RU2808567C2 - Invariant chain cancer vaccine from teleost fish - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: polypeptide having T-cell response enhancing activity is provided, the said polypeptide comprises the following: (a) a Teleostei invariant chain (INV) fragment comprising or consisting of 16 to 27 contiguous membrane proximal domain (MPD) amino acids from Teleostei INV, wherein the fragment has the activity enhancing T-cell response, and the fragment contains an amino acid sequence selected from the following SEQ ID NO: 11 and 4; and one or more cancer-specific antigens or cancer-specific neoantigens and/or one or more antigenic fragments thereof; or (b) a full-size Teleostei INV selected from the following SEQ ID NO: 2 or 9, and one or more cancer-specific antigens or cancer-specific neoantigens and/or one or more antigenic fragments thereof. A polynucleotide encoding the claimed polypeptide is proposed; an expression vector containing the said polynucleotide; a collection of two or more different vectors for preventing or treating a proliferative disease; a pharmaceutical composition for preventing or treating a proliferative disease; a kit for preventing or treating a proliferative disease; the use of a polypeptide, polynucleotide, vector or collection of vectors, or a pharmaceutical composition or kits containing such pharmaceutical compositions, for the prevention or treatment of a proliferative disease.

EFFECT: teleost invariant chain polypeptides or fragments thereof act as “T-cell enhancers” by converting non-immunogenic antigenic sequences into immunogenic T-cell antigens.

17 cl, 1 dwg, 1 tbl, 1 ex

Description

The present invention relates to polypeptides containing a fragment of a teleost invariant chain, optionally fused to one or more antigens, or a teleost invariant chain fused to one or more antigens or antigenic fragments thereof, a polynucleotide encoding such polypeptides, vectors containing such polynucleotides, a set of vectors containing such polynucleotides, and the use of such polypeptides, polynucleotides, vectors for the treatment or prevention of a proliferative disease, preferably cancer, a viral disease, a fungal disease or a bacterial disease. Invariant chain polypeptides from teleosts or fragments thereof act as “T cell enhancers” by converting non-immunogenic antigenic sequences into immunogenic T cell antigens.

BACKGROUND OF THE INVENTION

Sometimes vaccines produce a suboptimal or non-T cell immune response. This phenomenon of weak induction of T-cell immune responses is more often observed with vaccinations targeting antigens that are either entirely self-molecules, such as cancer-specific antigens, or partially self-molecules, such as cancer neoantigens. Cancer-specific neoantigens mainly originate from point mutations in the coding regions of genes that result in nonsynonymous single nucleotide variants resulting in a single amino acid change. A change in a single amino acid in a protein sequence very rarely results in a new epitope that can trigger a powerful immune response. In most cases, such a small change either does not generate a new epitope at all, or can generate a very weak one. Due to pre-existing central tolerance to self-antigens, induction of potent immune responses against cancer-specific antigens through vaccination remains challenging. To overcome the deficiency or low immunogenicity of cancer-specific antigens and neoantigens, several strategies have been used to restore the deficient/low immunogenicity of some genetic vaccines. The invariant chain (INV) has been shown to enhance CD8 ⁺ T cell induction in the context of genetic vaccination. The invariant chain is a chaperone protein belonging to class II molecules of the major histocompatibility complex (MHC), necessary for their maturation and assembly. INV also plays a role in the presentation of antigenic peptides and has been demonstrated to enhance T cell induction when fused to antigen in the context of genetic vaccination. Improved ability to immunize with a lentiviral vector expressing ovalbumin fused to INV has been described (Rowe et al, 2006 Mol Ther 13(2) 310-9). Subsequently, various reports have documented enhanced induction of CD8 ⁺ T cell responses by human adenovirus 5 and plasmid DNA vectors expressing INV fusion antigens.

During cancer vaccination, it is important to prevent tumors from evading the immunological response due to the emergence of new cancer-specific antigens that are not recognized by vaccine-induced T cells. The goal of a cancer vaccine in cancer treatment is to induce a diverse population of immune T cells that can recognize and eliminate as many cancer cells as possible at once, to reduce the chance that cancer cells will evade the T cell response. Therefore, it is desirable that the vaccine encodes a fairly large number of cancer-specific antigens. This is especially relevant for the recently described personalized vaccination approach based on cancer-specific neoantigens. To optimize the likelihood of success, the vaccine should target as many neoantigens as possible, however, the maximum insert size per vector is limited. Full-length INV sequences or large fragments thereof occupy a relatively large portion of the vaccine antigen insert. Therefore, the use of a short polypeptide as a T cell enhancer is preferable in the context of cancer vaccination, especially when multiple cancer-specific antigens are used in the vaccine.

Adenovirus-based genetic vaccination platforms, particularly the Great Apes derived Adenovirus (GAd) viral vector, have proven to be very effective in inducing T cell responses, and great ape-derived adenoviruses are suitable for encoding large antigens in the format of artificial genes consisting of polynucleotides encoding fragments of different proteins linked to each other (Borthwick, N., et al., Mol Ther, 2014. 22(2): p.464-75). Surprisingly, when used in the context of cancer-specific neoantigens, a T cell-mediated immune response was not induced.

The present inventors have identified specific INV sequences capable of restoring immunogenicity. Such INV sequences were suitable to overcome the absence or weak immunogenicity of cancer-specific neoantigens. In particular, two short fragments of a non-human INV from teleosts, both lacking the transmembrane domain, were identified that acted as potent T cell enhancers.

The use of human INVs or INVs from phylogenetically related species may result in the unwanted induction of an immune response against a given self-sequence in the context of vaccination. In this case, the autoimmune response will be directed towards normal tissues in which INV is expressed. The present inventors have unexpectedly discovered that teleost INVs, although very different from mammalian INVs, enhance T cell responses to antigens in mammals, and that this effect of enhancing T cell responses occurs for multiple antigens fused to the teleost invariant chain. and that just a short fragment of teleost INV is sufficient to trigger such a response. Thus, the present invention provides, among other things: (i) an improved enhancer of T cell responses to antigens in mammals, characterized by a reduced likelihood of inducing an undesirable T cell response against healthy tissue, (ii) an enhancer of T cell responses against multiple antigens, and ( iii) a short fragment capable of inducing a T cell immune response, which maximizes the ability to fuse a large number of antigens or antigenic fragments thereof.

Summary of the invention

In a first aspect of the invention, the invention relates to a polypeptide comprising: (a) a Teleostei invariant chain (INV) fragment comprising or consisting of 16 to 27 contiguous amino acids of the membrane proximal domain (MPD) in the INV Teleostei, where the fragment has T cell response enhancing activity and the MPD is characterized by an amino acid sequence selected from:

(i) NQRX ₁ DIKSLEEQX ₂ SX ₃ LX ₄ X ₅ X ₆ X ₇ TX ₈ GRSX ₉ X ₁₀ (SEQ ID NO: 001) in which

X ₁ is G or N, X ₂ is H or N, X ₃ is G or N, X ₄ is N or Q, X ₅ is E or A, X ₆ is Q or E, X ₇ is L or M, X ₈ is K or R, X ₉ is A or V and X ₁₀ is S or A; and wherein the fragment is preferably at least 60% identical to SEQ ID NO: 3; or

(ii) DQKQQIQZ ₁ LQZ ₂ Z ₃ NQRZ ₄ EKQZ ₅ Z ₆ Z ₇ RZ ₈ RZ ₉ S (SEQ ID NO: 8) in which

Z ₁ is G or D, Z ₂ is T or A, Z ₃ is T or S, Z ₄ is L or M, Z ₅ is M or V, Z ₆ is G or S, Z ₇ is Q or L, Z ₈ is P or S, and Z ₉ is E or V;

and wherein the fragment is preferably at least 60% identical to SEQ ID NO: 10;

and optionally one or more antigens and/or one or more antigenic fragments thereof;

or

(b) a full-length Teleostei INV selected from SEQ ID NO: 2, 5, 9 or 12, or variants thereof, which has T cell response enhancing activity, wherein the amino acid sequence of the MPD variant is at least 60% identical to SEQ ID NO : 3 or 10, and one or more antigens and/or one or more antigenic fragments thereof.

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

In a third aspect, the invention relates to a vector containing a polynucleotide according to the second aspect of the invention.

In a fourth aspect, the invention provides a set of two or more different vectors, wherein each of the different vectors contains a polynucleotide according to the second aspect of the invention encoding a different polypeptide according to the first aspect of the invention.

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

In a sixth aspect, the invention relates to a kit of parts containing a pharmaceutical composition according to the fifth aspect of the invention and separately packaged at least one immunomodulatory polynucleotide or at least one polynucleotide encoding an immunomodulator, or a vector containing a polynucleotide encoding an immunomodulator.

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

List of figures

The following describes the contents of the figures contained herein. In this context, also refer to the detailed description of the invention above and/or below.

Figure 1; Immunogenicity of GAd-penta (penta) and GAd vectors encoding full-length invariant chains BP and PO (BP_INV FL [SEQ ID NO: 9] and PO_INV_FL [SEQ ID NO: 2]) or their fragments FRAG_A (BP_INV FRAG_A [SEQ ID NO: 10] and PO_INV FRAG_A [SEQ ID NO: 3]) and FRAG_B (BP_INV FRAG_B [SEQ ID NO: 11] and PO_INV FRAG_B [SEQ ID NO: 4]) associated with the N-terminus of the penta antigen. Mice (n=5-6/group) were immunized with 10 ⁸ vp of each vector and immune responses were measured 2 weeks later using a spleen ELISpot assay. Shown are the average responses (number of IFNγ-producing T cells per million splenocytes) to a pool of 5 synthetic peptides corresponding to the pentatope-encoded neoantigen sequences. Values shown are means +/- SEM of measurements taken on 5-6 mice/group.

Detailed Description of the Invention

Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as they may vary. It should also be understood that the terminology used herein is intended to describe specific embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by one skilled in the art.

It is preferred that terms used herein be 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) and as described in Pharmaceutical Substances: Syntheses, Patents, Applications) Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999; the "Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals", edited by Susan Budavari et al., CRC Press, 1996, and the United States Pharmacopeia-25/National Formulary-20, published by United States Pharmcopeial Convention, Inc ., Rockville Md., 2001.

Throughout this specification and the claims that follow it, unless the context otherwise requires, the word “comprise” and variations such as “includes” and “comprising” will be understood to imply the inclusion of the specified feature, integer, step or group attributes, integers or stages, but not to the exclusion of any other attribute, integer, stage or group of integers or stages. The following passages describe various aspects of the invention in more detail. Each aspect so defined may be combined with any other aspect or aspects unless expressly stated otherwise. In particular, any feature indicated as preferred or advantageous may be combined with any other feature or features indicated as preferred or advantageous.

Several documents are cited throughout this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), both above and below, is incorporated herein by reference in its entirety. Nothing herein shall be construed as an admission that an invention is not entitled to backdate such disclosure by virtue of a prior invention. Some of the documents cited herein are described as “incorporated by reference.” In the event of a conflict between the definitions or ideas of such incorporated references and the definitions or ideas set forth in this specification, the text of this specification shall control.

Next, elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any quantity to create additional embodiments. The various examples and preferred embodiments described should not be construed as limiting the present invention to only those embodiments expressly described. It should be understood that this description supports and covers embodiments that combine the expressly described embodiments with any number of disclosed and/or preferred elements. In addition, any permutations and combinations of all described elements in this application are to be deemed disclosed in the specification of this application unless the context indicates otherwise.

Definitions

For the practical application of the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry and recombinant DNA technology are used as described in the relevant literature (see, for example, Molecular Cloning: A Laboratory Manual, ^2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

The following are some definitions of terms frequently used in this specification. These terms, whenever used in the remainder of the specification, will have their respective meanings and preferred meanings.

As used in this specification and the accompanying claims, the singular forms include plural references unless the content clearly indicates otherwise.

The term transmembrane domain (TMD), used in the present invention to refer to the TMD of invariant chain (INV) sequences, is defined as the amino acid segment starting with 17 residues located N-terminal to the Gln (Q) residue, conserved in all INVs, and ending with 8 residues located C-terminal to the conserved Q, thus including a total of 26 residues.

The term “membrane proximal domain” (MPD) is used in the present invention to refer to the 27 residue segment immediately C-terminal to the TMD INV sequences.

The term "adjuvant" is used in the present invention as substances that enhance the immune response to an antigen. In addition, adjuvants are also substances that can help stabilize antigen formulations. Adjuvants are added to vaccines to stimulate the immune system's response to the target antigen, but do not provide immunity on their own. Adjuvants are necessary to improve the routing and adaptive immune response to antigens. The effects of adjuvants are due to various mechanisms. For example, by increasing the presence of antigen in the blood and/or by helping antigen-presenting cells take up antigen, and/or by activating macrophages and lymphocytes, and/or by supporting the production of cytokines. Some adjuvants, such as alum, act as delivery systems, generating depots that trap antigens at the injection site, providing a slow release that continues to stimulate the immune system. The types of adjuvants described include: i) Inorganic compounds: alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide ii) Mineral oil: paraffin oil iii) Bacterial products: killed Bordetella pertussis, Mycobacterium bovis bacteria, toxoids iv) Non-bacterial organic substances: squalene, v) Delivery systems: detergents (Quil A), vi) plant saponins from Quillaja (see Quillaia), soybean, Polygala senega, vii) Cytokines: IL-1, IL-2, IL-12, viii) Combination: complete adjuvant Freund's, incomplete Freund's adjuvant.

The term "immunomodulator" is used in the present invention to mean any drug or substance that affects the immune system. An immunomodulator can regulate the immune response to the desired level by: i) strengthening a weak immune system; ii) control of an overactive immune system. A special class of immunomodulators that can strengthen a weak immune system are immunological checkpoint molecule (ICM) modulators, consisting of

i) agonistic activator MSM, such as a member of the tumor necrosis factor (TNF) receptor superfamily, preferably CD27, CD40, OX40, GITR or CD137

ii) antagonistic inhibitory MSM such as PD-1, CD274, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KTR, LAG3, TIM-3, VISTA or a member of the B7-CD28 superfamily, CD28 or ICOS, or its ligand antagonist.

Another class of immunomodulators that can strengthen a weak immune system are cytokines, which act as T-cell growth factors. Preferred examples of such cytokines are IL-2, IL-12, IL-15 or TL-17.

The term "antigen" is used in the context of the present invention to refer to any structure recognized by molecules of the immune response, for example, antibodies, T cell receptors (TCRs) and the like. Preferred antigens are cellular or foreign (eg, viral, bacterial or fungal) proteins that are associated with a particular disease. Antigens are recognized by highly variable antigen receptors (B-cell receptor or T-cell receptor) of the adaptive immune system and can trigger a humoral or cellular immune response. Antigens that cause this response are also called immunogens. Some proteins within cells, whether foreign or cellular, are processed into smaller peptides and presented to the major histocompatibility complex (MHC).

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

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 molecules of the immune system, for example, B cell receptors, T cell receptors or antibodies. Epitopes associated with antibodies or B cells are called "B cell epitopes", and epitopes associated with T cells are called "T cell epitopes". As used herein, the term "binding" preferably refers to specific binding, which is defined as binding with an association constant between an antibody or T-cell receptor (TCR) and the corresponding epitope of 1 x 10 ⁵ M-1 or greater, preferably 1 x 10 ⁶ M-1, 10 ⁷ M-1, 1 x 10 ⁸ M-1 or higher. The skilled person is well aware of how to determine the association constant (see, for example, Caoili, SE (2012) Advances in Bioinformatics Vol.2012). Preferably, specific binding of an antibody to an epitope is mediated by the Fab 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) T-cell receptor region. T cell epitopes are presented on the surface of the antigen presenting cell, where they are associated with major histocompatibility molecules (MHC). There are at least two different classes of MHC molecules, called MHC class I and II, respectively. Epitopes presented through the MHC-I pathway induce a cytotoxic T lymphocyte (CD8+ cell) response, whereas epitopes presented through the MHC-II pathway induce a T helper cell (CD4+ cell) response. T cell epitopes presented by MHC class I molecules are typically peptides 8 to 11 amino acids long, and T cell epitopes presented by MHC class II molecules are typically peptides 13 to 17 amino acids long. MHC class III molecules also present non-peptide epitopes as glycolipids. Accordingly, the term “T cell epitope” preferably refers to a peptide of 8 to 11 or 13 to 17 amino acids in length, which may be an MHC class I or MHC class II molecule. Epitopes typically consist of reactive surface moieties of amino acids that may or may not contain sugar side chains and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes differ in that binding to the former, but not to the latter, is lost in the presence of denaturing solvents.

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 (usually expressed only in fetal tissues and cancer somatic cells); or

(ii) oncoviral (encoded by oncogenic cell-transforming viruses); or

(iii) overexpression/accumulation (expressed in both normal and neoplastic tissue, with high levels of expression in neoplasia), for example, tyrosinase in melanomas or Her-2 receptor in breast cancer; or

(iv) cancer-testis (expressed only by cancer cells and adult reproductive tissues such as testes and placenta); or

(v) lineage-restricted (expressed primarily by one cancer histotype); or

(vi) cancer-specific isoform (change in exon composition of the transcript).

The term “cancer-specific neoantigen” is used in the context of the present invention to refer to an antigen that is not present in normal germline cells/cells, but is found in transformed cells, in particular cancer cells. A cancer-specific neoantigen may contain one or more, for example, 2, 3, 4, 5 or more neoepitopes. Preferably, the length of each cancer-specific neoantigen included in the polypeptide of the present invention is selected to ensure that they have a low probability of including epitopes that are found in normal/germ line cells. This is usually confirmed by the fact that the cancer-specific neoantigen contains 12 or fewer amino acids at the C-terminus and/or at the N-terminus of the amino acid change(s) that led to the formation of the neoepitope.

The cancer-specific neoantigen is preferably generated by a mutation occurring at the DNA level, and where the mutated protein may include a) one or more single amino acid (aa) changes caused by a non-synonymous SNV point mutation; and/or

b) a non-wild type amino acid sequence resulting from insertions/deletions resulting in a frameshift peptide; and/or

c) a non-wild type amino acid sequence resulting from changes in exon boundaries or mutations causing intron retention; and/or

d) mutated oncoprotein formed as a result of gene fusion.

A neoantigen that results from one or more single amino acid changes caused by a genomic point mutation of a nonsynonymous SNV is referred to as a single amino acid mutant peptide in the context of the present invention.

The term "polynucleotide" or "nucleic acid" includes polymers of both single-stranded and double-stranded nucleotides. The nucleotides that make up a polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of any type of nucleotide. These modifications include base modifications such as bromuridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphodiselenoate, phosphoranylothioate, phosphoroaniladate and phosphoryladate. Examples of polynucleotides are DNA and RNA.

An "isolated polynucleotide" is DNA or RNA of genomic, mRNA, cDNA or synthetic origin, or a combination thereof, that is not associated with all or part of a polynucleotide in which the isolated polynucleotide occurs naturally, or is associated with a polynucleotide to which it is not associated in nature. nature.

The term "expression cassette" is used in the context of the present invention to mean a polynucleotide that contains at least one nucleic acid sequence to be expressed, for example, a nucleic acid encoding a chain of cancer-specific neoantigens fused to an invariant chain of the present invention or fragments thereof, operably linked to transcriptional and/or translational control sequences. Preferably, the expression cassette includes cis-regulatory elements for efficient expression of the gene, such as a promoter, an initiation site and/or a polyadenylation site. Preferably, the expression cassette contains all additional elements necessary for expression of the polynucleotide in the patient's cell. Thus, a typical expression cassette contains a promoter operably linked to the expressed polynucleotide sequence and the signals necessary for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. Additional elements of the cassette may include, for example, amplifiers. The expression cassette preferably also contains a transcription termination region downstream of the structural gene to ensure efficient termination. The terminator region may be derived from the same gene as the promoter sequence or may be derived from a different gene.

The term "operably linked" as used in the context of the present invention refers to an arrangement of elements in which the components are so described and configured to perform their normal functions. A polynucleotide is "operably linked" if it is in operably linked to another nucleic acid sequence. For example, a promoter is operably linked to one or more transgenes if it affects transcription of one or more transgenes. In addition, controls operably associated with a coding sequence are capable of affecting the expression of the coding sequence. The controls do not need to be adjacent to the coding sequence as long as they function to direct its expression. Thus, for example, intervening untranslated but transcribed sequences may be present between the promoter sequence and the coding sequence, and the promoter sequence may still be considered to be “operably linked” to the coding sequence.

The terms "vector" or "expression vector" are used interchangeably and refer to a polynucleotide, a polynucleotide in some type of envelope, such as a viral envelope or a liposome, or a polynucleotide complexed with proteins that can be introduced or introduce the polynucleotide of the present invention into a cell, preferably a cell mammal. Examples of vectors include, but are not limited to, plasmids, cosmids, phages, liposomes, viruses, or artificial chromosomes. In particular, the vector is used to transport the promoter and polynucleotide of the invention into a suitable host cell. Expression vectors may contain replicon polynucleotide sequences that facilitate autonomous replication of the expression vector in a host cell. Once in a host cell, the expression vector can replicate independently of or simultaneously with the host's chromosomal DNA, and multiple copies of the vector and its inserted DNA can be created. In the case of using expression vectors that are not capable of replication, which is often the case for safety reasons, the vector may not replicate but simply direct the expression of the polynucleotide. Depending on the type of expression vector, the expression vector may be lost from the cell, that is, it only transiently expresses the neoantigens encoded by the polynucleotide, or it may be stable in the cell. Expression vectors typically contain expression cassettes, i.e. necessary elements that allow the transcription of a polynucleotide into an mRNA molecule. If the polynucleotide is RNA, no transcription is required and therefore the RNA molecules only require translation control elements.

The term "T cell enhancer amino acid sequence" refers to a polypeptide sequence that, when fused to an antigenic sequence, enhances the induction of T cells in the context of genetic vaccination.

The terms “preparation” and “composition” as used in the context of the present invention are intended to include a composition of the active compound, for example, the ape adenovirus vector of the present invention, with a carrier and/or excipient.

"Pharmaceutically acceptable" as used in the context of the present invention means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopoeia or other generally accepted pharmacopoeia for use in animals and, more specifically, in humans.

The term “carrier” as used herein refers to a pharmacologically inactive substance, such as, but not limited to, diluent, excipient, surfactants, stabilizers, physiological buffers or carriers, with which the therapeutically active ingredient is administered. Such pharmaceutical carriers may be liquid or solid. Liquid carrier includes, but is not limited to, sterile liquids such as saline solutions in water and oils, including, but not limited to, liquids of petroleum, animal, vegetable or synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil oil and the like. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, especially for injection solutions. Saline is the preferred carrier for intravenous administration of a pharmaceutical composition. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" E.W. Martin.

Suitable pharmaceutical "excipients" include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, glycol, water, ethanol and the like.

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

"Stabilizers" include, but are not limited to, mannitol, sucrose, trehalose, albumin, and protease and/or nuclease antagonists.

"Physiological buffer solution" that can be used in the context of the present invention includes, but is not limited to, sodium chloride solution, demineralized water, and suitable organic or inorganic buffer solutions 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 the appropriate buffer usually depends on the desired buffer molarity. Phosphate buffer is suitable, for example, for solutions for injections and infusions.

An "effective amount" or "therapeutically effective amount" is an amount of a therapeutic agent sufficient to achieve the intended purpose. The effective amount of a given therapeutic agent will vary 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 for administration.

The effective amount in each individual case can be determined empirically by one skilled in the art in accordance with methods established in the art.

As used herein, the terms “treat,” “treat,” “cure,” or “therapy” for a disease or disorder mean doing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing the development of symptoms characteristic of the disorders being treated; (c) suppressing the exacerbation of symptoms characteristic of the disorders being treated; (d) limiting or preventing relapse of the disorder(s) in a person who previously suffered from the disorder(s); and (f) limiting or preventing the recurrence of symptoms in people who have previously exhibited symptoms of the disorder(s).

Embodiments

Various aspects of the invention are described in more detail below. Each aspect so defined may be combined with any other aspect or aspects unless expressly stated otherwise. In particular, any feature indicated as preferred or advantageous may be combined with any other feature or features indicated as preferred or advantageous.

In a first aspect of the invention, the invention relates to a polypeptide comprising: (a) a Teleostei invariant chain (INV) fragment comprising or consisting of 16 to 27 contiguous amino acids of the membrane proximal domain (MPD) in the INV from Teleostei, wherein the fragment has T cell response enhancing activity and the MPD is characterized by an amino acid sequence selected from:

(i) NQRX ₁ DIKSLEEQX ₂ SX ₃ LX ₄ X ₅ X ₆ X ₇ TX ₈ GRSX ₉ X ₁₀ (SEQ ID NO: 001)

wherein

X ₁ is G or N, X ₂ is H or N, X ₃ is G or N, X ₄ is N or Q, X ₅ is E or A, X ₆ is Q or E, X ₇ is L or M, X ₈ is K or R, X ₉ is A or V and X ₁₀ is S or A;

and wherein the fragment is preferably at least 60% identical to SEQ ID NO: 3; or

(ii) DQKQQIQZ ₁ LQZ ₂ Z ₃ NQRZ ₄ EKQZ ₅ Z ₆ Z ₇ RZ ₈ RZ ₉ S (SEQ ID NO: 8)

wherein

Z ₁ is G or D, Z ₂ is T or A, Z ₃ is T or S, Z ₄ is L or M, Z ₅ is M or V, Z ₆ is G or S, Z ₇ is Q or L, Z ₈ is P or S, and Z ₉ is E or V;

and wherein the fragment is preferably at least 60% identical to SEQ ID NO: 10;

and optionally one or more antigens and/or one or more antigenic fragments thereof;

or

(b) a full-length Teleostei INV selected from SEQ ID NO: 2, 5, 9 or 12, or variants thereof, which has T cell response enhancing activity, wherein the amino acid sequence of the MPD variant is at least 60% identical to SEQ ID NO : 3 or 10, and one or more antigens and/or one or more antigenic fragments thereof. It is generally desirable that the INV fragment be as short as possible while maintaining its T cell antigen stimulatory effect. Preferably, the fragment contains, more preferably consists of 16 to 27, 17 to 26, 18 to 25, 19 to 24, 20 to 23, 21 to 22 contiguous amino acids MPD INV, preferably MPD according to SEQ ID NO: 3 [MPD in PO] or 10 [MPD in BP].

The preferred minimum fragment according to alternative (i) of the first aspect of the invention has the amino acid sequence:

DTKSLEEQX ₂ SX ₃ LX ₄ X ₅ X ₆ X ₇ (SEQ ID NO: 28) with the above value for X ₂ to X ₇ .

A more preferred minimum fragment according to alternative (i) of the first aspect of the invention has the amino acid sequence DIKSLEEQHSGLNEQL (SEQ ID NO: 4) or DIKSLEEQNSNLQAEM (SEQ ID NO: 7), and the most preferred minimum fragment is the amino acid sequence DIKSLEEQHSGLNEQL (SEQ ID NO: 4) .

The preferred minimum fragment according to alternative (ii) of the first aspect of the invention has the amino acid sequence:

QIQZ ₁ LQZ ₂ Z ₃ NQRZ ₄ EKQZ ₅ (SEQ ID NO: 29) with the above value for Z ₁ to Z ₅ .

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

Although the INV fragment may contain additional sequences at the N- and/or C-terminus of the MPD, it is preferable that such sequences are not contained in the fragment, and thus it is preferable that the fragment consists of a corresponding contiguous stretch of MPD amino acids.

If the INV fragment contains additional sequences at the N- and/or C-terminus of the MPD, it is preferable that the fragment contains the entire MPD, i.e. 27 amino acids. Preferably, the fragment does not contain a TMD, but contains additional C-terminal amino acids of INV. Preferably, these C-terminal amino acids are located immediately after the MPD.

The MPD sequence is preferably based on the MPD sequence from Paralichthys olivaceus (PO) according to SEQ ID NO: 003 or Boleophthalmus pectinirostris (BP) according to SEQ ID NO: 010. Preferably the fragment contains or consists of 16 to 27 amino acids MPD, wherein the 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.

The fragment may contain additional N- and/or C-terminal amino acid sequences of INV. Thus, it is preferable that the total length of the INV fragment be from 16 to 80, from 17 to 72, from 18 to 55, from 19 to 50, from 20 to 45, from 21 to 40, from 22 to 35, from 23 to 30 adjacent amino acids.

In a preferred embodiment of the polypeptide of the first aspect of the invention, the polypeptide comprises a Teleostei invariant chain (INV) fragment containing or consisting of 16 to 27 contiguous amino acids of the membrane proximal domain (MPD) of the Teleostei INV, wherein the fragment has T cell response enhancing activity, and MPD is characterized by the amino acid sequence DIKSLEEQX ₂ SX ₃ LX ₄ X ₅ X ₆ X ₇ (SEQ ID NO: 28), where X ₂ is H or N, X ₃ is G or N, X ₄ is N or Q, X ₅ is E or A, X ₆ is Q or E, X ₇ is L or M, wherein the fragment is preferably at least 60% identical to SEQ ID NO: 003 and optionally wherein the polypeptide contains one or several antigens and/or one or more antigenic fragments thereof. In a preferred embodiment of the polypeptide of the first aspect of the invention, the polypeptide comprises a Teleostei invariant chain (INV) fragment containing or consisting of 16 to 27 contiguous amino acids of the Teleostei INV membrane proximal domain (MPD), wherein the fragment has T cell response enhancing activity, hMPD characterized by the amino acid sequence QIQZ ₁ LQZ ₂ Z ₃ NQRZ ₄ EKQZ ₅ (SEQ ID NO: 29), where Z ₁ is G or D, Z ₂ is T or A, Z ₃ is T or S, Z ₄ is L or M, Z ₅ is M or V, and the fragment is preferably at least 60% identical to SEQ ID NO: 10 and, optionally, wherein the polypeptide contains one or more antigens and/or one or more antigenic fragments thereof.

In a preferred embodiment of the polypeptide of the first aspect of the invention, the amino acid sequence is:

(a) the MPD, according to alternative (a) (i) according to claim 1, is any of SEQ ID NO: 3 [MPD PO] and 6 [MPDZ1];

(b) the MPD, according to alternative (a) (ii) according to claim 1, is any of SEQ ID NO: 10 [MPD BP] and 13 [MPD Z2]; and/or

(c) the Teleostei invariant chain, according to alternative (b) according to claim 1, is any of SEQ ID NO: 2 and 9.

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

In each case described above, the fragment or variant has T cell response enhancer activity. T cell response enhancer activity can be measured as known in the art or as set forth in the accompanying experiments. Preferably, the T cell response enhancer activity is 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 associated with the same antigen or chain of antigens.

In a preferred embodiment of the polypeptide of the first aspect of the 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 INV Teleostei transmembrane domain (TMD) immediately at the N-terminus of the MPD, the TMD of the INV Teleostei preferably characterized by the following amino acid sequence (SEQ ID NO: 15)

AB ₁ KB ₂ B ₃ GB ₄ TB ₅ LB ₆ CB ₇ LB ₈ B ₉ B ₁₀ QB ₁₁ B ₁₂ B ₁₃ B ₁₄ YB ₁₅ B ₁₆ B ₁₇

wherein

B ₁ is V, L or Y, B ₂ is V or I, B ₃ is V or A, B ₄ is L, I or F, B ₅ is V, T or L, B ₆ is A or V, B ₇ is V or L, B ₈ is V or I, B ₉ 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 ₁₄ is I or A, B ₁₅ is F or M, B ₁₆ is L, M or V, B ₁₇ is V, F or L. If the fragment contains additional N- and/or C-terminal amino acid sequences from INV, it is preferred that the total length of the INV fragment be from 28 to 72, from 30 to 65, or from 35 to 46 contiguous amino acids.

The inventors of the present invention have unexpectedly discovered that a strong T cell response to two or more antigens, preferably neoantigens, can be induced by fusing a fragment of INV alternative (a) of the first aspect of the invention or INV alternative (b) of the first aspect of the invention with two or more antigens and /or their antigenic fragments. This allows for the simultaneous induction of a T cell response against multiple antigens. Thus, with respect to both alternative (a) of the first aspect of the invention and alternative (b) of the first aspect of the invention, it is preferable that the polypeptide contains multiple antigens and/or antigenic fragments thereof. For example, it is preferable that the polypeptide 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 or antigenic fragments thereof. antigenic fragments, 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 and even more preferably at least 300 different antigens and/or antigenic fragments thereof antigenic fragments.

To accommodate the maximum number of different antigens in a single polypeptide, it is particularly preferred that the polypeptide contains antigenic fragments of the antigens.

Antigens are selected depending on the respective therapeutic application. If therapeutic or prophylactic vaccination against a proliferative disease is desired, the antigen is selected from a cancer-specific antigen or a cancer-specific neoantigen. As stated above, particularly in the context of cancer vaccination, it is preferred that the polypeptide of the first aspect comprises two or more different antigens. Preferably, the polypeptide 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, and even more preferably at least 300 different cancer-specific antigens or antigenic fragments thereof. Alternatively, it is preferred that the polypeptide comprises 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 fragments, 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, and even more preferably at least 300 different cancer-specific neoantigens or antigenic fragments thereof. Alternatively, it is preferred that the polypeptide comprises 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, and even more preferably at least 300 different cancer-specific antigens, or neoantigens, or antigenic fragments thereof.

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.

As a rule, to be effective, preventive or therapeutic vaccination against viral, bacterial or fungal infection does not require as many different antigens as vaccination for the treatment of proliferative diseases. However, there are some viruses, such as HIV, that are characterized by a large diversity of epitopes, particularly in their envelope proteins. Multiple antigens can be included to elicit a broad immune response. Thus, it is preferable that the polypeptide contains at least 5 different viral antigens or an antigenic fragment thereof, more preferably at least 20 different viral antigens or an antigenic fragment thereof, even more preferably at least 50 different viral antigens or an antigenic fragment thereof, even more preferably at least 50 different viral antigens or an antigenic fragment thereof, and more preferably at least 100 different viral antigens or an antigenic fragment thereof, even more preferably at least 200 different viral antigens or an antigenic fragment thereof, and even more preferably at least 300 different viral antigens or an antigenic fragment thereof. The antigens may be selected from different strains of the same virus and/or from different viral species. In the latter case, the vaccine allows immunization against many different types of viruses.

Alternatively, it is preferred that said polypeptide comprises at least 5 different bacterial antigens or an antigenic fragment, more preferably at least 20 different bacterial antigens or an antigenic fragment, even more preferably at least 50 different bacterial antigens or an antigenic fragment, even more preferably at least 50 different bacterial antigens or an antigenic fragment, still more preferably at least 100 different bacterial antigens or an antigenic fragment thereof, even more preferably at least 200 different bacterial antigens or an antigenic fragment thereof, and even more preferably at least 300 different bacterial antigens or an antigenic fragment thereof.

Alternatively, it is preferable that said polypeptide comprises at least 5 different fungal antigens or an antigenic fragment, more preferably at least 20 different fungal antigens or an antigenic fragment, even more preferably at least 50 different fungal antigens or an antigenic fragment, even more preferably preferably at least 100 different fungal antigens or an antigenic fragment thereof, even more preferably at least 200 different fungal antigens or an antigenic fragment thereof, and even more preferably at least 300 different fungal antigens or an antigenic fragment thereof.

In all of the above embodiments, it is preferred that the antigens are T cell antigens. T cell antigens are antigens that are presented by the MHC and trigger a T cell response.

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

In a preferred embodiment of the polypeptide of the first aspect of the invention, one or more antigens and/or one or more antigenic fragments thereof are located at the C-terminus of an INV fragment according to alternative (a) of the first aspect of the invention or a full-length INV according to alternative (b) of the first aspect of the invention. It is particularly preferred that the antigens and/or antigenic fragments thereof are located directly at the C-terminus of the INV according to alternative (a) or (b) of the first aspect of the invention.

Preferably, the polypeptides of the invention are produced within the cells of the patient being vaccinated. Intracellular expression is a prerequisite for MHC presentation and thus stimulation of T cell responses. Accordingly, in a second aspect, the present invention provides a polynucleotide encoding a polypeptide according to the first aspect of the present invention. Preferably the polynucleotide is DNA or RNA. RNA is preferably used to directly cause translation of the encoded polypeptide. DNA encoding the polypeptide of the first aspect is typically inserted into expression cassettes that direct transcription of the mRNA encoding the polypeptides of the invention. However, a polynucleotide can also be RNA if the polynucleotide is contained in a vector and the vector is an RNA virus. The preferred RNA for direct use is self-amplifying RNA (SAM). Most preferably, the polynucleotide is DNA.

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

Two or more vectors are used if the number of different antigens or antigenic fragments thereof to be delivered to the patient is so large that the polynucleotide encoding the fusion polypeptide of INV and all antigens or antigenic fragments thereof cannot be accommodated in the selected vector. Accordingly, in a fourth aspect, the present invention provides a set of two or more different vectors, wherein each of the different vectors contains a polynucleotide according to the second aspect of the present invention encoding a different polypeptide according to the first aspect of the present invention.

A vector of the third aspect or a set of vectors of the fourth aspect, wherein the vector in each case is independently selected from the group consisting of a plasmid; cosmids; liposomal particle, viral vector or virus-like particle; preferably alphavirus vector, Venezuelan equine encephalitis (VEE) virus vector, sindbis virus vector (SIN), Semliki Forest virus (SFV) virus vector, simian or human cytomegalovirus (CMV) vector, lymphocytic choriomeningitis virus (LCMV) vector, retroviral vector, lentiviral vector, adenoviral vector, adeno-associated viral vector, poxvirus vector, vaccinia virus vector or modified vaccinia vector (MVA). Preferably, a vector set in which each member of the set contains a polynucleotide encoding a different antigen or fragments thereof, and which is thus typically administered simultaneously, uses a vector of the same type, for example, an adenovirus-derived vector.

Most preferred vectors are adenoviral vectors, in particular adenoviral vectors derived from apes or non-human apes. The preferred apes from which adenoviruses originate are chimpanzees (Pan), gorillas (Gorilla) and orangutans (Pongo), preferably bonobos (Pan paniscus) and common chimpanzees (Pan troglodytes). Commonly naturally occurring non-human ape adenoviruses are isolated from stool samples from the respective monkey. The most preferred vectors are non-replicating adenoviral vectors based on the vectors 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 replication-competent vectors Ad4 and Ad7. Human adenoviruses hAd4, hAd5, hAd7, hAd11, hAd26, hAd35 and hAd49 are well known in the art. Vectors based on naturally occurring ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63 and ChAd63 and ChAd63 are described in detail in WO 2005/071093. Vectors based on the naturally occurring PanAd1, PanAd2, PanAd3, ChAd55, ChAd73, ChAd83, ChAd146 and ChAd147 are described in detail in WO 2010/086189.

In a fifth aspect, the present invention provides a pharmaceutical composition comprising a polypeptide according to the first aspect of the present invention, a polynucleotide according to the second aspect of the present invention or a vector according to the third aspect of the present invention or a set of vectors according to the fourth aspect of the invention and a pharmaceutically acceptable excipient and optionally one or more adjuvants.

The present inventors have discovered that administration of at least one immunomodulator, such as a checkpoint molecule modulator (MCM), further increases the strength of the T cell response to an antigen or fragment thereof. Thus, in a preferred embodiment of the fifth aspect, the pharmaceutical composition contains at least one immunomodulator, for example, MSM, or at least one polynucleotide encoding an immunomodulator, for example, MSM, or a vector or liposomal particle containing a polynucleotide encoding an immunomodulator, for example, MSM.

In a sixth aspect, the present invention provides a kit of parts containing a pharmaceutical composition according to the fifth aspect of the present invention and separately packaged at least one immunomodulator, for example, MSM, or at least one polynucleotide encoding an immunomodulator, for example, MSM or a vector containing the polynucleotide , encoding an immunomodulator, such as MSM.

In a preferred embodiment of the fifth or sixth aspect, the immunomodulator is MSM and is selected from the group consisting of:

(a) an agonist of a member of the tumor necrosis factor (TNF) receptor superfamily, preferably CD27 (eg, Varlilumab), CD40 (eg, CP-870,893), OX40 (eg, INCAGN01949 or MEDI0562), GITR (eg, MEDI1873) or CD137 (eg , Utomilumab);

(b) PD-1 antagonist (eg, pembrolizumab or nivolumab), CD274 (atezolizumab or durvalumab), A2AR (eg, Preladenant), B7-H3 (eg, MGA271), B7-H4, BTLA, CTLA-4 (eg, Tremelimumab or AGEN1884), IDO, KIR, LAG3, TGM-3 (eg CA-327 or RMT3-23) or VISTA (eg CA-170) or an antagonist of a member of the B7-CD28 superfamily, preferably CD28 or ICOS, or an antagonist thereof ligand.

Other preferred immunomodulators are cytokines that act as T cell growth factors, in particular IL-2, IL-12 or IL-15.

In a seventh aspect, the present 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 set 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 kits containing such pharmaceutical compositions according to the sixth aspect of the invention, or their use in the prevention or treatment of a proliferative disease, preferably cancer, a viral disease, a fungal disease or a bacterial disease.

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

In a preferred embodiment, a polypeptide according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, or a vector or set of vectors according to the third or fourth aspect of the invention, or a pharmaceutical composition or kits containing such pharmaceutical compositions according to the fifth aspect of the invention, wherein at least one immunomodulator, for example, MSM, or at least one polynucleotide encoding an immunomodulator, for example, MSM, or a vector or liposomal particle containing a polynucleotide encoding an immunomodulator, for example, MSM is administered before, simultaneously with, or after administration of a polypeptide according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, or a vector or set of vectors according to the third or fourth aspect of the invention, or a pharmaceutical composition or kits containing such pharmaceutical compositions according to the fifth aspect of the invention.

In a preferred embodiment of the seventh aspect of the invention, administration of the checkpoint molecule modulator begins before the start of vaccine administration, or administration of the checkpoint inhibitor begins after the start of vaccine administration, or administration of the checkpoint inhibitor begins simultaneously with the start of vaccine administration.

In a preferred embodiment of the seventh aspect of the invention, the vaccination regimen is a heterologous primary immunization with two different viral vectors. Preferred combinations are an ape-derived adenoviral vector for priming and a poxvirus vector, vaccinia virus vector, or modified vaccinia vector (MVA) for boosting. Preferably they are administered sequentially, at an interval of at least 1 week, preferably 6 weeks.

In an eighth aspect, the present invention provides a polypeptide according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, or a vector or set of vectors according to the third or fourth aspect of the invention for use as a drug.

Examples

Invariant chains from teleost fish and their fragments convert non-immunogenic cancer antigens into iiviviyiioi snnys antigens.

The immunogenicity of ape adenovectors encoding five cancer neoantigens (Table 1, SEQ ID NO: 16-20) derived from the CT26 mouse tumor cell line was assessed in inbred BalBC mice. The GAd-penta vector, encoding only five cancer neoantigens linked in a head-to-tail (penta) direction, plus the original Met and C-terminal HA tag (SEQ ID NO: 21), was not found to be immunogenic until then , until the full-length sequences of Teleostei invariant chains (BP_INV FL [SEQ ID NO: 9] or PO_INV FL [SEQ ID NO: 2]) or their 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]). Mice were immunized with a single intramuscular immunization of GAd at doses of 10 ⁸ viral particles (vp). Splenocytes were collected two weeks after immunization and tested using the TFN-γ ELISpot by stimulating T cells in the presence of synthetic peptides corresponding to the amino acid sequence of five neoantigens. Negative control cultures included cells stimulated with culture medium alone but containing the solvent dimethyl sulfoxide (DMSO) used to produce peptides. Immune responses (number of T cells producing IFN-γ per million splenocytes) are shown in Figure 1. Responses were considered positive if (i) at least 20 specific spots per million splenocytes were detected; (ii) the number of spots detected in positive wells was three times the number of spots detected in mock control wells (DMSO). As shown in Figure 1, binding of BP_INV FL or PO_INV FL or their fragments FRAG_A or FRAG_B to the N-terminus of the pentaantigen converted the non-immunogenic Gad-Penta vaccine construct into highly immunogenic constructs, with 100% of vaccinated animals showing positive immunological responses.

Materials and methods

Immunization of mice

Six-week-old female BalBC mice were purchased from Envigo laboratories. All mice were vaccinated intramuscularly (quadriceps muscle) by injecting 50 μl of viral vectors into each side (total volume 100 μl) at doses of 10 ⁸ vp.

IFNγ ELISpot

IFNγ-producing T cells were assessed by ELISPOT assay 2 weeks after vaccination. MSIP S4510 plates (Millipore) were coated with 10 μg/ml anti-mouse IFN-γ (U-CyTech Utrecht, The Netherlands) overnight at 4°C. After washing and blocking, mouse splenocytes were plated in duplicate at two different densities (2.5 x ¹⁰⁵ and 5 x ¹⁰⁵ cells/well) and stimulated overnight with a pool of 25-mer synthetic peptides corresponding to the antigen vaccine sequence (Figure 1). , at a final concentration of 2 μg/ml of each peptide. Peptide diluent DMSO (Sigma-Aldrich, Milan, Italy) and ConA (Sigma-Aldrich, Milan, Italy) were used as negative and positive controls, respectively. Plates were treated by subsequent incubation with biotinylated anti-mouse IFN-γ antibody (U-CyTech Utrecht, The Netherlands), streptavidin-alkaline phosphatase conjugate (BD Biosciences, NJ), and finally with BCIP/NBT 1-step solution (Thermo Fisher Scientific , Rockford, IL). The plates were read and analyzed by a CTL automated plate reader. The ELISpot response was considered positive when all of the following conditions were met: presence of IFN-y production in Con-A stimulated wells; at least 20 specific spots per million splenocytes; the number of spots detected in positive wells is three times greater than the number detected in false control wells (DMSO); and decreased responses when diluting cells. ELISpot data were expressed as IFN-γ spot-forming cells (SFC) per million splenocytes.

Claims (33)

1. A polypeptide having activity that enhances the T-cell response, containing: (a) a Teleostei invariant chain (INV) fragment comprising or consisting of 16 to 27 contiguous membrane proximal domain (MPD) amino acids from the Teleostei INV, wherein the fragment has T cell response enhancing activity and the fragment contains an amino acid sequence selected from SEQ ID NO: 11 and 4; and one or more cancer-specific antigens or cancer-specific neoantigens and/or one or more antigenic fragments thereof; or (b) a full-length Teleostei INV selected from SEQ ID NO: 2 or 9, and one or more cancer-specific antigens or tumor-specific neoantigens and/or one or more antigenic fragments thereof. 2. Polypeptide according to claim 1, where the amino acid sequence is: (a) the MPD, according to alternative (a) according to claim 1, is SEQ ID NO: 3; or (b) The MPD, according to alternative (a) according to claim 1, is SEQ ID NO: 10. 3. The polypeptide of claim 1(a) or 2(a) or (b), wherein 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 the INV Teleostei directly at the N-terminus of the MPD, with the TMD from INV Teleostei preferably characterized by the following amino acid sequence (SEQ ID NO: 15) AB ₁ KB ₂ B ₃ GB ₄ TB ₅ LB ₆ CB ₇ LB ₈ B ₉ B ₁₀ QB ₁₁ B ₁₂ B ₁₃ B ₁₄ YB ₁₅ B ₁₆ B ₁₇ , wherein B ₁ is V, L or Y, B ₂ is V or I, B ₃ is V or A, B ₄ is L, I or F, B ₅ is V, T or L, B ₆ is A or V, B ₇ is V or L, B ₈ is V or I, B ₉ 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 ₁₄ is I or A, B ₁₅ is F or M, B ₁₆ is L, M or V, B ₁₇ is V, F or L. 4. Polypeptide according to any one of paragraphs. 1-3, where (i) the polypeptide includes at least five different cancer-specific antigens or cancer-specific neoantigens and/or antigenic fragments thereof; and/or (ii) one or more cancer-specific antigens or tumor-specific neoantigens and/or one or more antigenic fragments thereof are located at the C-terminus of the INV fragment according to alternative (a) according to claim 1. 5. A polynucleotide encoding a polypeptide according to any one of claims. 1-4, wherein the polynucleotide is DNA or RNA, preferably DNA. 6. An expression vector containing a polynucleotide according to claim 5. 7. A collection of two or more different vectors for preventing or treating a proliferative disease, each of the different vectors comprising a polynucleotide of claim 5 encoding a different polypeptide of claim 5. 1-4. 8. The vector according to claim 6 or a collection of vectors according to claim 7, where the vector in each case is independently selected from the group consisting of a plasmid; cosmids; liposomal particle, viral vector or virus-like particle; preferably alphavirus vector, Venezuelan equine encephalitis (VEE) virus vector, sindbis virus vector (SIN), Semliki Forest virus (SFV) virus vector, simian or human cytomegalovirus (CMV) vector, lymphocytic choriomeningitis virus (LCMV) vector, retroviral vector, lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector, a vaccinia virus vector or a modified vaccinia virus vector (MVA), wherein the adenoviral vector is preferably an ape adenoviral vector, more preferably a chimpanzee, bonobo or gorilla adenoviral vector. 9. Pharmaceutical composition for the prevention or treatment of a proliferative disease, containing a polypeptide according to any one of paragraphs. 1-4, a polynucleotide according to claim 5 or a vector/collection of vectors according to paragraphs. 6-8, a pharmaceutically acceptable excipient. 10. Pharmaceutical composition according to claim 9, additionally containing one or more adjuvants. 11. The pharmaceutical composition according to claim 9 or 10, containing at least one immunomodulator or at least one polynucleotide encoding an immunomodulator, or a vector or liposomal particle containing a polynucleotide encoding an immunomodulator. 12. A kit for the prevention or treatment of a proliferative disease, containing a pharmaceutical composition according to any one of paragraphs. 9, 10 or 11 and separately packaged at least one immunomodulator or at least one polynucleotide encoding an immunomodulator, or a vector containing a polynucleotide encoding an immunomodulator. 13. Pharmaceutical composition according to claims. 9-11 or set according to clause 12, where: (i) the immunomodulator is MSM and is selected from the group consisting of: (a) an agonist of a member of the tumor necrosis factor (TNF) receptor superfamily, preferably CD27, CD40, OX40, GITR or CD137; 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 member of the B7-CD28 superfamily, preferably CD28 or ICOS, or an antagonist of its ligand; and/or (ii) the immunomodulator is a T cell growth factor, preferably IL-2, IL-12 or IL-15. 14. Use of a polypeptide according to claims. 1-4, polynucleotide according to claim 5, vector or collection of vectors according to paragraphs. 6-8, or a pharmaceutical composition or kits containing such pharmaceutical compositions according to claims. 9-13, for the prevention or treatment of a proliferative disease. 15. Use according to claim 14, wherein the proliferative disease is cancer. 16. Use according to claim 15, where the cancer is selected from the group consisting of malignant neoplasms of the lip, oral cavity, pharynx, digestive organ, respiratory organ, intrathoracic organ, 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, lymphoid tissue and hematopoietic tissue. 17. Application according to any one of paragraphs. 14-16, where at least one immunomodulator or at least one polynucleotide encoding an immunomodulator, or a vector or liposomal particle containing a polynucleotide encoding an immunomodulator, is administered before, simultaneously or after administration of the polypeptide according to claims. 1-4, polynucleotide according to claim 5, vector or collection of vectors according to paragraphs. 6-8, or a pharmaceutical composition or kits containing such pharmaceutical compositions according to claims. 9-13.