Vaccine composition comprising HERV derived epitopes and their use in treatment of aml

Abstract

Provided are immunogenic agents and compositions tailored to induce immunity against hERV expression products in a high percentage of cancer patients as well. Also provided are methods for immune induction against cancer cells

Description

[0001] VACCINE COMPOSITION COMPRISING HERV DERIVED EPITOPES AND THEIR USE IN TREATMENT OF AML

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of cancer immunotherapy. In particular, the present invention relates to improved means and methods for designing and producing ready-to-use anti-cancer vaccines that match and target expression products of hERV sequences, which are not or only to a very limited degree expressed in normal tissues, but which are found in patients' cancer tissue. Also, the invention relates to methods for induction of immunity in cancer patients and for treatment of cancer.

[0004] BACKGROUND OF THE INVENTION

[0005] Treatment of malignant neoplasms in patients has traditionally focussed on eradication / removal of the malignant tissue via surgery, radiotherapy, and / or chemotherapy using cytotoxic or cytostatic drugs in dosage regimens that aim at preferential killing of malignant cells over killing of non-malignant cells.

[0006] In addition to the use of cytotoxic drugs, more recent approaches have focussed on targeting of specific biologic markers in the cancer cells to reduce systemic adverse effects exerted by classical chemotherapy. Monoclonal antibody therapy targeting cancer associated antigens has proven quite effective in prolonging life expectancy in a number of malignancies. While being successful drugs, monoclonal antibodies can due to their nature only be developed to target expression products that are known and appear in a plurality of patients, meaning that the vast majority of cancer specific antigens cannot be addressed by this type of therapy, because a large number of cancer specific antigens only appear intracellularly or in tumours from one single patient, cf. below.

[0007] As early as in the late 1950’ies the theory of immunosurveillance was formulated and suggested that lymphocytes recognize and eliminate autologous cells, such as cancer cells that exhibit altered antigenic determinants, and it is today generally accepted that the immune system inhibits carcinogenesis to a high degree. Nevertheless, immunosurveillance is not 100% effective and it is a continuing task to develop cancer therapies where the immune system's ability to eradicate cancer cells is sought improved / stimulated. One approach has been to induce immunity against cancer-associated antigens, but even though this approach has potential, it suffers the same drawback as antibody therapy that only a limited number of antigens can be addressed.

[0008] Many, if not all, tumours express mutations. These mutations may create new targetable antigens (neoantigens), which are potentially useful in specific T cell immunotherapy if it is possible to identify the neoantigens and their antigenic determinants (neoepitopes) within a clinically relevant timeframe. With current technology it is indeed possible to fully sequence the genome of cells and to analyse for existence of altered or new expression products within a few days, hence, within this timeframe it is possible to design personalized vaccines based on neoantigens and their neoepitopes.

[0009] WO 2022 / 023521 discloses methods for selection of epitopes to include in individualized cancer vaccines; focus is put on identification and utilisation of neoepitopes encoded by somatic variants of expressed genes in cancer cells. The methods disclosed in WO 2022 / 023521 hence rely on an identification of short peptides present in expression products that differ from the normal expression products in the patient, and as such the method in WO 2022 / 023521 will always require an individual evaluation of the potential usefulness of such short peptides.

[0010] WO 2023 / 111306 discloses a cancer therapeutic approach, which relies on immunization against expression products of genomic sequences (typically endogenous viral elements (" EVEs"), such as endogenous retroviral sequences (" ERVs")), where the expression products appear in a small or negligible percentage of samples of normal tissue.

[0011] To date, it does not appear that anyone has provided a cancer therapeutic approach that combines targeting of multiple expression products from hERVs, where selection of the targeted antigens is optimized vis-a-vis the population so as the aim at an optimized coverage of patients.

[0012] OBJECT OF THE INVENTION

[0013] It is an object of embodiments of the invention to provide immunogenic agents and compositions tailored to induce immunity against hERV expression products in a high percentage of cancer patients as well as to provide methods for immune induction against cancer cells. SUMMARY OF THE INVENTION

[0014] It has been found by the present inventor(s) that it is possible to design vaccine agents based on hERV expression products that are found in cancers of multiple patients in such a way that a predefined vaccine composition will have a high likelihood of being both immunogenic and able to target hERVs expressed in a given patient across a large proportion of the population to which the patient belongs.

[0015] By carefully evaluating previously recorded data from a given cancer type, the abundancy of hERV expression levels can be used to design ready-to-use vaccine agents that each provide a high degree of population coverage. In turn, multiple such vaccine agents can be prepared, meaning that one or more of the vaccine agents will match the hERV expression profile of the patient's cancer; this allows selection and use of a tumour specific vaccine immediately after the hERV expression profile of the patient's cancer has been determined but it is even possible to instigate treatment with a "shared vaccine" without this knowledge. Since the vaccine agent is designed to exhibit a high population coverage, the likelihood of a beneficial effect is likewise high.

[0016] So, in a 1staspect the present invention relates to a nucleic acid expression vector encoding a plurality of hERV epitope hotspots, wherein

[0017] 1) each hERV epitope hotspot is comprised of an amino acid sequence of at least 8 amino acid residues;

[0018] 2) each hERV epitope hotspot is comprised in at least one proteinaceous hERV expression product identified among expression products from malignant cells in a group of cancer patients;

[0019] 3) each hERV epitope hotspot exhibits a density of predicted ligands for allelic variants of HLA molecules, said allelic variants being found in a population to which the group of cancer patients belong, where said density is in the upper three quartiles of the average density of predicted ligands in the population for the same allelic variants of HLA molecules, said average density of predicted ligands being found in amino acid sequences of the same length as the hERV epitope hotspot; and

[0020] 4) the plurality of hERV epitope hotspots comprises a plurality of predicted ligands for allelic variants of HLA molecules in said population, whereby the probability is at least 50% that at least one of the predicted HLA ligands is an HLA ligand from at least one hERV expression product in a randomly selected individual in said population, preferably where the individual's malignant cells express the hERV as a proteinaceous expression product comprising the amino acid sequence of the predicted ligand. In a 2ndaspect, the invention relates to a viral or bacterial expression vector which comprises and is capable of expressing the nucleic acid expression vector of the first aspect of the invention and any embodiment of the 1staspect disclosed herein.

[0021] In a 3rdaspect, the invention relates to polypeptide comprising a plurality of hERV epitope hotspots, wherein

[0022] 1) each hERV epitope hotspot is comprised of an amino acid sequence of at least 8 amino acid residues;

[0023] 2) each hERV epitope hotspot is comprised in at least one proteinaceous hERV expression product identified among expression products from malignant cells in a group of cancer patients;

[0024] 3) each hERV epitope hotspot exhibits a density of predicted ligands for allelic variants of HLA molecules, said allelic variants being found in a population to which the group of cancer patients belong, where said density is in the upper three quartiles of the average density of predicted ligands in the population for the same allelic variants of HLA molecules, said average density of predicted ligands being found in amino acid sequences of the same length as the hERV epitope hotspot; and

[0025] 4) the plurality of hERV epitope hotspots comprises a plurality of predicted ligands for allelic variants of HLA molecules in said population, whereby the probability is at least 50% that at least one of the predicted HLA ligands is an HLA ligand from at least one hERV expression product in a randomly selected individual in said population, preferably where the individual's malignant cells express the hERV as a proteinaceous expression product comprising the amino acid sequence of the predicted ligand.

[0026] In a 4thaspect, the present invention relates to composition comprising either the polypeptide of the 3rdaspect of the invention or comprises a plurality of hERV epitope hotspots, wherein

[0027] 1) each hERV epitope hotspot is comprised of an amino acid sequence of at least 8 amino acid residues;

[0028] 2) each hERV epitope hotspot is comprised in at least one proteinaceous hERV expression product identified among expression products from malignant cells in a group of cancer patients;

[0029] 3) each hERV epitope hotspot exhibits a density of predicted ligands for allelic variants of HLA molecules, said allelic variants being found in a population to which the group of cancer patients belong, where said density is in the upper three quartiles of the average density of predicted ligands in the population for the same allelic variants of HLA molecules, said average density of predicted ligands being found in amino acid sequences of the same length as the hERV epitope hotspot; and

[0030] 4) the plurality of hERV epitope hotspots comprises a plurality of predicted ligands for allelic variants of HLA molecules in said population, whereby the probability is at least 50% that at least one of the predicted HLA ligands is an HLA ligand from at least one hERV expression product in a randomly selected individual in said population, preferably where the individual's malignant cells express the hERV as a proteinaceous expression product comprising the amino acid sequence of the predicted ligand.

[0031] In a 5thaspect, the present invention relates to a method for treatment of human patient suffering from cancer, the method comprising administering to said patient an immunologically effective amount of a 1) nucleic acid expression vector of the 1staspect of the present invention as well as of any embodiment of the 1staspect disclosed herein, 2) the viral or bacterial expression vector of the 2ndaspect of the invention as well as of any embodiment of the 2ndaspect disclosed herein, 3) the polypeptide of the 3rdaspect of the present invention as well as any embodiment thereof disclosed herein, or 4) a composition of the 4thaspect of the invention or any embodiment thereof disclosed herein.

[0032] Finally, in a 6thaspect, the present invention relates to a kit comprising a plurality of distinct nucleic acid expression vectors of the 1staspect of the invention as well as any embodiment thereof disclosed herein, distinct viral or bacterial expression vectors according to the second aspect of the invention as well as any embodiment thereof disclosed herein, distinct polypeptides of the 3rdaspect of the invention as well as any embodiment thereof disclosed herein, or distinct compositions of the 4thaspect of the invention as well as any embodiment thereof disclosed herein.

[0033] LEGENDS TO THE FIGURE

[0034] Fig. 1: IFNy responses of in vitro primed human PBMCs from healthy donors towards hERV hotspot antigens.

[0035] A: Bar plot showing the Spot forming units (SFU) per 1 million cells after subtracting unspecific background. The hotspot antigen pools were tested for pre-existing responses in naive HD PBMCs with a complete negative readout (data not shown).

[0036] B: Summary of the data presented in Fig. 1A. The heatmap shows SFU per 1 million cells after subtraction of background. All hotspot-donor combinations were tested. The SFU values are annotated in the hotspot-donor that displayed an immunogenic response. Immunogenic responses are defined according to the criteria: [Mean SFU ANTIGEN STIMULATED] > 2 x [Mean SFU UNSTIMULATED] + 10 SFU pr million stimulated cells. C: Representative example of the individual IFNy ELISpot wells from healthy donor 1 (HD-1). D: Comparison between observed hotspot ligand coverage probability and calculated theoretical ligand coverage probabilities.

[0037] Fig. 2: Graphs showing cytotoxicity exerted by cytotoxic T lymphocytes (CTLs) specific for different ERV hotspots at a CTL-to-target cell ratio of 5:1.

[0038] A: Healthy donor 1.

[0039] B: Healthy donor 7.

[0040] C: Healthy donor 3.

[0041] Fig. 3: Induction of antigen-specific T-cell responses using mERV hotspot antigens and observed tumour growth control.

[0042] A: Tumour growth over time (Mean + / - SEM) in C57BL / 6 mice after challenge with B16F10 tumour cells.

[0043] B: Tumour growth over time (Mean + / - SEM) in BALB / c mice after challenge with CT26 tumour cells.

[0044] C: T-cell immunogenicity measured by IFNy ELISpot upon re-stimulation with vaccine mERV peptide pools (Mean) in BALB / c.

[0045] DETAILED DISCLOSURE OF THE INVENTION

[0046] Definitions

[0047] An "endogenous retroelement" (" ERE") is a genetic element. EREs constitute nearly 50% of the human genome. These elements are present in almost all organisms and believed to be remnants of transposable elements that integrated in germline cells millions of years ago. Most ERE sequences contain mutated or truncated open reading frames and have lost their capacity to transpose in the genome. EREs comprise short and long interspersed retro-transposable elements (SINE and LINE), and these are collectively known as non-LTR elements. The remaining endogenous retroelements comprise LTR-bound elements comprising two major groups occupying comparable fractions of the genome: endogenous retroviruses (ERVs, in humans termed hERVs) and mammalian apparent LTR retrotransposons (MaLRs) (Kassiotis & Stoye, 2016, doi: 10.1038 / nri.2016.27). An "endogenous viral element" (" EVE") is an ERE, which is member of a subset of genes, which is a result of an in silico filtering process based on the presence of viral motifs in the gene. As such, this group is mostly composed of ERVs but can also contain members of the other different subcategories. ERVs in the human genome (hERVs) are of particular interest due to their abundance and the fact that they are well characterized.

[0048] A "malignant neoplasm" (also termed a cancer or malignant tumour) denotes a group of cells in a multicellular organism, which exhibit uncontrolled growth, invasive growth, and, normally, the ability to metastasize.

[0049] A "cancer specific antigen" is an antigen, which does not appear as an expression product in an individual's non-malignant somatic cells, but which appears as an expression product in cancer cells in the individual. This is in contrast to "cancer-associated" antigens, which also appear - albeit typically at low abundance - in normal somatic cells but are found in higher levels in at least some malignant tumour cells. In general, the peptides identified according to the present invention are considered to be cancer specific.

[0050] The term "adjuvant" has its usual meaning in the art of vaccine technology, i.e. a substance or a composition of matter which is 1) not in itself capable of mounting a specific immune response against the immunogen of the vaccine, but which is 2) nevertheless capable of enhancing the immune response against the immunogen. Or, in other words, vaccination with the adjuvant alone does not provide an immune response against the immunogen, vaccination with the immunogen may or may not give rise to an immune response against the immunogen, but the combined vaccination with immunogen and adjuvant induces an immune response against the immunogen which is stronger than that induced by the immunogen alone.

[0051] An MHC molecule (major histocompatibility molecule) is a tissue antigen expressed by nucleated cells in vertebrates, which binds to peptide antigens and displays ("presents") the antigens to T-cells carrying T-cell receptors. MHC class I is expressed by all nucleated cells and primarily present proteolytically degraded protein fragments derived from proteins present in the cell. MHC class II is expressed by professional antigen presenting cells that typically take up extracellular protein, degrade it with lysosomal proteases, and present protein fragments on the surface. In humans, the MHC molecules are encoded at the human leukocyte antigens (HLA) loci, which in the present invention are the preferred MHC molecules to evaluate binding to. Hence, the human MHC molecules are termed " HLA molecules" herein. An MHC ligand (or, in humans, an HLA ligand), is a peptide, which 1) binds to an MHC / HLA molecule and 2) can be eluted from antigen presenting cells that have subjected a protein to antigen processing and presented it on their surfaces (in the context of a Class I or Class II MHC molecule). Hence an MHC ligand is not merely a peptide that can bind to one of the 2 MHC class molecules; it also has to be a molecule which actually is presented by MHC molecules on living cells.

[0052] A " T-cell epitope" is peptide or a smaller stretch of amino acids in a protein, which is a ligand for an MHC (in humans: HLA) molecule, and which is recognized as foreign (non-self) by a T-cell in a vertebrate due to specific binding between a T-cell receptor and the cell carrying the MHC-peptide complex on its surface. Hence, a peptide, which constitutes a T-cell epitope in one individual will not necessarily be a T-cell epitope in a different individual of the same species. First of all, two individuals having differing MHC molecules that bind different sets of peptides, do not necessarily present the same peptides complexed to MHC, and further, if a peptide is autologous in one of the individuals it may not be able to bind any T-cell receptor.

[0053] A "neoepitope" is an antigenic determinant (typically an MHC Class I or II restricted epitope), which does not exist as an expression product from normal somatic cells in an individual due to the lack of a gene encoding the neoepitope, but which exists as an expression product in mutated cells (such as cancer cells) in the same individual. As a consequence, a neoepitope is from an immunological viewpoint truly non-self despite its autologous origin and it can therefore be characterized as a tumour specific antigen in the individual, where it constitutes an expression product. Being non-self, a neoepitope has the potential of being able to elicit a specific adaptive immune response in the individual, where the elicited immune response is specific for antigens and cells that harbour the neoepitope. Neoepitopes are on the other hand specific for an individual as the chances that the same neoepitope will be an expression product in other individuals is minimal. Several features thus contrast a neoepitope from, e.g., epitopes of tumour specific antigens: the latter will typically be found in a plurality of cancers of the same type (as they can be expression products from activated oncogenes) and / or they will be present - albeit in minor amounts - in non-malignant cells because of over-expression of the relevant gene(s) in cancer cells.

[0054] A "neopeptide" is a peptide ( / .e. a polyaminoacid of up to about 50 amino acid residues), which includes within its sequence a neoepitope as defined herein. A neopeptide is typically "native", i.e. the entire amino acid sequence of the neopeptide constitutes a fragment of an expression product that can be isolated from the individual, but a neopeptide can also be "artificial", meaning that it is constituted by the sequence of a neoepitope and 1 or 2 appended amino acid sequences of which at least one is not naturally associated with the neoepitope. In the latter case, the appended amino acid sequences may simply act as carriers of the neoepitope or may even improve the immunogenicity of the neoepitope (e.g. by facilitating processing of the neopeptide by antigen-presenting cells, improving biologic half-life of the neopeptide, or modifying solubility). The terms "neopeptide" and "neopeptide" are used interchangeably.

[0055] The term "amino acid sequence" is the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides and proteins. Sequences are conventionally listed in the N to C terminal direction.

[0056] " An immunogenic carrier" is a molecule or moiety to which an immunogen or a hapten can be coupled to enhance or enable the elicitation of an immune response against the immunogen / hapten. Immunogenic carriers are in classical cases relatively large molecules (such as tetanus toxoid, KLH, diphtheria toxoid etc.) which can be fused or conjugated to an immunogen / hapten, which is not sufficiently immunogenic in its own right - typically, the immunogenic carrier is capable of eliciting a strong T-helper lymphocyte response against the combined substance constituted by the immunogen and the immunogenic carrier, and this in turn provides for improved responses against the immunogen by B-lymphocytes and cytotoxic lymphocytes. More recently, the large carrier molecules have to a certain extent been substituted by so-called promiscuous T-helper epitopes, i.e. shorter peptides that are recognized by a large fraction of HLA haplotypes in a population, and which elicit T-helper lymphocyte responses.

[0057] A " T-helper lymphocyte response" is an immune response elicited on the basis of a peptide, which is able to bind to an MHC class II molecule (e.g. an HLA class II molecule) in an antigen-presenting cell and which stimulates T-helper lymphocytes in an animal species as a consequence of T-cell receptor recognition of the complex between the peptide and the MHC Class II molecule presenting the peptide.

[0058] An "immunogen" is a substance of matter, which is capable of inducing an adaptive immune response in a host, whose immune system is confronted with the immunogen. As such, immunogens are a subset of the larger genus "antigens", which are substances that can be recognized specifically by the immune system (e.g. when bound by antibodies or, alternatively, when fragments of the antigens bound to MHC molecules are being recognized by T-cell receptors) but which are not necessarily capable of inducing immunity - an immunogen is, however, always capable of eliciting immunity, meaning that a host that has an established memory immunity against the immunogen will mount a specific immune response against the immunogen. An "adaptive immune response" is an immune response in response to confrontation with an antigen or immunogen, where the immune response is specific for antigenic determinants of the antigen / immunogen - examples of adaptive immune responses are induction of antigen specific antibody production or antigen specific induction / activation of T helper lymphocytes or cytotoxic lymphocytes.

[0059] A "protective, adaptive immune response" is an antigen-specific immune response induced in a subject as a reaction to immunization (artificial or natural) with an antigen, where the immune response is capable of protecting the subject against subsequent challenges with the antigen or a pathology-related agent that includes the antigen. Typically, prophylactic vaccination aims at establishing a protective adaptive immune response against one or several pathogens. In the present context the immune responses induced by the peptides identified are typically therapeutic immune responses against a cancer in a patient.

[0060] " Stimulation of the immune system" means that a substance or composition of matter exhibits a general, non-specific immunostimulatory effect. A number of adjuvants and putative adjuvants (such as certain cytokines) share the ability to stimulate the immune system. The result of using an immunostimulating agent is an increased "alertness" of the immune system, meaning that simultaneous or subsequent immunization with an immunogen induces a significantly more effective immune response compared to isolated use of the immunogen.

[0061] The term "polypeptide" is in the present context intended to mean both short peptides of from 2 to 50 amino acid residues, oligopeptides of from 50 to 100 amino acid residues, and polypeptides of more than 100 amino acid residues. Furthermore, the term is also intended to include proteins, i.e. functional biomolecules comprising at least one polypeptide; when comprising at least two polypeptides, these may form complexes, be covalently linked, or may be non-covalently linked. The polypeptide(s) in a protein can be glycosylated and / or lipidated and / or comprise prosthetic groups. An " HLA ligand" is a peptide defined by an amino acid sequence, which has a length and amino acid distribution that will allow to bind at least one HLA molecule. If nothing else is indicated herein, peptides, oligopeptides and polypeptides are constituted by L-amino acids joined by eupeptide bonds i.e. an amide bond joining the α-carboxyl and α-amino groups of two neighbouring amino acid residues), where the L-amino acids are selected from the 22 proteinogenic amino acids: alanine (ala; A), arginine (arg; R), asparagine (asn; N), aspartic acid (asp; D), cysteine (cys; C), glutamine (gin; Q), glutamic acid (glu; E), glycine (gly; G), histidine (his; H), isoleucine (ile; I), leucine (leu; L), lysine (lys; K), methionine (met; M), phenylalanine (phe; F), proline (pro; P), serine (ser; S), threonine (thr; T), tryptophan (trp; W), tyrosine (tyr; Y), valine (val; V ), selenocysteine (sec; U), and pyrrolysine (pyl, O). A "hotspot sequence" denotes an amino acid sequence which comprises HLA ligand amino acid sequences with a high density, i.e. an above-normal number of HLA ligand amino acid sequences per base pair.

[0062] A "potential HLA ligand" (also termed a "predicted" HLA ligand) is a peptide defined by an amino acid sequence, which has a length and amino acid distribution that will allow it to bind at least one HLA molecule in a patient.

[0063] A "true HLA ligand" is a peptide which in a given patient binds to an HLA molecule and is presented by antigen presenting cells.

[0064] Specific embodiments of the invention

[0065] The 1staspect of the invention and embodiments thereof

[0066] The 1staspect relates to a nucleic acid expression vector encoding a plurality of hERV epitope hotspots, wherein 1) each hERV epitope hotspot is comprised of an amino acid sequence of at least 8 amino acid residues; 2) each hERV epitope hotspot is comprised in at least one proteinaceous hERV expression product identified among expression products from malignant cells in a group of cancer patients; 3) each hERV epitope hotspot exhibits a density of predicted ligands for allelic variants of HLA molecules, said allelic variants being found in a population to which the group of cancer patients belong, where said density is in the upper three quartiles of the average density of predicted ligands in the population for the same allelic variants of HLA molecules, said average density of predicted ligands being found in amino acid sequences of the same length as the hERV epitope hotspot; and 4) the plurality of hERV epitope hotspots comprises a plurality of predicted ligands for allelic variants of HLA molecules in said population, whereby the probability is at least 50% that at least one of the predicted HLA ligands is an HLA ligand in a randomly selected individual in said population, preferably where the individual's malignant cells express the hERV as a proteinaceous expression product comprising the amino acid sequence of the predicted ligand.

[0067] It will be understood in the following that the predicted ligands are selected from HLA Class 1 and 2 ligands, and that the expression vector of the first aspect may comprise both types of ligands as well as one single type. Preferably the HLA ligands encoded by the vector is a mix of the two types.

[0068] Such a vector can e.g. be in the form of a plasmid, but the exact configuration depends on the ultimate host cell, which is to express the vector. Such an expression vector finds several uses. It can - when tailored for a particular expression system - be of use in recombinant production of the hERV epitope hotspots or proteinaceous expression products comprising these. Alternatively, the expression vector of the first aspect of the invention is tailored for delivery as a nucleic acid vaccine (as a DNA or RNA vaccine vector), which results in transient expression of the coding regions in the host to whom the vector has been administered. In addition, the expression vector of the first aspect of the invention also finds use in preparation of bacterial or viral vaccine vectors. Each of these approaches are generally well understood in the art.

[0069] The nucleic acid expression vector preferably include the plurality of hERV epitope hotspots to comprise a plurality of predicted ligands for allelic variants of HLA molecules in said population, whereby the probability is

[0070] - at least 50% that at least 2 of the ERV derived predicted HLA ligands are ERV derived HLA ligands in a randomly selected individual in said population, or

[0071] - at least 50% that at least 3 of the ERV derived predicted HLA ligands are ERV derived HLA ligands in a randomly selected individual in said population, or

[0072] - at least 50% that at least 4 of the ERV derived predicted HLA ligands are ERV derived HLA ligands in a randomly selected individual in said population, or

[0073] - at least 50% that at least 5 of the ERV derived predicted HLA ligands are ERV derived HLA ligands in a randomly selected individual in said population, or

[0074] - at least 50% that at least 6 of the ERV derived predicted HLA ligands are ERV derived HLA ligands in a randomly selected individual in said population, or

[0075] - at least 50% that at least 7 of the ERV derived predicted HLA ligands are ERV derived HLA ligands in a randomly selected individual in said population, or

[0076] - at least 50% that at least 8 of the ERV derived predicted HLA ligands are ERV derived HLA ligands in a randomly selected individual in said population, or

[0077] - at least 50% that at least 9 of the ERV derived predicted HLA ligands are ERV derived HLA ligands in a randomly selected individual in said population, or

[0078] - at least 50% that at least 10 of the ERV derived predicted HLA ligands are ERV derived HLA ligands in a randomly selected individual in said population.

[0079] In this embodiment, the at least 50% can be at least 60%, at least 70%, at least 80%, and preferably at least 90%.

[0080] In other words, the plurality of hERV epitope hotspots is selected (see Example 1) to obtain a high theoretical population coverage in the sense that a hight percentage of the population will be able to mount a T-cell response against 1 or more of the predicted HLA ligands. To ensure that the predicted ligands in the hERV epitope hotspots are indeed capable of inducing immune response, the selected predicted HLA ligands can conveniently be subjected to testing in vitro to verify their suitability as immunogens (see Example 2) for details concerning in vitro assays for this purpose.

[0081] Hence, preferably, each hERV epitope hotspot (or the predicted ligands present therein) are preferably selected so that it, when tested in vitro, exhibits one or more of the following properties:

[0082] - ability to induce secretion of IFN-y from PBMCs of human donors as determined by stimulating the PBMCs with a hERV peptide pool corresponding to the hERV epitope hotspot; or

[0083] - ability to prime antigen-specific T-cells as determined by 1) incubating the hERV epitope hotspot with PBMCs treated to induce antigen-presenting cell differentiation and maturation and 2) re-stimulating with the hotspot to induce IFN-y secretion.

[0084] Different cancers exhibit different characteristics - for instance, cells of acute myeloid leukaemia are known to express a relative high number of hERVs, and it is generally believed that the pattern of such hERV expression varies from cancer to cancer. Hence, in order to further optimize the expression vector of the first aspect of the invention, each hERV epitope hotspot is comprised in at least one proteinaceous hERV expression product identified among expression products from malignant cells in a group of cancer patients suffering from the same histological type of cancer.

[0085] Generally, the histology of cancers provides for a division into 3 categories: epithelial tumours, non-epithelial tumours, and mixed tumours.

[0086] Examples of epithelial tumours, from where the hERV expression products are identified, are carcinoma and adenocarcinoma, and examples of non-epithelial tumours or mixed tumours are liposarcoma, fibrosarcoma, chondrosarcoma osteosarcoma leiomyosarcoma rhabdomyosarcoma glioma neuroblastoma medulloblastoma malignant melanoma malignant meningioma neurofibrosarcoma leukaemia, myeloproliferative disorder lymphoma hemangiosarcoma Kaposi's sarcoma, malignant teratoma, dysgerminoma, seminoma, and choriocarcinoma. Preferably the histological type of cancer, from where the hERV expression products are identified, is a leukaemia, in particular acute myeloid leukaemia (AML), or the histological type of cancer is myelodysplastic syndrome (MDS).

[0087] The design of the expression vector may vary. For instance, it may express the hERV epitope hotspots as one single polypeptide or as several polypeptides, wherein at least one of the several polypeptides includes at least 2 hERV epitope hotspots; this is in contrast to a design, where each hERV epitope hotspot is a separate expression product, meaning that the vector expresses multiple peptides / polypeptides. In turn, the latter result can be achieved by letting the hotspots be under control of separate expression control regions (including separate promoters / enhancers) or by letting 2 or more hotspots be under control of the same expression control region. In configurations of the vector, where the portion expression product may contain more than one hERV epitope hotspot, the vector may preferably encode linking amino acid sequences ( / .e. peptide linkers) to separate the hotspots. Encoded peptide linkers can be either "flexible" or "rigid", Also, it is envisaged that the linker(s) used in the invention in some embodiments can be cleavable, that is, include (a) recognition site(s) for endopeptidase(s), e.g. endopeptidases such as furin, caspases, cathepsins etc.

[0088] Typically, a nucleic acid expression vector of the first aspect encodes a number of hERV epitope hotspots which is in the range of 2-50.

[0089] As indicated above, the nucleic acid expression vector is designed to ensure a high population coverage in terms of immunogenicity of the selected predicted HLA ligands. In this context the choice of population has a certain impact, since there are genetic and geographic differences in the HLA expression. One approach to take is to consider the HLA expression on a global scale, meaning that the hERV epitope hotspots in the expression vector of the 1staspect of the invention are selected to include predicted HLA ligands that will address a high percentage of human individuals, irrespective of their genetic or geographic background; in this embodiment of the 1staspect, the population referred to above is the world population. Alternatively, the hotspots can be selected to match a more limited (but well-defined) population: a genetically delimited population or a geographically delimited population. In this context, cf. Arrieta-Bolanos E. etal. (2023), Front. Genet. 14, doi.org / 10.3389 / fgene.2023.866407, in particular Fig. 9, which provides an HLA map of the world.

[0090] Hence, the geographically delimited population can be selected from a continent's population, a population of a region, or a population of a country / nation / state. Further, the genetically delimited population is defined by its HLA profile.

[0091] The above-discussed average density of predicted ligands in the population for the same allelic variants of HLA molecules can be calculated as

[0092] v;- v n. U

[0093]

[0094] wherein Si is the length of the hERV epitope hotspot S,

[0095] P(L|Aᵢ) is the probability that a fragment of the hERV epitope hotspot is a ligand for HLA allele I,

[0096] f(Aᵢ) is the frequency of allele i in the population,

[0097] Σⱼ is the summation over all fragments contained in the hotspot S, and

[0098] Σᵢⁿ is the summation over all alleles in the population.

[0099] General discussion of expression vectors

[0100] A general disclosure relating to expression vectors is provided in the following:

[0101] When a nucleic acid vaccine is administered to a patient, the corresponding gene product (such as a desired antigen) is produced in the patient's body. In some embodiments, nucleic acid vaccine vectors that include optimized recombinant polynucleotides can be delivered to a human to induce a therapeutic or prophylactic immune response. In such cases, the nucleic acid expression vector can be DNA (for DNA vaccination) or RNA (for RNA vaccination), both generally composed as generally known in the art.

[0102] Plasmid and other naked DNA vectors are typically more efficient for gene transfer to muscle tissue. The potential to deliver DNA vectors to mucosal surfaces by oral administration has also been reported and DNA plasmids have been utilized for direct introduction of genes into other tissues than muscle. DNA vaccines have been introduced into animals primarily by intramuscular injection, by gene gun delivery, by jet injection (using a device such as a Stratis® device from PharmaJet), or by electroporation; each of these modes of administration apply to the presently disclosed method. After being introduced, the plasmids are generally maintained episomally without replication. Expression of the encoded proteins has been shown to persist for extended time periods, providing stimulation of both B and T cells.

[0103] In determining the effective amount of the vector to be administered in the treatment method disclosed herein (cf. below), the physician evaluates vector toxicities, progression of the cancer to be treated, and the production of anti-vector antibodies, if any. Administration can be accomplished via single or divided doses and typically as a series of time separated administrations. In the methods disclosed herein, the effective human dose per immunization in a time-separated series is between 0.1 pg and 500 mg, with dosages between 0.1 pg and 25 mg of the expression vector being preferred. That is, in the practice of the method disclosed herein dosages of between 0.5 pg and 20 mg in humans are typically used, and dosages are normally between 5 pg and 15 mg, between 50 pg and 10 mg, and between 500 pg and 8 mg, and particular interesting dosages are of about 0.0001, about 0.0005, about 0.001, about 0.005, about 0.01, about 0.05, about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7 and about 8 mg.

[0104] A series of immunizations with effective dosages will typically constitute a series of 2, 3, 4, 5, 6, or more dosages. Multiple (e.g. >6) dosages may for instance be relevant to keep a malignant neoplasm in check for a prolonged period of time.

[0105] The vaccine used in the method disclosed herein comprises one or more expression vectors; for instance, the vaccine may comprise a plurality of expression vectors each capable of autonomous expression of a nucleotide coding region in a mammalian cell to produce at least one immunogenic polypeptide. An expression vector often includes a eukaryotic promoter sequence, such as the nucleotide sequence of a strong eukaryotic promoter, operably linked to one or more coding regions. The compositions and methods herein may involve the use of any particular eukaryotic promoter, and a wide variety are known to the skilled person.

[0106] Examples are CMV and RSV promoters. The promoter can be heterologous with respect to the host cell. The promoter used may be a constitutive promoter. The promoter used may include an enhancer region and an intron region to improve expression levels, such as is the case when using a CMV promoter.

[0107] Numerous plasmids known in the art may be used for the production of nucleic acid vaccines. Suitable embodiments of the nucleic acid vaccine employ constructs using the plasmids VR1012 (Vical Inc., San Diego Calif.), pCMVI. UBF3 / 2 (S. Johnston, University of Texas), pTVG4 (Johnson etal., 2006, Vaccine 24(3); 293-303), pVAXl (Thermo Fisher Scientific, see above and the Examples below), or pcDNA3.1 (InVitrogen Corporation, Carlsbad, Calif.) as the vector.

[0108] In addition, the vector construct can according to the present invention advantageously contain immunostimulatory sequences (ISS). The aim of using such sequences in the vaccine vector is to enhance T-cell response towards encoded neo-epitopes, in particular Thl cell responses, which are elicited by adjuvants that incorporate agonists of the toll-like receptors TLR3, TLR7-TLR8, and TLR9. and / or cytosolic RNA receptors such as, but not limited to, RIG-1, MDA5 and LGP2 (Desmet eta / . 2012. Nat. Rev. Imm. 12(7), 479-491)

[0109] One possibility of employing ISS is to mimic a bacterial infection activating TLR9 by stimulating with unmethylated CG-rich motifs (so-called CpG motifs) of six bases with the general sequence NNCGNN (which have a 20-fold higher frequency in bacterial DNA than in mammalian DNA) either as directly administered small synthetic DNA oligos (ODNs), which contain partially or completely phosphorothioated backbones, or by incorporating the CpG motifs in the DNA vector backbone. Immunostimulatory CpGs can be part of the DNA backbone or be concentrated in an ISS where the CpG sequence(s) typically will be positioned between the stop codon in the neo-epitope coding sequence and the poly-A tail encoding sequence ( / .e. the ISS is located between the stop codon and the polyadenylation signal). However, since CpG sequences exert an effect irrespectively of their position in a longer DNA molecule, their position could in principle be anywhere in the vaccine vector as long as the presence of the CpG motif does not interfere with the vector's ability to express the coding regions of the vaccine antigen.

[0110] If present in the vaccine as separate ODNs, where the ODNs function as immunological adjuvants, CpG motif containing oligonucleotides are typically to be co-administered / formulated together with the DNA vaccine by the selected delivery technology and will typically constitute hexamers or longer multimers of DNA comprising the sequence NNCGNN or the reverse complementary sequence. Useful ODNs for this purpose are e.g. commercially available from InivoGen, 5 Rue Jean Rodier, F-31400, Toulouse, France, which markets a range of Class A, B, and C ODNs.

[0111] For details pertaining to RNA vaccine vectors, reference can e.g. be made to the following review: Zhang Z et al. (2023) Signal Transduction and Targeted Therapy 8:365 (doi.org / 10.1038 / s41932-023-01579-1)

[0112] For recombinant production of protein, the nucleic acid expression vector is typically DNA and will be tailored to best fit with the cell chosen for recombinant production of the proteinaceous expression product. In this context, numerous vectors and expression systems exist, cf. e.g. Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4thEdition.

[0113] The 2ndaspect of the invention and embodiments thereof

[0114] This aspect relates to a viral or bacterial expression vector which comprises and is capable of expressing the nucleic acid expression vector of the first aspect of the invention. As such, the viral or bacterial expression vector can be used as a vaccine vector, or it may be used as a agent in recombinant production.

[0115] Viral expression vectors for vaccine purposes are typically selected from a pox virus vector such as a vaccinia vector, an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a lentivirus vector, a herpesvirus vector, and a bacteriophage. For recombinant production purposes, the range of vectors is larger and also includes bacteriophages and virus that infect plants. Bacterial expression vectors useful in vaccination include bacterial vaccine vectors typically used for this purpose, see for instance da Silva A. J. et al. Braz J Microbiol. 2015 Mar 4;45(4):1117-1129. doi: 10.1590 / S1517-83822014000400001. Particularly interesting bacterial vaccine vectors are mycobacteria, including Mycobacterium bovis BCG and derivatives thereof. Again, for recombinant production, the number of useful organisms is larger and include E. coll, various lactobacteria, etc. Alternatives to E. coll are for instance selected among Bacillus spp., including Bacillus subtilis, Pseudomonas spp. including Pseudomonas fluorescens, Ralstonia spp. including Ralstonia eutropha, Lactococcus spp., and Lactobacillus spp.

[0116] The 3rdaspect of the invention and embodiments thereof

[0117] This aspect relates to a polypeptide comprising a plurality of hERV epitope hotspots as these are defined in respect of the 1staspect of the invention and the embodiments thereof discussed above. So, each an every aspect of the 1staspect of the invention insofar as it relates to expression products from the nucleic acid expression vector that include >1 hERV epitope hotspot apply mutatis mutandis to the 3rdaspect of the invention. In this context, some embodiments of the 3rdaspect of the invention entail that 2 or more of, and preferably all of, the hERV epitope hotspots are separated by peptide linkers.

[0118] The 4thaspect of the invention and embodiments thereof

[0119] This aspect relates to compositions comprising as active ingredient(s) either the polypeptide of the 3rdaspect of the invention or a plurality of hERV epitope hotspots ( / .e. the separate hotspots which are detailed in respect of the 1staspect of the invention). In both cases, further includes an immunological adjuvant. In addition, the composition (vaccine) will normally comprise at least one of a pharmaceutically acceptable carrier, vehicle, diluent, and excipient

[0120] The practical preparation of a proteinaceous vaccine composition entails that the immunogenic composition prepared will comprise standard components for a vaccine as those that are well-known in the art. The protein-based compositions prepared according to the invention thus typically contain an immunological adjuvant, which is commonly an aluminium based adjuvant or one of the other adjuvants described in the following:

[0121] Adjuvants to enhance effectiveness of an immunogenic composition include, but are not limited to: (1) aluminium salts (alum), such as aluminium hydroxide, aluminium phosphate, aluminium sulphate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (WO 90 / 14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE, although not required) formulated into submicron particles using a microfluidizer such as Model HOY microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DetoxTM); (3) saponin adjuvants such as Stimulon™ (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (eg. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg. gamma interferon), macrophage colony stimulating factor (M-CSF), tumour necrosis factor (TNF), etc.; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.

[0122] As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl- L-alanine-2"-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

[0123] The immunogenic compositions (e.g. the immunising antigen or immunogen or polypeptide or protein or nucleic acid, pharmaceutically acceptable carrier (and / or diluent and / or vehicle), and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

[0124] The compositions can thus contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

[0125] Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991).

[0126] Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.

[0127] Immunogenic compositions used as vaccines comprise an immunologically effective amount of the relevant immunogen, as well as any other of the above-mentioned components, as needed. By "immunologically effective amount", it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies or generally mount an immune response, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount of immunogen will fall in a relatively broad range that can be determined through routine trials. However, for the purposes of protein vaccination, the amount administered per immunization is typically in the range between 0.5 pg and 500 mg (however, often not higher than 5,000 pg), and very often in the range between 10 and 200 pg.

[0128] The immunogenic compositions are conventionally administered parenterally, e.g., by injection, either subcutaneously, intramuscularly, or transdermally / transcutaneously (cf. e.g. W0 98 / 20734). Additional formulations suitable for other modes of administration include oral, pulmonary and nasal formulations, suppositories, and transdermal applications. In the case of nucleic acid vaccination and antibody treatment, also the intravenous or intraarterial routes may be applicable. Dosage treatment may be a single dose schedule or a multiple dose schedule, for instance in a prime-boost dosage regimen or in a burst regimen. The vaccine may be administered in conjunction with other immunoregulatory agents as may be convenient or desired.

[0129] When the precise composition and format of a vaccine has been determined as set forth above, the invention relies generally on methods well known to the medical practitioner for inducing immunity and follow up on patients. This also entails dosing of the vaccines (which in the case protein / peptide-based vaccines typically entails administration of between 0.5 pg and 500 pg per dosage, typically provided as at least a priming dosage followed by one or several booster immunizations).

[0130] The 5thaspect of the invention and embodiments thereof

[0131] This is a method for treatment of human patient suffering from cancer, the method comprising administering to said patient an immunologically effective amount of a 1) nucleic acid expression vector of the 1staspect of the present invention as well as of any embodiment of the 1staspect disclosed herein, 2) the viral or bacterial expression vector of the 2ndaspect of the invention as well as of any embodiment of the 2ndaspect disclosed herein, 3) the polypeptide of the 3rdaspect of the present invention as well as any embodiment thereof disclosed herein, or 4) a composition of the 4thaspect of the invention or any embodiment thereof disclosed herein

[0132] This aspect relates to a method of immunizing / vaccinating, cf. above. The immunogenic substance can be a protein or peptide as detailed under the 4thaspect of the invention but may also be a vector as detailed under the 1stand 2ndaspects of the invention.

[0133] DNA vaccines, including the DNA encoding the desired antigen, can be introduced into a host cell in any suitable form including, the fragment alone, a linearized plasmid, a circular plasmid, a plasmid capable of replication, an episome, RNA, etc. Preferably, the gene is contained in a plasmid. In certain embodiments, the plasmid is an expression vector.

[0134] Individual expression vectors capable of expressing the genetic material can be produced using standard recombinant techniques.

[0135] Routes of administration include, but are not limited to, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterially, intraocularly and oral as well as topically, transdermally, by inhalation or suppository or to mucosal tissue such as by lavage to vaginal, rectal, urethral, buccal and sublingual tissue. In other words, the route of administration can be selected from any one of parenteral routes, such as via the intramuscular route, the intradermal route, transdermal route, the subcutaneous route, the intravenous route, the intra-arterial route, the intrathecal route, the intramedullary route, the intrathecal route, the intraventricular route, the intraperitoneal, the intranasal route, the vaginal route, the intraocular route, or the pulmonary route; is administered via the oral route, the sublingual route, the buccal route, or the anal route; or is administered topically.

[0136] Typical routes of administration include intramuscular, intraperitoneal, intradermal and subcutaneous injection. Genetic constructs may be administered by means including, but not limited to, traditional syringes, needleless injection devices, "microprojectile bombardment gene guns", or other physical methods such as electroporation (" EP"), "hydrodynamic method", or ultrasound. DNA vaccines can be delivered by any method that can be used to deliver DNA as long as the DNA is expressed and the desired antigen is produced in the cell.

[0137] In some embodiments, a DNA vaccine composition disclosed herein is delivered via or in combination with known transfection reagents such as cationic liposomes, fluorocarbon emulsion, cochleate, tubules, gold particles, biodegradable microspheres, or cationic polymers. Cochleate delivery vehicles are stable phospholipid calcium precipitants consisting of phosphatidyl serine, cholesterol, and calcium; this nontoxic and noninflammatory transfection reagent can be present in a digestive system. Biodegradable microspheres comprise polymers such as poly(lactide-co-glycolide), a polyester that can be used in producing microcapsules of DNA for transfection. Lipid-based microtubes often consist of a lipid of spirally wound two layers packed with their edges joined to each other. When a tubule is used, the nucleic acid can be arranged in the central hollow part thereof for delivery and controlled release into the body of an animal.

[0138] A DNA vaccine can also be delivered to mucosal surfaces via microspheres. Bioadhesive microspheres can be prepared using different techniques and can be tailored to adhere to any mucosal tissue including those found in eye, nasal cavity, urinary tract, colon and gastrointestinal tract, offering the possibilities of localized as well as systemic controlled release of vaccines. Application of bioadhesive microspheres to specific mucosal tissues can also be used for localized vaccine action. In some embodiments, an alternative approach for mucosal vaccine delivery is the direct administration to mucosal surfaces of a plasmid DNA expression vector which encodes the gene for a specific protein antigen.

[0139] The DNA plasmid vaccines disclosed are formulated according to the mode of administration to be used. Typically, DNA plasmid vaccines are injectable compositions, they are sterile, and / or pyrogen free and / or particulate free. In some embodiments, an isotonic formulation is preferably used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some embodiments, isotonic solutions such as phosphate buffered saline are preferred; one preferred solution is Tyrode's buffer. In some embodiments, stabilizers include gelatine and albumin. In some embodiments, a stabilizing agent that allows the formulation to be stable at room or ambient temperature for extended periods of time, such as LGS or other poly-cations or poly-anions is added to the formulation.

[0140] The second constituent in the nucleic acid vaccine composition disclosed herein is the pharmaceutically acceptable carrier, diluent, or excipient, which is preferably in the form of a buffered solution. Parenteral vehicles include sodium chloride solution, Ringer's dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and antimicrobials include antioxidants, chelating agents, inert gases and the like. Preferred preservatives include formalin, thimerosal, neomycin, polymyxin B and amphotericin B.

[0141] Vaccination in general is discussed in the following:

[0142] When immunization methods entail that an immunogenic protein disclosed herein or a composition comprising it is administered the subject typically receives between 0.5 and 5,000 pg of the immunogenic protein disclosed herein per administration.

[0143] In preferred embodiments of this aspect, the immunization scheme includes that the patient (human) receives a priming administration and one or more booster administrations.

[0144] Preferred embodiments of this aspect disclosed herein comprise that the administration is a therapeutic treatment of cancer.

[0145] Vaccines disclosed herein induce cellular immunity, so it is preferred that the administration is for the purpose of inducing T lymphocytes that target the cancer.

[0146] Pharmaceutical compositions can as mentioned above comprise polypeptides or nucleic acids disclosed herein. The pharmaceutical compositions will comprise a therapeutically effective amount thereof.

[0147] The term "therapeutically effective amount" or "prophylactically effective amount" as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance.

[0148] Reference is however made to the ranges for dosages of immunologically effective amounts of polypeptides, cf. above.

[0149] However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician.

[0150] For purposes of the present invention, an effective dose will be from about 0.01 mg / kg to 50 mg / kg or 0.05 mg / kg to about 10 mg / kg of DNA or RNA constructs in the individual to which it is administered.

[0151] A pharmaceutical composition can as described herein also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

[0152] Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991).

[0153] Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier. The 6thaspect of the invention and embodiments thereof

[0154] This aspect relates to a kit comprising a plurality of 1) distinct nucleic acid expression vectors of the first or second aspects of the invention, 2) distinct polypeptides according to the 3rdaspect, or distinct compositions of the 4thaspect.

[0155] The rationale behind this aspect is to provide a "battery" of vaccine agents, which each have a high likelihood of being immunogenic in a vaccinated individual in a given population, but where one or more the vaccine agents are superior relative to the individual and the particular individual's cancer and its expression profile in terms of hERV expression products. Hence the kit allows that the cancer patient is optimally treated with one (or more) of the components of the kit.

[0156] EXAMPLE 1

[0157] Designing hERV-based anti-AML vaccine

[0158] Identification of hERVs expressed by AML patients

[0159] RNA sequencing data from 150 patients with Acute Myeloid Lymphoma (AML) was downloaded from The Cancer Genome Atlas (TCGA). Each patient's RNA sequencing data was mapped to the human reference genome with the addition of all known hERV sequences and patient expressions were quantified in the form of transcripts per million (TPM). Each hERV was treated as a single isoform / transcript. A quantitative hERV expression matrix was generated by extracting and concatenating TPM values of hERVs for each patient. Binary hERV expression matrices were generated from these quantitative expression matrices by defining a cutoff of 1 TPM. All hERVs with a TPM above or equal to 1 were considered expressed in a patient, whereas those with a TPM below 1 considered not expressed.

[0160] Population HLA frequency distribution

[0161] HLA allele counts in different populations were scraped from allelefrequencies.net.

[0162] Specifically, counts were scraped for HLA alleles in the list at raw.githubusercontent.com / ANHIG / IMGTHLA / Latest / Allelelist.txt. From these counts, a general population HLA frequency distribution was calculated by first summing counts of HLA alleles at a 2-field resolution across populations and then dividing by the total number of counts for each of the HLA loci A, B, C and DRB1.

[0163] HERV ligand hotspot generation

[0164] A set of hERVs expressed in AML were determined from the binary hERV expression matrix. hERVs found to be expressed in normal tissue were removed from the set using the method defined in PCT / EP2022 / 086444. Furthermore, a list of HLA alleles found in the general population were determined from the HLA frequency distribution data. Ligands of these HLA alleles were predicted for hERVs expressed in AML using the MHC ligand prediction tool disclosed in detail in EP patent application no: 24163828.7. Ligands calculated to exhibit a ligand probability <0.15 were discarded, while ligands found in multiple hERVs were combined. A patient expression fraction was calculated for each ligand based on the binary hERV expression matrices.

[0165] These ligands were used to generate all possible ligand hotspots with a minimum length of 8 amino acids (AA) and - in this case - a maximum length of 64 AAs. A "ligand hotspot" is in this context an AA sequence with one or more ligands. As an example, if one considers a hERV with 3 ligands A, B and C, one after the other, within a stretch of 64 AAs, a total of 6 ligand hotspots of varying length can be generated: 3 single ligand hotspots A, B and C, 2 double ligand hotspots AB and BC and 1 triple ligand hotspot ABC.

[0166] Ligand hotspot deduplication and Vaccine design generation

[0167] Ligand hotspots were then ranked based on the density of predicted HLA ligands across 10,000 sampled individuals with the HLA genotypes taken from the previously described population HLA frequency distribution. The individual hotspots' ligand density was in this way estimated as a single, comparable number for a simulated, representative population. See the " Algorithms" section for more information.

[0168] After ranking the ligand hotspots, they were deduplicated based on ligand overlap which was defined as the intersection over union (IOU) of ligands between two hotspots to avoid selecting two hotspots covering an almost identical set of ligands. The final vaccine was then designed by selecting a set of hotspots that optimized the ligand density calculated across the full set of hotspots in the vaccine. AML vaccine designs have been developed from four different ligand hotspot sets created by varying the IOU ligand overlap threshold, specifically, 0%, 20%, 40% and 60%. Scoring algorithm

[0169] The algorithm takes hERVs, hotspots, ligands and HLA allele population frequency tables as inputs. Below these terms are described and an example of such data is given in a table.

[0170] hERV

[0171] Definition: A dormant retroviral gene in the genome left over by an infection of a retrovirus.

[0172] Data representation: sequence and expressed (yes or no)

[0173] ID sequence expressed 1 QTVRSSDEMKIAVRLDEMSMLSLNYEFPMQIAISPYEW Yes (SEQ ID NO: 27)

[0174] 2 WDNEILDVRAFRILWLAAMQCRLGAAYKIGHLPANKKGM No (SEQ ID NO: 28)

[0175] 3 MACHSDEMKIAVRLDEMSMISLNYEFPMQIAISP Yes

[0176]

[0177] (SEQ ID NO: 29)

[0178] A table like this is available for each patient

[0179] Hotspot

[0180] Definition: A subsequence of an hERV that contains a single or multiple HLA ligands. Note that a hotspot can be present in multiple hERVs, as the subsequence may not be unique to a single hERV but can be found in multiple.

[0181] Data representation: sequence, sources (ERV ID:start position) and ligands

[0182] ID sequence sources ligands

[0183] 1 DEMKIAVRLDEMSM {1:7, 3:6} {DEMKIAVRL, AVRLDEMSM}

[0184] (SEQ ID NO: 30) (SEQ ID NO: 30, residues 1-9 and 6-14) 2 FPMQIAISPY {1:27} {FPMQIAISPY}

[0185] (SEQ ID NO: 31) (SEQ ID NO: 31)

[0186] 3 DVRAFRILWLAAMQCRL {2:7} {DVRAFRILW, RILWLAAMQCRL}

[0187] (SEQ ID NO: 32) (SEQ ID NO: 32, residues 1-9 and 6-17) 4 MSMISLNYE {3:17} {MSMISLNYE}

[0188]

[0189] (SEQ ID NO: 33) (SEQ ID NO: 33)

[0190] Ligand

[0191] Definition: An amino acid sequence which binds an MHC protein. Note that a ligand can be present in multiple hotspots. Data representation: sequence, allele, probability, and expression fraction

[0192] ID sequence allele probability expression fraction 1 DEMKIAVRL HLA-A*01:01 0.923 0.192 (SEQ ID NO: 30, residues 1-9)

[0193] 2 AVRLDEMSM HLA-C*01:01 0.621 0.084 (SEQ ID NO: 30, residues 6-14)

[0194] 3 FPMQIAISPY HLA-B*01:02 0.875 0.093 (SEQ ID NO: 31)

[0195] 4 DVRAFRILW HLA-A*01:02 0.492 0.206 (SEQ ID NO: 32, residues 1-9)

[0196] 5 RILWLAAMQCRL HLA-DRB1*01:01 0.749 0.148 (SEQ ID NO: 32, residues 6-17)

[0197] 6 MSMISLNYE HLA-B*01:01 0.985 0.114

[0198]

[0199] (SEQ ID NO: 33)

[0200] Note that expression fraction is determined based on the fraction of patient's that has the ligand expressed via any of their expressed ERVs.

[0201] Vaccine design

[0202] Definition: A collection of hotspots.

[0203] Data representation: hotspots

[0204] ID hotspots

[0205] 1 {1,2,3}

[0206] 2 {1,3,4}

[0207]

[0208] HLA allele population frequency

[0209] Definition: A frequency distribution of HLA alleles in the general population split by loci, so that each locus' frequencies sum to 1 (loci: HLA-A, HLA-B, HLA-C and HLA-DRB1).

[0210] Data representation: Allele, locus, and frequency

[0211] Allele Locus Frequency Locus frequency sum HLA-A*01:01 HLA-A 0.5 - HLA-A*01:02 HLA-A 0.5 1.0

[0212] HLA-B*01:01 HLA-B 0.3 - HLA-B*01:02 HLA-B 0.7 1.0

[0213]

[0214] Allele Locus Frequency Locus frequency sum HLA-C*01:01 HLA-C 1.0 1.0

[0215] HLA-DRB1*01:01 HLA-DRB1 1.0 1.0

[0216]

[0217] Note that the " Locus frequency sum" column is there to underline that the frequencies of a locus must sum to one.

[0218] The Algorithm

[0219] The algorithm can score a vaccine design by doing a population simulation of ligand hits. It requires an HLA allele frequency table of the population of interest, the ligands in the vaccine design and the number of individuals to simulate.

[0220] Here follows an example of how the scoring algorithm functions where it is set to simulate just 3 individuals and the HLA population allele frequency and the ligand table from the data examples above are used.

[0221] Step 1:

[0222] Sample HLA types for the 3 simulated individuals by sampling two alleles for each locus based on locus allele frequencies in the HLA allele frequency table.

[0223] Below is a table of 3 sampled individuals using the HLA population allele frequency table above.

[0224] Individual HLA expression

[0225] 1 2 x HLA-A*01:01

[0226] HLA-B*01:01, HLA-B*01:02

[0227] 2 x HLA-C*01:01

[0228] 2 x HLA-DRB1*01:01

[0229] 2 HLA-A*01:01, HLA-A*01:02

[0230] 2 x HLA-B*01:02

[0231] 2 x HLA-C*01:01

[0232] 2 x HLA-DRB1*01:01

[0233] 3 HLA-A*01:01, HLA-A*01:02

[0234] HLA-B*01:01, HLA-B*01:02

[0235] 2 x HLA-C*01:01

[0236]

[0237] 2 x HLA-DRB1*01:01

[0238] Note that these are sampled based on allele frequencies. It could very well be that two or all three of the HLA types were the same. The chances of this would diminish when using a more diverse HLA population allele frequency table, which would be the case with real data. Step 2:

[0239] Simulate the number of ligand hits for each individual by limiting the ligand table to ligands that bind to alleles in the individual's HLA type and determining ligand hits based on the ligand probability and expression fraction. A ligand hit is determined by selecting a random number between 0-1. If the number is below the product of a ligand's MHC binding probability and expression fraction, then it is a hit.

[0240] Below is an example for individual 1. The individual rows are potential ligand hits and the final "hit" denotes whether a potential ligand has been deemed an actual ligand in the simulation. The table therefore shows that in this simulation, individual 1 has 2 ligand hits.

[0241] ID allele probability expression prob * random hit (prob) fraction (exp f) exp f number

[0242] 1 HLA-A*01:01 0.923 0.192 0.177 0.428 no 2 HLA-C*01:01 0.621 0.084 0.052 0.012 yes 3 HLA-B*01:02 0.875 0.093 0.081 0.823 no 5 HLA-DRB1*01:01 0.749 0.148 0.110 0.198 no 6 HLA-B*01:01 0.985 0.114 0.112 0.110 yes

[0243]

[0244] The tables for individuals 2 and 3 are not shown but they have 1 and 3 ligand hits, respectively.

[0245] Step 3:

[0246] Determine ligand coverage probabilities from the number of ligand hits of the simulated individuals.

[0247] Individual Number of ligand hits

[0248] 1 2

[0249] 2 1

[0250] 3 3

[0251]

[0252] To determine the ligand coverage probabilities across the population simply go from 1 to the highest number of ligand hits in the table and calculate the fraction of individuals that has exactly that number of ligand hits or more. Ligand hits Fraction of

[0253] above or equal individuals

[0254] 1 1.00

[0255] 2 0.66

[0256] 3 0.33

[0257]

[0258] The result of the scoring algorithm is a cumulative probability distribution of having 1 or more, 2 or more, and so on, up to the highest number of ligand hits found in a simulated individual. To have a single comparable score the cumulative probability distribution can be summed.

[0259] EXAMPLE 2

[0260] Immunogenicity testing of hotspots

[0261] Reference is made to Fig. 1.

[0262] Al-Immunology™ predicted hotspot antigens for 19 different human ERV (hERV) hotspots (Hotspots 1-19), which were probed for their potential to induce specific T-cell responses. Peripheral blood mononuclear cells (PBMCs) from 8 healthy human donors were primed in an IVS assay and re-stimulated with peptides representing 19 hERV hotspots. T-cell responses were assessed using interferon y (IFN-y) ELISpot analysis. Additionally, hERV-specific T cells with a matching HLA were co-cultured with hERV-loaded leukemia cells to determine their cytotoxicity.

[0263] The study's objective was to investigate the ability of T-cell epitope hotspots to induce specific T cells in healthy donor-derived PBMCs and show their ability to eliminate cancer cells.

[0264] Materials

[0265] hERV peptide pools: 20-28-mer peptides overlapping the span of an hERV hotspot were used for PBMC stimulation.

[0266] hERV minimal peptides: 9-11-mer peptides derived from selected hERV hotspots were used for T2 loading. Peripheral blood mononuclear cells (PBMCs): PBMCs were isolated from buffy coats from healthy donors by ficoll gradient separation. The PBMCs were HLA typed.

[0267] T2 cells: A*0201 Leukemic human tumor cell line acquired from ATCC. Herein referred to as target cells.

[0268] Study plan

[0269] Analysis of pre-existing immune response:

[0270] To investigate the presence of pre-existing immune responses against the hERV hotspots, PBMCs from healthy donors with different HLA types were re-stimulated with hERV peptide pools. T-cell activation was evaluated by secretion of IFN-y by ELISpot analysis.

[0271] Induction of de-novo immune responses:

[0272] To investigate the immunogenicity of the hERV hotspots, an optimized in vitro priming assay (IVS) on PBMCs from healthy donors was carried out.

[0273] The protocol was designed to induce of de novo immune responses against tumour-associated antigens. The protocol was first described by Bozkus etal[l] and later validated as a tool to demonstrate the immunogenicity of hERVs [2].

[0274] Briefly, PMBCs were cultured for 24 hours with a cocktail of cytokines designed to induce antigen-presenting cell differentiation and maturation. Subsequently, the PBMCs were incubated for 24 hours with the hERV peptide pools and a combination of adjuvants designed to induce antigen presentation and T-cell priming. During the following 10 days, the cell cultures received cytokines feeding the expansion of antigen-specific T cells.

[0275] Evaluation of the functionality of antigen-specific T cells

[0276] After the priming phase, the presence of antigen-specific T cells was evaluated by restimulation with the same hERV peptide pools used during priming, followed by measurement of cytokine secretion. The presence of hERV-specific T cells was assessed by IFN-y secretion in ELISpot assays. Alternatively, the functionality of the newly primed functional T cells was evaluated by means of cytotoxicity in a tumour-cell-killing assay in a selection of healthy donors.

[0277] Following the protocol described by Friedmann et al [3], the in vitro expanded T cells were co-cultured with the human leukaemia tumour cell line T2, which was loaded with relevant hERV minimal peptides for 2 hours. Tumour-cell killing was measured by Calcein-AM release over a span of 150 minutes. The positive control corresponds to a chemical lysing agent (Triton-X). The irrelevant peptides used as a negative control are peptides derived from human housekeeping gene

[0278] Correlation between theoretical calculated ligand coverage probabilities and measured probabilities

[0279] As the experimental data presented above and in Fig. 1 reveals no information about which ligands are immunogenic, a cumulative probability was calculated for each hotspot by calculating the probability that at least one ligand was a true ligand for each hotspot and for each allele. The formula for calculating at least one true prediction is 1 - product(1 - p), where p is the probability of a single ligand and product(1 - p) is the product of 1 minus p for each ligand prediction for the given allele. The Allele-Hotspot cumulated predictions where then multiplied by 0.2, which approximates the positive predictive value for a true ligand to be immunogenic in a random individual. Finally, the Allele-Hotspot predictions where fed into the population coverage calculation in lieu of ligand predictions.

[0280] The Bootstrap samples were calculated by drawing from the 8 healthy donors in vitro samples 10,000 times with replacement, and calculating the fraction if >1, >2, >3 and >4 immunogenic hotspots in the sample. 95% CI intervals were then extracted from this data.

[0281] From Figure 1D it is evident that the observed number of immunogenic Hotspots observed in the 8 healthy doners are in line with the expected number given the theoretical ligand coverage probability.

[0282] Fig.2 shows the results in the above-described tumour-cell-killing assay: hERV-specific primary cytotoxic T cells derived from three different healthy donors (HD-1, HD-7, and HD-3) were generated using the above-presented in vitro priming assay. Cytotoxic T cells specific for different ERV hotspots in all cases exhibit cytotoxicity against hERV-loaded T2 cells at a CTL-to-target ratio of 5:1.

[0283] EXAMPLE 3

[0284] Murine study of immunization effect

[0285] A mouse-specific vaccine was designed using the approach disclosed further exemplified in example 1. Contrary to example 1, the population was defined by MHC alleles from Balb / c and C57BL / 6 mice and the hotspots discovered from the env protein of MuLV, which is a well described mERV. Reference is made to Fig. 3.

[0286] Groups of n=13 mice (BALB / c or C57BL / 6) were immunized prophylactically at days -14, -7, and 0 (relative to tumour inoculation) with 25 pg pDNA_mERV_Hotspot_gp70 dissolved in 100 pl or "immunized" with 100 pl vehicle before sub-cutaneous inoculation with 200,000 CT26 (BALB / c) or 150,000 B16F10 (C57BL / 6) tumour cells formulated in PBS and Matrigel. After tumour cell inoculation, follow-up immunizations were made at day 7 and 14; all immunizations were made intramuscularly supplied with electroporation at the injection sites. After euthanization (when tumours are too large and / or ulcerates) splenocytes were isolated for analysis of the immune responses. Data are shown in Fig. 3. The plasmid DNA sequence is set forth in SEQ ID NO: 1 and the amino acid sequence of the encoded hotspot-containing protein is set forth in SEQ ID NO: 2; the individual hotspots are set forth in SEQ ID NOs: 3-26 and are the full-length protein separated by GGG linkers.

[0287] As appears, the growth of the tumour over time is controlled by the immunizations.

[0288] Sequences 1-26 are set forth in the following:

[0289] SEQ ID NO: 1:

[0290] tggccattgc atacgttgta tccatatcat aatatgtaca tttatattgg ctcatgtcca acattaccgc catgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctccgcggc cgggaacggt gcattggaac gcggattccc cgtgccaaga gtgacgtaag taccgcctat agactctata ggcacacccc tttggctctt atgcatgcta tactgttttt ggcttggggc ctatacaccc ccgcttcctt atgctatagg tgatggtata gcttagccta taggtgtggg ttattgacca ttattgacca ctccaacggt ggagggcagt gtagtctgag cagtactcgt tgctgccgcg cgcgccacca gacataatag ctgacagact aacagactgt tcctttccat gggtcttttc tgcagtcacc gtcgtcgacg gtatcgataa gcttgatatc gaattccgcc gccaccatgg ccccccgtgt gaccccactc ctggccttca gcctgctggt tctctggacc ttcccagccc caactctggg gggtgctaat gatgcggaag actgctgcct gtctgtgacc cagcgcccca tccctgggaa catcgtgaaa gccttccgct accttcttaa tgaagatggc tgcagggtgc ctgctgttgt gttcaccaca ctaaggggct atcagctctg tgcacctcct gaccagccct gggtggatcg catcatccga agactgaaga agtcttctgc caagaacaaa ggcaacagca ccagaaggag ccctgtgtct gagctcaaaa ccccacttgg tgacacaact cacacagagc ccaaatcttg tgacacacct cccccgtgcc caaggtgccc aggcggtgga agcagcggag gtggaagtgg aggacagccc cgagaaccac aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc agcgggcagc cggagaacaa ctacaacacc acgcctccca tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacatcttc tcatgctccg tgatgcatga ggctctgcac aaccgcttca cgcagaagag cctctccctg tctccgggta aaggcctcgg tggcctgtct ggtccacctt actatgaagg cgttgcagtg ttgggtacct atagcaacgg cggaggcgtc gctctcggga actctcccca tcaagttttc aacctcggcg ggggaaaaag tataaccaac cttgaaaagt cacttggcgg cggcgctggg gttgagaaca gactgttgaa cctgggtggc ggtaccggcc ctcctaacgg ggctgcccgg aacctcgaga ctacaggtgg cggggtcctc acacaacaat atcatcaact gaaggggggt ggagctactc agcaatttca gcaattgggg ggggggctga aaattaccga ctctggacct cgggtcccaa ttgggcctaa tcctgtcttg agcgaccgag gaggcggtaa gccaagctca tcttgggact atataggtgg tggtcacaat gagggttttt acgtctgtcc cggcccccac agacctcggg gcgggggcac acaacaatat catcaactca aaactattgg aggaggtggg gtctatcatc aattcgagcg ccgcgccaag tacaagagag aaggtggagg gggcgccgca aggaacctgg aaactacagg gggcgggtgg cccagggtca cctaccactc cccaagttac gtctatcatc agttcgaagg agggggtttc tatgccgacc acaccggctt ggtgggtggt ggggattaca tcactgtatc aaacaacctc ggtggtggag atagggagac agtatgggcc atcactggaa accacggcgg cggaaaaagc ccatggttta ctacacttgg tggggggctc actattagat tcacaagttt tggcgggggg gttcttacac aacaatatca ccaacttaag actattggcg acgggggggg cgaacccttg acatcataca ccccccgctg taacacagct tggaatcgcg gcggaggcgc tgcccctact gggactactt gggctggagg cggggaagaa cccctgacta gctacactcc acgctgcaac actgcctgga acagaggtgg gggctggggg ctggaatatc gcgcaccatt ctcaccccct cccggatagg atccagatct aacgacaaaa cgacaaaacg acaaggcgcc agatctggcg tttcgttttg tcgttttgtc gttagatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat atgcccattc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tcgggggggg ggggcgctga ggtctgcctc gtgaagaagg tgttgctgac tcataccagg cctgaatcgc cccatcatcc agccagaaag tgagggagcc acggttgatg agagctttgt tgtaggtgga ccagttggtg attttgaact tttgctttgc cacggaacgg tctgcgttgt cgggaagatg cgtgatctga tccttcaact cagcaaaagt tcgatttatt caacaaagcc gccgtcccgt caagtcagcg taatgctctg ccagtgttac aaccaattaa ccaattctga ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctcgagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca gagattttga gacacaacgt ggctttcccc ccccccccat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcagattg gctat SEQ ID NO: 2:

[0291] SGPPYYEGVA VLGTYSNGGG VALGNSPHQV FNLGGGKSIT NLEKSLGGGA GVENRLLNLG GGTGPPNGAA RNLETTGGGV LTQQYHQLKG GGATQQFQQL GGGLKITDSG PRVPIGPNPV LSDRGGGKPS SSWDYIGGGH NEGFYVCPGP HRPRGGGTQQ YHQLKTIGGG GVYHQFERRA KYKREGGGGA ARNLETTGGG WPRVTYHSPS YVYHQFEGGG FYADHTGLVG GGDYITVSNN LGGGDRETVW AITGNHGGGK SPWFTTLGGG LTIRFTSFGG GVLTQQYHQL KTIGDGGGEP LTSYTPRCNT AWNRGGGAAP TGTTWAGGGE EPLTSYTPRC NTAWNRGGGW GLEYRAPFSP PPG

[0292] SEQ ID NO: 3:

[0293] SGPPYYEGVA VLGTYSN

[0294] SEQ ID NO: 4:

[0295] VALGNSPHQV FNL

[0296] SEQ ID NO: 5:

[0297] KSITNLEKSL

[0298] SEQ ID NO: 6:

[0299] AGVENRLLNL

[0300] SEQ ID NO: 7:

[0301] TGPPNGAARN LETT

[0302] SEQ ID NO: 8:

[0303] VLTQQYHQLK

[0304] SEQ ID NO: 9:

[0305] ATQQFQQL

[0306] SEQ ID NO: 10:

[0307] LKITDSGPRV PIGPNPVLSD R

[0308] SEQ ID NO: 11:

[0309] KPSSSWDYI

[0310] SEQ ID NO: 12:

[0311] HNEGFYVCPG PHRPR SEQ ID NO: 13:

[0312] TQQYHQLKTI G

[0313] SEQ ID NO: 14:

[0314] VYHQFERRAK YKRE

[0315] SEQ ID NO: 15:

[0316] GAARNLETT

[0317] SEQ ID NO: 16:

[0318] WPRVTYHSPS YVYHQFE

[0319] SEQ ID NO: 17:

[0320] FYADHTGLV

[0321] SEQ ID NO: 18:

[0322] DYITVSNNL

[0323] SEQ ID NO: 19:

[0324] DRETVWAITG NH

[0325] SEQ ID NO: 20:

[0326] KSPWFTTL

[0327] SEQ ID NO: 21:

[0328] LTIRFTSF

[0329] SEQ ID NO: 22:

[0330] VLTQQYHQLK TIGD

[0331] SEQ ID NO: 23:

[0332] EPLTSYTPRC NTAWNR

[0333] SEQ ID NO: 24:

[0334] AAPTGTTWA

[0335] SEQ ID NO: 25:

[0336] EEPLTSYTPR CNTAWNR SEQ ID NO: 26:

[0337] WGLEYRAPFS PPPG

[0338] LIST OF REFERENCES

[0339] 1. Cimen Bozkus C, Blazquez AB, Enokida T, Bhardwaj N. A T-cell-based immunogenicity protocol for evaluating human antigen-specific responses. STAR Protoc. 2021 Sept 17;2(3): 100758.

[0340] 2. Bonaventura P, Alcazer V, Mutez V, Tonon L, Martin J, Chuvin N, Michel E, Boulos RE, Estornes Y, Valladeau-Guilemond J, Viari A, Wang Q, Caux C, Depil S. Identification of shared tumor epitopes from endogenous retroviruses inducing high-avidity cytotoxic T cells for cancer immunotherapy. Sci Adv. 2022 Jan 26;8(4):eabj3671.

[0341] 3. Friedmann KS, Kaschek L, Knörck A, Cappello S, Lünsmann N, Küchler N, Hoxha C, Schäfer G, Iden S, Bogeski I, Kummerow C, Schwarz EC, Hoth M. Interdependence of sequential cytotoxic T lymphocyte and natural killer cell cytotoxicity against melanoma cells. J Physiol. 2022;600(23):5027-54.

Claims

CLAIMS1. A nucleic acid expression vector encoding a plurality of hERV epitope hotspots, wherein1) each hERV epitope hotspot is comprised of an amino acid sequence of at least 8 amino acid residues;2) each hERV epitope hotspot is comprised in at least one proteinaceous hERV expression product identified among expression products from malignant cells in a group of cancer patients;3) each hERV epitope hotspot exhibits a density of predicted ligands for allelic variants of HLA molecules, said allelic variants being found in a population to which the group of cancer patients belong, where said density is in the upper three quartiles of the average density of predicted ligands in the population for the same allelic variants of HLA molecules, said average density of predicted ligands being found in amino acid sequences of the same length as the hERV epitope hotspot; and4) the plurality of hERV epitope hotspots comprises a plurality of predicted ligands for allelic variants of HLA molecules in said population, whereby the probability is at least 50% that at least one of the predicted HLA ligands is an HLA ligand in a randomly selected individual in said population, preferably where the individual's malignant cells express the hERV as a proteinaceous expression product comprising the amino acid sequence of the predicted ligand.

2. The nucleic acid expression vector according to claim 1, wherein the plurality of hERV epitope hotspots comprises a plurality of predicted ligands for allelic variants of HLA molecules in said population, whereby the probability is at least 50% that at least 2 of the predicted HLA ligands are HLA ligands in a randomly selected individual in said population.

3. The nucleic acid expression vector according to claim 1 or 2, wherein the plurality of hERV epitope hotspots comprises a plurality of predicted ligands for allelic variants of HLA molecules in said population, whereby the probability is at least 50% that at least 3 of the predicted HLA ligands are HLA ligands in a randomly selected individual in said population.

4. The nucleic acid expression vector according to any one of the preceding claims, wherein the plurality of hERV epitope hotspots comprises a plurality of predicted ligands for allelic variants of HLA molecules in said population, whereby the probability is at least 50% that at least 4 of the predicted HLA ligands are HLA ligands in a randomly selected individual in said population.

5. The nucleic acid expression vector according to any one of the preceding claims, wherein the plurality of hERV epitope hotspots comprises a plurality of predicted ligands for allelic variants of HLA molecules in said population, whereby the probability is at least 50% that at least 5 of the predicted HLA ligands are HLA ligands in a randomly selected individual in said population.

6. The nucleic acid expression vector according to any one of the preceding claims, wherein the plurality of hERV epitope hotspots comprises a plurality of predicted ligands for allelic variants of HLA molecules in said population, whereby the probability is at least 50% that at least 6 of the predicted HLA ligands are HLA ligands in a randomly selected individual in said population.

7. The nucleic acid expression vector according to any one of the preceding claims, wherein the plurality of hERV epitope hotspots comprises a plurality of predicted ligands for allelic variants of HLA molecules in said population, whereby the probability is at least 50% that at least 7 of the predicted HLA ligands are HLA ligands in a randomly selected individual in said population.

8. The nucleic acid expression vector according to any one of the preceding claims, wherein the plurality of hERV epitope hotspots comprises a plurality of predicted ligands for allelic variants of HLA molecules in said population, whereby the probability is at least 50% that at least 8 of the predicted HLA ligands are HLA ligands in a randomly selected individual in said population.

9. The nucleic acid expression vector according to any one of the preceding claims, wherein the plurality of hERV epitope hotspots comprises a plurality of predicted ligands for allelic variants of HLA molecules in said population, whereby the probability is at least 50% that at least 9 of the predicted HLA ligands are HLA ligands in a randomly selected individual in said population.

10. The nucleic acid expression vector according to any one of the preceding claims, wherein the plurality of hERV epitope hotspots comprises a plurality of predicted ligands for allelic variants of HLA molecules in said population, whereby the probability is at least 50% that at least 10 of the predicted HLA ligands are HLA ligands in a randomly selected individual in said population.

11. The nucleic acid expression vector according to any one of the preceding claims, wherein the percentage- in claim 1 is at least 60%, or- in claim 2 is at least 60%, or- in claim 3 is at least 60%, or- in claim 4 is at least 60%, or- in claim 5 is at least 60%, or- in claim 6 is at least 60%, or- in claim 7 is at least 60%, or- in claim 8 is at least 60%, or- in claim 9 is at least 60%, or- in claim 10 is at least 60%.

12. The nucleic acid expression vector according to any one of the preceding claims, wherein the percentage- in claim 1 is at least 70%, or- in claim 2 is at least 70%, or- in claim 3 is at least 70%, or- in claim 4 is at least 70%, or- in claim 5 is at least 70%, or- in claim 6 is at least 70%, or- in claim 7 is at least 70%, or- in claim 8 is at least 70%, or- in claim 9 is at least 70%, or- in claim 10 is at least 70%.

13. The nucleic acid expression vector according to any one of the preceding claims, wherein the percentage- in claim 1 is at least 80%, or- in claim 2 is at least 80%, or- in claim 3 is at least 80%, or- in claim 4 is at least 80%, or- in claim 5 is at least 80%, or- in claim 6 is at least 80%, or- in claim 7 is at least 80%, or- in claim 8 is at least 80%, or- in claim 9 is at least 80%, or- in claim 10 is at least 80%.

14. The nucleic acid expression vector according to any one of the preceding claims, wherein the percentage- in claim 1 is at least 85%, or- in claim 2 is at least 85%, or- in claim 3 is at least 85%, or- in claim 4 is at least 85%, or- in claim 5 is at least 85%, or- in claim 6 is at least 85%, or- in claim 7 is at least 85%, or- in claim 8 is at least 85%, or- in claim 9 is at least 85%, or- in claim 10 is at least 85%.

15. The nucleic acid expression vector according to any one of the preceding claims, wherein the percentage- in claim 1 is at least 90%, or- in claim 2 is at least 90%, or- in claim 3 is at least 90%, or- in claim 4 is at least 90%, or- in claim 5 is at least 90%, or- in claim 6 is at least 90%, or- in claim 7 is at least 90%, or- in claim 8 is at least 90%, or- in claim 9 is at least 90%, or- in claim 10 is at least 90%.

16. The nucleic acid expression vector according to any one of the preceding claims, wherein the density is higher than the average density of predicted ligands in the population for the same allelic variants of HLA molecules.

17. The nucleic acid expression vector according to any one of the preceding claims, wherein each hERV epitope hotspot, when tested in vitro, exhibits one or more of the following properties:- ability to induce secretion of IFN-y from PBMCs of human donors as determined by stimulating the PBMCs with a hERV peptide pool corresponding to the hERV epitope hotspot; or- ability to prime antigen-specific T-cells as determined by 1) incubating the hERV epitope hotspot with PBMCs treated to induce antigen-presenting cell differentiation and maturation and 2) re-stimulating with the hotspot to induce IFN-y secretion.

18. The nucleic acid expression vector according to any one of the preceding claims, wherein each hERV epitope hotspot is comprised in at least one proteinaceous hERVexpression product identified among expression products from malignant cells in a group of cancer patients suffering from the same histological type of cancer.

19. The nucleic acid expression vector according to claim 18, wherein the histological type of cancer is an epithelial tumour, a non-epithelial tumour, and a mixed tumour.

20. The nucleic acid expression vector according to claim 19, wherein the epithelial tumour is selected from a carcinoma and an adenocarcinoma, and wherein the non-epithelial tumour or mixed tumour is selected from a liposarcoma, a fibrosarcoma, a chondrosarcoma, an osteosarcoma, a leiomyosarcoma, a rhabdomyosarcoma, a glioma, a neuroblastoma, a medulloblastoma, a malignant melanoma, a malignant meningioma, a neurofibrosarcoma, a leukaemia, a myeloproliferative disorder, a lymphoma, a hemangiosarcoma, a Kaposi's sarcoma, a malignant teratoma, a dysgerminoma, a seminoma, and a choriocarcinoma.

21. The nucleic acid expression vector according to claim 19, wherein the histological type of cancer is a leukemia.

22. The nucleic acid expression vector according to claim 21, wherein the non-epithelial is acute myeloid leukaemia (AML).

23. The nucleic acid expression vector according to claim 19, wherein the histological type of cancer is myelodysplastic syndrome (MDS).

24. The nucleic acid expression vector according to any one of the preceding claims, which expresses the hERV epitope hotspots as one single polypeptide or as several polypeptides, wherein at least one of the several polypeptides includes at least 2 hERV epitope hotspots.

25. The nucleic acid expression vector according to claim 24, wherein 2 or more of, and preferably all of, the hERV epitope hotspots present in the same polypeptide are separated by peptide linkers.

26. The nucleic acid expression vector according to any one of claims 1-23, wherein each hERV epitope hotspot is a separate expression product.

27. The nucleic acid expression vector according to any one of the preceding claims, wherein the number of hERV epitope hotspots encoded are from 2 to 50.

28. The nucleic acid expression vector according to any one of the preceding claims, wherein the population is the world population.

29. The nucleic acid expression vector according to any one of claims 1-27, wherein the population to which the patient belongs is a genetically delimited population or a geographically delimited population.

30. The nucleic acid expression vector according to claim 29, wherein the geographically delimited population is selected from a continent's population, a population of a region, or a population of a country / nation / state.

31. The nucleic acid expression vector according to claim 29, wherein the genetically delimited population is defined by its HLA profile.

32. The nucleic acid expression vector according to any one of the preceding claims, wherein the average density of predicted ligands in the population is calculated as∑ⁿᵢ ∑ⱼ P(L|Aᵢ) · f(Aᵢ) / SₗwhereinSi is the length of the hERV epitope hotspot S,P(L|Aᵢ) is the probability that a fragment of the hERV epitope hotspot is a ligand for HLA allele I,f(Ai) is the frequency of allele i in the population,Σⱼ is the summation over all fragments contained in the hotspot S, andΣᵢⁿ is the summation over all alleles in the population.

33. The nucleic acid expression vector, wherein the predicted HLA ligands are ligands for HLA Class 1, ligands for HLA Class 2, or a mix of ligands for HLA Class 1 and 2.

34. A viral or bacterial expression vector which comprises and is capable of expressing the nucleic acid expression vector according to any one of the preceding claims.

35. The viral expression vector according to claim 34, which is selected from a pox virus vector such as a vaccinia vector, an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a lentivirus vector, a herpesvirus vector, and a bacteriophage.

36. The bacterial expression vector according to claim 34, which is selected from E. coli, Mycobacterium bovis, Bacillus spp., including Bacillus subtilis, Pseudomonas spp. including Pseudomonas fluorescens, Ralstonia spp. including Ralstonia eutropha, Lactococcus spp., and Lactobacillus spp.

37. A polypeptide comprising a plurality of hERV epitope hotspots, wherein1) each hERV epitope hotspot is comprised of an amino acid sequence of at least 8 amino acid residues;2) each hERV epitope hotspot is comprised in at least one proteinaceous hERV expression product identified among expression products from malignant cells in a group of cancer patients;3) each hERV epitope hotspot exhibits a density of predicted ligands for allelic variants of HLA molecules, said allelic variants being found in a population to which the group of cancer patients belong, where said density is in the upper three quartiles of the average density of predicted ligands in the population for the same allelic variants of HLA molecules, said average density of predicted ligands being found in amino acid sequences of the same length as the hERV epitope hotspot; and1) the plurality of hERV epitope hotspots comprises a plurality of predicted ligands for allelic variants of HLA molecules in said population, whereby the probability is at least 50% that at least one of the predicted HLA ligands is an HLA ligand from at least one hERV expression product in a randomly selected individual in said population, preferably where the individual's malignant cells express the hERV as a proteinaceous expression product comprising the amino acid sequence of the predicted ligand.

38. The polypeptide according to claim 37, wherein the hERV epitope hotspots are as defined in any one of claims 18-23 and 27-33.

39. The polypeptide according to claim 37 or 38, wherein 2 or more of, and preferably all of, the hERV epitope hotspots are separated by peptide linkers.

40. A composition comprising the polypeptide according to any one of claims 37-39 in combination with an immunological adjuvant.

41. The composition according to claim 40, which comprises at least one of a pharmaceutically acceptable carrier, vehicle, diluent, and excipient.

42. A composition comprising a plurality of hERV epitope hotspots, wherein1) each hERV epitope hotspot is comprised of an amino acid sequence of at least 8 amino acid residues;2) each hERV epitope hotspot is comprised in at least one proteinaceous hERV expression product identified among expression products from malignant cells in a group of cancer patients;3) each hERV epitope hotspot exhibits a density of predicted ligands for allelic variants of HLA molecules, said allelic variants being found in a population to which the group of cancer patients belong, where said density is in the upper three quartiles of the average density of predicted ligands in the population for the same allelic variants of HLA molecules, said average density of predicted ligands being found in amino acid sequences of the same length as the hERV epitope hotspot; and5) the plurality of hERV epitope hotspots comprises a plurality of predicted ligands for allelic variants of HLA molecules in said population, whereby the probability is at least 50% that at least one of the predicted HLA ligands is an HLA ligand from at least one hERV expression product in a randomly selected individual in said population, preferably where the individual's malignant cells express the hERV as a proteinaceous expression product comprising the amino acid sequence of the predicted ligand.

43. The composition according to claim 42, which comprises an immunological adjuvant.

44. The composition according to claim 42 or 43, which comprises at least one of a pharmaceutically acceptable carrier, diluent, vehicle, and excipient.

45. The composition according to any one of claims 42-44, wherein the hERV epitope hotspots are as defined in any one of claims 2-23 and 28-33.

46. A method for treatment of human patient suffering from cancer, the method comprising administering to said patient an immunologically effective amount of a nucleic acid expression vector according to any one of claims 1-33, the viral or bacterial expression vector according to any one of claims 34-36, the polypeptide according to any one of claims 37-39, or a composition according to any one of 40-45.

47. A kit comprising a plurality of distinct nucleic acid expression vectors according to any one of claims 1-33, distinct viral or bacterial expression vectors according to any one of claims 34-36, distinct polypeptides according to any one of claims 37-39, or distinct compositions according to any one of 40-45.

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