELISPOT results of HHDDR1 HLA-A2 transgenic mice vaccinated with the antigen peptide ANKRD30A-B1 (ENT169_B1) in Example 2 (as shown), and the cross-reaction with the human corresponding peptide ANKRD30A-H1 (ENT169-H). Data are expressed as 1×10 6 The number of total T cells is provided.

[0552] Fig.11 : Shows the ELISPOT results of HHDDR1 HLA-A2 transgenic mice vaccinated with the antigen peptide CDH17-B1 (ENT176_B1) in Example 2 (as shown), and the cross-reaction with the human corresponding peptide CDH17-H1 (ENT176-H). Data are expressed as 1×10 6 The number of total T cells is provided.

[0553] Fig.12 : Shows the ELISPOT results of HHDDR1 HLA-A2 transgenic mice vaccinated with the antigen peptide TOP2A-B2 (ENT205_B1) in Example 2 (as shown in the figure), and the cross-reaction with the corresponding human peptide TOP2A-H2 (ENT205-H). Data are expressed as 1×10 6 The number of total T cells is provided.

[0554] Fig.13 : shows CDC20-B1, KIF2C-B1 and UBE2C-B1 peptide-specific CD8+ T cells detected in peripheral blood from healthy donors (HLA-A2 positive) in Example 3.

[0555] Fig.14 : Shows the in vitro affinity of the antigenic peptide KIF2C-B11 (ENT207_B2) compared with the corresponding human KIF2C epitope KIF2C-H1 (ENT207-H) in Example 1.

[0556] Fig.15 : Shows the in vitro affinity of the antigenic peptide KIF2C-B12 (ENT207_B3) compared to the corresponding human KIF2C epitope KIF2C-H1 (ENT207-H) in Example 1.

[0557] Fig.16 : Shows the in vitro affinity of the antigenic peptide KIF2C-B13 (ENT207_B4) compared to the corresponding human KIF2C epitope KIF2C-H1 (ENT207-H) in Example 1.

[0558] Fig.17: Shows the in vitro affinity of the antigenic peptide UBE2C-B11 (ENT168_B2) compared to the corresponding human UBE2C epitope UBE2C-H1 (ENT168-H) in Example 1.

[0559] Fig.18 : The cytotoxicity of the CDC20-B1, KIF2C-B1 and UBE2C-B1 peptide-specific human T cell clones expanded in vitro in Example 3 under stimulation with peptides derived from microbiota is shown. CDC20-B1, KIF2C-B1 and UBE2C-B1 peptide-specific T cells have the ability to kill T2 cells loaded with bacterial peptides or human peptides.

[0561] Example

[0562] Below, will show the specific examples of various embodiments and aspects of the present invention. However, the scope of the present invention should not be limited by the specific embodiments described herein. The preparation and examples given below are to enable those skilled in the art to more clearly understand and practice the present invention. However, the scope of the present invention is not limited by the exemplary embodiments, which are only intended to be an illustration of a single aspect of the present invention, and functionally equivalent methods are within the scope of the present invention. In fact, in addition to those modifications described herein, various modifications of the present invention will easily become apparent to those skilled in the art from the foregoing description, the accompanying drawings and the following examples. All such modifications fall within the scope of the appended claims.

[0563] Example 1: Antigenic peptide has superior affinity for HLA-A*0201 allele.

[0564] Next, the binding affinity of various selected antigenic peptides and corresponding human tumor antigen fragments (human reference peptides) to the HLA-A*0201 allele was determined in vitro. That is, the antigenic peptide of sequence 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). In addition, the antigenic peptide of sequence 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). In addition, the antigenic peptide of sequence 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). In addition, the antigenic peptide of sequence SEQ ID NO: 4 ("YLAFVPLAL"; also referred to herein as UBE2C-B11) was compared with the corresponding reference human peptide derived from UBE2C ("ALYDVRTIL"; SEQ ID NO: 19, also referred to herein as UBE2C-H1). In addition, the antigenic peptide of sequence 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). In addition, the antigenic peptide of sequence 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). In addition, the antigenic peptide of sequence 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). In addition, the antigenic peptide of sequence 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).In addition, the antigenic peptide of sequence 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). In addition, the antigenic peptide of sequence 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). In addition, the antigenic peptide of sequence 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). In addition, the antigenic peptide of sequence 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). In addition, the antigenic peptide of sequence SEQ ID NO: 13 ("IVWSDVTYV"; also referred to herein as MMP11-B1) was compared with the corresponding reference human peptide derived from MMP11 ("KVWSDVTPL", SEQ ID NO: 28, also referred to herein as MMP11-H1). In addition, the antigenic peptide of sequence SEQ ID NO: 14 ("AVIGIVAAV"; also referred to herein as OR51E2-B1) was compared with the corresponding reference human peptide derived from OR51E2 ("AQIGIVAVV", SEQ ID NO: 29, also referred to herein as OR51E2-H1). In addition, 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).

[0565] A. Materials and Methods

[0566] A1. Measurement of peptide affinity for the T2 cell line.

[0567] The experimental scheme is similar to that validated 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. 2000 December; 30(12): 3411-21). Peptide affinity measurements were performed using human tumor cells T2, which express HLA-A*0201 molecules but are TAP1 / 2 negative and cannot present endogenous peptides.

[0568] T2 cells (5×10 per well 4 Cells) were incubated in serum-free medium (TexMacs) supplemented with 100 ng / μl 2-microglobulin, where the peptide concentration was reduced from 100 μM to 0.1 μM (4 points: 100 μM, 10 μM, 1 μM, 0.1 μM), and incubated at 37° C. for 16 hours. The cells were then washed twice and labeled with anti-HLA-A2 antibody conjugated to PE (clone REA517, Miltenyi).

[0569] Analysis was achieved by FACS (Macsquant Analyzer 10 or Macsquant Analyzer 16 - Miltenyi).

[0570] For each peptide concentration, the geometric mean of labeling associated with the peptide of interest was 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. The relative affinity was then determined as follows:

[0571] Relative affinity = concentration of each peptide that induces 20% HLA-A*0201 expression / concentration of reference peptide that induces 20% HLA-A*0201 expression.

[0572] A2. Dissolution of Peptide

[0573] Each peptide was solubilized taking into account its amino acid composition. For peptides that did not contain any cysteine, lysine, or tryptophan, DMSO was added up to 10% of the total volume. Other peptides were resuspended in water or NH4OH.

[0574] B. Results

[0575] Table 3 below shows the mean relative fluorescence intensity values ​​of T2 cells obtained for different concentrations of each peptide (the data have been normalized to the mean fluorescence of the HIV peptide, i.e. a value of 100 equals the best binding observed to the HIV peptide):

[0576] Table 3.

[0577]

[0578]

[0579] Table 4 below summarizes the concentration required to induce 20% HLA-A2 expression and the in vitro binding affinity of each tested peptide (*normalized to the HIV-pol concentration of the peptide that induced 20% HLA-A2 expression during the same experiment).

[0580] Table 4.

[0581]

[0582]

[0583] also, Figure 1-Figure 6 and Figure 14-17 Results are shown for a selected example, i.e., the antigenic peptide CDC20-B1 compared to the corresponding human CDC20 fragment CDC20-H1 ( Figure 1 ), antigen peptide KIF2C-B1 was compared with the corresponding human KIF2C fragment KIF2C-H1 ( Figure 2 ), antigen peptide KIF2C-B11 was compared with the corresponding human KIF2C fragment KIF2C-H1 ( Fig.14 ), antigen peptide KIF2C-B12 compared with the corresponding human KIF2C fragment KIF2C-H1 ( Fig.15 ), antigen peptide KIF2C-B13 compared with the corresponding human KIF2C fragment KIF2C-H1 ( Fig.16 ), the antigenic peptide UBE2C-B1 was compared with the corresponding human UBE2C fragment UBE2C-H1 and with another human UBE2C epitope UBE2C-H11 ( Figure 3 ), antigenic peptide UBE2C-B11 compared with the corresponding human UBE2C fragment UBE2C-H1 ( Fig.17 ), antigenic peptide ANKRD30A-B1 was compared with the corresponding human ANKRD30A fragment ANKRD30A-H1 ( Figure 4 ), antigenic peptide CDH17-B1 was compared with the corresponding human CDH17 fragment CDH17-H1 ( Figure 5 ), antigenic peptide TOP2A-B2 was compared with the corresponding human TOP2A fragment TOP2A-H2 ( Figure 6 ).

[0584] In summary, the results show that the antigenic peptides of the present invention exhibit binding affinity to HLA-A*0201 that is at least similar to that of the corresponding human tumor antigen fragments. In most cases, the observed binding affinity of the antigenic peptides of the present invention is stronger than the binding affinity of the corresponding human epitope. Without being bound by any theory, it is assumed that this strong binding affinity of the antigenic peptides of the present invention reflects their ability to elicit an immune response (i.e., their immunogenicity).

[0585] Example 2: 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- Immunogenicity in A2 transgenic mice and cross-reactivity with the corresponding human peptide.

[0586] A. Materials and Methods

[0587] A.1 Mouse Model

[0588] 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 mouse sex and age) to experimental groups, where each group was immunized with a specific vaccination peptide (vacc-pAg) in combination with a common helper peptide (h-pAg UCP2; sequence: KSVWSKLQSIGIRQH; SEQ ID NO: 39) (as shown in Table 6 below).

[0589] Table 6. Composition of experimental groups. h-pAg: "helper" peptide; vacc-pAg: vaccination peptide. The number of booster injections is indicated in brackets.

[0590]

[0591]

[0592] Peptides are provided as follows:

[0593] vacc-pAg: UBE2C-B1, ANKRD30A-B1, CDH17-B1, CDC20-B1, TOP2A-B2, and KIF2C-B1 were all produced and provided at 4 mM concentration;

[0594] h-pAg:UCP2 was resuspended in pure distilled water at a concentration of 10 mg / mL

[0595] On each day of injection and for each group, the peptide formulation (emulsion) to be injected was freshly prepared. 2 mL Luer lock syringes (4606701V, B BRAUN) and Luer lock female-female The mixture for 10 animals was prepared using an adapter (B. Braun, 5206634): 500 μL of the peptide mixture in syringe 1 was emulsified with 500 μL Montanide ISA 51VG (Seppic) contained in syringe 2. The emulsification process was first performed by pushing the peptide mixture contained in syringe 1 into syringe 2 (containing Montanide) at a very low speed. The mixture was then transferred from one syringe to the other, in 6 cycles at a very low speed (one cycle corresponds to one transfer from one syringe to the other), and then at a very high speed as fast as possible for 1 minute. Each emulsion was prepared in excess to compensate for the dead volume during injection.

[0596] Animals were immunized with a primary injection on day 0 (d0) and a booster injection on day 14. Each mouse was injected subcutaneously on the loose skin of the neck with 100 μL of an oil-based emulsion containing:

[0597] · 30 nmol vacc-pAg and 30 or 100 μg UCP2 helper peptide dissolved in 50 μL of dedicated final solvent

[0598] Montanide ISA 51VG (Seppic), added at a 1:1 (v:v) ratio (50 μL per mouse).

[0599] A.2 Analysis

[0600] Seven days after the booster injection (i.e., day 21), the animals were euthanized and spleens were harvested. Splenocytes were prepared by mechanical disruption of the organ followed by 70 μm filtration and lysis of red blood cells.

[0601] The cell suspension was further used for ELISPOT-IFN assay (Table 5). Cells were cultured in 200 μL complete T cell culture medium. The experimental conditions (repeated in duplicate) were as follows: the total number of cells per well was 2×10 5 When cultured in the presence of the positive control PMA / ionomycin (PMA: Sigma P8139: final concentration 0.1 μM; ionomycin: Sigma I0634: final concentration 1 μM), the total cell number was 2x10 4 The ability of the cultures to secrete IFN was assessed (Mouse IFN-ELISpotPLUS Kit, Mabtech 3321-4APT-10) according to the manufacturer's instructions (incubation time before assay was approximately 12-48 hours). The peptides used for restimulation are listed in Table 7.

[0602] Table 7. Setup for ELISPOT-IFNγ assay.

[0603]

[0604] Spots were counted on the iSpot Fluorospot Reader System (AID). Data plotting and statistical analysis were performed using Prism-9 software (GraphPad Software Inc.).

[0605] B. Results

[0606] All mice were 8 to 15 weeks old at the start of the experiment. Males and females were used in the study. Animals were housed in groups, with a maximum of 6 per cage. At sacrifice, spleen T cell populations were analyzed by flow cytometry, showing that the vast majority belonged to the CD4+ T cell subset.

[0607] After plating and incubation with appropriate stimuli, IFNγ-producing cells were visualized and counted. Data are expressed as 1 × 10 6 The number of spots per total T cell is provided. The group mean is then plotted using the individual mean values ​​(obtained from duplicates). Statistical analysis was performed using an unpaired nonparametric test (MannWhitney) for comparison (compared to an irrelevant peptide condition) (**: p<0.01; *: p<0.05).

[0608] In general, vaccination with the antigenic peptides of the present invention (CDC20-B1, KIF2C-B1, UBE2C-B1, ANKRD30A-B1, CDH17-B1, and TOP2A-B2) induced significant T cell responses in the ELISPOT-IFNγ assay in HHDDR1 mice ( Figure 7-Figure 12 ).

[0609] result( Figure 7 ) showed that immunization of HHD-DR1 mice with CDC20-B1 allowed the induction of T cells that could respond strongly after challenge with CDC20-B1 or the human corresponding peptide CDC20-H1. Therefore, CDC20-B1 is highly immunogenic and is able to generate an effective immune response against the corresponding human peptide.

[0610] result( Figure 8 ) showed that immunization of HHD-DR1 mice with KIF2C-B1 allowed the induction of T cells that could respond strongly after challenge with KIF2C-B1 or the human corresponding peptide KIF2C-H1. Therefore, KIF2C-B1 is highly immunogenic and is able to generate an effective immune response against the corresponding human peptide.

[0611] result( Fig. 9 ) showed that immunization of HHD-DR1 mice with UBE2C-B1 allowed the induction of T cells that could strongly respond after challenge with UBE2C-B1 or the human corresponding peptide UBE2C-H1 or another human UBE2C epitope UBE2C-H11. Therefore, UBE2C-B1 is highly immunogenic and is able to generate an effective immune response against the corresponding human peptide and another human UBE2C epitope UBE2C-H11.

[0612] result( Fig.10 ) showed that immunization of HHD-DR1 mice with ANKRD30A-B1 allowed the induction of T cells that could respond strongly after challenge with ANKRD30A-B1 or the human corresponding peptide ANKRD30A-H1. Therefore, ANKRD30A-B1 is highly immunogenic and is able to generate an effective immune response against the corresponding human peptide.

[0613] result( Fig.11 ) showed that immunization of HHD-DR1 mice with CDH17-B1 allowed the induction of T cells that could respond strongly after challenge with CDH17-B1 or the human corresponding peptide CDH17-H1. Therefore, CDH17-B1 is highly immunogenic and is able to generate an effective immune response against the corresponding human peptide.

[0614] result( Fig.12 ) showed that immunization of HHD-DR1 mice with TOP2A-B2 allowed the induction of T cells that could respond strongly after being challenged with TOP2A-B2 or the human corresponding peptide TOP2A-H2. Therefore, TOP2A-B2 is highly immunogenic and is able to generate an effective immune response against the corresponding human peptide.

[0615] In summary, these immunogenicity studies performed in HHDDR1 mice described in Example 2 showed that the six antigenic peptides of the present invention, CDC20-B1, KIF2C-B1, UBE2C-B1, ANKRD30A-B1, CDH17-B1 and TOP2A-B2, induced a strong immune response. Cross-reactivity of T cells generated against CDC20-H1, KIF2C-B2, UBE2C-B1, ANKRD30A-B1, CDH17-B1 and TOP2A-B2 with the corresponding human peptides was demonstrated in HHDDR1 mice.

[0616] These results therefore provide experimental evidence that antigen-based immunotherapy can enhance T cell responses in vivo and that the antigenic peptides of the present invention are particularly effective for this purpose.

[0617] Example 3: UBE2C-B1 (ENT_168-B1), CDC20-B1 (ENT_204-B1), KIF2C-B1 (ENT_207- B1) Ex vivo cytotoxic effect of specific CD8 human T cells.

[0618] Several studies support the idea that there is a pool of T cells specific for microbial peptides. It is expected that the number of microbial-specific T cells against peptides is small but sufficient to be reactivated by vaccine challenge.

[0619] 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 to investigate their cytotoxic capacity.

[0620] 3.1 Identification of antigen peptide-specific CD8 T cells in humans

[0621] In vitro expansion methods and specific pMHC multimers have been used to identify 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 corresponding 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 expansion using T2 cells loaded with EO4010 peptide to increase the number of specific T cell clones. Cytometry analysis using fluorescent multimers was performed to detect OMP peptide-specific CD8 T cells on the enriched CD8 T cell population.

[0622] Fig.13 The results obtained using one HLA-A2 healthy donor are illustrated. For this donor, cell expansion allowed 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%).

[0623] Together, these results suggest the presence of CD8 T cells in the blood of healthy HLA-A2 donors that are able to recognize peptides derived from the microbiota.

[0624] 3.2 Antigenic peptide-specific CD8 T cell cytotoxicity

[0625] Cytotoxicity assays were performed using the CD8+ T cells expanded as described above in the presence of different ratios of target cells and effector cells to assess their cytotoxic capacity using flow cytometry readout. Target cells were T2 cell lines loaded with bacterial peptides or human counterparts. As negative controls, unloaded T2 cells and T2 cells loaded with irrelevant peptides were used. Fig.18 As shown, the antigen peptide-specific human T cell clones expanded in vitro 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, the in vitro expanded UBE2C-B1 (ENT_168-B1), CDC20-B1 (ENT_204-B1) and KIF2C-B1 (ENT_207-B1) specific human T cell clones can kill T2 cells loaded with human UBEC2, CDC20 and KIF2C peptides.

[0626] Overall, these results indicate that T cell clones that can recognize microbial peptides and kill targets with both microbial peptides and human counterparts exist in healthy volunteers. These data are particularly encouraging because T cell clones have been obtained in healthy donors, so we can expect that specific T cell clones can be effectively expanded in patients exposed to immunization with the antigenic peptides of the present invention.

Claims

1. An antigenic peptide comprising or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 1-16 and SEQ ID NOs: 40-42, wherein optionally, one or two amino acid residues may be replaced, deleted or added.

2. The antigenic peptide according to claim 1, wherein the antigenic peptide consists of the amino acid sequence shown in any one of SEQ ID NOs: 1-16 and SEQ ID NOs: 40-42.

3. The antigenic peptide according to any one of the preceding claims, wherein the antigenic peptide is 8 to 15 amino acids or 8 to 11 amino acids in length, preferably the antigenic peptide is 9 or 10 amino acids in length. 4 . The antigenic peptide according to claim 1 , wherein the antigenic peptide comprises or consists of the amino acid sequence shown in SEQ ID NO:

1. 5 .

5. The antigenic peptide according to any one of claims 1 to 3, wherein the antigenic peptide comprises or consists of the amino acid sequence shown in SEQ ID NO:

2.

6. The antigenic peptide according to any one of claims 1 to 3, wherein the antigenic peptide comprises or consists of the amino acid sequence shown in SEQ ID NO:

3.

7. An immunogenic compound comprising the antigenic peptide according to any one of the preceding claims.

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

9. A pharmaceutical composition comprising -Antigenic peptide according to any one of claims 1 to 6, - The immunogenic compound according to claim 7, or - The nucleic acid according to claim 8, and, optionally, one or more pharmaceutically acceptable excipients or carriers.

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

11. The pharmaceutical composition according to claim 10, wherein the different components are related to - 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.

12. The pharmaceutical composition according to any one of claims 9 to 11, wherein the pharmaceutical composition further comprises - an antigenic peptide comprising or consisting of the amino acid sequence shown 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.

13. The pharmaceutical composition according to any one of claims 9 to 12, further comprising an auxiliary peptide, preferably a peptide comprising or consisting of the amino acid sequence according to SEQ ID NO:

39.

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

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

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