Cancer immunotherapy

Abstract

The present invention relates to carriers comprising mRNA encoding a HORMAD1 polypeptide, in particular mRNA-based vaccines, more in particular dendritic cell (DC) based vaccines. The present invention further relates to methods for preparing an immunotherapy agent and / or the use of the vaccines for inducing immune responses and preventing and / or treating subjects having cancer cells or tumors that express HORMAD1.

Description

[0001] CANCER IMMUNOTHERAPY

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to carriers comprising mRNA encoding a HORMAD1 polypeptide, in particular mRNA-based vaccines, more in particular dendritic cell (DC) based vaccines. The present invention further relates to methods for preparing an immunotherapy agent and / or the use of the vaccines for inducing immune responses and preventing and / or treating subjects having cancer cells or tumors that express HORMAD1 .

[0004] BACKGROUND TO THE INVENTION

[0005] Lung cancer is the most common cancer worldwide with 1 .200.000 new cases annually (source: WHO). Lung cancer is also the deadliest type of cancer, with a higher cause-specific mortality than breast, colorectal and prostate cancer combined. More than 80% of lung cancer cases are of the non-small cell type (NSCLC). Almost half of NSCLC patients are diagnosed with advanced (metastatic or stage IV) disease, and an alarming number of patients develop metastatic relapse after resection or chemoradiation with curative intent. Stage IV lung cancer is considered incurable. A minority of advanced NSCLC harbours oncogenic driver mutations and gene re-arrangements (e.g. EGFR, ALK, ROS1 , BRAF), and can be successfully controlled for sometimes several years using matched targeted therapies (oral kinase inhibitors).

[0006] For the majority of “wildtype” NSCLC, remaining options are immunotherapy in the shape of immune checkpoint inhibitors (ICI), including anti-PD-1 , anti-PD-L1 and anti-CTLA-4, in many cases combined with chemotherapy. ICI act by re-activating exhausted tumor-reactive T-cells. Results so far have been encouraging, but only a minority of patients derive clinical benefit from these ICI-based regimen: in a first-line metastatic lung cancer setting more than half of treated patients does not respond to immune checkpoint blockade and in a second or higher line setting 4 out of 5 patients do not respond to this therapy.

[0007] These clinical results clearly suggest that the full anti-tumoral power of the immune system is not being leveraged yet. From the mechanism of action of ICI, it can be derived that efficacy is low to absent in tumors that have intrinsically poor or absent T-cell infiltration (immune “cold” tumors).

[0008] A large body of preclinical evidence suggests that the success of immune checkpoint inhibition against established tumors can be enhanced by combining with vaccination. Vaccination aims to prime and / or expand tumor antigen-targeting T-cells and induce immunological memory against later disease relapse. In contrast to immune checkpoint blockade, which impacts the full T-cell repertoire including potentially self-reactive lymphocytes, a judicious selection of antigenic target(s) in the vaccine design results in a highly tumor-focused immune response. However, on their own, vaccines have failed to deliver substantial clinical benefit in NSCLC so far. This calls for a more advanced strategy to direct clinically meaningful immune responses against cancerous foci. Vaccination with tumor antigen-loaded dendritic cells (DCs) is nowadays one of the most advanced forms of cancer immunotherapy. Dendritic cells (DCs) are specialized antigen presenting leukocytes that are now recognized to be the main controllers of the immune response. DCs continuously sample their environment for antigens and must judiciously decide between maintaining immune tolerance (e.g. against self-antigens) versus active immune response (e.g. against microbial pathogens or cancer cells). Classical DC-based cancer vaccines consist of DCs derived in vitro from autologous peripheral blood monocytes, exposed to activating factors and subsequently loaded with tumor-derived antigens. The source of these antigens can be: lysates from autologous tumor or representative tumor cell lines, (synthetic) protein antigen, peptide-based tumor antigen, mRNA(s) encoding selected tumor antigen(s) or autologous whole-tumor-derived mRNA. In humans, several early-phase trials have shown that DCbased immunotherapy to be feasible and safe.

[0009] A need exists for identifying additional cancer antigens for use in therapeutic cancer vaccines, in particular HLA-independent and clinically relevant antigens with a high level of tumor-restricted expression. Moreover, a vaccine-based approach must achieve the important biological objective of turning a T-cell poor or immune “cold” tumor into a “hot” tumor, characterized by a strong infiltration of competent killer T cells, resulting in suppressed tumor growth or tumor elimination. As emphasized earlier, Immune hot tumors are also inherently more sensitive to immune checkpoint inhibition, providing the basis for a synergistic combination of cancer vaccines with ICI.

[0010] The present invention addresses all these needs and provides an effective HORMAD1 -targeting mRNA vaccine wherein autologous T-cells can be primed against the HORMAD1 antigen to specifically induce cell death in HORMAD1 -expressing cancer cells.

[0011] SUMMARY OF THE INVENTION

[0012] Accordingly, in a first aspect, the present invention provides a carrier comprising one or more mRNA encoding a HORMAD1 polypeptide, in particular wherein the HORMAD1 polypeptide is expressed at an effective level that exceeds expression by promoter leakage. In particular, the carrier is selected from the group consisting of liposomes, lipid micro- or nanoparticles, polymeric micro- or nanoparticles, exosomes, cationic nano-emulsions, cationic peptides or cationic polymers, antigen presenting cells, plasmids and viral vectors. In a specific embodiment the carrier is a polymeric nanoparticle or a lipid nanoparticle (LNP).

[0013] In a particular embodiment of the invention, said antigen presenting cell (APC) is selected from the group comprising: dendritic cell (DC) or B-cell, preferably isolated from or generated from the blood of a subject, a dendritic cell-line, a B-cell line, a macrophage, or any other leukocyte with antigen- presenting properties. The cells or cell lines can be autologous or allogenic. In a specific embodiment, the APC is a DC or a B-cell, or a combination thereof. In a particular embodiment, said HORMAD1 polypeptide comprises or consist of an amino acid sequence characterized by any one of SEQ ID NOs: 1 -7, or a sequence having at least 85% sequence identity thereto, including an immunogenic fragment thereof. The invention further provides a nucleic acid molecule encoding said polypeptide or immunogenic fragment, and a vector or host cell comprising said nucleic acid molecule. The nucleic acid molecule can be either DNA or RNA, and in particular is mRNA.

[0014] In another embodiment, the mRNA further comprises a 5’-cap or cap analog, a 5-untranslated region (5’UTR), and / or a 3-untranslated region (3’UTR), and optionally intron (non-coding) sequences and / or a poly(A) sequence.

[0015] In another specific embodiment, the carrier is loaded with synthetic mRNA (molecules). “Carrier-loaded mRNA" refers to mRNA that has been encapsulated by or associated with a delivery system, or carrier, to protect it from degradation and facilitate its intracellular delivery into cells. The term synthetic mRNA refers to mRNA introduced into cells / carriers from an external source (also referred to as exogeneous RNA), rather than being naturally produced by the cell's own genetic material. It is particularly pointed out that the carrier according to the invention expressed the HORMAD1 polypeptide in an effective level that exceeds expression by promoter leakage.

[0016] In a further embodiment, the carrier, in particular DC or nanoparticle, of the invention may additionally comprise one or more mRNA molecules, in particular synthetic mRNA molecules, encoding an immunostimulatory polypeptide, such as a PRAME polypeptide; in particular a PRAME polypeptide characterized by any one of SEQ ID NOs: 8 -13, or a sequence having at least 85% sequence identity thereto.

[0017] The present invention further provides a composition comprising a carrier as defined herein, and further comprising one or more carriers comprising one or more mRNA molecules encoding a immunostimulatory polypeptide, such as a PRAME polypeptide; in particular a PRAME polypeptide characterized by any one of SEQ ID NOs: 8-13, or a sequence having at least 85% sequence identity thereto, including immunogenic fragments thereof.

[0018] The present invention also provides a combination comprising at least two different mRNA molecules, one encoding for a HORMAD1 polypeptide and one for a PRAME polypeptide, in particular wherein said HORMAD1 polypeptide is characterized by any one of SEQ ID NOs: 1 - 7, or a sequence having at least 85% sequence identity thereto, and wherein said PRAME polypeptide is characterized by any one of SEQ ID NOs: 8 - 13, or a sequence having at least 85% sequence identity thereto. The two or more mRNAs, in particular synthetic mRNAs, can be incorporated in or associated with the same or a separate carrier. The invention also provides a combination therapy of a carrier comprising mRNA molecules encoding a HORMAD1 polypeptide and a checkpoint inhibitor (such as a PD-1-, PD-L1-, PD-L2- or CTLA-4 inhibitor) and / or chemotherapeutic agent. Said combination therapy can further comprise other immunostimulatory polypeptides or carriers comprising mRNA molecules encoding immunostimulatory polypeptides, e.g. a combination of mRNA molecules encoding for a HORMAD1 polypeptide and a PRAME polypeptide.

[0019] In a particular embodiment, the combination as defined herein is used for the introduction of said mRNA molecules in a carrier, more particular in an antigen presenting cell or nanoparticle.

[0020] The invention further provides a pharmaceutical composition comprising the mRNA(s) of the invention, in particular a carrier-based mRNA formulation, the composition or the combination as defined herein, and at least one pharmaceutically acceptable excipient; in particular wherein said pharmaceutical composition is a vaccine.

[0021] The present invention also provides the mRNA(s) of the invention, in particular a carrier-based mRNA formulation, the composition, the combination, or the pharmaceutical composition as defined herein, for use in human and / or veterinary medicine.

[0022] The present invention further provides the mRNA(s) of the invention, in particular a carrier-based mRNA formulation, the composition, the combination, or the pharmaceutical composition as defined herein, for use in eliciting an immune response, in particular a T cell response, in a subject in need thereof.

[0023] The present invention further provides the mRNA(s) of the invention, in particular a carrier-based mRNA formulation, the composition, the combination, or the pharmaceutical composition as defined herein, for use in stabilizing tumor growth or inducing tumor regression in a subject in need thereof.

[0024] The present invention further provides the mRNA(s) of the invention, in particular a carrier-based mRNA formulation, the composition, the combination, or the pharmaceutical composition as defined herein, for use in therapy such as cancer therapy, in particular immunotherapy in a subject in need thereof, more in particular in a subject having cancer.

[0025] The present invention further provides the mRNA(s) of the invention, in particular a carrier-based mRNA formulation, the composition, the combination, or the pharmaceutical composition as defined herein, for use in the treatment of cancer, in particular wherein cancer cells express HORMAD1 and optionally PRAME; in particular wherein said cancer is lung cancer, more in particular NSCLC. In a further aspect, the present invention provides an ex vivo or in vitro method for improving the immunostimulatory characteristics of antigen presenting cells comprising the introduction of one or more mRNA molecules encoding one or more immunostimulatory polypeptides in said antigen presenting cell, characterized in that amongst the polypeptide at least HORMAD1 and optionally PRAME are expressed.

[0026] In a further embodiment, the present invention provides a method for preparing an immunotherapy agent comprising the steps of: a) obtaining a carrier; b) ex vivo modifying said carrier comprising the introduction / loading of a mRNA molecule encoding a HORMAD1 polypeptide, and c) harvesting the obtained carrier. In another further embodiment, the method further includes ex vivo modifying another or the same carrier by introduction of one or more mRNA molecules encoding another immunostimulatory polypeptide, such as a PRAME polypeptide. In a particular embodiment, the carrier is a nanoparticle or an antigen presenting cell.

[0027] The present invention also provides a method for the treatment of cancer, said method comprising the step of administering to a patient in need thereof, the mRNA(s) of the invention, in particular a carrierbased mRNA formulation, the composition, the combination, or the pharmaceutical composition as defined herein.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] With specific reference now to the figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention only. They are presented in the cause of providing what is believed to be the most useful and readily description of the principles and conceptual aspects of the invention. In this regard no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0030] Figure 1 (referred to as FIG. 1 ): In vitro HORMAD1 -specific tumor killing assay. Apoptotic tumor cell counts over time, measured from co-incubation of H-1650 NSCLC target cells together with T-cells primed by mRNA-DCs as indicated. Values represent a mean Caspase-3 / 7 reporter-positive object count of 4 different images / well. Shapiro-Wilk normality test was performed. Data were anlysed using ANOVA followed by Tukey’s multiple comparison with * p < 0.05 and ** p < 0.01 .

[0031] Figure 2 (referred to as FIG. 2): Therapeutic tumor control using a HORMAD1 mRNA-DC vaccine in a NSCLC tumor-bearing humanized mouse model. Immunodeficient NSG mice were intravenously injected with human peripheral blood lymphocytes (PBL) to engraft a human immune system. At the same day, mice were subcutaneously inoculated with human NSCLC H-1650 tumor cells. When palpable tumors were observed, mice were injected twice with indicated treatment condition. A. Graph shows the volumetric tumor size evolution for indicated treatment groups (6-8 mice / group). B. Representative images of the tumor size at day 21 after tumor inoculation are shown. Shapiro-Wilk normality test was performed. Data were analysed using ANOVA Kruskal-Wallis with Dunn’s multiple comparisons test was performed with * p < 0.05.

[0032] Figure 3 (referred to as FIG. 3): In vitro tumor killing assay using T cells primed with autologous DCs loaded with HORMAD1 mRNA and DCs loaded with PRAME mRNA. Apoptotic tumor cell counts over time, measured from co-incubation of H-1650 NSCLC target cells together with T-cells primed by mRNA-DCs as indicated. Apoptotic cells detected by a Caspase-cleavable fluorescent reporter as described in FIG 1. Values represent a mean Caspase-3 / 7 reporter-positive object count of 4 different images / well. Shapiro-Wilk normality test was performed. Data were anlysed using ANOVA followed by Tukey’s multiple comparison with * p < 0.05 and ** p < 0.01 .

[0033] Figure 4 (referred to as FIG. 4): Therapeutic tumor control using combination therapy of HORMAD1 mRNA-DC + PRAME mRNA-DC vaccine in a NSCLC tumor-bearing humanized mouse model. Similar experimental setup as described under FIG 2. Immunodeficient NSG mice were intravenously injected with human peripheral blood lymphocytes (PBL) to engraft a human immune system. At the same day, mice were subcutaneously inoculated with human NSCLC H-1650 tumor cells. When palpable tumors were observed, mice were vaccinated twice with saline (open rounds), Mock DC (open squares), DC loaded with an irrelevant control antigen (CTRL DC, closed rounds) or combined vaccination with DC loaded with mRNA encoding HORMAD1 + DC loaded with mRNA encoding PRAME (closed squares) as indicated by the arrows below the X-axis. Graph shows the volumetric tumor size evolution for indicated treatment groups (6-8 mice / group). Shapiro-Wilk normality test was performed. Data were analysed using ANOVA Kruskal-Wallis with Dunn’s multiple comparisons test was performed with * p < 0.05.

[0034] Figure 5 (referred to as FIG. 5): HORMAD1 -specific T-cell response in a patient with advanced NSCLC treated with HORMAD1-mRNA-loaded DCs, as measured using IFN-gamma ELISPOT. Peripheral blood was collected at different timepoints before (pre-vax), during and after vaccination (post-vax). Cells were subject to in vitro stimulation whereupon IFN-gamma production was determined and represented as spot forming units (SFU). Data are normalized against number of live responder lymphocytes seeded and are represented relative to assay background.

[0035] Figure 6 (referred to as FIG. 6): Flow-cytometry analysis of CD8 T-cell responses to HORMAD1 mRNA-DCs vaccination in a patient with advanced NSCLC. Peripheral blood was collected at different timepoints before (pre-vax), during and after vaccination (post-vax). Cells were restimulated in vitro whereupon IFN-gamma production was determined using flow cytometry. A. Representative flow cytometry plots. The numbers in the plots indicate the frequencies of IFN-y producing cells within the CD8+ T cell population. B. Summary of the IFN-gamma secretion by CD8+ T cells upon in vitro stimulation with relevant antigen. Data are represented relative to assay background.

[0036] Figure 7 (referred to as FIG. 7): Flow-cytometry analysis of CD4 T-cell responses to HORMAD1 mRNA-DCs vaccination in a patient with advanced NSCLC. Peripheral blood was collected at different timepoints before (pre-vax), during and after vaccination (post-vax). Cells were restimulated in vitro whereupon IFN-gamma production was determined using flow cytometry. A. Representative flow cytometry plots. The numbers in the plots indicate the frequencies of IFN-y producing cells within the CD4+ T cell population. B. Summary of the IFN-gamma secretion by CD4+ T cells upon in vitro stimulation with relevant antigen. Data are represented relative to assay background.

[0037] Figure 8 (referred to as FIG. 8): Expression of HORMAD1 in archival formalin-fixed, paraffin- embedded tumor sample of advanced NSCLC patient treated with HORMAD1-mRNA-DCs (same subject as FIG. 5, 6, 7) using A. qPCR with HORMAD1 -specific primers and B. RNA-NGS.

[0038] Figure 9 (referred to as FIG. 9): In vitro tumor killing assay using T cells primed against the HORMAD1 target of the invention by autologous HORMAD1 mRNA-LNP loaded DCs. Apoptotic tumor cell counts over time, measured from co-incubation of H-1650 NSCLC target cells together with T cells primed beforehand by autologous HORMAD1 mRNA-LNP loaded DCs. Apoptotic tumor cells were detected by a Caspase-cleavable fluorescent reporter. Values represent a mean Caspase-3 / 7 reporter-positive object count of 4 different images / well. Shapiro-Wilk normality test was performed. Data were analysed using unpaired t-test with * p < 0.05, ** p < 0.01 , *** p < 0.001 and **** p < 0.0001 . Figure 10 (referred to as FIG. 10): In vitro tumor killing assay using T cells primed against the HORMAD1 target of the invention by HORMAD1 mRNA loaded B cells. Apoptotic tumor cell counts over time, measured from co-incubation of H-1650 NSCLC target cells together with T cells primed beforehand by autologous HORMAD1 mRNA-loaded B cells. Apoptotic tumor cells were detected by a Caspase-cleavable fluorescent reporter. Values represent a mean Caspase-3 / 7 re porter- positive object count of 4 different images / well. Shapiro-Wilk normality test was performed. Data were analysed using unpaired t-test with * p < 0.05, ** p < 0.01 , *** p < 0.001 and **** p < 0.0001 .

[0039] Figure 11 (referred to as FIG.11): In vitro tumor killing assay using T cells primed against the HORMAD1 splice variant HORMAD1-204. Apoptotic tumor cell counts over time, measured from co- incubation of H-1650 NSCLC target cells together with T cells primed beforehand by autologous mRNA- loaded DCs. The mRNA encode the HORMAD1 splice variant 204. Apoptotic tumor cells were detected by a Caspase-cleavable fluorescent reporter. Values represent a mean Caspase-3 / 7 reporter-positive object count of 4 different images / well. Shapiro-Wilk normality test was performed. Data were analysed using unpaired t-test with * p < 0.05.

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise. The terms "comprising", "comprises" and "comprised of as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open- ended and do not exclude additional, non-recited members, elements or method steps. The term "about" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / -20% or less, preferably +1-10% or less, more preferably + / -5% or less, of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed. Whereas the terms "one or more" or "at least one", such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0042] All references, and teachings specifically referred to, cited in the present specification are hereby incorporated by reference in their entirety. Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0043] In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention.

[0044] The present invention identified HORMAD1 as a powerful target in cancer immunotherapy. More specific, the invention provides a HORMAD1 mRNA vaccine inducing effective T-cell mediated anti- tumoral effects.

[0045] The present invention is directed to a cancer vaccine aiming to direct an adaptive immune response towards cancer or tumor cells, in particular expressing one or more of the antigens of the invention. The aim is to achieve tumor elimination or at least tumor growth suppression and / or the inducement of immunological memory capable of protecting against relapse of said cancer. To achieve this, cancer vaccines need to induce a specific pattern of immunity which is a T-cell response comprising cytotoxic T-cells directed against epitopes exclusively displayed by cancer cells.

[0046] Accordingly, in a first aspect, the present invention provides a carrier comprising one or more nucleic acid molecules (such as DNA, cDNA or RNA), in particular mRNA, encoding a HORMAD1 polypeptide, or an immunogenic fragment thereof.

[0047] As used herein, the human HORMAD1 gene encodes for the HORMA domain-containing protein 1 , also named Cancer / testis antigen 46 (CT46). CT46 / HORMAD1 is a single-copy gene on chromosome 1q21 .3, encoding a putative protein of 394 aa. Conserved protein domain analysis identified a HORMAD domain involved in chromatin binding. CT46 / HORMAD1 is expressed in testis and in several cancers, such as lung cancer, breast cancer, esophageal cancer, endometrial cancer, bladder cancer, bone cancer, hematopoietic or lymphoid cancer, gastrointestinal cancer (including colon cancer), ovarian cancer, skin cancer, neuroblastoma, testicular cancer, thymoma, uterine carcinoma, melanoma, sarcoma, cervix cancer, head or neck cancer.

[0048] Alternatively spliced transcript variants encoding multiple isoforms of HORMAD1 have been observed for this gene, as given in the following Table 1 :

[0049] A splice variant can be selected based on the maximum number of potentially generated epitopes, e.g. translating into the longest amino acid sequence.

[0050] Particularly relevant for lung cancer are HORMAD1-202 (Q86X24-1) represented by SEQ ID NO:1 and HORMAD1-204 (Q86X24-4) represented by SEQ ID NO: 2.

[0051] With respect to non-small cell lung cancer, prevalence for each specific splice variant of HORMAD1 was analyzed using the TCGA (The Cancer Genome Atlas Program) public genomic expression database. Our analysis shows that the splice transcript resulting in the longest amino acid sequence (SEQ ID NO:1) is also the most prevalent in patients with NSCLC, and this holds for both dominant histological subtypes, i.e. squamous as well as as non-squamous lung cancer.

[0052] In a further embodiment, the HORMAD1 is HORMAD1-201 (Q86X24-2) represented by SEQ ID NO: 3, Q86X24-3 (represented by SEQ ID NO: 4), or Q86X24-5 (represented by SEQ ID NO: 5).

[0053] In a further embodiment, the HORMAD1 is HORMAD1-203 (X6R751) represented by SEQ ID NO: 6 and HORMAD1-205 (X6RG95) represented by SEQ ID NO: 7

[0054] In a particular embodiment, the invention provides a HORMAD1 polypeptide comprising or consisting of a sequence having at least 85%, at least 90%, or at least 95% sequence identity to any one of the HORMAD1 isoforms provided in Table 1 , in particular to any one of SEQ ID NO: 1 to SEQ ID NO: 7, or an immunogenic fragment thereof. The invention furthermore provides an isolated nucleic acid molecule encoding one or more of the polypeptides or fragments of the invention.

[0055] The term "nucleic acid encoding a polypeptide” encompasses a polynucleotide which includes only coding sequences for the polypeptide as well as a polynucleotide which includes additional coding and / or non-coding sequences. Polynucleotides can be in the form of RNA, in particular mRNA, or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand. The present invention further relates to a nucleic acid molecule, encoding the polypeptides or fragments according to the present invention. The term “nucleic acid” as used herein means a polymer of any length composed essentially of nucleotides, e.g., deoxyribonucleotides and / or ribonucleotides. The term “nucleic acid” further preferably encompasses DNA, RNA and DNA / RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g. chemically synthesized) DNA, RNA or DNA / RNA hybrids.

[0056] A nucleic acid can be naturally occurring, e.g., present in or isolated from nature, can be recombinant, i.e., produced by recombinant DNA technology, and / or can be, partly or entirely, chemically or biochemically synthesized. In some specific embodiments, the nucleic acids are synthetic or exogenous nucleic acid molecules meaning that they are synthetic or engineered molecules introduced into a living cell from outside the cell, rather than being naturally transcribed from the original cell. Exogenous molecules are typically used therapeutically or in vaccines to provide cells with a genetic "instruction manual" to produce specific proteins, which can prevent or treat diseases by replacing missing proteins, eliciting an immune response, or delivering therapeutic proteins. It is particularly pointed out that exogenous nucleic acids can express (poly)peptides in an effective level that exceeds expression by promoter leakage. Further, the “nucleic acid” can be double-stranded, partly double stranded, or singlestranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear. By “encoding” it is meant that a nucleic acid sequence or part(s) thereof corresponds, by virtue of the genetic code of an organism in question to a particular amino acid sequence, e.g., the amino acid sequence of one or more desired polypeptides.

[0057] The present invention further relates to an expression vector capable of expressing and / or expressing a nucleic acid molecule according to the present invention. The present invention further relates to a host cell comprising a nucleic acid molecule according to the present invention or an expression vector as described before. The present invention further relates to a method for producing a protein or peptide according to the present invention, said method comprising culturing the host cell according to the present invention, and isolating the peptide from said host cell or its culture medium.

[0058] In a specific embodiment, the nucleic acid molecule is RNA, in particular mRNA. More in particular, the RNA as taught herein may be capable of being expressed in a mammalian subject or cell, such as a human subject or cell. The term “mammal” includes any animal, such as, for example, mice, rats, hamsters, rabbits, dogs, cats, guinea pigs, gerbils, cattle, cows, sheep, horses, pigs and primates, e.g., monkeys and apes (e.g., chimpanzee, baboon, or monkey). Particularly preferred are human subjects. In one embodiment, the invention provides an “expression cassette”, comprising a nucleic acid construct capable of directing the transcription of an RNA or the expression of a gene / coding sequence of interest which is operably linked to a (gene) promoter. Expression cassettes are generally DNA constructs preferably including (5’ to 3’ in the direction of transcription): a (gene) promoter region, a polynucleotide sequence of interest with a transcription initiation region, and a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal; all these elements being operably or operatively linked meaning that all of these regions should be capable of operating (being expressed) in a cell, such as prokaryotic (e.g. bacterial) or eukaryotic (e.g. mammalian, yeast, insect, fungal, plant, algal) cells, when transformed into that cell. The promoter region comprising the transcription initiation region, which preferably includes the RNA polymerase binding site, and the polyadenylation signal may be native to the cell to be transformed, may be derived from an alternative source, or may be synthetic, as long as it is functional in the cell. Such expression cassettes can be constructed in e.g. a “vector” or “expression vector” (linear or circular nucleic acids, plasmids, cosmids, viral vectors (defective or infectious), phagemids, etc.). Said vectors may include a cloning or expression vector, as well as a delivery vehicle such as a viral, lentiviral or adenoviral vector. Expression vectors may comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems as described before. In particular, an expression vector as described herein may comprise a nucleic acid molecule as described herein operably linked to at least one regulatory sequence. Regulatory sequences are selected to direct the expression of the RNA in a suitable host cell, and include promoters, enhancers, and other expression control elements as known to the skilled person. Hence, in embodiments, the vector includes a promoter for driving expression of the nucleic acid of interest, optionally a nucleic acid sequence encoding a signal peptide that secretes the RNA, and optionally a nucleic acid sequence encoding a terminator. The construction of expression vectors for use in transfecting cells is also well known in the art, and thus can be accomplished via standard techniques (see, for example, Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clif ton, N.J.).

[0059] As used herein, the term “ribonucleic acid” or “RNA” or “mRNA” refers to a nucleic acid molecule which is a polymer of nucleotides, these nucleotides being usually adenosine monophosphate, uridinemonophosphate, guanosine monophosphate and cytidine monophosphate which are connected to each other along a backbone, formed by phosphodiester bonds between a sugar (ribose) of a first and a phosphate moiety of a second, adjacent monomer. Usually, messenger RNA may be obtainable by transcription of a DNA sequence, e.g. inside a cell. In eukaryotic cells, transcription is performed inside the nucleus and results in a premature RNA which is then processed in a messenger RNA (abbreviated as mRNA). Processing of premature RNA generally comprises a variety of different posttranscriptional modifications such as splicing, 5’-capping, polyadenylation, export from the nucleus or the mitochondria. Accordingly, a mature mRNA typically comprises a 5’-cap, a 5- untranslated region (UTR), an open reading frame (ORF), a 3’ UTR, and a poly(A) sequence.

[0060] The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application may recite “T”s in a representative RNA or mRNA sequence but understands that “T”s would be substituted for “U”s. Further, any of the RNA or mRNA sequences disclosed herein and identified by a particular sequence identification number, is also intended to disclose its corresponding DNA sequence complementary to the RNA, where each “U” of the RNA sequence is substituted with “T”.

[0061] As used herein, the term “RNA” or “mRNA”, in reference to the (immunogenic) compositions or vaccines of the present disclosure, preferably refers to a synthetic or exogenous RNA, including chemically modified RNA molecules, in particular for stabilization of the RNA molecule, typically to render them more stable to disintegration or degradation, the latter a consequence of e.g. RNA-triggered innate cellular immune response, or environmental factors such as extracellular enzymatic digest. For example, the RNA is a “nucleoside-modified” nucleic acid comprising at least one modified nucleoside that are capable of being translated by translational machinery in a cell. For example, an mRNA may be modified by replacement of some or all of the uridines with pseudouridine, 1 -methyl pseudouridine or other modified uridine. In other embodiments, the mRNA may include some or all cytidines replaced by methylated cytidines. Hence the RNA provided herein may contain from 0% to 100% of modified nucleotides, including any intervening percentage. The RNA molecule for use in the immunogenic composition should direct the expression of the (HORMAD1) polypeptide and generate an immune response against the antigen or epitope(s) therein. In some embodiments, the mRNA molecules as used in the carrier described herein are exogenous mRNA molecules which needs to be understood that the mRNA molecules express the polypeptides such as HORMAD1 , and optionally PRAME, in an effective level that exceeds expression by promoter leakage or other background low-level transcription noise.

[0062] In one embodiment, the amino acid sequence of the antigen or fragment described herein is encoded by a coding sequence which is codon-optimized and / or the G / C content of which is increased compared to wild type coding sequence. This also includes embodiments, wherein one or more sequence regions of the coding sequence are codon-optimized and / or increased in the G / C content compared to the corresponding sequence regions of the wild type coding sequence. In one embodiment, the codon optimization and / or the increase in the G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0063] The term "codon-optimized" refers to the alteration of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present invention, coding regions are preferably codon-optimized for optimal expression in a subject to be treated using the RNA molecules described herein. Codon-optimization is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, the sequence of RNA may be modified such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons". The mRNA can be modified to optimize translational efficiency and presentation on MHC class I and class II molecules.

[0064] In some embodiments of the invention, the guanosine / cytosine (G / C) content of the coding region of the RNA described herein is increased compared to the G / C content of the corresponding coding sequence of the wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence encoded by the wild type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that mRNA. Sequences having an increased G (guanosine)ZC (cytosine) content are more stable than sequences having an increased A (adenosine)ZU (uracil) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favourable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by the RNA, there are various possibilities for modification of the RNA sequence, compared to its wild type sequence. In particular, codons which contain A andZor U nucleotides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A andZor U or contain a lower content of A andZor U nucleotides. For example, the GZC content of the coding region of the RNA described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the GZC content of the coding region of the wild type RNA.

[0065] The term “antigen” or “immunostimulatory polypeptide” as used herein refers to a substance such as a polypeptide or the like, which elicits an immune response, e.g., elicits an immune response when present in a subject (for example, when present in a human or mammalian subject). The instant invention is based at least in part on the understanding that mRNA-encoded antigens, when expressed from mRNA administered to a cell or subject, can cause the immune system to produce an immune response to the expressed antigen, for example can trigger the production of antibodies against the expressed antigen, e.g., binding andZor neutralizing antibodies, can trigger B and or T cell responses specific to the expressed antigen, and ultimately can cause a therapeutic andZor a protective (prophylactic) response against (subsequent) encounter with the antigen. As used herein the term "immunogenic fragment" refers to a fragment of a protein that is immunogenic, i.e., capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor. Preferably, an immunogenic fragment of the present invention is immunodominant for antibody andZor T cell receptor recognition. In a particular embodiment, an immunogenic fragment as referred to herein is a fragment of the antigen provided herein that retains at least 50%, 60%, 70%, 80%, or 90% of the immunogenicity of the full-length protein. Fragments can be as small as 8 amino acids or at the other extreme, be large fragments that are missing as little as a single amino acid from the full-length protein as provided herein. In a particular embodiment the fragment comprises 8 to all minus one of the amino acid residues of the full-length protein. In other embodiments, the fragment comprises or consists of 8 to 500, 8 to 400, 8 to 300, 8 to 250, 8 to 200, 8 to 150, 8 to 100 or 8 to 50 amino acid residues. Such fragments will include at least one epitope (or antigenic determinant) of the native polypeptide. In one embodiment, they have a length of at least 8 amino acids, preferably at least 9, 10, 11 , 12, 13, 14, 15, 20 or 50 amino acids. In some embodiments, fragments can comprise at least 8 amino acids, at least 10 amino acids, at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, of a polypeptide sequence disclosed herein.

[0066] The vaccine technology described herein features nucleic acids, particularly messenger RNA (mRNA) designed to encode an antigen of interest. The compositions of the present disclosure comprise a (at least one) mRNA having an open reading frame (ORF) encoding a HORMAD1 antigen. An open reading frame (ORF) is a continuous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG or AUG)) and ending with a stop codon (e.g., TAA, TAG orTGA, or UAA, UAG or UGA). An ORF typically encodes a protein. It will be understood that the sequences disclosed herein may further comprise additional elements, e.g., 5' and 3' UTRs, but that those elements, unlike the ORF, need not necessarily be present in an mRNA of the present disclosure. The mRNAs provided herein may include any 5' untranslated region (UTR) and / or any 3' UTR. A variety of 5'UTR and 3'UTR sequences are known and available in the art. As known by the skilled person, 5'UTRs that are heterologous or synthetic may be used with any desired 3' UTR sequence. For example, a heterologous 5'UTR may be used with a synthetic 3'UTR with a heterologous 3’ UTR. Exemplary UTR sequences are UTRs derived from alphaglobins or beta-globins, amino-terminal enhancer of split (AES) or mitochondrially encoded 12S rRNA (mtRNRI), however, other UTR sequences may be used as can be determined by the skilled person.

[0067] In specific embodiments, the RNA molecule is a mRNA comprising at least, from 5’ to 3’,

[0068] (i) a 5’-cap or cap analog,

[0069] (ii) a 5-untranslated region (UTR),

[0070] (iii) an open reading frame (ORF) including coding sequences as described herein (e.g. sequences encoding for SEQ ID NOs 1-7) and, optionally intron (non-coding) sequences,

[0071] (iv) a 3’ UTR, and

[0072] (v) optionally, a poly(A) sequence.

[0073] A poly(A) tail or sequence may contain 10 to 300 adenosine monophosphates. More specifically, the poly(A) tail comprises at least 20 adenosine monophosphates. The poly(A) tail can contain chemical modifications to enhance translational efficiency, or can contain segments wherein at least two adenosines are separated by a spacer element of a different length.

[0074] In a further embodiment, the invention provides an nucleic acid, in particular mRNA, that comprises or consists of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the nucleotide sequence of the HORMAD1 transcripts ENST00000361824.7, ENST00000368995.8, ENST00000322343.11 , ENST00000368987.5, or ENST00000442853.5; or encoding for SEQ ID NO: 1-7. In particular, the invention provides a nucleic acid that comprises or consists of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the nucleotide sequence of 14 or 16 (HORMAD1-202 Q86X24-1), or SEQ ID NO: 40 or 41 (HORMAD1-204 Q86X24-4). SEQ ID NOs

[0075] 16 and 41 represent a full mRNA construct including signal peptide, MITD, UTRs and a poly A tail.

[0076] As mentioned before, the skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application may recite “T”s in a representative RNA or mRNA sequence but understands that “T”s would be substituted for “U”s. Further, any of the RNA or mRNA sequences disclosed herein and identified by a particular sequence identification number herein, is also intended to disclose its corresponding DNA sequence complementary to the RNA, where each “U” of the RNA sequence is substituted with “T

[0077] In a further embodiment, the RNA molecule is flanked by 5’ and 3’ untranslated regions (UTRs) designed to optimize protein translation, in addition to a poly-A tail of specific length and possibly containing alterations in nucleotide content in order to optimize RNA stability. In still a further embodiment, the antigen-encoding RNA can be flanked by sequences designed to increase presentation of peptides on MHC class I and class II molecules, with the aim of stimulating both CD8 as well as CD4 / T-helper responses to the RNA-encoded antigen. These sequences include on the 5’ side a signal peptide sequence, which directs protein translation into the lumen of the endoplasmic reticulum from which the protein has access to endolysosomal compartments. This can be further combined by trafficking sequences on the 3’ side of the RNA with the aim of directing vesicles containing newly translated protein to the lysosomal compartments. These sequences include the cytoplasmic trafficking domain of lysosomal-associated proteins such as LAMP1 , DC-LAMP, MHC class I intracellular trafficking domain (MITD), or other trafficking domains from proteins that localize to the (endo)lysosomal compartments.

[0078] In one embodiment of the present invention, the amino acid sequence of the polypeptide of the invention (e.g. HORMAD1 or PRAME) has at least 85% sequence identity (thus including at least 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity), preferably at least 95% sequence identity, more preferably at least 99%, most preferably 100% sequence identity with the amino acid sequence as provided herein or as represented by the respective accession numbers (Tables 1 & 2). The term “sequence identity” as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base or the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, “sequence identity” will be understood to mean the “match percentage” calculated by an appropriate method. The percentage identity of nucleic acid and polypeptide sequences can be calculated using commercially available algorithms, which compare a reference sequence with a query sequence. The following programs (provided by the National Center for Biotechnology Information) may be used to determine homologies / identities: BLAST, gapped BLAST, BLASTN and PSI BLAST, which may be used with default parameters.

[0079] Thus, 100% identity to SEQ ID Nos provided herein is not necessary, meaning that one or more amino acid modifications are possible. For example, the polypeptide may have up to 30 amino acids removed from the N- or C-terminal ends of the protein, e.g. up to about 1 , 5, 10, 15, 20, or 30 amino acids removed. As noted elsewhere in the present disclosure, 85% sequence identity is likely to be sufficient to provide suitable level of the required immune response. As used herein, the term "amino acid modification" refers to an amino acid addition, amino acid deletion, and / or to an amino acid substitution as compared to the reference sequence. Preferably, said one or more amino acid substitution is a ‘conservative’ amino acid substitution, i.e. the substitution of an amino acid by another amino acid of the same class, in which the classes are as follows:

[0080] Class Amino acid examples

[0081] Nonpolar Ala, Vai, Leu, Pro, Met, Phe, Trp, He

[0082] Uncharged polar Gly, Ser, Thr, Cys, Tyr, Asn, Gin

[0083] Acidic Asp, Gly

[0084] Basic Lys, Arg, His

[0085] As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of the HORMAD1 or PRAME antigens of interest. For example, provided herein is any protein fragment (meaning a polypeptide sequence at least one amino acid residue shorter than a reference antigen sequence but otherwise identical) of a reference protein, provided that the fragment is immunogenic and confers an immune response. Antigens / antigenic polypeptides can range in length from about 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids to full length proteins.

[0086] Diverse approaches in delivering nucleic acid-based vaccines can be employed in the context of the present invention, including carrier-based delivery and directly injectable DNA / RNA. The DNA / RNA can be RNA is formulated as a liquid or as a solid.

[0087] In a particular embodiment of the present invention, the nucleic acid molecule may be delivered by a physical delivery system known in the art for the delivery of nucleic acids. Such physical delivery systems include electroporation, ultrasound, photoporation, gene gun, microneedles, and pressurebased systems.

[0088] Diverse approaches in delivering mRNA-based vaccines can be employed in the context of the present invention, including carrier-based delivery and directly injectable mRNA. In a further embodiment, the nucleic acid molecule as taught herein may be formulated with other macromolecules such as a carrier to improve cellular uptake. As used herein, a “carrier” includes a delivery vehicle encapsulating the nucleic acid, or a vehicle associated / complexed with the nucleic acid, such as liposomes, (lipid or polymeric) micro- or nanoparticles, exosomes, cationic nano-emulsions, cationic peptides or cationic polymers, and antigen presenting cells. Lipid nanoparticles typically comprise a cationic lipid, optionally an ionizable cationic lipid, a neutral lipid, a sterol, and / or a polyethylene glycol (PEG)-modified lipid. Other suitable delivery vehicles are plasmids or a viral based system (e.g., an adenovirus system, an adeno associated virus (AAV) vector, a poxvirus, or a lentivirus).

[0089] In a particular embodiment of the invention, the carrier is an antigen presenting cell (APC), more specific an APC selected from the group consisting of: a dendritic cell (DC) or a B-cell, a dendritic cell-line, a 13- cell line, a macrophage, and a leukocyte with antigen-presenting properties. In particular, the cells are isolated from or generated from the blood of a subject. The cells or cell lines can be autologous or allogenic. In a preferred embodiment, the APC is a DC, or a B-cell. Among professional antigen- presenting cells, DCs feature the strongest immunogenic power. DCs are a heterogenous family comprising different subsets of “conventional DCs” or eDCs (cDC1 , cDC2, cDC3), plasmacytoid DCs (PDCs) and monocyte-derived DCs (moDCs). Hence in specific embodiments, the DC may be resp. a eDC, a plasmacytoid DC or a monocyte DC. In a specific embodiment, the carrier is DC, B-cell or a nanoparticle, more particularly a lipid nanoparticle.

[0090] In particular, the combination as defined herein is used for the introduction of said mRNA molecules in an antigen presenting cell.

[0091] Antigen-presenting cells (APCs) are a crucial component of the immune system that play a key role in initiating and regulating immune responses. These cells are responsible for capturing, processing, and presenting antigens to other immune cells, particularly T cells. There are several types of antigen- presenting cells, with the most notable ones being dendritic cells, macrophages, and B cells. These cells may carry out any of the following functions:

[0092] Antigen capture: APCs capture antigens from their surrounding environment. Dendritic cells, for example, are specialized in capturing antigens at sites of infection or inflammation.

[0093] Antigen processing: Once captured, APCs process antigens by breaking them down into smaller fragments. This process typically occurs within cellular compartments called lysosomes.

[0094] Antigen Presentation: processed antigen fragments are then displayed on the surface of the APC, bound to molecules known as major histocompatibility complex (MHC) molecules. MHC molecules with attached antigens serve as a signal for T cells, enabling them to recognize and respond to the specific antigen.

[0095] Activation of T-cells: the interaction between the antigen-MHC complex on the APC and the T cell receptor on a T cell is a crucial step in activating T cells. This interaction helps initiate adaptive immune responses, leading to the elimination of pathogens or abnormal cells.

[0096] CD8 or “cytotoxic” T-cells can directly engage and eliminate cancer cells or virus-infected cells. CD4 or “helper” T-cells can boost this CD8 response, and also stimulate antibody production from B-cells. For these reasons, DCs represent an attractive target for transfection by mRNA vaccines, both in vivo and ex vivo. A dendritic cell as used herein, is a type of immune cell that plays a crucial role in the initiation and regulation of immune responses. These cells are named for their distinctive branch-like projections called dendrites, which extend from their cell bodies. The main function of dendritic cells is to capture, process, and present antigens to other immune cells, particularly T cells. Dendritic cells serve as a bridge between the innate and adaptive immune systems, playing a key role in the activation of T cells and the coordination of immune responses. They are crucial for the recognition of foreign substances and the development of an effective immune defense. Antigens are molecules that can stimulate an immune response. Dendritic cells act as antigen-presenting cells (APCs) by taking up antigens, digesting them into smaller fragments, and then presenting these fragments on their cell surfaces, while providing high levels of T-cell costimulatory signals. The high cell surface expression of T-cell costimulatory receptors is a key distinctive feature of DCs and instrumental to the DC’s capacity to overcome immunological tolerance. The antigen presentation by DCs is essential for activating and directing the immune system's responses against pathogens, such as bacteria, viruses, and other foreign substances, as well as against cancer cells. In a particular embodiment, isolated DCs are loaded with one or more (synthetic) mRNA molecules encoding one or more HORMAD1 polypeptides as provided herein. The term “isolated” is used to indicate that a cell, peptide or nucleic acid is separated from its native environment. Isolated cells, polypeptides and nucleic acids may be substantially pure, i.e. essentially free of other substances with which they may bound in nature. ‘Isolated’ is not limited to the cells as directly separated from their native environment, but may also refer to cultured cells, as well as cells obtained from precursor cells.

[0097] Combinations

[0098] In a second aspect of the invention, it was demonstrated that it can be of particular advantage to combine mRNA encoded HORMAD1 with other mRNA encoded antigens, in particular antigens and / or antigens expressed in certain cancer types (tumor antigens).

[0099] An example of a preferred polypeptide is a PRAME polypeptide; in particular a PRAME polypeptide characterized by SEQ ID NOs: 8-13, or a sequence having at least 85% sequence identity thereto.

[0100] PRAME, which stands for "Preferentially Expressed Antigen in Melanoma," is a tumor antigen associated with various types of cancers. Tumor antigens are substances expressed on the surface of cancer cells that can be recognized by the immune system. PRAME was initially identified in melanoma, a type of skin cancer, but it has since been found to be expressed in other cancer types as well. PRAME is a member of the cancer / testis antigen family, which means that its expression is typically restricted to cancer cells and normal cells in the testis. It is generally not expressed in normal tissues outside the testis, making it a potentially attractive target for cancer immunotherapy.

[0101] Alternatively spliced transcript variants encoding multiple isoforms of PRAME have been observed for this gene, as given in the following Table 2.

[0102] A splice variant can be selected based on the maximum number of potentially generated epitopes, e.g. translating into the longest amino acid sequence.

[0103] Particularly relevant for lung cancer are PRAME-201 (A0A024R1 E6, P78395); PRAME-202 (A0A024R1 E6, P78395); PRAME-203 (A0A024R1 E6, P78395); PRAME-205 (A0A024R1 E6, P78395) and PRAME-214 (A0A024R1 E6, P78395) represented by SEQ ID NO: 8. The mRNA sequence corresponding to PRAME-201 (A0A024R1 E6) is represented by SEQ ID NO 15.

[0104] In a further embodiment, the invention provides an mRNA that comprises or consists of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 15 or 17. SEQ ID NO 17 represent a full mRNA construct including signal peptide, MITD, UTRs and a poly A tail.

[0105] With respect to non-small cell lung cancer, prevalence for each specific splice variant of PRAME was analyzed using the TOGA public genomic expression database. Our analysis shows that the splice transcript resulting in the longest amino acid sequence (SEQ ID NO: 8) are also the most prevalent in patients with NSCLC, and this holds for both dominant histological subtypes, i.e. squamous as well as non-squamous lung cancer.

[0106] In a further embodiment, the PRAME polypeptide is PRAME-209 (E7EW99) represented by SEQ ID NO: 9, PRAME-206 (B5MCY4) represented by SEQ ID NO: 10, PRAME-207 (E7EMH2) represented by SEQ ID NO:11 , PRAME204 (B5MD04) represented by SEQ ID NO:12 or PRAME-208 (H7C2P3) represented by SEQ ID NO: 13.

[0107] Where a combination of HORMAD1 and other antigens, e.g. PRAME, is used, the invention provides that these are either used as a combination of polypeptides or (synthetic) mRNA molecules encoding these antigens, either as one naked mRNA or introduced in the same carrier, e.g. DC, B-cells or LNPs, or a combination of mRNA molecules encoding these antigens, yet introduced as separate naked mRNAs or in different carriers, e.g. DCs, B-cells or LNPs. The antigens or mRNAs may then be combined and administered as a single vaccine composition (e.g., comprising multiple RNA encoding multiple antigens) or may be administered separately.

[0108] Accordingly, the present invention also provides a kit, combination or composition comprising a carrier comprising an mRNA encoding for a HORMAD1 polypeptide, and one or more carriers each independently comprising a mRNA molecule encoding a (immunostimulatory) polypeptide or antigen, in particular a PRAME polypeptide as provided herein. In said embodiment, the carrier is as provided herein such as a nanoparticle or other delivery means, in particular an antigen presenting cell, more in particular a dendritic cell, a B-cell, or a nanoparticle such as an LNP.

[0109] Additionally, the invention provides a combination comprising a HORMAD1 polypeptide and a PRAME polypeptide, in particular an mRNA molecules encoding for a HORMAD1 polypeptide and an mRNA molecule encoding for a PRAME polypeptide, more in particular wherein said HORMAD1 polypeptide is characterized by SEQ ID NO: 1-7, or a sequence having at least 85% sequence identity thereto, and wherein said PRAME polypeptide is characterized by SEQ ID NO: 8-13, or a sequence having at least 85% sequence identity thereto.

[0110] In a further embodiment, the HORMAD1 polypeptide and combinations of the present invention can induce an effective immune response which can be further strengthened by combination of the vaccine platform (mRNA loaded carrier as provided herein) with state-of-the art immunotherapies (e.g. checkpoint inhibitors) or chemotherapy. In a specific embodiment, the HORMAD1 mRNA vaccine is combined with a second therapeutic or agent such as one or more of a chemotherapeutic or an immunotherapeutic agent. A specific immunotherapeutic agent for use in the combination therapies disclosed herein includes a so called “checkpoint inhibitor”. During the last few years, in addition to therapy concepts based on oncolytic viruses, the field of immuno-oncology has become a valuable approach in the fight against cancer. One of the most recent promising approaches to activate therapeutic antitumor immunity is the blockade of immune checkpoints. Immune checkpoints refer to a plethora of inhibitory pathways hardwired into the immune system that are crucial for maintaining selftolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues in order to minimize collateral tissue damage. It is now clear that tumors co-opt certain immune- checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumor antigens. Because many of the immune checkpoints are initiated by ligand-receptor interactions, they can be readily blocked by antibodies or modulated by recombinant forms of ligands or receptors. An important immune checkpoint receptor as defined herein is cytotoxic T-lymphocyte- associated antigen 4 (CTLA4; also known as CD152), which down modulates the amplitude of T cell activation. The approved anti-CTLA4 antibody is known under the name "ipilimumab" (BMS) and tremelimumab (AstraZeneca).

[0111] Another important immune-checkpoint receptor as defined herein is programmed cell death protein 1 (PD1), and limits T cell effector functions within tissues. The humanized monoclonal antibody pembrolizumab (also known as MK-3575 (MSD)) is directed against the target PD-1. Further anti-PD1 antibodies are nivolumab (BMS) and cemiplimab (Regeneron). In addition, anti-PD-L1 monoclonal antibodies are used as immune checkpoint inhibitors. These include atezolizumab (Roche), durvalumab (AstraZeneca) and avelumab (Merck KGaA).

[0112] Hence, the invention also provides a combination therapy of (i) a carrier comprising one or more mRNA molecules encoding a HORMAD1 polypeptide; or (ii) one or more mRNA molecules encoding for a HORMAD1 polypeptide and one or more PRAME polypeptide; together with a checkpoint inhibitor (such as but not exclusively a PD-1-, PD-L1-, PD-L2- or CTLA-4 inhibitor) and / or chemotherapeutic agent.

[0113] In a particular embodiment, the checkpoint inhibitor is a CTLA-4 inhibitor or antagonist, specifically binding CTLA-4. In a further embodiment, the checkpoint inhibitor is a PD-1 and CTLA-4 bispecific molecule. Such bispecific molecules are capable of specifically binding to PD-1 and CTLA-4 molecules that are present on the surfaces of exhausted and tolerant tumor-infiltrating lymphocytes and other cell types. In a particular embodiment, the checkpoint inhibitor is a programmed cell death protein 1 (PD-1) inhibitor, or a programmed death ligand (PD-L1) or (PD-L2) inhibitor. The term “inhibitor” or “antagonist” refers to any chemical compound or biological molecule that impairs the ability of such cell-surface molecules to respond to their respective ligand, e.g. a compound or molecule that blocks binding of PD- L1 expressed on a cancer cell to PD-1 expressed on an immune cell (T-cell, B-cell, or NKT-cell) and / or blocks binding of PD-L2 expressed on a cancer cell to the immune-cell expressed PD-1 . Alternative names or synonyms for PD-1 and its ligands include: PDCD1 , PD1 , CD279, and SLEB2 for PD-1 ; PDCD1 L1 , PDL1 , B7H1 , B7-4, CD274, and B7-H for PD-L1 ; and PDCD1 L2, PDL2, B7-DC, Btdc, and CD273 for PD-L2. In one embodiment, the PD-1 inhibitor blocks binding of human PD-L1 to human PD- 1 , and preferably blocks binding of both human PD-L1 and PD-L2 to human PD-1. Human PD-1 amino acid sequences can be found in NCBI Locus No. : NP005009. Human PD-L1 and PD-L2 amino acid sequences can be found in NCBI Locus No.: NP054862 and NP079515, respectively.

[0114] In one embodiment, said inhibitor includes antibodies and antigen-binding fragments thereof. In the alternative, PD-1 or PD-L1 (2) binding moieties or antagonists can be used which include a variety of different types of molecules including those that specifically bind resp. PD-1 or PD-L1 (2). Such ligands include small molecules, polypeptides (e.g. a fusion protein) or nucleic acids (aptamers, siRNA, shRNA, etc), and the like. In a further embodiment, the checkpoint inhibitor is a bispecific molecule containing an anti-PD1 or anti-PD-L1 moiety and an anti-VEGF moiety, e.g. ivonescimab (AK112, Summit / Akeso / Pfizer) and pumitamig (BioNTech / BMS).

[0115] Examples of chemotherapeutic agents are cisplatin, carboplatin, pemetrexed, vinorelbine, gemcitabine, paclitaxel (Taxol), and docetaxel (Taxotere).

[0116] Immunogenic compositions

[0117] In another aspect, the present invention provides a pharmaceutical composition, e.g. an immunogenic composition, or kit containing an RNA molecule as described herein, and one or more pharmaceutically acceptable excipients. In one embodiment, the pharmaceutical composition is a vaccine, more in particular a mRNA vaccine, even more particular a carrier-based mRNA vaccine such as a dendritic cell vaccine, a B-cell vaccine, or nanoparticle loaded mRNA vaccine, as provided herein. It may be used in medicine, more specific in cancer immunotherapy.

[0118] A dendritic cell vaccine is a type of cancer vaccine that utilizes dendritic cells, a specialized type of immune cell, to stimulate an immune response against cancer cells. In the context of a dendritic cell vaccine for cancer and as known to the skilled person, dendritic cells are provided as an autologous or allogeneic cell product, manipulated ex vivo, and then reintroduced into the patient to enhance the immune system's ability to recognize and attack cancer cells. The process typically involves the following steps:

[0119] - Provision of dendritic cells: autologous dendritic cells are often generated in the laboratory from precursor cells such as a (human) peripheral blood mononuclear cell, a monocyte, or another myeloid progenitor cell obtained from the patient's blood. Alternatively differentiated DCs can be isolated directly from patient’s blood or, in some cases, from tissues. Allogeneic dendritic cells typically consist of a transformed leukocytic cell line with antigen-presenting capacity.

[0120] - Loading dendritic cells with antigens: the isolated dendritic cells are then loaded with tumor-specific antigens, such as the antigens as provided herein. These antigens can be derived from the patient's own cancer cells or synthesized versions of tumor-associated antigens in different formats such as mRNA molecules, protein or peptides. In the alternative, the dendritic cells are loaded with the antigen after maturation.

[0121] - Maturation of dendritic cells: the dendritic cells are matured or activated in the laboratory to enhance their ability to stimulate an immune response. This step is important for ensuring that the dendritic cells effectively present the tumor antigens to the T-cells of the immune system while providing strong costimulatory signals to overcome immune tolerance.

[0122] - Vaccination: the matured dendritic cells, loaded with tumor antigens, are reintroduced into the patient through injection. Once inside the body, these dendritic cells present the tumor antigens to T cells, initiating an immune response specifically targeted against the cancer cells.

[0123] Dendritic cell vaccines are being explored as a personalized and targeted approach to cancer immunotherapy. By using the patient's own cells and tailoring the vaccine to the individual's cancer profile, researchers aim to enhance the specificity and effectiveness of the immune response against the cancer. While dendritic cell vaccines are still an area of active research, some promising results have been observed in clinical trials for certain types of cancer.

[0124] The invention provides DC-based vaccination, but also provides that the mRNA molecules as defined herein are directly used in vivo, i.e. introduced into the patient. Accordingly, the invention provides a pharmaceutical composition comprising the mRNA encoding the polypeptides as specified herein optionally incorporated in a carrier, such as a DC, a B-cell, or nanoparticle, or the composition or the combination as defined herein, and at least one pharmaceutically acceptable excipient. In specific embodiments, the composition comprises suitable delivery vehicles for in vivo delivery. In particular, because mRNA is large and negatively charged, it cannot pass through the anionic lipid bilayer of cell membrane. Moreover, inside the body, it is engulfed by cells of the innate immune system and degraded by nucleases. In vivo delivery requires vehicles that can transfect cells, without causing toxicity or unwanted immunogenicity.

[0125] As mentioned before, there are two basic approaches for the delivery of mRNA molecules that have been described to date and well known to the skilled person. Both approaches can be used in the present invention. In one embodiment, the mRNA is loaded ex vivo into an antigen presenting cell, such as dendritic cells (DCs), B cell or macrophages, followed by administering the transfected cells to a subject.

[0126] Another approach encompasses direct parenteral injection of mRNA with or without a carrier / delivery system. The carrier can be a nanoparticle such as a liposome, a lipid nanoparticle, a (biodegradable) polymeric nanoparticle, polyplexes or viral-based delivery systems, wherein the mRNA molecule is encapsulated, entrapped or complexed.

[0127] Pharmaceutically acceptable excipients must have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a person to be treated. Some examples of compounds which can be used as pharmaceutically acceptable excipients are sugars, such as, for example, lactose, glucose, trehalose and sucrose; starches, such as, for example, corn starch or potato starch; dextrose; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, groundnut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from theobroma; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences.

[0128] The choice of a pharmaceutically acceptable excipient is determined, in principle, by the manner in which the composition or vaccine is administered. Compositions / vaccines are preferably formulated in liquid or solid form. For example, the compositions suitable for parenteral administration may comprise the active ingredient combined with a pharmaceutically active carrier, such as sterile water, or sterile isotonic saline. It may also be prepared as injectable formulations, packaged in unit dosage form, such as in ampules or in multi-dose containers. It may also be provided in dry form (powder or granular) for reconstitution with a suitable vehicle prior to parenteral administration. Pharmaceutical compositions may be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical agents, such as vaccine compositions, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety). Uses

[0129] The present invention provides the antigen, the nucleic acid molecule, in particular mRNA(s), vector or host cell of the invention, preferably in combination with a carrier (incorporated in or associated with) as provided herein, as well as the composition, the combination, or the pharmaceutical composition as defined herein, for use as a medicament, in particular for use in human and / or veterinary medicine. In addition, the invention provides the use of the antigen, nucleic acid molecule, vector or host cell of the present disclosure for the manufacture of a medicament for treating cancer. The aim is to invoke an immune response against cancer cells expressing one or more tumor antigens of the invention, and to treat a cancer disease involving cells expressing one or more tumor antigens as provided herein.

[0130] The present invention further provides the mRNA(s) of the invention, preferably in combination with a carrier as provided herein, the composition, the combination, or the pharmaceutical composition as defined herein, for use in eliciting an immune response, in particular a T cell response, in a subject in need thereof. A subject may be any mammal, including non-human primate and human subjects. Typically, a subject is a human subject.

[0131] In the context of the invention, “eliciting an immune response” refers to the process of activating the immune system in response to the presence of a foreign substance, such as a pathogen or an antigen. The immune system is a complex network of cells, tissues, and organs that work together to defend the body against infections and other harmful entities. When a foreign substance enters the body, various components of the immune system are mobilized to recognize, neutralize, and eliminate the threat. The immune response can be broadly categorized into two types: innate and adaptive.

[0132] Innate immune response: this is the immediate, non-specific defense mechanism that provides the first line of defense against pathogens. It includes physical barriers like the skin, as well as cellular and molecular components such as white blood cells and proteins that recognize and attack a wide range of pathogens.

[0133] Adaptive immune response: this is a more specific and targeted response that develops over time. It involves the activation of lymphocytes (B cells and T cells) that are capable of recognizing and remembering specific antigens. The adaptive immune response provides long-lasting protection against specific pathogens.

[0134] In particular, a sufficient cancer vaccine requires induction of a type 1 polarized effector T-cell response. The immune response is controlled by the cytokines produced by T-helper cells (Th) type 1 or 2. Th1 cells produce IL-2 and IFN-y, which are crucial for inducing anti-tumor cytotoxic T cells (CTLs), which express CD8. The CD8+ population also produces IFN-y. The stimulation of T lymphocytes can be investigated by detecting their cytokine (IFN-y, IL-2 and TNF-a) production. In the present examples, enzyme-linked immune absorbent spot (ELISpot) and flow cytometry was performed to assess the activation of the T cells based on different parameters. For the vaccine to elicit an antitumoral response, the antigen needs to be expressed in the tumor. It was shown for the first time herein that the HORMAD1 mRNA-loaded carriers induced antigen-specific cytotoxic T cells (CD8+), in vitro as well as in vivo. Thus, in a particular embodiment, the present invention provides a pharmaceutical composition or cancer vaccine comprising a mRNA molecule encoding for HORMAD1 , or comprising a nucleic acid molecule encoding the amino acid sequence as set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 7 or a sequence having at least 85%, at least 90% or at least 95% sequence identity thereto, including immunogenic fragments thereof, and optionally another immunostimulatory polypeptide, particularly incorporated or associated with a carrier such as a DC for use to induce a cytotoxic T lymphocyte (CD8+) (Tc) response against the malignant cells. In another embodiment, the present invention provides a pharmaceutical composition or cancer vaccine further comprising one or more mRNA molecules encoding for PRAME (cf. Table 2), or comprising one or more nucleic acid molecules encoding the amino acid sequences as set forth in any one of SEQ ID NO: 8 to SEQ ID NO: 13 or a sequence having at least 85%, at least 90% or at least 95% sequence identity thereto, including immunogenic fragments thereof.

[0135] The present invention further provides the antigen, the nucleic acid molecule, in particular mRNA(s), vector or host cell of the invention, preferably in combination with a carrier as provided herein, the composition, the combination, orthe pharmaceutical composition as defined herein, for use in stabilizing tumor growth and / or for use in stabilizing or inhibiting tumor size in a subject in need thereof, more specific in a subject having cancer. In one embodiment, the tumor size is reduced by at least 10% in the treated subject, in particular at least 20%, 25%, 30%, 35%, 40%, 50%, or even more. The effect on tumor growth can be quantified by the “Tumor Growth Inhibition (TGI)” index which is a widely used indicator to measure the effect of treatment on tumor development in preclinical in vivo experiments. Preferably, the TGI is more than 40%, in particular more than 50%, and even more particular more than 60%. In the clinic, RECIST (Response Evaluation Criteria in Solid Tumors) provides a pragmatic methodology to evaluate the activity and efficacy of new cancer therapeutics in solid tumors, using validated and consistent criteria to assess changes in tumor burden in patients, and can be used in the present invention.

[0136] The present invention further provides the antigen, the nucleic acid molecule, in particular mRNA(s), vector or host cell of the invention, preferably in combination with a carrier as provided herein, the composition, the combination, or the pharmaceutical composition as defined herein, for use in therapy, in particular immunotherapy in a subject in need thereof.

[0137] Immunotherapy is a type of medical treatment that harnesses the body’s own immune system to recognize, target, and combat diseases, particularly cancer. The goal of immunotherapy is to boost or modify the immune response, enabling it to more effectively identify and destroy abnormal or infected cells. In the context of cancer treatment, cancer cells can sometimes evade detection by the immune system. Immunotherapy seeks to overcome these evasive mechanisms and enhance the body’s natural ability to fight cancer. For example, HORMAD1 mRNA vaccines can be used to elicit an immune response against HORMAD1 expressed on cancer cells. They may be used as vaccines to prevent disease (“prophylactic vaccination”), or to activate the immune system to treat ongoing disease (“therapeutic vaccination”). The vaccines or compositions are administered in therapeutically effective amounts sufficient to elicit an immune response.

[0138] The present invention further provides the antigen, the nucleic acid molecule, in particular mRNA(s), vector to host cell of the invention, preferably in combination with a carrier as provided herein, the composition of DCs, the combination, or the pharmaceutical composition as defined herein, for use in the treatment of cancer, in particular wherein cancer cells express HORMAD1 and optionally another immunostimulatory polypeptide, such as PRAME; in particular wherein said cancer is lung cancer, more in particular NSCLC (non-small cell lung cancer). In one embodiment, the cancer is unresectable Stage III or metastatic Stage IV NSCLC.

[0139] In the context of the present invention, the term “cancer” refers to any kind of disease provoked by a malignant tumor. Included are malignancies of the various organ systems, such as affecting lung, breast, thyroid, blood, lymphoid tissues and bone marrow, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. Examples of cancers expressing HORMAD1 are lung cancer, in particular non-small cell lung cancer (NSCLC) of both non-squamous and squamous cell histological subtypes, breast cancer (including triple-negative breast cancer), esophageal cancer, endometrial cancer, cervical cancer, ovarian cancer, testicular cancer, colorectal cancer, squamous cell skin cancer, and head and neck cancer.

[0140] Examining the presence of the antigen / polypeptide, e.g. in the tumor or cancer cells, can be carried out at the transcript level by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) or bulk RNA-level next-generation sequencing (RNA-Seq), or single-cell RNA-Seq. At the protein level, detection can be performed on tissue sections by immunohistochemistry or immunofluorescence, on tissue extracts by Western blot or mass spectrometry-based techniques, on single-cell preparations by flow-cytometry, mass-cytometry or cellular indexing of transcriptomes and epitopes (CITE-Seq). Finally, presented epitopes derived from the antigen can be detected using mass spectrometry-based methods (HLA-immunopeptidomics).

[0141] In one embodiment, the cancer is found to express an antigen, e.g. HORMAD1 or PRAME, when a threshold of 1 transcript per mill is determined e.g. by using the method of the present examples. Non-small cell lung cancer (NSCLC) is a type of lung cancer that accounts for the majority of lung cancer cases. It is named "non-small cell" to distinguish it from small cell lung cancer, another major type of lung cancer. NSCLC is further classified into several subtypes based on cell morphology and molecular features.

[0142] The three main subtypes of non-small cell lung cancer are:

[0143] Non-Squamous Cell Carcinoma (Adenocarcinoma): this is the most common subtype of NSCLC, often found in the outer regions of the lungs. Adenocarcinoma is more common in non-smokers. Squamous Cell Carcinoma: this type of NSCLC typically arises in the central airways of the lungs. It is strongly associated with a history of smoking and may be found in the bronchi.

[0144] Large Cell Carcinoma: large cell carcinoma is a less common subtype of NSCLC and can occur in any part of the lung. It belongs to the spectrum of neuro-endocrine tumors of the lung which also includes small-cell lung cancer. It tends to grow and spread quickly.

[0145] Non-small cell lung cancer is often diagnosed at more advanced stages because symptoms may not be apparent in the early stages. Treatment options for NSCLC include surgery, radiation therapy, chemotherapy, targeted therapies, and immunotherapy. The choice of treatment depends on the stage of the cancer, the specific subtype (including the presence of targetable oncogenic genome alterations), and other individual factors of the patient. Advances in treatment modalities, including combination regimen, have improved outcomes for some patients with non-small cell lung cancer.

[0146] The present invention also provides a method for reducing the symptoms of or for treatment of cancer, in particular wherein the cancer cells express the polypeptides of the invention, more in particular NSCLC, said method comprising administering to a subject in need thereof the polypeptide, the nucleic acid molecule (e.g. the mRNA), the vector or host cell, the combination, or the (pharmaceutical) composition as defined herein.

[0147] Alternatively, the invention provides a method of preventing, reducing and / or inhibiting the recurrence, growth, proliferation, migration and / or metastasis of a cancer cell or population of cancer cells / a tumor in a subject in need thereof, comprising administering to the subject an effective amount of the polypeptide, the nucleic acid molecule (e.g. the mRNA), the vector or host cell, preferably in combination with a carrier as provided herein, the combination orthe (pharmaceutical) composition as defined herein.

[0148] The invention further provides a method of inducing an immune response in a subject comprising: administering to a subject in need of such treatment, a nucleic acid, in particular mRNA, preferably incorporated in or associated with a carrier as defined herein, such as an antigen presenting cell, wherein the nucleic acid encodes HORMAD1 , more specific encoding a polypeptide characterized by an amino acid sequence set forth as any one of SEQ ID NO: 1 - SEQ ID NO: 7, or a sequence having at least 85% identity thereto, or an immunogenic fragment thereof, in an amount effective to induce an immune response in the subject. The terms "treatment" and "treating" as used herein generally mean to obtain a desired pharmacologic and / or physiologic effect, and covers any treatment of a disease in a mammal, particularly a human, including:

[0149] (1) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom, but has not yet been diagnosed as having it;

[0150] (2) inhibiting the disease symptom, i.e., arresting its development; or

[0151] (3) relieving the disease symptom, i.e., causing regression of the disease or symptom, e.g. regression of a tumor or of tumor growth.

[0152] The assessment of tumor growth or regression is important in evaluating the efficacy of anti-cancer therapies. In 2000, the Response Evaluation Criteria in Solid Tumors (RECIST) criteria were created as the international standard for measurement of tumor response, or lack-thereof, to cancer therapies. Radiographically identified target lesions are measured serially across standard diameters to identify best response to therapy.

[0153] The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. In addition, the vaccine can be used as “adjuvant therapy” given in addition to a primary or initial therapy to maximize its effectiveness in a curative setting, or as a “maintenance” or “consolidative” therapy subsequent to and initial therapy to maximize disease control and delay disease recurrence.

[0154] In a further aspect, the present invention provides an in vitro or ex vivo method for improving the immune stimulating characteristics of antigen presenting cells comprising the introduction, either by transduction, transfection or any other method known to the skilled person, of one or more mRNA molecules encoding one or more polypeptides in said antigen presenting cell, characterized in that amongst the polypeptide at least HORMAD1 and optionally PRAME are introduced. In one aspect, the polypeptides are functional, meaning that they can elicit de novo or memory adaptive immune response against epitopes present in the polypeptide and / or capable of triggering an adaptive immune response against epitopes present in the polypeptide.

[0155] In particular, the invention also provides a preparation of antigen presenting cells, such as dendritic cells, for use in eliciting an immune response in a subject in need thereof, characterized in that an mRNA molecule encoding HORMAD1 is introduced in said dendritic cells; wherein optionally a further mRNA molecule encoding PRAME is introduced in the same or another dendritic cell.

[0156] Cell transduction refers to the introduction of foreign genetic material, such as DNA or RNA, into a cell. This process is commonly used in various research and therapeutic applications, including gene therapy, cell-based therapies, and the study of cellular functions. There are several methods for achieving cell transduction, and the choice of method depends on the type of cells, the nature of the genetic material being introduced, and the intended outcome. In the context of the present invention, the term “transfecting or transfection” is meant to be a process for deliberately introducing naked or purified nucleic acids, such as vectors (DNA or RNA) or mRNA molecules into eukaryotic cells. The term “transducing or transduction” is meant to be a type of transfection process using virus-mediated gene transfer, e.g. by using a retroviral or lentiviral vector. However, in the context of the present invention, any suitable method for transfection / transduction of antigen presenting cells may be used, such as electroporation, viral transduction, photoporation or lipofection. Some common methods of cell transduction include amongst others:

[0157] Viral Vectors:

[0158] Adenoviruses: adenoviral vectors are derived from adenoviruses, which are viruses that can infect a broad range of dividing and non-dividing cells. Adenoviral vectors are often used for transient gene expression in both in vitro and in vivo applications.

[0159] Lentiviruses: lentiviral vectors are derived from lentiviruses, and they are capable of infecting both dividing and non-dividing cells. Lentiviral vectors are commonly used for stable and long-term gene expression in both dividing and non-dividing cells.

[0160] Retroviral Vectors: retroviruses can integrate their genetic material into the host cell's genome. Retroviral vectors are often used for stable gene expression in dividing cells.

[0161] Adeno-Associated Viruses (AAV): AAVs are small viruses that can infect both dividing and nondividing cells. They are commonly used for gene delivery in research and clinical applications due to their relatively low pathogenicity and ability to provide long-term gene expression.

[0162] Electroporation: Electroporation involves applying an electric field to cells, creating temporary pores in the cell membrane. This allows foreign genetic material to enter the cells. Electroporation is commonly used for various cell types, including bacteria, yeast, and mammalian cells.

[0163] Lipofection: Lipofection involves the use of lipid-based reagents to form complexes with genetic material, creating liposomes. These liposomes can fuse with the cell membrane, delivering the genetic material into the cell. Lipofection is commonly used for transient transfection in mammalian cells.

[0164] Microinjection: Microinjection involves using a fine needle to directly inject genetic material into the cell. This method is highly precise but is generally used for specific applications, such as the creation of transgenic animals.

[0165] The choice of method depends on factors such as the type of cells, the desired duration of gene expression, and the intended application. Each method has its advantages and limitations, and researchers select the most appropriate method based on the specific requirements of their experiments or therapeutic goals.

[0166] The invention thus also provides a method for preparing an immunotherapy agent comprising the steps of: a) obtaining a carrier; b) ex vivo modifying or loading said carrier of step a) comprising the introduction of a mRNA encoding HORMAD1 , in particular encoding an amino acid sequence having at least 85% sequence identity to the amino acid sequence selected from the group comprising of any one of : SEQ ID NO: 1 to SEQ ID NO: 7, or an immunogenic fragment thereof; c) harvesting the obtained carriers.

[0167] More specific, the invention provides a method for preparing an immunotherapy agent comprising the steps of: a) obtaining or culturing antigen presenting cells; b) ex vivo modifying said antigen presenting cells of step a) comprising the introduction of a mRNA encoding HORMAD1 , in particular encoding an amino acid sequence having at least 85% sequence identity to the amino acid sequence selected from the group comprising of any one of : SEQ ID NO: 1 to SEQ ID NO: 7, or an immunogenic fragment thereof; c) harvesting the obtained antigen presenting cells.

[0168] In particular, the antigen presenting cells of step a) are dendritic cellsor B-cells, more in particular mature dendritic cells.

[0169] In a further embodiment, the carrier or antigen presenting cell may additionally comprise one or more mRNA molecules encoding an immunostimulatory polypeptide, such as a PRAME polypeptide; in particular a PRAME polypeptide characterized by any one of SEQ ID NOs: 8 -13, or a sequence having at least 85% sequence identity thereto.

[0170] More in particular, the method of introduction used in step b) is selected from transfection or transduction, more specific electroporation, viral transduction, photoporation, or lipofection of mRNA encoding HORMAD1 and optionally PRAME.

[0171] Administration

[0172] The nucleic acid (e.g. mRNA), vector, host cel, carrier (e.g. nanoparticle, APC, DC, etc.), the composition, the combination, or the pharmaceutical composition as provided herein can be administered by a variety of methods, such as, but not limited to, injection (e.g., subcutaneous, intradermal, intravenous, intralymphatic, intraarticular, intramuscular, intraperitoneal), by continuous infusion, sustained release from implants, etc. The composition can be administered at specific intervals, e.g. as a prime-boost regimen. The term “booster” refers to an extra administration of the (vaccine) composition. A booster (or booster vaccine) may be given after an earlier administration of the composition. The time of administration between the initial administration of the composition and the booster may be from 5 minutes to 1 hour, to 1 day, to 1 week, to 1 month, to 1 year or even more, including all the periods in between. A composition may be administered with other prophylactic or therapeutic compounds. As used herein, when referring to a prophylactic composition, such as a vaccine.

[0173] In a particular embodiment, the composition, e.g. the dendritic cell-based vaccine, can be administered with physiologically acceptable carriers, buffers, diluents, adjuvants, immunomodulators, etc. Preferably, the dendritic cell vaccine is autologous to the patient it is administered to, or is maximally HLA-matched. The dose of cells administered to a subject is in an effective amount, effective to achieve the desired beneficial therapeutic response in the subject over time, or to inhibit growth of cancer cells, while maintaining a good tolerability profile (minimal toxicity). An amount adequate to accomplish this is defined as a "therapeutically effective dose". The dose will be determined by the biological and / or clinical activity of dendritic cell produced and optionally the condition of the patient. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular cell in a particular patient. In determining the effective amount of the cell to be administered in the treatment or prophylaxis of diseases such as cancer (e.g., lung cancer, metastatic melanoma, prostate cancer, etc.), the physician (or investigator) needs to evaluate immune responses against HORMAD1 or the respective antigens included in the vaccine (i.e. immunomonitoring), along with the clinical evolution of the tumor using measurable parameters (radiological tumor burden by regular or immune-related RECIST criteria, tumor markers, circulating tumor cells, plasma circulating tumor DNA or other surrogate markers of disease load or disease activity).

[0174] It is well known to those skilled in the art that there is no evidence for a preferred dose of the vaccine or DCs to be administered to achieve a specific level of biological and / or clinical effect. Likewise no clear dose-limiting toxicity (DLT) has been observed and accordingly no maximal tolerated dose (MTD) has been observed. The doses most commonly administered are dictated by the yield of DCs obtained from one round of leukapheresis and the desired number of subsequent vaccinations. In one embodiment, doses fall within 5-100x106DCs per vaccination round, repeated 2 to 8 times, in particular 2 to 6 times, more in particular 2 to 4 times. Likewise, there is no relationship between the number of cells injected and toxicity. Toxicity with DC vaccination is usually low, and rather linked to the route of administration (more acute side effects with intravenous route as compared to intradermal route). The injections may be e.g. 2, 3, 4, 5 or 6 times repeated in a 1 , 2 or 3 weeks interval and should be given either intravenously or near lymph nodes by intradermal or subcutaneous injections or injected directly into the lymph nodes. Booster injections may be performed after a pause, e.g. of 1 to several months.

[0175] All of the features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0176] The invention will be further described by the following figures, tables and examples, which are not intended to limit the scope of protection as defined in the claims. EXAMPLES

[0177] Materials and Methods

[0178] Monocyte-derived Autologous Dendritic Cell culture

[0179] Monocyte-derived dendritic cells were generated according to a proprietary accelerated culture protocol as described (Brabants et al, 2018; WO2019243537). For pre-clinical evaluation, material from anonymous healthy HLA-A2+donors was used. Peripheral blood mononuclear cells (PBMC) are separated into monocytes (CD14+fraction) and peripheral blood lymphocytes (PBL) using immunomagnetic separation (MACS) with anti-CD14 microbeads, according to the manufacturer’s instruction. Starting from buffy coats obtained from local blood transfusion center, a density gradient centrifugation using Ficoll-paque was performed prior to immunomagnetic separation. No prior density centrifugation was performed when apheresis material was used as starting material.

[0180] PBLs were frozen in RPMI containing 20% Alburex20 (Human serum Albumin 20 g / l, CSC Behring) and 10% Cryosure dimethyl sulfoxide (DMSO).

[0181] CD14+monocytes were cultured in GMP cell differentiation bags at a density of 1 x106cells / ml in serum- free GMP CellGro medium containing 1000 U / ml pharmaceutical-grade granulocyte macrophage colony-stimulating factor (GM-CSF), 500 U / ml GMP-certified recombinant human interleukine-4 (hulL- 4). On day 3 of the culture, 2,5 |j.g / ml synthetic TLR4 agonist Monophosphoryl lipid A (MPLA) and 1000 lU / ml pharmaceutical-grade IFN-y were added to the culture medium for another 24h. Mature DCs (mDCs) were harvested on day 4.

[0182] Target antigen cloning and mRNA production

[0183] The sequence of the antigens of interest i.e. HORMAD1 and PRAME were selected via the Ensembl human genome browser. Coding sequence cDNA of the selected transcript was analyzed for presence of BamHI or BspQI restriction sites. If present, bases of the specific codon were changed according to codon optimization to circumvent the specific restriction site. Subsequently, the selected sequence was checked for proper RNA folding. The sequence of the different antigens of interest were custom- synthetized as gBIocks (IDT) and cloned into an mRNA vector backbone using Gibson assembly method. Transformation into competent cells was performed followed by plasmid DNA purification. Quality control includes Sanger sequencing as well as gel electrophoresis upon incubation with specific restriction enzymes. DNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000. The obtained DNA was linearized using the BspQI restriction enzyme (NEB) for further in vitro transcription into mRNA using the Hiscribe T7 mRNA kit with CleanCap reagent AG (NEB). RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND- 1000 and RNA integrity was assessed using a Bioanalyzer 2100.

[0184] Used mRNA sequences are HORMAD1 - splice variant 202 - represented by SEQ ID NO:14; HORMAD1 - splice variant 204 - represented by SEQ ID NQ:40; and PRAME - splice variant 201 - represented by SEQ ID NO:15. Full mRNA constructs, which include sequences that regulate amongst others stability, location and translation efficiency, are as represented by SEQ ID NO: 16, 17 and 41.

[0185] The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application may recite “T”s in a representative RNA or mRNA sequence but understands that “T”s would be substituted for “U”s. Further, any of the RNA or mRNA sequences disclosed herein and identified by a particular sequence identification number, is also intended to disclose its corresponding DNA sequence complementary to the RNA, where each “U” of the RNA sequence is substituted with “T”.

[0186] Electroporation of DCs

[0187] Cells were harvested and washed twice with Opti-MEM prior to electroporation. For large scale electroporation, 50x106DCs were resuspended in 350 pd Opti-MEM and transferred to a 4 mm gap cuvette whereupon 50 pd nuclease-free water containing the mRNA at a dosage of 1 .g / 106cells was added to the cell suspension. For small scale electroporation, 5x106DCs were resuspended in 170 pd Opti-MEM and transferred to a 4 mm gap cuvette, whereupon a mixture of 5 pd nuclease-free water containing the mRNA at a dosage of 1 .g / 106cells supplemented with 25 pd Opti-MEM was added to the cell suspension. Electroporation with nuclease-free water (Mock) or with mRNA-encoding an irrelevant antigen (CTRL AG) served as a negative control. Electroporation was performed using the Gene Pulser XCell Electroporation System according to following settings: square wave protocol with a pulse time of 1 msec and a Voltage of 500 V. Immediately after electroporation, DCs were transferred to serum-free GMP CellGro medium containing 1000 U / ml pharmaceutical-grade granulocyte macrophage colony-stimulating factor (GM-CSF) and 250 U / ml GMP-certified recombinant human interleukine-4 (hulL-4). Cells were transferred to GMP cell differentiation bags or ultra-low attachment plates at a density of 106DC / ml and cultured for 4 hours at 37°C and 5% CO2.

[0188] Four hours after electroporation, DC phenotype and electroporation efficiency were evaluated using flow cytometry and DCs were cryopreserved in cryopreservation medium.

[0189] The following mRNA sequences were used: HORMAD1 - splice variant 202 - represented by SEQ ID NO: 14 and PRAME - splice variant 201 - represented by SEQ ID NO: 15. Full mRNA constructs are as represented by SEQ ID NO: 16 and 17 resp.

[0190] Generation of lipid nanoparticles

[0191] In vitro transcribed (IVT) mRNA was formulated into lipid nanoparticles (LNPs) composed of the ionizable lipid SM-102, cholesterol, DSPC (1 ,2-distearoyl-sn-glycero-3-phosphocholine), and DMG- PEG2000 (1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) at a molar ratio of 50:10:38.5:1 .5, respectively. LNPs were prepared via ethanol dilution using an automated T-junction microfluidic mixing device to ensure reproducible nanoparticle formation.

[0192] The resulting mRNA-LNP formulations underwent physicochemical characterization for particle size and zeta potential using a Malvern Zetasizer Nano-ZS (Malvern Instruments). The encapsulation efficiency and mRNA concentration were quantified using the Quant-iT™ RiboGreen® RNA Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. To assess total encapsulated mRNA, LNPs were diluted in TE buffer containing 1 % (v / v) Triton X-100 (Sigma-Aldrich) and incubated for 10 min at 37 °C to lyse nanoparticles. Unencapsulated (free) mRNA was determined by measuring the signal of mRNA diluted directly in TE buffer without detergent treatment. Encapsulation efficiency was calculated as the ratio of encapsulated to total mRNA.

[0193] DC loading with mRNA formulated in lipid nanoparticles

[0194] Immature DCs were seeded in ultra-low attachment (ULA) plates at a density of 1 x106cells / ml in serum- free GMP CellGro medium containing 1000 U / ml pharmaceutical-grade granulocyte macrophage colony-stimulating factor (GM-CSF) (Leukine sargramostim) and 500 U / ml GMP-certified recombinant human interleukine-4 (hulL-4) whereupon mRNA-LNP at a concentration of 2 .g / 106cells was added to the cell suspension. Cells were cultured for 24 hours at 37°C and 5% CO2. DC phenotype and mRNA- LNP loading efficiency were evaluated using flow cytometry and DCs were cryopreserved in cryopreservation medium.

[0195] Isolation of B cells

[0196] CD19+B cells were isolated from peripheral blood mononuclear cells (PBMC) derived from apheresis of a healthy HLA-A2+volunteer using immunomagnetic separation (MACS) with anti-CD19 microbeads, according to the manufacturer’s instruction.

[0197] Electroporation of B cells

[0198] Cells were harvested and washed twice with Opti-MEM prior to electroporation. To that end, 50x106B cells were resuspended in 350 pd Opti-MEM and transferred to a 4 mm gap cuvette, whereupon 50 pd nuclease-free water containing the mRNA at a dosage of 1 .g / 106cells was added to the cell suspension. Electroporation with mRNA-encoding an irrelevant antigen (CTRL AG) served as a negative control. Electroporation was performed using the Gene Pulser XCell Electroporation System according to following settings: exponential decay protocol 225 V and 300 .F. Immediately after electroporation, B cells were transferred to IMDM medium. Cells were transferred to ultra-low attachment plates at a density of 106cells / ml and cultured for 4 hours at 37°C and 5% CO2.

[0199] Four hours after electroporation, B cell phenotype and electroporation efficiency were evaluated using flow cytometry and B cells were cryopreserved in cryopreservation medium.

[0200] Flow cytometry

[0201] To perform surface staining, cells were washed and resuspended in phosphate buffered saline (PBS) supplemented with 0,5 mM ethylenediaminetetraacetic acid (EDTA); 0,25% bovine serum albumin (BSA); and 0,05% sodium azide (NaN3) further referred to as flow cytometry buffer. To prevent nonspecific binding, cells were pre-incubated for 30 min at 4°C with anti-human FcR-blocking reagent after which cells were washed with flow cytometry buffer. To identify dead cells, a fixable viability eFluor506 dye was used. Surface staining of PBMC, PBL CD14+fraction after ferromagnetic isolation and CD19+fraction after ferromagnetic isolation was performed by staining for 30 min at 4°C with a cocktail of following fluorochrome-conjugated anti-human monoclonal antibodies: anti-CD14-FITC ; anti-CD3-BV421 ; anti- CD19-PE-Cy7 and anti-CD56-PE after which cells were washed and resuspended in flow cytometry buffer.

[0202] Dendritic cell phenotype was evaluated by staining for 30 min at 4°C with following anti-human antibody cocktails: anti-CD11 c-APC (clone S-HCL-3); anti-HLA-DR-APC-Cy7 (clone L243) supplemented with anti-CD40-PE (clone SC3); antiCD80-PE (clone 2D10.4); anti-CD83-PE (clone HB15c) ; CD86-PE (clone IT2.2); anti-CD70-PE (clone REA292); anti-CD274-PE (clone MIH1) or anti-CCR7-PE (clone REA108).

[0203] Samples were acquired on a Fortessa LSR and analyzed using FlowJo software.

[0204] Tumor cell lines

[0205] H-1650 were obtained from American Type Culture Collection (ATCC). Full cell line authentication was performed). H-1650 cells were expanded according to recommended culture conditions, kept free of mycoplasma contamination and stored in cryopreservation medium at -150°C until further use.

[0206] Mice

[0207] NSG mice were housed in individually ventilated cages under pathogen-free conditions in a temperature- and humidity-controlled environment with 12 / 12h light / dark cycle and received food and water ad libitum.

[0208] Animals were treated according to the Federation of European Laboratory Animal Science Association (FELASA) guidelines. Experiments were reviewed and approved by the Ethical Committee of Ghent University (ECD20 / 102k, ECD21 / 11). Where possible, the investigators were (double) blinded during data collection and analysis.

[0209] In vitro cancer antigen-specific T-cell cytotoxicity study

[0210] The mRNA loaded DCs, mRNA-LNP loaded DC or mRNA loaded B-cells are subsequently used to prime naive autologous T cells in vitro. To sustain T cell fitness, T cell supporting cytokines were added to the culture. Two rounds of“ / n vitro vaccination” were performed by which mRNA-loaded DCs, mRNA- LNP loaded DC or mRNA loaded B cells are added to the T cell culture (i.e. at dO and d7) at a 1 :10 ratio. At day 14, stimulated T cells are used as effector cells for an in vitro killing assay with HLA-A2 matched H-1650 NSCLC cancer cells loaded with an apoptosis reporter dye (Caspase-3 / 7). The effector-target co-culture is imaged over time using the Incucyte incubator / imaging system, which detects and quantifies the number of apoptotic cancer cells in real time.

[0211] In vivo therapeutic cancer vaccination study

[0212] The in vivo demonstration is provided using a humanized mouse model in which tumor and immune system are of human origin and partially HLA-matched, while the therapeutic DCs and the pre-engrafted lymphocytes are autologous to each other. In contrast to other models such as syngeneic mouse tumor models that can only demonstrate mouse T-cell responses, this humanized mouse model delivers demonstration of anti-cancer efficacy in a manner that is as close as possible to the clinical setting. Immunodeficient Nod Scid gamma (NSG) mice were injected intravenously at day 0 with 10x106PBLs, prepared as above, to induce engraftment of a human adaptive immune system. The same day, mice were inoculated subcutaneously with 2x106H-1650 NSCLC tumor cells in the right flank.

[0213] A tail vein blood sample was taken to determine the engraftment of a human immune system. Mice were randomized and assigned to the different experimental groups according to gender, age and tumor size. When a palpable tumor (+-50 mm3) was detected, mice were injected intravenously with 5x105mRNA- DC, twice with a one-week interval.

[0214] Tumor size was monitored longitudinally by bidimensional caliper measurements and tumor volume was estimated using the formula for a prolate ellipsoid i.e. (length x widthA2) / 2.

[0215] Nine days after the last mRNA-DC injection, mice were sacrificed for cross-sectional analysis. Tumors were analyzed for human immune cell engraftment as well as T cell phenotype and functionality.

[0216] The investigators were blinded for treatment randomization as well as tumor measurements and data analysis.

[0217] Human study

[0218] An investigational therapy consisting of autologous DCs, manufactured according to our proprietary method described above, and loaded with the HORMAD1 mRNA-encoded antigen was evaluated in a refractory stage IV NSCLC adult patient with heavily pre-treated metastatic NSCLC not receiving anti- tumoral therapy at the time of enrolment. After leukapheresis, the patient received up to five biweekly intravenous infusions of the mRNA-DC vaccine following an intra-patient dose escalation regimen up to a maximum dose of 100 x 106DCs.

[0219] Immunomonitoring in vitro stimulation protocol

[0220] To study vaccine-induced T-cell responses, peripheral blood was collected before each vaccination dose starting from the 2nddose and if feasible, 3, 12 and 24 weeks (+ / - 1 week) after the last vaccine dose. Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved. To obtain baseline samples, surplus of the leukapheresis product used for vaccine production was cryopreserved.

[0221] Patient PBMC were thawed and rested overnight at a concentration of 10x106cells / ml in IMDM supplemented with 10% human AB serum, 2 mM L-glutamine (Invitrogen), 100 lU / ml penicillin and 100 lU / ml streptomycin , further referred to as completed IMDM (cIMDM). The following day, PBMC were plated in 96-well u-bottom plates at 1x106PBMC / 100 pl cIMDM. Antigen-specific peptide pools were added to the PBMC at a concentration of 1 pg peptide / ml. The peptide pool consisted of overlapping 15-mers together covering the full protein sequence. The peptides were custom-made and lyophilized by JPT. At day 3, the cells were diluted 1 :10 in cIMDM supplemented with 2,5 ng / ml IL-7 and IL-15 and transferred to a 24-well plate. IL-7 and IL-15 were replenished at day 7 and cells were further incubated until day 10. On day 10 the cells were restimulated with the antigen peptide pools and T-cell responses were evaluated by measuring IFN-y production by performing IFN-y ELISpot and intracellular cytokine staining followed by flow cytometry. PBMC of the corresponding timepoints to which a buffer containing DMSO was added at day 0 and pulsed with the overlapping 15-mers together covering the full protein sequence at d10 served as the assay background condition.

[0222] Immunomonitoring: Intracellular IFN-y staining

[0223] As a positive control, T cells were stimulated with ImmunoCult Human CD3 / CD28 / CD2 T Cell Activator. After 2 hours of stimulation, GolgiPlug was added, and the cells were incubated for another 4 hours. After incubation, the cells were stained with fixable Live / Dead stain and with anti-CD3-PE-Cy7, anti- CD8-APC-Fire750 and anti-CD4-PerCP-Cy5.5 antibodies for 30 min at 4°C. Next, the cells were fixed and permeabilized and stained intracellularly with an anti-IFN-y-FITC antibody for 30 minutes on 4°C. Cells were measured on a flow cytometer with Facs Diva software. The resulting data was analyzed by using FlowJo v.10 software. Cells were hierarchically gated on lymphocytes (FSC-A vs SSC-A), living cells (FSC-A versus Live / Dead), singlets (FSC-A vs FSC-H), IVS T cells (FSC-A vs CellTrace in Pacific Blue), CD3+CD4+T cells and CD3+CD8+T cells. IFN-y staining was plotted for each of the indicated populations. CD4+IFN-y+or CD8+IFN-y+T cell frequencies were calculated as the mean from duplicates. T cell responses were calculated by subtracting the CD4+IFN-y+or CD8+IFN-y+T cell frequencies observed in the DMSO control from the respective frequencies detected after peptide stimulation. Negative values were set to zero. A T cell response was considered positive if the mean frequency of CD4+IFN-y+or CD8+IFN-y+T cells after peptide stimulation was at least 2-fold higher than the mean frequency in the corresponding DMSO-control and if at least 25 events were present in the CD4+IFNy+or CD8+IFN-y+gate. To be defined as a vaccine-induced T cell response, positive T cell responses had to be detected in at least one time point during the treatment phase and the frequency of the positive T cell response had to be at least 2-fold higher than the frequency of the response observed at baseline. Due to limited samples sizes, no statistical analyses were performed.

[0224] Immunomonotoring: IFN-y ELISpot

[0225] An ELISpot plate pre-coated with IFN-y antibodies (Human IFN-y) was conditioned with cIMDM for 30 minutes at room temperature. The in vitro stimulated cells were harvested and seeded at a concentration of 50 000 - 150 000 cells in 100 pl cIMDM per well of the ELISpot plate. Antigen-specific peptide pools were added to the wells at a final concentration of 1 pg peptide / ml or DMSO as negative control. As a positive control, T cells were stimulated with anti-CD3 antibody (mAb CD3-2,). The plate was incubated for 16-24 hours at 37°C and 5% CO2. Stimulations were done in duplicate. After incubation, plates were washed with PBS and a secondary anti-IFN-y antibody directly conjugated with alkaline phosphatase (7-B6-ALP) was added. After incubation for 2 hours, plates were washed and BCIP / NBT-plus substrate was added. When clear spots had emerged, the reaction was stopped by washing the plate in tap water and the plates were left to air-dry in the dark. The plates were scanned using an ELISpot plate reader (CTL) and spot counts were analyzed by ImmunoSpot software (CTL). RNA extraction of patient residual tumor material

[0226] RNA was extracted from residual tumor material formalin-fixed, paraffin-embedded (FFPE) samples using the Qiagen miRNeasy FFPE kit according to manufacturer’s instructions. In brief, tumor material was scraped from the freshly prepared glass slides using an RNAse-free sterile scraper. The average scraped surface was calculated as 375 mm2 with an average tumor percentage of 20-30% as determined by the pathology department. Next, the sample was treated with deparaffinization solution to remove any residual paraffin upon which samples are incubated in an optimized lysis buffer containing proteinase K allowing release of the RNA followed by short heat treatment which reverses formalin crosslinking of the released nucleic acids. Subsequently, the supernatant is treated with DNAse upon which the lysate is mixed with RBC buffer and ethanol. The mixture is transferred to an RNeasy MinElute spin column and any contaminants are washed away. Total RNA is eluted using RNAse-free water. RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000.

[0227] NGS library preparation and sequencing

[0228] Per sample, an amount of 10 ng of total RNA was used as input for the SMART-Seq Stranded Kit. RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 and RNA integrity was assessed using a Bioanalyzer 2100. Positive and negative controls were included in the experimental design using 10ng Control RNA and 7pl RRI water, respectively. First, RNA is converted to cDNA using random priming (scN6 Primer) and SMART (Switching Mechanism At 5’ end of RNA Template) technology and then full-length adapters for Illumina sequencing (including specific barcodes for dual-indexing libraries) are added through PCR using a limited number of cycles (5 cycles). The PCR products are purified after which ribosomal cDNA is selectively depleted by cleaving the ribosomal cDNAs by scZapR in the presence of mammalian-specific scR-Probes which target nuclear and mitochondrial rRNA sequences. This process leaves the library fragments originating from non- rRNA molecules untouched. The remaining cDNA fragments are further amplified with primers universal to all libraries (15 cycles). Lastly, the PCR products are purified once more to yield the final cDNA library. All libraries were finally quantified using Qubit dsDNA HS kit (Thermo Fisher Scientific) and their size distribution was checked using a Bioanalyzer 2100 (Agilent). Sequence-libraries of each sample were equimolarly pooled and sequenced on Illumina NextSeq500 v2.5 flow-cell (76-8-8-0, 1 % PhiX).

[0229] NGS data analysis

[0230] Preprocessing: Low quality ends and adapter sequences were trimmed off from the Illumina reads with FastX 0.0.14 and Cutadapt 1.15 (Galaxy). Subsequently, small reads (length < 35 bp), polyA-reads (more than 90 % of the bases equal A), ambiguous reads (containing N), low-quality reads (more than 50 % of the bases < Q25) and artifact reads (all but three bases in the read equal one base type) were filtered using using FastX 0.0.14 and ShortRead 1.44.3 . With Bowtie2 2.3.3.1 reads that align to phixjllumina were identified and removed. Mapping: The preprocessed reads were aligned with STAR aligner v2.5.2b to the reference genome of Homo sapiens (GRCh38) (AD et al, 2013). Default STAR aligner parameter settings were used, except for ‘--outSAMprimaryFlag OneBestScore --twopassMode Basic --alignlntronMin 50 --alignlntronMax 500000 --outSAMtype BAM SortedByCoordinate’. Using Samtools 1 .5, reads with a mapping quality smaller than 20 were removed from the alignments.

[0231] Counting: The number of reads in the alignments that overlap with gene features were counted with featurecounts 1.5.. Following parameters were chosen: -Q 0 -s 2 -t exon -g genejd. Genes for which all samples had less than 1 count-per-million were removed. Raw counts were further corrected within samples for GC-content and between samples using full quantile normalization, as implemented in the EDASeq package from Bioconductor.

[0232] Differential gene expression: With the EdgeR 3.28.1 package of Bioconductor, a negative binomial generalized linear model (GLM) was fitted against the normalized counts. We did not use the normalized counts directly but worked with offsets. Differential expression was tested for with a GLM likelihood ratio test, also implemented in the EdgeR package. The resulting p-values were corrected for multiple testing with Benjamini-Hochberg to control the false discovery rate

[0233] A threshold for positivity of expression was set at 1 transcript per million (tpm). All samples above were considered as positive.

[0234] RT-qPCR

[0235] RNA extracted from the residual patient tumor material was subject to reverse transcription (RT) using the Superscript II RT PCR kit, followed by an RNAseH treatment (20 min 37°C). The resulting cDNA served as a template for quantitative polymerase chain reaction (qPCR) using Lightcycler 480 SYBR Green I master mix . Following RT-qPCR protocol was applied: Pre-incubation 5 min at 95°C; Amplification: 40 amplification cycles of denaturation 10 sec at 95°C, annealing 20 sec at 60°C and elongation 10 sec at 72°C; a melting curve step 5 sec at 95°C, 1 min at 65°C and a final step at 97°C. cDNA preamplification was performed (Sso advanced pre-amp System, BioRad) prior to qPCR. The assay was performed with four technical replicates. Data were analysed using qBasePlus software and normalized to the reference genes Actb, Gapdh, Ywhaz. A positive CNRQ value after qBasePlus analysis reflects a meaningful result.

[0236] Primers: Ten candidate reference genes were selected based on prior publications (Hellemans et al., 2011 ; Saviozzi et al., 2006). These reference genes include Actb, b2m, Esd, Folr2A, gapdh, Plodl , rRNA18S, Tbp, Yap1 , Ywhaz. Most optimal housekeeping genes were determined using human lung cancer tumor cell lines as well a reference tumor samples and were selected based on qBasePlus GeNORM analysis for efficiency value and stability analysis.

[0237] Target specific primers were designed using the IDT PrimerQuest online tool. At least four different primers sets were selected and evaluated for HORMAD1. Primers were further evaluated using BioTools-OligoCalc and UnaFod-mFOLD for difference in melting temperature, (self-) complementarity, seundary structures and hairpin formation. Finally, primer sets were BLASTED using NCBI BLAST. The designed primer sets were first evaluated on human lung cancer tumor cell lines as well a reference tumor sample.

[0238] The primer sequences are listed in Table 4.

[0239] Statistical analysis

[0240] Shapiro-Wilk Normality test was performed to determine Gaussian distribution (a = 0,05) of the data. When data were normally distributed according to Shapiro-Wilk testing, unpaired two-tailed student t- test or ANOVA followed by Tukey’s multiple comparisons test was performed. If data were not normally distributed according to Shapiro-Wilk testing, unpaired nonparametric Mann-Whitney test or ANOVA Kruskal-Wallis test with Dunn’s multiple comparisons test was performed. Statistical analyses were performed using the GraphPad Prism software. Statistical significance levels are indicated using following p-values: * < 0.05; ** < 0.01 ; *** < 0.001 ; **** < 0.0001 . The numbers of independent biological replicates or the numbers of individual mice have been indicated in the figure legends. Results

[0241] The value of HORMAD1 as a target antigen for cancer immunotherapy is demonstrated in the examples herein, generated in a stepwise manner using an mRNA-modified dendritic cell-based vaccine approach: (1) preclinical in vitro and (2) in vivo experiments using HORMAD1 as single target, (3) preclinical in vitro and (4) in vivo experiments using HORMAD1 combined with an additional cancer antigenic target, and (5) data highlighting effects in one treated cancer patient.

[0242] Example 1 : In vitro T cell-mediated tumor killing when targeting HORMAD1 alone as target antigen

[0243] The capacity of T-cells primed against the HORMAD1 antigen to specifically induce cell death in HORMAD1 -expressing cancer cells was demonstrated using the following experiment. DCs generated from HLA-A2-positive anonymous healthy donor leukocytes according to the aforementioned method were loaded with mRNA encoding the full open reading frame of HORMAD1 , and used as antigen- presenting cells to prime autologous T-cells. T-cells were subsequently co-incubated with H1650 cells, an HLA-A2-positive non-small cell lung cancer cell line. H1650 is known to express HORMAD1 mRNA, as documented in the CCLE public database (RPKM 3.28) and subsequently confirmed in our RNAseq analysis (normalized cpm 10.5). Tumor cell killing, quantified as the cell count of apoptotic tumor cells, was monitored in real-time. The data (Fig. 1) shows selective killing of tumor cells when T-cells were primed beforehand with HORMAD1 mRNA-loaded DCs. The effect is clearly above background cytotoxicity measured when T-cells were primed with DCs loaded with an irrelevant antigen, or T-cells not contacted with DCs, indicating an antigen-specific effect.

[0244] Example 2: In vivo tumor control when targeting HORMAD1 alone as target antigen

[0245] To assess the capacity of HORMAD1 mRNA-DCs to induce anti-tumoral T-cell responses in vivo, we established a humanized mouse model in which HLA-A2 lymphocytes were engrafted systemically, together with subcutaneous implantation of a tumor xenograft of the abovementioned HLA-A2-positive NSCLC cell line. Mice were treated with systemic injections of HORMAD1 mRNA-DCs autologous to the engrafted immune system, and tumor growth evolution was monitored. Treatment was started when tumors were clinically palpable. Mice were euthanized when tumor size reached 500 mm3or at signs of graft-versus-host-disease.

[0246] The main endpoint was tumor growth inhibition (%TGI), defined as (1 -{Tt / TO I Ct / C0} 1 1 -{C0 / Ct}) X 100 where

[0247] Tt = median tumor volume of treated at time t

[0248] TO = median tumor volume of treated at time 0

[0249] Ct = median tumor volume of control at time t

[0250] CO = median tumor volume of control at time 0

[0251] TGI is usually calculated when Ct = or > 1000 mm3. TGI >50% is considered clinically meaningful.

[0252] The data in Fig. 2 shows profound suppression of tumor growth in mice treated with HORMAD1-mRNA loaded DCs. Fig 2A shows evolution of lung cancer xenograft volumes over time in different treatment groups. Fig 2B provides a cross-sectional picture of xenograft sizes at day 21 after tumor engraftment. The effect is highly antigen-specific as mice injected with DCs loaded with an irrelevant antigen showed no response. This is reflected in the TGI% indices as shown in Table 5.

[0253] Example 3: In vitro T cell-mediated tumor killing when targeting HORMAD1 in combination with an additional cancer antigen

[0254] Antigen-specific immunotherapy benefits from simultaneous targeting of multiple antigens to avoid emergence of escape variants. Hence, we explored the effect of targeting HORMAD1 in combination with an additional cancer antigen. Using the same in vitro assay as described in Example 1 , T-cells were primed with both DCs loaded with HORMAD1-mRNA, as well as DCs loaded with mRNA encoding an additional protein belonging to the cancer-germline antigen class, i.e. PRAME (CT130). The HLA-A2+ non-small cell lung cancer cell line used in the experiments has been documented to express PRAME (CCLE database: 71.87 RPKM, our own RNAseq analysis: 127 normalized cpm). The data (Fig. 3) clearly shows powerful tumor killing effects compared to control conditions.

[0255] Example 4: In vivo tumor control targeting for HORMAD1 in combination with an additional cancer antigen

[0256] Having established the in vitro efficacy of priming cytotoxic T-cells against HORMAD1 and PRAME in combination, we sought to investigate this approach in vivo using the humanized mouse platform described under Example 2. The data (Fig. 3) clearly shows profound tumor inhibitory effects of treating humanized mice with both autologous HORMAD1-mRNA DCs as well as PRAME-mRNA DCs in combination. The corresponding %TGI indices are shown in Table 6.

[0257] Example 5: Clinical activity of HORMAD1 -targeted vaccine approach in a cancer patient

[0258] DCs of the invention, loaded with HORMAD1 mRNA, were administered to a refractory stage IV NSCLC patient. An important endpoint of biological activity in human subjects is the capacity to induce cellular immune responses against the mRNA-encoded antigens loaded in the injected DCs. The example provided from one treated patient shows that injection of HORMAD1-mRNA DCs elicits antigen-specific T-cell responses as detected using ELISPOT on serial blood samples (Fig 5). Responses emerge over baseline / pre-vaccine levels starting from the lowest injected DC dose (containing 2.5x106HORMAD1- mRNA-DCs), and peaks 3 weeks after the last administered dose in this patient. Responses persisted over baseline levels 3 months afterthe last DC dose and progressively decreased (no booster dose was given). Additional analysis of this example subject’s response by flow-cytometry shows that HORMAD1- specific responses are elicited both in the CD8+ as well as the CD4+ T-cell compartment (resp. Fig 6, Fig 7). Analysis of archival tumor sample from this example patient confirmed the expression of HORMAD1 antigen (resp. Fig 8A, Fig 8B), as defined by a positive signal on qPCR (calibrated normalized relative quantity > 0) and an expression level of >1 transcripts per million (TPM) by RNAseq.

[0259] Example 6: In vitro tumor killing assay using T cells primed against the HORMAD1 target of the invention by autologous HORMAD1 mRNA-LNP loaded DCs

[0260] The main cellular target of mRNA-LNP-based vaccination is the dendritic cell network in injected tissues: the DCs take up the mRNA-LNP particles, translate the mRNA cargo into the corresponding polypeptide, which is subsequently processed to smaller peptides (epitopes) for presentation on surface MHC molecules to T-cells. To provide evidence for this mechanism in a well-controlled environment, the following in vitro experiment was designed. DCs generated from HLA-A2-positive anonymous healthy donor leukocytes according to the aforementioned method were loaded with HORMAD1 mRNA-LNPs and used as antigen-presenting cells to prime autologous T cells. DC are central to antigen presentation due to their ability to internalize, process and present antigens and stimulating robust adaptive immune responses. Lipid nanoparticles can be internalized by multiple mechanisms including macropinocytosis, clathrin-mediated endocytosis and caveolae-mediated endocytosis. DCs are major executors of these mechanisms. The HORMAD1 mRNA encodes the sequence resulting in the longest polypeptide, represented by HORMAD1 splice variant 202. T cells were subsequently co-incubated with H1650 cells, an HLA-A2-positive non-small cell lung cancer cell line. H1650 is known to express HORMAD1 , as documented in the CCLE public database (RPKM 3.28) and subsequently confirmed in our RNAseq analysis (normalized cpm 10.5). Tumor cell killing, quantified as the cell count of apoptotic tumor cells, was monitored in real-time. Apoptotic tumor cells were detected by a Caspase-cleavable fluorescent reporter. The data (Fig. 9) shows selective killing of tumor cells when T cells were primed beforehand with HORMAD1 mRNA-LNP loaded DCs. The effect is clearly above background cytotoxicity measured when T cells were primed with DCs loaded with an irrelevant antigen, indicating an antigen-specific effect.

[0261] Example 7: In vitro T cell mediated tumor killing when T cells were primed using HORMAD1 mRNA loaded B cells.

[0262] B cells generated from HLA-A2-positive anonymous healthy donor leukocytes according to the aforementioned method were loaded with HORMAD1 mRNA and used as antigen-presenting cells to prime autologous T cells. The HORMAD1 mRNA encodes the full open reading frame of HORMAD1 , represented by HORMAD1 splice variant 202. T cells were subsequently co-incubated with H1650 cells, an HLA-A2-positive non-small cell lung cancer cell line. H1650 is known to express HORMAD1 mRNA, as documented in the CCLE public database (RPKM 3.28) and subsequently confirmed in our RNAseq analysis (normalized cpm 10.5). Tumor cell killing, quantified as the cell count of apoptotic tumor cells, was monitored in real-time, effect. Apoptotic tumor cells were detected by a Caspase-cleavable fluorescent reporter. The data (Fig. 10) shows selective killing of tumor cells when T cells were primed beforehand with HORMAD1 mRNA loaded B cells. The effect is clearly above background cytotoxicity measured when T cells were primed with B cells loaded with an irrelevant antigen, indicating an antigenspecific effect.

[0263] Example 8: In vitro T cell mediated tumor killing when T cells were primed using HORMAD1 mRNA loaded DC cells. The capacity of T cells primed against an alternative and shorter isoform of HORMAD1 to specifically induce cell death in HORMAD1 -expressing cancer cells was demonstrated using the following experiment. DCs generated from HLA-A2- positive anonymous healthy donor leukocytes according to the aforementioned method were loaded with mRNA encoding the HORMAD1 splice variant 204 and used as antigen-presenting cells to prime autologous T cells. T cells were subsequently co-incubated with H1650 cells, an HLA-A2-positive non-small cell lung cancer cell line. H1650 is known to express HORMAD1 mRNA, as documented in the CCLE public database (RPKM 3.28) and subsequently confirmed in our RNAseq analysis (normalized cpm 10.5). Tumor cell killing, quantified as the cell count of apoptotic tumor cells, was monitored in real-time. Apoptotic tumor cells were detected by a Caspase-cleavable fluorescent reporter. The data (Fig. 11) shows selective killing of tumor cells when T cells were primed beforehand with mRNA encoding HORMAD1 splice variant 204 loaded DCs. The effect is clearly above background cytotoxicity measured when T cells were primed with DCs loaded with mRNA encoding an irrelevant antigen, indicating an antigen-specific effect.

[0264] In conclusion, the examples provided demonstrate the effect of using HORMAD1 as a target for antigenspecific immunotherapy. To provide this demonstration, we used an mRNA loaded carrier such as an LNP or B-cell as well as a mRNA-modified dendritic cell vaccine approach to elicit HORMAD1 -specific tumor T-cells, resulting in highly target-specific killing of cancer cells in vitro, and deep suppression of a HORMAD1 -expressing human cancer xenograft in vivo.

[0265] In addition, the clinical example provided demonstrates that targeting HORMAD1 , as part of an mRNA- modified autologous DC therapy, is capable of triggering HORMAD1 -specific T-cell responses (CD8 and CD4) in a heavily pretreated advanced cancer patient. This effect is observed even after low doses of the vaccine where the tumor was also confirmed to express the HORMAD1 target antigen. REFERENCES

[0266] Brabants E, Heyns K, De Smet S, Devreker P, Ingels J, De Cabooter N, Debacker V, Dullaers M, VAN Meerbeeck JP, Vandekerckhove B, Vermaelen KY. An accelerated, clinical-grade protocol to generate high yields of type 1 -polarizing messenger RNA-loaded dendritic cells for cancer vaccination. Cytotherapy. 2018 Sep;20(9):1164-1181 .

[0267] Hellemans J, Vandesompele J, qPCR data analysis: unlocking the secret to successful results. PCR Troubleshooting and optimization: the essential guide. 2011.

[0268] Saviozzi S, Cordero F, Lo lacono M, Novello S, Scagliotti GV, Calogero RA, Selection of suitable reference genes for accurate normalization of gene expression profile studies in non-small cell lung cancer. BMC Cancer. 2006 Vol 6(200).

Claims

-46-CLAIMS1 . A carrier comprising mRNA encoding a HORMAD1 polypeptide.

2. The carrier according to claim 1 , wherein said carrier is selected from the group consisting of: a (lipid or polymeric) nanoparticle, a polymer, a peptide, and an antigen presenting cell (APC).

3. The carrier according to claim 2, wherein the APC is selected from the group consisting of: a dendritic cell (DC), a B-cell, a dendritic cell-line, a B-cell line, a macrophage, and a leukocyte with antigen- presenting properties.

4. The carrier according to any one of claims 1 to 3, wherein said carrier is a DC, in particular an autologous or allogenic DC.

5. The carrier according to any one of claims 1 to 4, wherein said HORMAD1 polypeptide is characterized by any one of SEQ ID NOs: 1-7, or a sequence having at least 85% sequence identity thereto.

6. The carrier according to any one of claim 1 to 5, wherein the mRNA is synthetic mRNA.

7. A combination comprising at least two different mRNA molecules each independently encoding for a HORMAD1 polypeptide and a PRAME polypeptide, in particular wherein said HORMAD1 polypeptide is characterized by any one of SEQ ID NOs: 1-7, or a sequence having at least 85% sequence identity thereto, and wherein said PRAME polypeptide is characterized by any one of SEQ ID NOs: 8-13, or a sequence having at least 85% sequence identity thereto.

8. The combination according to claim 7, wherein said mRNA molecules encoding for HORMAD1 and PRAME polypeptides are independently introduced in or associated with the same or a separate carrier, in particular a (lipid or polymeric) nanoparticle, a polymer, a peptide or an antigen presenting cell.

9. A pharmaceutical composition comprising the carrier according to any one of claims 1 to 6, or the combination of claims 7 or 8, and at least one pharmaceutically acceptable excipient; in particular wherein said pharmaceutical composition is a vaccine.

10. The carrier according to any one of claims 1 to 6, the combination of claims 7 or 8, or the pharmaceutical composition of claim 9, for use in human and / or veterinary medicine, in particular for use in therapy, more in particular for use in immunotherapy in a subject in need thereof.

11. The carrier according to any one of claims 1 to 6, the combination of claims 7 or 8, or the pharmaceutical composition of claim 9, for use in eliciting an immune response in cancer therapy, in particular a T cell response, in a subject in need thereof.

12. The carrier according to any one of claims 1 to 6, the combination of claims 7 or 8, or the pharmaceutical composition of claim 9, for use in stabilizing tumor growth or inducing tumor regression in a subject in need thereof.

13. The carrier according to any one of claims 1 to 6, the combination of claims 7 or 8, or the pharmaceutical composition of claim 9, for use in the treatment of cancer, in particular wherein cancer cells express HORMAD1 and optionally PRAME, as defined by a threshold of 1 transcript per mill; in particular wherein said cancer is lung cancer, more in particular NSCLC.

14. A method for preparing an immunotherapy agent comprising the steps of: a) obtaining a carrier; b) ex vivo modifying or loading said carrier comprising the introduction of an mRNA molecule encoding a HORMAD1 polypeptide; and c) harvesting the obtained carrier.

15. A method for the treatment of cancer, said method comprising the step of administering to a patient in need thereof, the carrier according to any one of claims 1 to 6, the combination of claims 7 or 8, or the pharmaceutical composition of claim 9.

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