Teleost fish invariant chain cancer vaccine

Abstract

The present invention relates to fragments of teleost invariant chain optionally fused to one or more antigens, or polypeptides comprising teleost invariant chain fused to one or more antigens or antigenic fragments thereof, polynucleotides encoding such polypeptides, vectors comprising such polynucleotides, collections of vectors comprising such polynucleotides, and the use of such polypeptides, polynucleotides, and vectors to treat or prevent disease, particularly neoplastic disease. The teleost invariant chain polypeptides or fragments thereof act as "T cell enhancers" that convert non-immunogenic antigen sequences into immunogenic T cell antigens.

Description

Technical Field

[0001] The present invention relates to a fragment of an invariant chain of a teleost fish optionally fused to one or more antigens, or a polypeptide comprising a teleost fish invariant chain fused to one or more antigens or antigenic fragments thereof, a polynucleotide encoding such a polypeptide, a vector comprising such a polynucleotide, a collection of vectors comprising such a polynucleotide, and the use thereof for treating or preventing a proliferative disease, preferably cancer, a viral disease, a fungal disease or a bacterial disease of such a polypeptide, polynucleotide, vector. The teleost fish invariant chain polypeptide or a fragment thereof acts as a "T cell enhancer" that converts a non-immunogenic antigen sequence into an immunogenic T cell antigen.

Background Art

[0002] Vaccines may induce a suboptimal T cell immune response or no T cell immune response at all. This phenomenon of insufficient induction of the T cell immune response is more frequently observed in the case of vaccination targeting antigens that are either completely self-molecules, such as cancer-specific antigens, or partially self, such as cancer-specific neoantigens. Cancer-specific neoantigens usually result from point mutations in the coding region of genes, which lead to non-synonymous single nucleotide variants and, as a result, a change in one amino acid. It is rare for a single amino acid change in a protein sequence to generate a novel epitope capable of inducing a strong immune response. In most cases, this small change may not generate any novel epitopes or may generate very weak epitopes. Due to the existing central immune tolerance to self-antigens, the induction of a strong immune response against cancer-specific antigens by vaccination remains a difficult task. To overcome the lack or insufficiency of immunogenicity of cancer-specific antigens and neoantigens, several strategies have been adopted to remedy the lack / insufficiency of immunogenicity of some genetic vaccines. The invariant chain (INV) is associated with genetic vaccination and CD8 +It has been shown to enhance the induction of T cells. The invariant chain is a chaperone protein for major histocompatibility complex (MHC) class II molecules and is required for their maturation and assembly. INV also plays a role in presenting antigen peptides, and it has been demonstrated that when fused to an antigen in the context of gene vaccination, it increases the induction of T cells. Improvement of immune function by a lentiviral vector expressing ovalbumin fused to INV has been reported (Non-Patent Document 1). Subsequently, various reports have demonstrated enhanced induction of CD8 + T cell responses with plasmid DNA vectors expressing human adenovirus type 5 and INV fusion antigens.

[0003] In cancer vaccination, it is important to avoid tumor escape due to the emergence of novel cancer-specific antigens not recognized by T cells induced by the vaccine. The challenge of cancer vaccines in cancer cure is to induce a diverse population of immune T cells that can recognize and eliminate as many cancer cells as possible at once in order to reduce the chance for cancer cells to "escape" the T cell response. Therefore, it is desirable for the vaccine to encode a very large number of cancer-specific antigens. This is particularly relevant to the recently described personalized vaccine approach based on cancer-specific neoantigens. To optimize the probability of success, it is necessary to target as many neoantigens as possible with the vaccine, but the maximum insert size of the vector is limited. The full-length INV sequence or large fragments thereof occupy a relatively large portion of the vaccine antigen insert. Therefore, the use of short polypeptides as T cell enhancers is preferable in the context of anti-cancer vaccination, especially when using several cancer-specific antigens in the vaccine.

[0004] Gene vaccination platforms based on adenoviruses, particularly great ape-derived adenovirus (GAd) viral vectors, have been shown to be very potent in inducing T cell responses, and great ape-derived adenoviruses are suitable for encoding large antigens in the form of artificial genes composed of polynucleotides encoding fragments from different proteins linked successively (Non-Patent Document 2). Unexpectedly, when used in the context of cancer-specific neoantigens, no T cell-mediated immune response was induced.

[0005] The inventors have identified specific INV sequences that can restore immunogenicity. Such INV sequences are suitable for overcoming the lack or insufficiency of immunogenicity of cancer-specific neoantigens. In particular, two short fragments of non-human teleost INV were identified, neither of which contained a transmembrane domain that acts as a strong T cell enhancer.

[0006] The use of human INV or INV from species phylogenetically closely related is likely to lead to the undesirable induction of an immune response to this self-sequence in relation to vaccination. In this case, the autoimmune reaction is directed against normal tissues in which the INV is expressed. The inventors have surprisingly found that although teleost INV is quite different from mammalian INV, it increases the T cell response to antigens in mammals, and this T cell response enhancing effect is exerted on multiple antigens fused to teleost invariant chain, and short fragments of teleost INV are already sufficient to induce this reaction. Therefore, the present invention provides, inter alia, (i) an improved enhancer of the T cell response to antigens in mammals that reduces the possibility of inducing an undesirable T cell response against healthy tissues, (ii) an enhancer of the T cell response to multiple antigens, and (iii) a short fragment capable of maximizing the ability to induce T cells that can fuse a large number of antigens or antigenic fragments thereof.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

[0008] In a first aspect of the present invention, the present invention provides (a) A fragment of an invariant chain (INV) of teleost fish, comprising or consisting of 16 to 27 consecutive amino acids of the membrane-proximal domain (MPD) of the INV of teleost fish, the fragment having T cell response enhancer activity, and the MPD is (i) NQRX1DIKSLEEQX2SX3LX4X5X6X7TX8GRSX9X 10 (SEQ ID NO: 001) (where X1 is G or N, X2 is H or N, X3 is G or N, X4 is N or Q, X5 is E or A, X6 is Q or E, X7 is L or M, X8 is K or R, X9 is A or V, and X 10 is S or A, the fragment is preferably at least 60% identical to SEQ ID NO: 3), or (ii) DQKQQIQZ1LQZ2Z3NQRZ4EKQZ5Z6Z7RZ8RZ9S (SEQ ID NO: 8) (where Z1 is G or D, Z2 is T or A, Z3 is T or S, Z4 is L or M, Z5 is M or V, Z6 is G or S, Z7 is Q or L, Z8 is P or S, Z9 is E or V, the fragment is preferably at least 60% identical to SEQ ID NO: 10), a fragment characterized by an amino acid sequence selected from, and optionally, one or more antigens and / or one or more antigenic fragments thereof, or, (b) A full-length teleost INV selected from SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 9 or SEQ ID NO: 12, or a variant thereof having T cell response enhancer activity, wherein the amino acid sequence of the MPD of the variant is at least 60% identical to SEQ ID NO: 3 or SEQ ID NO: 10, and one or more antigens and / or one or more antigenic fragments thereof, relates to a polypeptide comprising

[0009] In a second aspect, the invention further relates to a polynucleotide encoding the polypeptide according to the first aspect of the invention, which is DNA or RNA, preferably DNA.

[0010] In a third aspect, the invention relates to a vector comprising the polynucleotide according to the second aspect of the invention.

[0011] In a fourth aspect, the invention relates to a collection of two or more different vectors, each comprising a polynucleotide according to the second aspect of the invention encoding a different polypeptide according to the first aspect of the invention.

[0012] In a fifth aspect, the invention relates to a pharmaceutical composition comprising the polypeptide of the first aspect of the invention, the polynucleotide of the second aspect of the invention, or the vector / vector collection of the third or fourth aspect of the invention, a pharmaceutically acceptable excipient, and optionally one or more adjuvants.

[0013] In a sixth aspect, the invention relates to a kit of parts comprising the pharmaceutical composition of the fifth aspect of the invention and, separately packaged, at least one immunomodulatory substance, or at least one polynucleotide encoding the immunomodulatory substance, or a vector comprising a polynucleotide encoding the immunomodulatory substance.

[0014] In a seventh aspect, the present invention relates to a polypeptide according to the first aspect, a polynucleotide according to the second aspect, a vector or collection of vectors according to the third or fourth aspect, or a pharmaceutical composition according to the fifth or sixth aspect, or a kit comprising such a pharmaceutical composition, for use in the prevention or treatment of a proliferative disease, preferably cancer, a viral disease, a fungal disease or a bacterial disease.

[0015] The content of the drawings included in this specification is described below. In this regard, also refer to the detailed description of the invention above and / or below.

Brief Description of the Drawings

[0016]

Figure 1

Modes for Carrying Out the Invention

[0017] Before the present invention is described in detail below, it should be understood that since the specific methodologies, protocols and reagents described herein can be changed, the present invention is not limited thereto. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0018] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland, as well as in "Pharmaceutical Substances: Syntheses, Patents, Applications" by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999, the "Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals", Susan Budavari et al. eds., CRC Press, 1996, and the United States Pharmacopeia-25 / National Formulary-20, 2001 published by the United States Pharmcopeial (Pharmacopeial) Convention, Inc., Rockville, Maryland, 2001.

[0019] Throughout the following specification and claims, unless the context requires otherwise, the terms "comprise", "comprises" and "comprising" and other variants thereof are to be construed as meaning that they include the stated features, integers or steps or groups of features, integers or steps, but are not to be construed as excluding any other features, integers or steps or groups of features, integers or steps. In the following sections, various aspects of the invention are defined in more detail. Each aspect so defined can be combined with any one or more other aspects, unless it is clearly indicated otherwise. In particular, any feature shown to be preferred or advantageous can be combined with any one or more other features shown to be preferred or advantageous.

[0020] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions for use, etc.), whether above or below, is hereby incorporated by reference in its entirety to form a part of this specification. No admission is made that the present invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as "being incorporated by reference to form a part of this specification". In case of conflict between the definition or teaching of such a reference forming a part of this specification and the definition or teaching set forth herein, the text of this specification shall prevail.

[0021] The elements of the present invention are described below. These elements are presented with specific embodiments, but it should be understood that these elements can be combined in any way and in any number to create further embodiments. The variously described examples and preferred embodiments are not to be construed as limiting the present invention to only the exemplified embodiments. This specification is to be understood as supporting and encompassing the exemplified embodiments and embodiments that combine any number of the disclosed and / or preferred elements. Further, unless otherwise specified in the context, any permutations and combinations of all the elements described in this application are considered to be disclosed by the specification of this application.

[0022] Definitions For carrying out the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, and recombinant DNA technology are used, which are described in the literature of the relevant technical field (see, for example, Molecular Cloning: A Laboratory Manual, 2 nd nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0023] Some definitions of terms frequently used in this specification are shown below. These terms will have the defined meanings and preferred meanings respectively in the remainder of this specification when they are each used.

[0024] As used in this specification and the appended claims, the singular forms "a", "an", and "the" specifying a quantity include a plurality of referents unless otherwise clearly indicated in the context.

[0025] The term transmembrane domain (TMD) is used in the present invention to refer to the TMD of the invariant chain sequence (INV), and is defined as an amino acid segment that starts with the 17 residues N-terminal of the Gln (Q) residue conserved in all INVs and ends with the 8 residues C-terminal of the conserved Q, thus containing a total of 26 residues.

[0026] The term membrane-proximal domain (MPD) is used in the present invention to refer to a 27-residue segment immediately C-terminal to the TMD of INV.

[0027] The term "adjuvant" is used in the present invention as a substance that enhances the immune response to an antigen. In addition, adjuvants have also evolved as substances that can assist in stabilizing antigen formulations. Adjuvants are added to vaccines to stimulate the immune system's response to the target antigen, but they do not provide immunity themselves. Adjuvants are necessary to improve the routing and adaptive immune response to antigens. Adjuvants apply their effects through various mechanisms. For example, by keeping the antigen present in the blood for a long time and / or by helping antigen-presenting cells absorb the antigen and / or by activating macrophages and lymphocytes and / or by assisting in the production of cytokines. Some adjuvants, such as alum, function as delivery systems by generating a depot that captures the antigen at the injection site and provide sustained release to continuously stimulate the immune system. The types of adjuvants described include: i) inorganic compounds: alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, ii) mineral oils: paraffin oil, iii) bacterial products: killed Bordetella pertussis, Mycobacterium bovis, toxoids, iv) non-bacterial organics: squalene, v) delivery systems: detergents (Quil A, vi) Quillaja (see Quillaia), soybean, plant saponins of Polygala senega, vii) cytokines: IL-1, IL-2, IL-12, viii) combinations: Freund's complete adjuvant, Freund's incomplete adjuvant.

[0028] The term "immunomodulatory substance" is used in the present invention to refer to any drug or substance that affects the immune system. Immunomodulatory substances can i) strengthen a weak immune system and ii) regulate the immune response to the correct level by controlling an overactive immune system. Specific classes of immunomodulatory substances that can strengthen a weak immune system are i) A modulator (MCM) of immunological checkpoint molecules, which is a member of the tumor necrosis factor (TNF) receptor superfamily, preferably an activating activator MCM such as CD27, CD40, OX40, GITR or CD137. ii) An antagonistic inhibitory MCM, which is a member of the superfamily of PD-1, CD274, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3, VISTA or B7-CD28, CD28 or ICOS, or an antagonist of its ligand. It is a modulator (MCM) of immunological checkpoint molecules consisting of.

[0029] Another class of immunomodulatory substances that can enhance a weak immune system are cytokines that act as T cell growth factors. Preferred examples of such cytokines are IL-2, IL-12, IL-15, or IL-17.

[0030] The term "antigen" is used in the context of the present invention to refer to any structure recognized by molecules of the immune response, such as antibodies, T cell receptors (TCRs), etc. Preferred antigens are cellular or foreign proteins associated with a particular disease, such as viral, bacterial or fungal proteins. Antigens can be recognized by a very diverse set of antigen receptors (B cell receptors or T cell receptors) of the adaptive immune system and can induce a humoral or cellular immune response. Antigens that induce such a response are also referred to as immunogens. Some intracellular proteins are processed into smaller peptides and presented by the major histocompatibility complex (MHC), regardless of whether they are foreign or cellular.

[0031] The term "antigenic fragment thereof" refers to a part of a given antigen that is still recognized by molecules of the immune system. The antigenic fragment will contain at least one epitope or antigenic determinant. Preferably, the antigenic fragments of the present invention contain at least one T cell epitope.

[0032] The term "epitope", also known as an antigenic determinant, is used in the context of the present invention to refer to a segment of an antigen, preferably a peptide that is bound by a molecule of the immune system, such as a B-cell receptor, a T-cell receptor or an antibody. An epitope to which an antibody or B cell binds is referred to as a "B-cell epitope", and an epitope to which a T cell binds is referred to as a "T-cell epitope". In this context, the term "binding" preferably refers to a 1×10 5 M -1 or greater, preferably 1×10 6 M -1 or greater, 1×10 7 M -1 or greater, 1×10 8 M -1Relating to specific binding defined as binding by the above binding constants. Those skilled in the art are well aware of how to determine binding constants (see, for example, Caoili, S.E. (2012) Advances in Bioinformatics Vol. 2012). Preferably, specific binding of an antibody to an epitope is mediated by the Fab (Fragment, antigen binding) region of the antibody, specific binding of a B cell is mediated by the Fab region of the antibody contained in the B cell receptor, and specific binding of a T cell is mediated by the variable (V) region of the T cell receptor. T cell epitopes are presented on the surface of antigen-presenting cells, where they bind to major histocompatibility (MHC) molecules. There are at least two different classes of MHC molecules, called MHC class I and class II, respectively. Epitopes presented via the MHC-I pathway induce a response by cytotoxic T lymphocytes (CD8+ cells), and epitopes presented via the MHC-II pathway induce a response by T helper cells (CD4+ cells). T cell epitopes presented by MHC class I molecules are usually peptides 8 to 11 amino acids in length, and T cell epitopes presented by MHC class II molecules are usually peptides 13 to 17 amino acids in length. MHC class III molecules also present non-peptide epitopes as glycolipids. Thus, the term "T cell epitope" preferably refers to peptides 8 to 11 or 13 to 17 amino acids in length that can be presented by either MHC class I or MHC class II molecules. Epitopes usually consist of chemically active surface groupings of amino acids, may or may not have sugar side chains, and usually have specific charge characteristics along with specific three-dimensional structural characteristics. Conformational epitopes and non-conformational epitopes are distinguished in that binding to the former is lost in the presence of a denaturing solvent while binding to the latter is not lost.

[0033] The term "cancer-specific antigen" is used in the context of the present invention to refer to a protein that is specifically expressed in cancer cells or is more abundant in cancer cells than in healthy cells. Cancer-specific antigens include the following types of antigens: (i) Oncofetal (typically expressed only in fetal tissue and cancerous somatic cells), or, (ii) Oncovirus (encoded by a tumorigenic transforming virus), or, (iii) Overexpression / accumulation (expressed by both normal and neoplastic tissues, with a very high expression level in neoplasms), for example, melanoma tyrosinase or breast cancer Her-2 receptor, or, (iv) Cancer-testis (expressed only by cancer cells and adult reproductive tissues such as the testis and placenta), or, (v) Lineage-restricted (expressed mainly by a single cancer tissue type), or, (vi) Cancer-specific isoform (changes in transcriptional exon composition).

[0034] The term "cancer-specific neoantigen" is used in the context of the present invention to refer to an antigen that does not exist in normal / germline cells but occurs in transformed, particularly cancerous cells. A cancer-specific neoantigen may contain one or more, for example, 2, 3, 4, 5 or more neoepitopes. The length of each cancer-specific neoantigen contained in the polypeptides of the present invention is preferably selected such that they are less likely to contain epitopes that occur in normal / germ cells. Typically, this can be confirmed by the cancer-specific neoantigen containing 12 or fewer amino acids at the C-terminus and / or N-terminus of the amino acid change(s) that created the neoepitope.

[0035] Cancer-specific neoantigens are preferably generated by mutations occurring at the DNA level, and the mutated proteins are a) one or more single aa changes caused by non-synonymous SNV point mutations, and / or, b) non-wild-type amino acid sequences caused by insertions / deletions that result in frameshift peptides, and / or, c) a non-wild-type amino acid sequence caused by a mutation that generates an exon boundary change or intron retention, and / or d) a mutant cancer protein generated by a gene fusion event, may be included.

[0036] Neoantigens that are the result of one or more single amino acid changes caused by genomic point mutation non-synonymous SNVs are referred to in the context of the present invention as single amino acid mutation peptides.

[0037] The terms "polynucleotide" or "nucleic acid" include both single-stranded and double-stranded nucleotide polymers. The nucleotides that make up a polynucleotide can be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphorosenoate, phosphorodiselenoate, phosphoranilothioate, phosphoramidate, and phosphoramidate. Examples of polynucleotides are DNA and RNA.

[0038] An "isolated polynucleotide" is DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, that is not associated with all or part of a polynucleotide found in nature or that is linked to a polynucleotide not linked in nature.

[0039] The term "expression cassette" is used in the context of the present invention to refer to a polynucleotide comprising at least one nucleic acid sequence to be expressed, for example, a nucleic acid encoding a series of cancer-specific neoantigens fused to the invariant chain of the present invention or a fragment thereof operably linked to transcriptional and / or translational control sequences. Preferably, the expression cassette comprises cis-regulatory elements for the efficient expression of a given gene, such as a promoter, an initiation site, and / or a polyadenylation site. Preferably, the expression cassette comprises all additional elements necessary for the expression of the polynucleotide in the cells of the patient. Thus, a typical expression cassette comprises a promoter operably linked to the polynucleotide sequence to be expressed, as well as signals necessary for efficient polyadenylation of the transcript, ribosome binding site, and translation termination. Additional elements of the cassette may include, for example, enhancers. The expression cassette also preferably includes a transcription termination region downstream of the structural gene to provide efficient termination. The termination region may be obtained from the same gene as the promoter sequence or from a different gene.

[0040] The term "operably linked" as used in the context of the present invention refers to the arrangement of elements such that the components so described are configured to perform their normal functions. A polynucleotide is "operably linked" when placed in a functional relationship with another nucleic acid sequence. For example, a promoter is operably linked to one or more transgenes if it affects the transcription of the one or more transgenes. Further, control elements operably linked to a coding sequence can affect the expression of the coding sequence. The control elements need not be adjacent to the coding sequence so long as they function to direct the expression of the coding sequence. Thus, for example, intervening untranslated but transcribed sequences can be present between the promoter sequence and the coding sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence.

[0041] The terms "vector" or "expression vector" are used interchangeably and refer to a polynucleotide, a polynucleotide within a particular envelope such as a viral coat or liposome, or a polynucleotide complexed with a protein that can be introduced into a cell, preferably a mammalian cell, or that can introduce the polynucleotide of the present invention. Examples of vectors include, but are not limited to, plasmids, cosmids, phages, liposomes, viruses, or artificial chromosomes. In particular, a vector is used to transport a promoter and the polynucleotide of the present invention into a suitable host cell. An expression vector may contain a "replicon" polynucleotide sequence that promotes autonomous replication of the expression vector in a host cell. Upon entry into a host cell, the expression vector can replicate independently of or simultaneously with the host chromosomal DNA and generate multiple copies of the vector and its inserted DNA. When a non-replicating expression vector is used (which is often the case for safety reasons), the vector may only direct the expression of the polynucleotide without replicating. Depending on the type of expression vector, the expression vector may be lost from the cell, i.e., it may only transiently express the neoantigen encoded by the polynucleotide or it may be stable within the cell. An expression vector typically contains an expression cassette, i.e., the necessary elements to enable transcription of the polynucleotide into an mRNA molecule. If the polynucleotide is RNA, transcription is not required, and the RNA molecule only requires translational control elements.

[0042] The term "T cell enhancer amino acid sequence" refers, in the context of genetic vaccination, to a polypeptide sequence that, when fused to an antigen sequence, increases the induction of T cells.

[0043] As used in the context of the present invention, the terms "preparation" and "composition" are intended to include formulations of active compounds, such as the great ape adenovirus vector of the present invention, that contain a carrier and / or excipient.

[0044] As used in the context of the present invention, "pharmaceutically acceptable" means approved by the regulatory authorities of the federal or state government, or listed in the United States Pharmacopeia, or other generally recognized pharmacopeias for use in animals, more specifically in humans.

[0045] The term "carrier", as used herein, refers to a pharmacologically inert substance such as a diluent, excipient, surfactant, stabilizer, physiological buffer or vehicle, etc., which is administered together with the therapeutically active ingredient, although not limited thereto. Such pharmaceutical carriers can be liquid or solid. Examples of liquid carriers include, but are not limited to, sterile solutions such as physiological saline solutions in water and oil, including those of petroleum origin, animal origin, plant origin or synthetic origin such as groundnut oil, soybean oil, mineral oil, sesame oil, etc. Physiological saline solutions, as well as aqueous solutions of dextrose and glycerol, can also be used as liquid carriers, especially in the case of injection solutions. Physiological saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

[0046] Suitable pharmaceutical "excipients" include starch, glucose, lactose, sucrose, gelatin, malt, rice flour, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc.

[0047] Examples of "surfactants" include, but are not limited to, anionic, cationic and nonionic surfactants such as sodium deoxycholate, sodium dodecyl sulfate, Triton X-100, and polysorbates such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80.

[0048] Examples of the "stabilizer" include, but are not limited to, mannitol, sucrose, trehalose, albumin, and antagonists of protease and / or nuclease.

[0049] Examples of the "physiological buffer" that can be used in the context of the present invention include, but are not limited to, sodium chloride solution, deionized water, and appropriate organic or inorganic buffers such as, but not limited to, phosphate buffer, citrate buffer, Tris buffer (tris(hydroxymethyl)aminomethane), HEPES buffer ([4-(2-hydroxyethyl)piperazino]ethanesulfonic acid), or MOPS buffer (3-morpholino-1-propanesulfonic acid). The choice of each buffer generally depends on the molar concentration of the desired buffer. Phosphate buffer is suitable, for example, for injection solutions and infusion solutions.

[0050] "Effective amount" or "therapeutically effective amount" is the amount of a therapeutic agent sufficient to achieve the intended purpose. The effective amount of a given therapeutic agent varies depending on factors such as the nature of the agent, the route of administration, the size and species of the animal receiving the therapeutic agent, and the purpose of administration. The effective amount in an individual case can be determined empirically by one of ordinary skill in the art according to methods established in the art.

[0051] As used herein, "treating", "treatment", "treat", or "therapy" of a disease or disorder means achieving one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing the onset of symptoms characteristic of the disorder being treated (which may be plural); (c) suppressing the worsening of symptoms characteristic of the disorder being treated (which may be plural); (d) limiting or preventing the recurrence of the disorder (which may be plural) in an individual who has previously had the disorder (which may be plural); and (e) limiting or preventing the recurrence of symptoms in an individual who has previously shown signs of the disorder (which may be plural).

[0052] Embodiments The various aspects of the present invention are defined in more detail below. Each aspect so defined can be combined with any other aspect(s), unless the contrary is explicitly stated. In particular, any feature shown as being preferred or advantageous can be combined with any other feature(s) shown as being preferred or advantageous.

[0053] In a first aspect of the invention, the invention is (a) a fragment of an invariant chain (INV) of teleost fish comprising or consisting of 16 to 27 consecutive amino acids of the membrane-proximal domain (MPD) of the INV of teleost fish, said fragment having T cell response enhancer activity, and said MPD is (i) NQRX1DIKSLEEQX2SX3LX4X5X6X7TX8GRSX9X 10 (SEQ ID NO: 001) (where X1 is G or N, X2 is H or N, X3 is G or N, X4 is N or Q, X5 is E or A, X6 is Q or E, X7 is L or M, X8 is K or R, X9 is A or V, X 10 is S or A, said fragment is preferably at least 60% identical to SEQ ID NO: 3 (MPD of PO), or (ii) DQKQQIQZ1LQZ2Z3NQRZ4EKQZ5Z6Z7RZ8RZ9S (SEQ ID NO: 8) (where Z1 is G or D, Z2 is T or A, Z3 is T or S, Z4 is L or M, Z5 is M or V, Z6 is G or S, Z7 is Q or L, Z8 is P or S, Z9 is E or V, said fragment is preferably at least 60% identical to SEQ ID NO: 10 (MPD of BP)), a fragment characterized by an amino acid sequence selected from, and optionally, one or more antigens and / or one or more antigenic fragments thereof, or, (b) A full-length teleost INV selected from SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 9 or SEQ ID NO: 12, or a variant thereof having T cell response enhancer activity, wherein the amino acid sequence of the MPD of the variant is at least 60% identical to SEQ ID NO: 3 (MPD of PO) or SEQ ID NO: 10 (MPD of BP), and one or more antigens and / or one or more antigenic fragments thereof, Relating to a polypeptide comprising.

[0054] Generally, it is desirable for the fragment of INV to be as short as possible while retaining its T cell antigen-stimulating effect. Preferably, the fragment comprises 16 to 27, 17 to 26, 18 to 25, 19 to 24, 20 to 23, or 21 or 22 consecutive amino acids of the MDP by the MPD of INV, preferably SEQ ID NO: 3 (MPD of PO) or SEQ ID NO: 10 (MPD of BP), and more preferably consists of them.

[0055] The preferred minimum fragment described in option (i) of the first aspect of the present invention has the amino acid sequence: DIKSLEEQX2SX3LX4X5X6X7 (SEQ ID NO: 28), where X2 to X7 have the meanings shown above.

[0056] More preferred minimum fragments described in option (i) of the first aspect of the present invention have the amino acid sequences DIKSLEEQHSGLNEQL (SEQ ID NO: 4) or DIKSLEEQNSNLQAEM (SEQ ID NO: 7), and the most preferred minimum fragment has the amino acid sequence DIKSLEEQHSGLNEQL (SEQ ID NO: 4).

[0057] The preferred minimum fragment described in option (ii) of the first aspect of the present invention has the amino acid sequence: QIQZ1LQZ2Z3NQRZ4EKQZ5 (SEQ ID NO: 29), where Z1 to Z5 have the meanings shown above.

[0058] A more preferred minimum fragment according to option (ii) of the first aspect of the present invention is the amino acid sequence QIQGLQTSNQRLEKQM (SEQ ID NO: 11) or QIQDLQATNQRMEKQV (SEQ ID NO: 14), and the most preferred minimum fragment is the amino acid sequence QIQGLQTSNQRLEKQM (SEQ ID NO: 11).

[0059] The fragment of INV can include additional sequences at the N-terminus and / or C-terminus of MPD, but such sequences are preferably not included in the fragment, and thus the fragment preferably consists of a continuous sequence of amino acids of MPD respectively.

[0060] When the fragment of INV includes additional sequences at the N-terminus and / or C-terminus of MPD, it is preferred that the fragment includes the entire MPD, that is, 27 amino acids. The fragment preferably does not include the TMD but includes additional C-terminal amino acids of INV. Preferably, these C-terminal amino acids are directly contiguous to MPD.

[0061] The sequence of MPD is preferably based on the MPD sequence of Paralichthys olivaceus (PO) according to SEQ ID NO: 003 or Boleophthalmus pectinirostris (BP) according to SEQ ID NO: 010. Preferably, the fragment includes or consists of 16 to 27 amino acids of MPD, where MPD is at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 010.

[0062] The fragment may include additional N-terminal and / or C-terminal amino acid sequences of INV. Therefore, the full length of the fragment of INV is preferably 16 to 80, 17 to 72, 18 to 55, 19 to 50, 20 to 45, 21 to 40, 22 to 35, 23 to 30 consecutive amino acids.

[0063] In a preferred embodiment of the polypeptide of the first aspect of the present invention, the polypeptide comprises or consists of a fragment of the membrane-proximal domain (MPD) of the invariant chain (INV) of teleost fish, the fragment comprising 16 to 27 consecutive amino acids of the MPD of teleost fish INV, the fragment having T cell response enhancer activity, the MPD being characterized by the amino acid sequence DIKSLEEQX2SX3LX4X5X6X7 (SEQ ID NO: 28) (where X2 is H or N, X3 is G or N, X4 is N or Q, X5 is E or A, X6 is Q or E, and X7 is L or M), the fragment being preferably at least 60% identical to SEQ ID NO: 003, and optionally, the polypeptide comprises one or more antigens and / or one or more antigenic fragments thereof.

[0064] In a preferred embodiment of the polypeptide of the first aspect of the present invention, the polypeptide comprises or consists of a fragment of the membrane-proximal domain (MPD) of the invariant chain (INV) of teleost fish, the fragment comprising 16 to 27 consecutive amino acids of the MPD of teleost fish INV, the fragment having T cell response enhancer activity, the MPD being characterized by the amino acid sequence QIQZ1LQZ2Z3NQRZ4EKQZ5 (SEQ ID NO: 29) (where Z1 is G or D, Z2 is T or A, Z3 is T or S, Z4 is L or M, and Z5 is M or V), the fragment being preferably at least 60% identical to SEQ ID NO: 10, and optionally, the polypeptide comprises one or more antigens and / or one or more antigenic fragments thereof.

[0065] In a preferred embodiment of the polypeptide of the first aspect of the present invention, (a) the amino acid sequence of the MPD according to option (a)(i) of claim 1 is any one of SEQ ID NO: 3 (MPD PO) and SEQ ID NO: 6 (MPD Z1), (b) the amino acid sequence of the MPD according to option (a)(ii) of claim 1 is any one of SEQ ID NO: 10 (MPD BP) and SEQ ID NO: 13 (MPD Z2), and / or, (c) the amino acid sequence of the teleost invariant chain according to option (b) of claim 1 is any one of SEQ ID NO: 2 and SEQ ID NO: 9.

[0066] When the fragment consists of 16 to 27 amino acids of the MPD of INV, preferred fragments have a length of 16 to 27, 17 to 26, 18 to 25, 19 to 24, 20 to 23, 21 or 22 consecutive amino acids of the MPD of INV according to SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 13.

[0067] In each case outlined above, the fragment or variant has T cell response enhancer activity. The T cell response enhancer activity can be measured as known in the art or as shown in the following experiments. The T cell response enhancer activity is preferably at least 50%, preferably at least 80% of the T cell response enhancer activity of the INV fragment according to SEQ ID NO: 004 when bound to the same antigen or a series of antigens.

[0068] In a preferred embodiment of the polypeptide of the first aspect of the present invention, the fragment consists of at least 16 to 27 N-terminal amino acids of the MPD and 1 to 26 consecutive amino acids of the transmembrane domain (TMD) of teleost INV immediately N-terminal to the MPD, and the TMD of the teleost INV preferably has the following amino acid sequence (SEQ ID NO: 15) AB1KB2B3GB4TB5LB6CB7LB8B9B 10 QB 11 B 12 B 13 B 14 YB 15 B 16 B 17 (where B1 is V, L or Y, B2 is V or I, B3 is V or A, B4 is L, I or F, B5 is V, T or L, B6 is A or V, B7 is V or L, B8 is V or I, B9 is M, S or A, B 10 is S, A or G, B 11 is A or V, B 12 is M or F, B 13 is I or T, B 14is I or A, and B 15 is F or M, and B 16 is L, M or V, and B 17 is V, F or L) is characterized by

[0069] When the fragment contains an additional N-terminal and / or C-terminal amino acid sequence of INV, the full length of the fragment of INV is preferably 28 to 72, 30 to 65 or 35 to 46 consecutive amino acids.

[0070] Surprisingly, the inventors have found that a strong T cell response against two or more antigens, preferably neoantigens, can be induced by fusion to an INV fragment of option (a) of the first aspect of the invention or two or more antigens and / or antigenic fragments thereof of option (b) of the first aspect of the invention. This enables the simultaneous induction of T cell responses against multiple antigens. Therefore, for both option (a) of the first aspect of the invention and option (b) of the first aspect of the invention, the polypeptide preferably contains a plurality of antigens and / or antigenic fragments thereof. For example, the polypeptide preferably contains at least 5 different antigens and / or antigenic fragments thereof, more preferably at least 20 different antigens and / or antigenic fragments thereof, even more preferably at least 50 different antigens and / or antigenic fragments thereof, even more preferably at least 100 different antigens and / or antigenic fragments thereof, even more preferably at least 200 different antigens and / or antigenic fragments thereof, even more preferably at least 300 different antigens and / or antigenic fragments thereof.

[0071] To accommodate the maximum number of various antigens within one polypeptide, it is particularly preferred that the polypeptide contains antigenic fragments of the antigens.

[0072] The antigen is selected according to each therapeutic use. When therapeutic or prophylactic vaccination against a proliferative disease is desired, the antigen is selected from cancer-specific antigens or cancer-specific neoantigens. As particularly mentioned above in connection with cancer vaccination, the polypeptide of the first aspect preferably contains two or more different antigens. The polypeptide preferably contains at least 5 different cancer-specific antigens or antigenic fragments thereof, more preferably at least 20 different cancer-specific antigens or antigenic fragments thereof, even more preferably at least 50 different cancer-specific antigens or antigenic fragments thereof, even more preferably at least 100 different cancer-specific antigens or antigenic fragments thereof, even more preferably at least 200 different cancer-specific antigens or antigenic fragments thereof, even more preferably at least 300 different cancer-specific antigens or antigenic fragments thereof. Alternatively, the polypeptide preferably contains at least 5 different cancer-specific neoantigens or antigenic fragments thereof, more preferably at least 20 different cancer-specific neoantigens or antigenic fragments thereof, even more preferably at least 50 different cancer-specific neoantigens or antigenic fragments thereof, even more preferably at least 100 different cancer-specific neoantigens or antigenic fragments thereof, even more preferably at least 200 different cancer-specific neoantigens or antigenic fragments thereof, even more preferably at least 300 different cancer-specific neoantigens or antigenic fragments thereof. Alternatively, the polypeptide preferably contains at least 5 different cancer-specific antigens or neoantigens or antigenic fragments thereof, more preferably at least 20 different cancer-specific antigens or neoantigens or antigenic fragments thereof, even more preferably at least 50 different cancer-specific antigens or neoantigens or antigenic fragments thereof, even more preferably at least 100 different cancer-specific antigens or neoantigens or antigenic fragments thereof, even more preferably at least 200 different cancer-specific antigens or neoantigens or antigenic fragments thereof, even more preferably at least 300 different cancer-specific antigens or neoantigens or antigenic fragments thereof.

[0073] Alternatively, the antigen is a viral protein or an antigenic fragment thereof, a bacterial protein or an antigenic fragment thereof, or a fungal protein or an antigenic fragment thereof.

[0074] Generally, prophylactic or therapeutic vaccination against viral, bacterial or fungal infections does not require as many different antigens as vaccination in the treatment of proliferative diseases to be effective. Nevertheless, there are some viruses, such as HIV, that have a high epitope diversity, especially in their coat proteins. To induce a broad immune response, multiple antigens can be included. Therefore, the polypeptide preferably comprises at least 5 different viral antigens or antigenic fragments thereof, more preferably at least 20 different viral antigens or antigenic fragments thereof, even more preferably at least 50 different viral antigens or antigenic fragments thereof, even more preferably at least 100 different viral antigens or antigenic fragments thereof, even more preferably at least 200 different viral antigens or antigenic fragments thereof, even more preferably at least 300 different viral antigens or antigenic fragments thereof. The antigens can be selected from different strains of the same virus and / or from different virus species. In the latter case, the vaccine enables immunization against various different virus species.

[0075] Alternatively, the polypeptide preferably comprises at least 5 different bacterial antigens or antigenic fragments thereof, more preferably at least 20 different bacterial antigens or antigenic fragments thereof, even more preferably at least 50 different bacterial antigens or antigenic fragments thereof, even more preferably at least 100 different bacterial antigens or antigenic fragments thereof, even more preferably at least 200 different bacterial antigens or antigenic fragments thereof, even more preferably at least 300 different bacterial antigens or antigenic fragments thereof.

[0076] Alternatively, the polypeptide preferably comprises at least 5 different fungal antigens or antigenic fragments thereof, more preferably at least 20 different fungal antigens or antigenic fragments thereof, even more preferably at least 50 different fungal antigens or antigenic fragments thereof, even more preferably at least 100 different fungal antigens or antigenic fragments thereof, even more preferably at least 200 different fungal antigens or antigenic fragments thereof, and even more preferably at least 300 different fungal antigens or antigenic fragments thereof.

[0077] In all of the above embodiments, the antigen is preferably a T cell antigen. A T cell antigen is an antigen that is presented by MHC and induces a T cell response.

[0078] Preferably, each of the antigens or antigenic fragments thereof has a length of 8 to 100 amino acids, more preferably 8 to 50 amino acids, and even more preferably 8 to 30 amino acids.

[0079] In a preferred embodiment of the polypeptide of the first aspect of the present invention, one or more antigens and / or one or more antigenic fragments thereof are located at the fragment of INV described in option (a) of the first aspect of the present invention, or at the C-terminus of the full-length INV described in option (b) of the first aspect of the present invention. According to option (a) or (b) of the first aspect of the present invention, it is particularly preferred that the antigen and / or its antigenic fragment is immediately adjacent to the C-terminus of INV.

[0080] The polypeptide of the present invention is preferably produced intracellularly in the vaccinated patient. Intracellular expression is a prerequisite for MHC presentation and thus stimulation of the T cell response. Thus, in a second aspect, the present invention relates to a polynucleotide encoding the polypeptide according to the first aspect of the present invention. Preferably, the polynucleotide is DNA or RNA. The RNA is preferably used to directly induce translation of the encoded polypeptide. The DNA encoding the polypeptide of the first aspect is typically inserted into an expression cassette that directs transcription of the mRNA encoding the polypeptide of the present invention. However, if the polynucleotide is contained in a vector and the vector is an RNA virus, the polynucleotide may be RNA. The RNA preferably for direct application is self-amplifying RNA (SAM). Most preferably, the polynucleotide is DNA.

[0081] In a third aspect, the present invention relates to a vector comprising the polynucleotide according to the second aspect of the present invention. Preferably, the polynucleotide of the present invention is operably linked to an expression control sequence.

[0082] If the number of different antigens or antigenic fragments thereof to be delivered to the patient is very large and a polynucleotide encoding the fusion polypeptide of INV and all antigens or antigenic fragments thereof cannot be accommodated in the selected vector, two or more vectors are used. Thus, in a fourth aspect, the present invention relates to a collection of two or more different vectors each comprising a polynucleotide according to the second aspect of the present invention encoding different polypeptides according to the first aspect of the present invention.

[0083] In the collection of vectors of the third aspect or the vectors of the fourth aspect, in each case, the vector is a plasmid; a cosmid; a liposome particle, a viral vector or a virus-like particle; preferably, an alphavirus vector, a Venezuelan equine encephalitis (VEE) virus vector, a Sindbis (SIN) virus vector, a Semliki Forest virus (SFV) virus vector, a simian or human cytomegalovirus (CMV) vector, a lymphocytic choriomeningitis virus (LCMV) vector, a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a poxvirus vector, a vaccinia virus vector or a modified vaccinia Ankara (MVA) vector, independently selected from the group consisting of. Each member of the collection contains a polynucleotide encoding a different antigen or a fragment thereof, and thus a collection of vectors that are usually administered simultaneously preferably uses the same vector type, for example, an adenovirus-derived vector.

[0084] The most preferred vectors are adenoviral vectors, particularly adenoviral vectors derived from human or non-human great apes. Preferred great apes from which adenoviruses are derived are chimpanzees (Pan), gorillas (Gorilla), and orangutans (Pongo), preferably bonobos (Pan paniscus) and common chimpanzees (Pan troglodytes). Usually, natural non-human great ape adenoviruses are isolated from fecal samples of the respective great apes. The most preferred vectors are non-replicating adenoviral vectors based on the vectors of hAd5, hAd11, hAd26, hAd35, hAd49, ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd55, ChAd63, ChAd73, ChAd82, ChAd83, ChAd146, ChAd147, PanAd1, PanAd2, and PanAd3, or vectors of Ad4 and Ad7 with replication ability. Human adenoviruses hAd4, hAd5, hAd7, hAd11, hAd26, hAd35, and hAd49 are well known in the art. Vectors based on natural ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63, and ChAd82 are detailed in WO 2005 / 071093. Vectors based on natural PanAd1, PanAd2, PanAd3, ChAd55, ChAd73, ChAd83, ChAd146, and ChAd147 are detailed in WO 2010 / 086189.

[0085] In a fifth aspect, the present invention relates to a pharmaceutical composition comprising a polypeptide of the first aspect of the present invention, a polynucleotide of the second aspect of the present invention, or a collection of vectors of the third aspect of the present invention or vectors of the fourth aspect of the present invention, a pharmaceutically acceptable excipient, and optionally one or more adjuvants.

[0086] The inventors have found that administration of at least one immunomodulatory substance, for example a modulator of a checkpoint molecule (MCM), further improves the strength of the T cell response to an antigen or a fragment thereof. Thus, in a preferred embodiment of the fifth aspect, the pharmaceutical composition comprises at least one immunomodulatory substance, such as MCM, or at least one polynucleotide encoding an immunomodulatory substance, such as MCM, or a vector or liposomal particle comprising a polynucleotide encoding an immunomodulatory substance, such as MCM.

[0087] In a sixth aspect, the present invention relates to a kit of parts comprising the pharmaceutical composition of the fifth aspect of the present invention and at least one immunomodulatory substance, such as MCM, or at least one polynucleotide encoding an immunomodulatory substance, such as MCM, or a vector comprising a polynucleotide encoding an immunomodulatory substance, such as MCM, separately packaged.

[0088] In a preferred embodiment of the fifth or sixth aspect, the immunomodulatory substance is MCM and is selected from the group consisting of: (a) agonists of tumor necrosis factor (TNF) receptor superfamily members, preferably CD27 (e.g., Varlilumab), CD40 (e.g., CP-870,893), OX40 (e.g., INCAGN01949 or MEDI0562), GITR (e.g., MEDI1873) or CD137 (e.g., Utomilumab); (b) An antagonist of PD-1 (e.g., pembrolizumab or nivolumab), CD274 (e.g., atezolizumab or durvalumab), A2AR (e.g., preladenant), B7-H3 (e.g., MGA271), B7-H4, BTLA, CTLA-4 (e.g., tremelimumab or AGEN1884), IDO, KIR, LAG3, TIM-3 (e.g., CA-327 or RMT3-23) or VISTA (e.g., CA-170), or an antagonist of a B7-CD28 superfamily member, preferably an antagonist of CD28 or ICOS, or an antagonist of their ligands.

[0089] Other preferred immunomodulatory substances are cytokines that act as T cell growth factors, particularly IL-2, IL-12 or IL-15.

[0090] In a seventh aspect, the invention relates to a polypeptide according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, or a vector or collection of vectors according to the third or fourth aspect of the invention, or a pharmaceutical composition according to the fifth aspect of the invention, or a kit comprising such a pharmaceutical composition, for use in the prevention or treatment of a proliferative disease, preferably cancer, a viral disease, a fungal disease or a bacterial disease.

[0091] In a preferred embodiment, in a polypeptide according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, or a vector or collection of vectors according to the third or fourth aspect of the invention, or a pharmaceutical composition or a kit comprising such a pharmaceutical composition according to the fifth aspect of the invention, the cancer is selected from the group consisting of malignant neoplasms of the lip, oral cavity, pharynx, digestive tract, respiratory tract, intrathoracic organs, bone, articular cartilage, skin, mesothelial tissue, soft tissue, breast, female genitalia, male genitalia, urinary tract, brain and other parts of the central nervous system, thyroid, endocrine glands, lymphoid tissue, and hematopoietic tissue.

[0092] In a preferred embodiment, in the polypeptide according to the first aspect of the present invention, the polynucleotide according to the second aspect of the present invention, or the vector or collection of vectors according to the third or fourth aspect of the present invention, or the pharmaceutical composition according to the fifth aspect of the present invention or a kit comprising such a pharmaceutical composition, at least one immunomodulatory substance, such as MCM, or at least one polynucleotide encoding an immunomodulatory substance, such as MCM, or a vector or liposome particle comprising a polynucleotide encoding an immunomodulatory substance, such as MCM, is administered before, simultaneously with, or after the administration of the polypeptide according to the first aspect of the present invention, the polynucleotide according to the second aspect of the present invention, or the vector or collection of vectors according to the third or fourth aspect of the present invention, or the pharmaceutical composition according to the fifth aspect of the present invention or a kit comprising such a pharmaceutical composition.

[0093] In a preferred embodiment of the seventh aspect of the present invention, the administration of the modulator of the checkpoint molecule is initiated before the initiation of the administration of the vaccine, or the administration of the checkpoint inhibitor is initiated after the initiation of the administration of the vaccine, or the administration of the checkpoint inhibitor is initiated simultaneously with the initiation of the administration of the vaccine.

[0094] In a preferred embodiment of the seventh aspect of the present invention, the vaccination regimen is a heterologous prime-boost with two different viral vectors. Preferred combinations are an adenovirus vector derived from a great ape for priming and a poxvirus vector, a vaccinia virus vector or a modified vaccinia Ankara (MVA) vector for boosting. Preferably, these are administered sequentially at intervals of at least one week, preferably six weeks.

[0095] In an eighth aspect, the present invention relates to the polypeptide according to the first aspect of the present invention, the polynucleotide according to the second aspect of the present invention, or the vector or collection of vectors according to the third or fourth aspect of the present invention for use as a medicament.

Example

[0096] Teleost invariant chain and its fragments convert non-immunogenic cancer antigens into immunogenic antigens The immunogenicity of a great ape adenovirus encoding five cancer neoantigens (Table 1, SEQ ID NOs: 16 to 20) derived from the CT26 mouse tumor cell line was evaluated in BalBC inbred mice. In addition to the five head-to-tail linked cancer neoantigens (penta), the vector GAd-penta (SEQ ID NO: 21) encoding the initiating Met and C-terminal HA tag was found to be non-immunogenic unless the full-length teleost invariant chain sequence (BP_INV FL (SEQ ID NO: 9) or PO_INV FL (SEQ ID NO: 2)) or its fragments (fragment BP_FRAG_A (SEQ ID NO: 10) or BP_FRAG_B (SEQ ID NO: 11), or PO_FRAG_A (SEQ ID NO: 3) or PO_FRAG_B (SEQ ID NO: 4)) was added (SEQ ID NOs: 22 to 27) to the N-terminus of the penta antigen. Mice were immunized by a single intramuscular immunization with GAd at a dose of 10 8 individual virus particles (vp). Spleen cells were collected 2 weeks after immunization and tested by IFN-γ ELISpot by stimulating T cells in the presence of synthetic peptides corresponding to the amino acid sequences of the five neoantigens. The negative control cultures contained cells stimulated with culture medium alone (but containing the solvent dimethyl sulfoxide (DMSO) used in the preparation of the peptides). The immune response (number of T cells producing IFN-γ per million spleen cells) is shown in Figure 1. A response was considered positive if (i) at least 20 specific spots / one million spleen cells were detected and (ii) the number of spots seen in the positive wells exceeded three times the number detected in the mock control wells (DMSO). As shown in Figure 1, the binding of BP_INV FL or PO_INV FL, or their fragments FRAG_A or FRAG_B, to the N-terminus of the penta antigen converted the non-immunogenic vaccine construct Gad-Penta into a highly immunogenic construct, and 100% of the vaccinated animals showed a positive immune response.

[0097] Table 1: Penta-antigen: Composition of the penta-antigen. CT26 neoantigens are present in the penta-antigen assembled in the order shown. Mutated amino acids are underlined in bold for each neoantigen. [Table 1]

[0098] Materials and Methods Immunization of Mice Six-week-old female BalBC mice were purchased from Envigo laboratories. All mice were vaccinated intramuscularly (quadriceps femoris) by injecting 50 μl (total volume 100 μl) of the viral vector at a dose of 10 8 vp into one side.

[0099] IFNγ ELISpot IFNγ-producing T cells were evaluated by an ELISPOT assay 2 weeks after vaccination. MSIP S4510 plates (Millipore) were coated overnight at 4°C with anti-mouse IFNγ (U-CyTech, Utrecht, Netherlands) at 10 μg / ml. After washing and blocking, mouse splenocytes were added at two different densities (2.5×10 5 cells and 5×10 5Cells were plated in duplicate and stimulated overnight with a pool of 25-mer synthetic peptides corresponding to the antigen vaccine sequences at a final concentration of 2 μg / ml of each peptide (Figure 1). Peptide diluent DMSO (Sigma-Aldrich, Milan, Italy) and ConA (Sigma-Aldrich, Milan, Italy) were used as negative and positive controls, respectively. Plates were developed by subsequent incubation with biotinylated anti-mouse IFNγ antibody (U-CyTech, Utrecht, The Netherlands), streptavidin-alkaline phosphatase conjugated (BD Biosciences, New Jersey), and finally BCIP / NBT 1-step solution (Thermo Fisher Scientific, Rockford, Illinois). Plates were acquired and analyzed with a CTL automated plate reader. An ELISpot response was considered positive if all of the following conditions were met: presence of IFNγ production in wells stimulated with Con-A; at least 20 specific spots per million splenocytes; the number of spots seen in positive wells being three times the number detected in mock control wells (DMSO); and a decrease in response with cell dilution. ELISpot data were expressed as IFNγ spot-forming cells (SFC) per million splenocytes.

Claims

1. A fragment of the invariant chain (INV) of a teleost fish consisting of 16 amino acids of the membrane proximal domain (MPD) of the INV of the teleost fish, said MPD being characterized by an amino acid sequence selected from SEQ ID NOs: 4, 7, 11 and 14; and One or more cancer-specific antigens or cancer-specific neoantigens and / or one or more antigenic fragments thereof A polypeptide comprising: A polypeptide, wherein the one or more cancer-specific antigens or cancer-specific neoantigens and / or one or more antigenic fragments thereof are C-terminal to a fragment of the INV.

2. The polypeptide described in claim 1, wherein the polypeptide comprises at least five different cancer-specific antigens or cancer-specific neoantigens and / or antigenic fragments thereof.

3. 3. A polynucleotide encoding the polypeptide of claim 1 or 2, which is DNA or RNA.

4. A vector comprising the polynucleotide of claim 3.

5. A collection of two or more different vectors each comprising the polynucleotide of claim 3 encoding a different polypeptide of claim 1 or 2.

6. 6. The vector of claim 4 or the collection of vectors of claim 5, wherein the vectors are in each case independently selected from the group consisting of a plasmid, a cosmid, a liposomal particle, a viral vector or a virus-like particle.

7. 6. The vector of claim 4 or the collection of vectors of claim 5, wherein the vectors are, in each case, independently selected from the group consisting of an alphavirus vector, a Venezuelan equine encephalitis (VEE) virus vector, a Sindbis (SIN) virus vector, a Semliki Forest virus (SFV) virus vector, a simian or human cytomegalovirus (CMV) vector, a lymphocytic choriomeningitis virus (LCMV) vector, a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a poxvirus vector, a vaccinia virus vector or a modified vaccinia Ankara (MVA) vector.

8. 6. The collection of vectors according to claim 4 or claim 5, wherein the vectors are in each case great ape adenoviral vectors.

9. A pharmaceutical composition comprising a polypeptide according to claim 1 or 2, a polynucleotide according to claim 3, or a vector or collection of vectors according to any one of claims 4 to 8, a pharma- ceutically acceptable excipient and, optionally, one or more adjuvants.

10. 10. The pharmaceutical composition of claim 9, comprising at least one immunomodulatory agent, or at least one polynucleotide encoding said immunomodulatory agent, or a vector or liposomal particle comprising a polynucleotide encoding said immunomodulatory agent.

11. A kit of parts comprising the pharmaceutical composition according to claim 9 or 10 and separately packaged at least one immunomodulator, or at least one polynucleotide encoding said immunomodulator, or a vector comprising a polynucleotide encoding said immunomodulator.

12. (i) the immunomodulator is an MCM; (a) an agonist of a tumor necrosis factor (TNF) receptor superfamily member, and / or (b) an antagonist of PD-1, CD274, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3 or VISTA, or an antagonist of a B7-CD28 superfamily member; and / or (ii) the immunomodulatory agent is a T cell growth factor; A pharmaceutical composition according to claim 9 or 10 or a kit of parts according to claim 11.

13. A polypeptide according to claim 1 or 2, a polynucleotide according to claim 3, a vector or a collection of vectors according to any one of claims 4 to 8, or a pharmaceutical composition according to any one of claims 9 to 12, or a kit comprising such a pharmaceutical composition, for use in the prevention or treatment of cancer.

14. 13. The polypeptide according to claim 1 or 2, the polynucleotide according to claim 3, the vector or collection of vectors according to any one of claims 4 to 8, or the pharmaceutical composition according to any one of claims 9 to 12 or a kit comprising such a pharmaceutical composition for use in the prevention or treatment of cancer, wherein said cancer is selected from the group consisting of malignant neoplasms of the lips, oral cavity, pharynx, digestive tract, respiratory tract, intrathoracic organs, bone, articular cartilage, skin, mesothelial tissue, soft tissue, breast, female genital organs, male genital organs, urinary tract, brain and other parts of the central nervous system, thyroid gland, endocrine glands, lymphatic tissue, and hematopoietic tissue.

15. 15. The polypeptide of claim 1 or 2, the polynucleotide of claim 3, the vector or collection of vectors of any one of claims 4 to 8, or the pharmaceutical composition of any one of claims 9 to 12, or a kit comprising such a pharmaceutical composition, for use, in which at least one immunomodulator, or at least one polynucleotide encoding said immunomodulator, or a vector or liposome particle comprising a polynucleotide encoding said immunomodulator, is administered before, simultaneously with or after administration of the polypeptide of claim 1 or 2, the polynucleotide of claim 3, the vector or collection of vectors of any one of claims 4 to 8, or the pharmaceutical composition of any one of claims 9 to 12, or a kit comprising such a pharmaceutical composition.

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