Cancer immunotherapy

Abstract

The present invention relates to novel tumor-associated antigens (TAAs) useful in cancer vaccines. The present invention also relates to vaccines including such immunogens and / or nucleic acid molecules encoding the same. The present invention further relates to methods of using the vaccines for inducing immune responses and preventing and / or treating subjects having cancer cells or tumors that express these antigens, such as e.g. lung cancer.

Description

[0001] CANCER IMMUNOTHERAPY

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to novel tumor-associated antigens (TAAs) useful in cancer vaccines. The present invention also relates to vaccines including such immunogens and / or nucleic acid molecules encoding the same. The present invention further relates to methods of using the vaccines for inducing immune responses and preventing and / or treating subjects having cancer cells or tumors that express these antigens, such as e.g. lung cancer.

[0004] BACKGROUND TO THE INVENTION

[0005] Boosting a patient's immune system by immunotherapy represents a promising approach in cancer treatment. Cancer vaccines can prime the immune system to better recognize specific antigens, such as tumor-associated antigens (TAAs), for targeted destruction. In cancer vaccines, it is critical to obtain a strong cytotoxic T lymphocyte (CD8+) (Tc) response against the malignant cells. Naive Tc cells are selected and activated by dendritic cells (DC) to recognize selected antigens and, in the case of a vaccine, through cross-presentation. The activated Tc cells can then expand, traffic to tumors, recognize tumor cells displaying these antigens, and release chemical factors to induce cancer cell apoptosis. In conventional strategies, soluble TAAs (as recombinant proteins or peptides) are co-administered with DC activators / adjuvants to improve this T cell selection and activation process.

[0006] Cancer testis antigens (CTA, also known as “cancer germline antigens”) is a large protein family that is exclusively expressed in the testis, placenta and certain types of malignant tumor, and is involved in the regulation of critical processes during tumorigenesis and development. Shielded from the immune system by the blood-testis barrier, CTAs are categorized as immunogenic tumor-associated antigens and deemed optimal targets for the design of therapeutic cancer vaccines.

[0007] Immunotherapeutic strategies targeting CTAs include engineered T-cell receptor T-cell therapy, chimeric antigen receptor T-cell therapy and vaccine-based therapy. Cancer vaccines are an attractive complement or alternative to conventional cancer treatments with great prophylactic and therapeutic potential. Cancer vaccines stimulate tumor-specific immune responses through delivering tumor antigens into antigen-presenting cells (APCs) and induce vigorous anti-tumor immunity to inhibit tumor growth, recurrence and metastasis. Compared with other immunotherapeutic strategies, cancer vaccines provide specific, safe and tolerable control of cancer progression. Furthermore, nanomaterials have been utilized to design vaccine platforms, which improved the efficacy during antigen delivery, processing and presentation to T cells. A variety of cancer nanomaterial-based vaccines have been designed to deliver peptide / adjuvant or nucleic acid of CTAs.

[0008] In addition, vaccination with tumor antigen-loaded dendritic cells (DCs) represent 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 DC-based 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. Immune hot tumors are also inherently more sensitive to immune checkpoint inhibition (ICI), providing the basis for a synergistic combination of cancer vaccines with ICI.

[0010] The present invention provides a pharmaceutical composition directed to induce a specific adaptive immune response towards cancer cells expressing one or more antigens thereby resulting in tumor elimination or at least tumor growth stabilization or suppression.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention identified the proteins OIP5, PBK, CEP55, XAGE1 B, TTK and IGF2BP3 as cancer-testis antigens (CTAs), in particular as targets for therapy, more particular immunotherapy. Different protein-coding RNA splice variants for these proteins are provided in Tables 1-6 disclosed herein, and part of the present invention.

[0013] In an embodiment, the invention provides an antigen, and uses thereof, comprising or consisting of an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence selected from the group consisting of: SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 56; or an immunogenic fragment thereof, or a nucleic acid molecule encoding said antigen or fragment, and uses thereof.

[0014] In a further embodiment, the nucleic acid molecule comprises or consists of a nucleic acid sequence characterized by any one of SEQ ID NO: 19 to 30, 54 or 57. In an even further embodiment, the full nucleic acid constructs comprises or consists of a nucleic acid sequences characterized by any one of SEQ ID NO: 31 to 36, or 55.

[0015] In another embodiment, the invention provides a pharmaceutical composition comprising at least one antigen comprising or consisting of an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence selected from the group comprising SEQ ID NO: 1 to SEQ ID NO: 18 or SEQ ID NO: 56; or an immunogenic fragment thereof, wherein the immunogenic fragment comprises or consists of an amino acid sequence having 85% sequence identity to the amino acid sequence selected from the group comprising of SEQ ID NO: 37 to SEQ ID NO: 49; or a nucleic acid molecule encoding said antigen or immunogenic fragment.

[0016] Thus, herein provided is a pharmaceutical composition, optionally comprising a carrier, comprising a nucleic acid as characterized in Tables 1-6. More specific, the pharmaceutical composition comprises mRNA or splice variants encoding for the (full) sequence of OIP5, PBK, CEP55, XAGE1 B, TTK and IGF2BP3, which are cancer testis antigens expressed at high level in lung cancer. In one embodiment, provided herein is pharmaceutical composition comprising RNA molecules with splice variants encoding the longest polypeptide from the protein-coding gene, and / or wherein said RNA molecule is incorporated in a dendritic cell or nanoparticle. By introducing mRNA encoding for the full sequence of OIP5, PBK, CEP55, XAGE1 B, TTK and IGF2BP3 in a carrier such as dendritic cells, B-cells or nanoparticles, the present invention allows presentation of a broad range of epitopes from the full polypeptide sequence of each of these antigens, without requiring prior knowledge of donor HLA-type. In addition, the epitopes, e.g. when used as a part of a DC vaccine, are presented in a context of high T-cell costimulatory molecule expression as well as a cytokine / chemokine release driving type 1 polarized T-cell response, as required for effective anti-cancer immunity. Because of the expression pattern of these antigens, the resulting T-cell response is highly cancer-specific. In another specific embodiment, the mRNA molecules in the pharmaceutical composition or in the carrier are synthetic mRNA molecules.

[0017] In another embodiment, the invention provides a pharmaceutical composition comprising a nucleic acid molecule, in particular an mRNA, wherein the nucleic acid molecule comprises or consists of a nucleic acid sequence characterized by any one of SEQ ID NO: 19 to 36, 54, 55 or 57, in particular where SEQ ID NO: 31-36 and 55 are full mRNA constructs, which include sequences that regulate amongst others stability, location and translation efficiency.

[0018] In a further embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, and optionally an adjuvant. In particular, the pharmaceutical composition is an immunogenic composition, more in particular a vaccine, even more particular a cancer vaccine.

[0019] In one embodiment, the antigen is a recombinant protein.

[0020] The invention further provides a carrier, vector or host cell comprising the nucleic acids taught herein.

[0021] In another embodiment, the invention provides a carrier comprising the antigen or nucleic acid molecule taught herein. More 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 nanoparticle, such as a lipid nanoparticle (LNP) or polymeric nanoparticle, in particular a lipid nanoparticle. The carrier can be an antigen presenting cell (APC), e.g. as 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; in particular the carrier is a dendritic cell (DC), or B-cell.

[0022] In one embodiment the invention hence provides a carrier comprising a nucleic acid (such as DNA or RNA) encoding an antigen, wherein the antigen comprises an amino acid sequence at least 85%, at least 90%, or at least 95% identical to the amino acid sequence selected from the group consisting of: SEQ ID NO: 1 to SEQ ID NO: 18 or SEQ ID NO: 56; or a fragment thereof.

[0023] In a further specific embodiment, the present invention provides a pharmaceutical composition comprising a carrier, such as a DC, a B-cell or a nanoparticle, comprising a nucleic acid molecule encoding an amino acid sequence having at least 70% sequence identity to the amino acid sequence selected from the group comprising of: SEQ ID NO: 1 to SEQ ID NO: 18 or SEQ ID NO: 56.

[0024] The invention furthermore provides the antigen, nucleic acid molecule, carrier, vector, host cell, composition or vaccine for use in human or veterinary medicine.

[0025] The invention furthermore provides the antigen, nucleic acid molecule, carrier, vector, host cell, composition or vaccine for use in immunotherapy.

[0026] The invention furthermore provides the antigen, nucleic acid molecule, carrier, vector, host cell, composition or vaccine for use in treating cancer, in particular a cancer wherein the tumor cells express one or more of OIP5, PBK, CEP55, XAGE1 B, TTK and IGF2BP3. In a specific embodiment, the cancer is selected from the group consisting of: lung cancer, breast cancer, thyroid cancer, head and neck cancers, cancers of the gastro-intestinal tract, hepatocellular carcinoma, pancreatic cancer, renal cell carcinoma, bladder cancer, prostate cancer, testicular cancer, ovarian cancer, endometrial cancer, melanoma, glioblastoma, mesothelioma and hematological cancers.

[0027] The invention further provides a method of producing the recombinant proteins, nucleic acid molecules and vectors, in particular using a host cell. The invention also encompasses a method of preparing an immunotherapy agent, in particular a DC- or nanoparticle-based vaccine, said method comprising the step of loading isolated carriers such as nanoparticles or APCs, e.g. DCs, with a nucleic acid molecule encoding one or more of the antigens OIP5, PBK, CEP55, XAGE1 B, TTK or IGF2BP3, or antigens with an amino acid sequence having at least 85%, or at least 90%, in particular at least 95% sequence identity to the amino acid sequence selected from the group comprising of: SEQ ID NO: 1 to SEQ ID NO: 18 or SEQ ID NO: 56.

[0028] 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 or loading said carrier comprising the introduction of one or more mRNA molecules encoding an amino acid sequence having at least 85%, or at least 90%, in particular at least 95% sequence identity to the amino acid sequence selected from the group comprising of: SEQ ID NO: 1 to SEQ ID NO: 18 or SEQ ID NO: 56, or an immunogenic fragment thereof; c) harvesting the obtained carrier; and d) optionally formulating the carrier of (c) to be suitable for use in immunotherapy. In a particular embodiment, the carrier is a nanoparticle, such as a polymeric or lipid nanoparticle, or an antigen presenting cell, in particular a dendritic cell, T-cell or 13- cell.

[0029] 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 antigens and / or antigens expressed in certain cancer types (tumor antigens) selected from the list comprising: CLDN6, KK-LC-1 , MAGE- A3, MAGE-A4, PRAME, MAGE-CI, HORMAD1 , and NY-ESO-1.

[0030] The invention furthermore provides combinations of at least two antigens of the invention, in particular of at least three antigens of the invention (including nucleic acids encoding them), more in particular combinations comprising CEP55 and IGF2BP3, even more in particular combinations comprising CEP55, TTK and IGF2E3P3, or combinations comprising CEP55, XAGE1 E3, PE3K and IGF2E3P3. Said combinations are particularly relevant for the treatment of lung cancer.

[0031] The invention also provides a combination therapy of a pharmaceutical composition comprising one or more antigens of the present invention, or a pharmaceutical composition comprising one or more mRNA molecules encoding the antigens of the present invention; and a checkpoint inhibitor (such as a PD-1-, PD-L1-, PD-L2- or CTLA-4 inhibitor) and / or chemotherapeutic agent.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] 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.

[0034] Figure 1 : Expression of the antigens in H1650, a HLA-A2-positive human non-small cell lung cancer cell line, as measured by RNAseq.

[0035] Figure 2: Shotgun mass spectrometric analysis of human dendritic cells electroporated with mRNA encoding each antigen of the invention separately. Translated protein abundance is displayed as ranked iBAQ values.

[0036] Figures 3: In vitro tumor killing assay of H1650 NSCLC cells using T cells primed against targets of the invention by autologous mRNA-loaded DCs. Apoptotic tumor cell counts over time, measured from co-incubation of H1650 NSCLC target cells together with T-cells primed beforehand by autologous mRNA-loaded DCs. The mRNAs encode separate targets of the invention, as indicated: OIP5 (panel

[0037] A), PBK ( panel B), CEP55 ( panel C), XAGE1 B ( panel D), TTK ( panel E), or IGF2BP3 (panel F).

[0038] 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.

[0039] Figure 4. In vivo therapeutic tumor control using a mRNA-DC vaccine against targets of the invention. 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 non-small cell lung cancer (NSCLC) H1650 tumor cells. When palpable tumors were observed, mice were injected twice (indicated by the arrows below the X-axis) with the indicated DC conditions. Graph shows volumetric tumor size evolution for indicated treatment groups, i.e. treatment with DCs loaded with mRNA encoding: OIP5 ( panel A), PBK ( panel B), CEP55 ( panel C),

[0040] XAGE1 B ( panel D), TTK ( panel E) or IGF2BP3 ( panel F). Five to eight mice per treatment group were included. Shapiro-Wilk normality test was performed. Data were analyzed using Mann Whitney U- test or unpaired t-test with * p < 0.05, ** p < 0.01 , *** p < 0.001 and **** p < 0.0001 .

[0041] Figure 5. In vitro tumor killing assay of H2087 NSCLC cells using T cells primed against targets of the invention by autologous mRNA-loaded DCs. Apoptotic tumor cell counts over time, measured from co-incubation of H2087 NSCLC target cells together with T-cells primed beforehand by autologous mRNA-loaded DCs. The mRNAs encode separate targets of the invention, as indicated: OIP5 (panel A), PBK (panel B), CEP55 (panel C), XAGE1 B (panel D), TTK (panel E), IGF2BP3 (panel F). 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.

[0042] Figure 6. In vitro tumor killing assay of H1650 NSCLC cells using T cells primed against the OIP5 target of the invention by autologous OIP5 mRNA-LNP loaded DCs. Apoptotic tumor cell counts over time, measured from co-incubation of H1650 NSCLC target cells together with T cells primed beforehand by autologous mRNA-LNP loaded DCs. The mRNA encode the separate OIP5 target of the invention, covering the full open reading frame as represented by OIP5 splice variant 201 . 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.

[0043] Figure 7. In vitro tumor killing assay of H1650 NSCLC cells using T cells primed against the OIP5 target of the invention by autologous OIP5 mRNA-loaded B cells. Apoptotic tumor cell counts over time, measured from co-incubation of H1650 NSCLC target cells together with T cells primed beforehand by autologous mRNA-loaded B cells. The mRNA encode the separate OIP5 target of the invention, covering the full open reading frame as represented by OIP5 splice variant 201 . 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.

[0044] Figure 8. In vitro tumor killing assay using T cells primed against the OIP5 splice variant OIP5- 202. Apoptotic tumor cell counts over time, measured from co-incubation of H1650 NSCLC target cells together with T cells primed beforehand by autologous mRNA-loaded DCs. The mRNA encode the separate OIP5 target splice variant 202. 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 .

[0045] Figure 9. In vitro tumor killing assay using T cells primed against the OIP5 peptide VLADSVHLA. Apoptotic tumor cell counts over time, measured from co-incubation of H1650 NSCLC target cells together with T cells primed beforehand by autologous DC that were loaded with the OIP5-peptide VLADSVHLA (SEQ ID NO: 37). 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 .

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] Several benefits and advantages are provided by the present invention.

[0052] Usually, shared tumor antigens have been selected, which are over-expressed in the tumor but also expressed to some extent in normal tissues. The ideal cancer antigen should be highly cancer-restricted to ensure safety (no on-target I off-tumor effects) and efficacy (antigens shared with normal tissues are associated with high level of immune tolerance). The present invention makes use of an antigen which is part of the cancer-testis antigen class of proteins, which are not expressed in adult normal tissues except in the testis (an organ which is shielded from immune responses).

[0053] In addition, recombinant protein-based formulations mostly favor humoral (antibody-based) immune responses while effective tumor killing requires cellular immune responses. Also, antibodies can only potentially affect cancer cells if the target is displayed on the cell surface. Many cancer antigens, such as those specifically used in the present invention, are only expressed intracellularly and are only accessible for immune recognition following processing and presentation of derived fragments (epitopes) on MHC class I molecules. Of note, selecting for specific epitopes (peptides rather than full sequences) imposes restrictions in terms of the HLA-type and limits the number of patients that can be treated. By introducing mRNA encoding the full antigenic sequence in DCs obtained from a patient / donor, the antigen processing machinery of the DC can generate the full spectrum of epitopes that are intrinsically selected for that patients’ specific HLA type. These epitopes are presented at high densities on MHC class I and class II molecules in the context of high surface expression of T-cell costimulatory molecules (a key feature of activated DCs), which can avert immune tolerance and produce strong T-cell mediated immunity. Furthermore, classical vaccine formulations, when injected, target endogenous antigen-presenting cell system, foremost dendritic cells. However, in tumor-bearing hosts I cancer patients this system is known to be profoundly defective. The advantage of implementing the present invention as part of a dendritic cell vaccine approach is that it circumvents this defect by ensuring that antigen presentation is performed by highly immunogenic dendritic cells generated ex vivo and of which the immunogenicity is established prior to administration to the tumor-bearing host or cancer patient.

[0054] The present invention identified cancer-restricted target OIP5, PBK, CEP55, XAGE1 B, TTK and IGF2BP3 antigens, all with intracellular expression and no expression in adult normal tissues, except for gametogenic cells. Because of their highly cancer-restricted expression, and their role in several hallmarks oftumorigenesis these antigens are particularly useful in the development of cancer vaccines. Most of the CTA-targeted immunotherapies evaluated so far have focused on the use of MAGE- subfamily antigens, with variable success. Hence, the possibility to target non-MAGE CTAs may offer important additional options for cancer immunotherapy.

[0055] In a particular embodiment, the present invention provides a pharmaceutical composition comprising at least one non-MAGE cancer-testis antigen selected from the list comprising OIP5, PBK, CEP55, XAGE1 B, TTK and IGF2BP3, or an immunogenic fragment thereof. In a particular embodiment, the present invention also provides a carrier, or a pharmaceutical composition comprising such carrier, wherein the carrier includes one or more nucleic acid molecules encoding one or more of said non- MAGE cancer-testis antigens or immunogenic fragment thereof.

[0056] The pharmaceutical composition of the invention can comprise one or more of said antigens or nucleic acids encoding them, in particular two, more in particular three, even more in particular four antigens or nucleic acids encoding them. The pharmaceutical composition can comprise a carrier comprising one or more nucleic acid sequences, one or more amino acid sequences, or a combination thereof. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof. The antigen can be a recombinant antigen.

[0057] As described below, each ofthe targets is subject to alternative splicing, i.e. multiple RNA splice variants can be generated from one specific gene. Particularly when the vaccine consists of RNA, it is therefore important to select the most optimal splice variant for a specific disease indication. A splice variant can be selected based on the highest prevalence for a given tumor type. In addition, a splice variant can be selected based on the maximum number of potentially generated epitopes, e.g. translating into the longest amino acid sequence. Ideally, the optimal splice variant to be used in a vaccine maximizes both parameters.

[0058] 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”.

[0059] In one embodiment, the present invention identified OIP5 as a powerful target in cancer immunotherapy. As used herein, the human OIP5 gene encodes for the Opa interacting protein 5, also named Cancer / testis antigen 86 (CT86), HMIS18BETA or MIS18B. It is located on human chromosome 15q 15 and consists of five exons. Its mRNA sequence is 1249 bp long and encodes the OIP5 protein, which has a molecular weight of 25 kDa. OIP5 plays an important role in maintaining the structure and function of centromeres. Abnormal expression of OIP5 has been identified in several tumors, such as lung cancer, breast cancer, glioblastoma, hepatocellular carcinoma and bladder cancer.

[0060] Different protein-coding RNA splice variants have been found for this gene as shown in Table 1 .

[0061] Table 1

[0062] The longest amino acid sequence is generated from splice variant OIP5-201 (043482) represented by SEQ ID NO: 1.

[0063] The RNA sequence corresponding to OIP5-201 (043482) is represented by SEQ ID NO: 19. As mentioned before, and also relevant for the nucleic acid sequence further below, 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”.

[0064] In a further embodiment the invention provides a nucleic acid characterized by the Transcript ID given in Table 1 and uses thereof. More specific, the invention provides an RNA, in particular 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% sequence identity to the nucleotide sequence of SEQ ID NO: 19 or 25 or SEQ ID NO: 54.

[0065] With respect to non-small cell lung cancer, prevalence for each specific splice variant of OIP5 was analyzed using the TOGA public genomic expression database. Said 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 non-squamous lung cancer. Depending on the application and / or cancer type, a specific protein version or splice variant can be selected

[0066] In a further embodiment, the OIP5 polypeptide is OIP5-202 (H0YKL4) represented by SEQ ID NO: 2. The RNA sequence corresponding to OIP5-202 is represented by SEQ ID NO: 54.

[0067] In a further embodiment, the present invention identified PBK as a target in cancer immunotherapy.

[0068] As used herein, the human PBK gene encodes PDZ binding kinase, a TOPK_HUMAN Protein Lymphokine-activated killer T-cell-originated protein kinase, also named Cancer / testis antigen 84 (CT84), NORI-3, SPK or TOPK. Overexpression is found in breast cancer, lung cancer, gastric cancer and hepatocellular carcinoma. Overexpression of the PBK protein might be correlated with poor prognosis.

[0069] Different protein-coding RNA splice variants have been found for this gene as shown in Table 2.

[0070] Table 2

[0071] Particularly relevant is PBK-203 (Q96KB5-2) represented by SEQ ID NO: 4, PBK-205 (V9HWH0) represented by SEQ ID NO: 56, and PBK-201 (Q96KB5-1) represented by SEQ ID NO: 3.

[0072] The RNA sequence corresponding to PBK-203 (Q96KB5-2) is represented by SEQ ID NO: 26.

[0073] The RNA sequence corresponding to PBK-205 (V9HWH0) is represented by SEQ ID NO: 57. The RNA sequence corresponding to PBK-201 (Q96KB5-1) is represented by SEQ ID NO: 20.

[0074] In a further embodiment, the invention provides a nucleic acid characterized by the Transcript ID given in Table 2 and uses thereof. More specific, the invention provides an RNA, in particular 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% sequence identity to the nucleotide sequence of SEQ ID NO: 20, 26 or 57.

[0075] With respect to non-small cell lung cancer, prevalence for each specific splice variant of PBK was analyzed using the TOGA public genomic expression database. The analysis shows that the splice transcript PBK-201 (SEQ ID NO: 3) is 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.

[0076] In a further embodiment, the PBK polypeptide is, PBK-202 (E5RFX4) represented by SEQ ID NO: 5 In a further embodiment, the present invention identified CEP55 as a target in cancer immunotherapy. As used herein, the human CEP55 gene encodes a 464-amino acid centrosomal protein, also named Cancer / testis antigen 111 (CT111). Overexpression is found in lung cancer, colon cancer, liver cancer and oral cavity squamous cell carcinoma.

[0077] Different protein-coding RNA splice variants have been found for this gene as shown in Table 3.

[0078] Table 3

[0079] The longest amino acid sequence is generated from splice variant CEP55-201 (Q53EZ4-1) represented by SEQ ID NO: 6. The RNA sequence corresponding to CEP55-201 (Q53EZ4-1) is represented by SEQ ID NO: 21.

[0080] In a further embodiment, a nucleic acid characterized by the Transcript ID given in Table 3 and uses thereof. More specific, the invention provides the invention provides an RNA, in particular 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% sequence identity to the nucleotide sequence of SEQ ID NO: 21 or 27.

[0081] With respect to non-small cell lung cancer, prevalence for each specific splice variant of CEP55 was analyzed using the TOGA public genomic expression database. The analysis shows that the splice transcript resulting in the longest amino acid sequence (SEQ ID NO: 6) is 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.

[0082] In a further embodiment, the CEP55 polypeptide is CEP55-202 (H0Y432) represented by SEQ ID NO: 7. In a further embodiment, the CEP55 is Q53EZ4-2, represented by SEQ ID NO: 8.

[0083] In a further embodiment, the present invention identified XAGE1 B as a target in cancer immunotherapy. As used herein, the human XAGEI b gene encodes for X antigen family member 1 B, an 81-amino acid polypeptide, also named Cancer / testis antigen 12.1 (CT12.1). XAGEI b protein expression appears to be restricted to lung cancers, particularly adenocarcinomas.

[0084] Different protein-coding RNA splice variants have been found for this gene as shown in Table 4. Table 4

[0085] The longest amino acid sequences are generated from splice variants XAGE1 B-203 (Q9HD64-2) and XAGE1 B-205 (Q9HD64-2) represented by SEQ ID NO: 9. The RNA sequence corresponding to XAGE1 B-203 (Q9HD64-2) and XAGE1 B-205 (Q9HD64-2) is represented by SEQ ID NO: 22.

[0086] In a further embodiment, a nucleic acid characterized by the Transcript ID given in Table 4 and uses thereof. More specific, the invention provides the invention provides an RNA, in particular 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% sequence identity to the nucleotide sequence of SEQ ID NO: 22 or 28.

[0087] With respect to non-small cell lung cancer, prevalence for each specific splice variant of XAGE1 B was analyzed using the TOGA public genomic expression database. The analysis shows that of the two splice transcripts resulting in the longest amino acid sequence (SEQ ID NO: 9), mRNA variant ENST00000375616.6 is also the most prevalent in patients with NSCLC, in particular for the non- squamous histology where this protein is highly expressed.

[0088] In a further embodiment, the XAGE1 B polypeptide is XAGE1 B-202 (Q9HD64-4) represented by SEQ ID NO: 10.

[0089] In a further embodiment, the present invention identified TTK as a target in cancer immunotherapy.

[0090] As used herein, the human TTK gene encodes a dual specificity protein kinase TTK, also named Cancer / testis antigen 96 (CT96). Overexpression is found in 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), thyroid papillary cancer, hepatocellular cancer, pancreatic ductal adenocarcinoma, glioma and gastric cancer.

[0091] Different protein-coding RNA splice variants have been found for this gene as shown in Table 5. Table 5

[0092] The longest amino acid sequence is generated from splice variant TTK-202 (P33981-1) represented by SEQ ID NO: 11. The RNA sequence corresponding to TTK-202 (P33981-1) is represented by SEQ ID NO: 23. In a further embodiment, the invention provides a nucleic acid characterized by the Transcript ID given in Table 5 and uses thereof. More specific, the invention provides an RNA, in particular 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% sequence identity to the nucleotide sequence of SEQ ID NO: 23 or 29.

[0093] With respect to non-small cell lung cancer, prevalence for each specific splice variant of TTK was analyzed using the TOGA public genomic expression database. The analysis shows that the splice transcript resulting in the longest amino acid sequence (SEQ ID NO: 11) is 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.

[0094] In a further embodiment, the TTK polypeptide is TTK-208 (P33981-2) and TTK-201 (P33981-2) represented by SEQ ID NO: 12. In a further embodiment, the TTK polypeptide is TTK-205 (D6RIC6) represented by SEQ ID NO: 13, TTK-209 (D6REX1) represented by SEQ ID NO: 14, TTK-204 (D6RF82) represented by SEQ ID NO: 15 or TTK-211 (D6RFY1) represented by SEQ ID NO: 16. In a further embodiment, the present invention identified IGF2BP3 as a target in cancer immunotherapy. As used herein, the human IGF2BP3 gene encodes an insulin like growth factor 2 mRNA binding protein 3, also named Cancer / testis antigen 98 (CT98) or IMP-3, IMP3. Overexpression is found in non-small cell lung cancer (NSCLC) of both non-squamous and squamous cell histological subtypes, glioblastoma multiforma, head and neck squamous cell carcinoma, nasopharyngeal carcinoma, uterine corpus endometrial carcinoma, colon adenocarcinoma, esophageal cancer, mesothelioma and melanoma.

[0095] Different protein-coding RNA splice variants have been found for this gene as shown in Table 6.

[0096] Table 6

[0097] The longest amino acid sequence is generated from splice variant IGF2BP3-201 (000425-1) represented by SEQ ID NO: 17. The RNA sequence corresponding to IGF2BP3-201 (000425-1) is represented by SEQ ID NO: 24.

[0098] In a further embodiment, the invention provides a nucleic acid characterized by the Transcript ID given in Table 6 and uses thereof. More specific, the invention provides an RNA, in particular 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% sequence identity to the nucleotide sequence of SEQ ID NO: 24 or 30.

[0099] With respect to non-small cell lung cancer, prevalence for each specific splice variant of IGF2BP3 was analyzed using the TOGA public genomic expression database. The analysis shows that the splice transcript resulting in the longest amino acid sequence (SEQ ID NO: 17) is 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.

[0100] In a further embodiment, the IGF2BP3 polypeptide is IGF2BP3-218 (000425-2) represented by SEQ ID NO: 18.

[0101] In one embodiment, the invention provides proteins comprising or consisting of the amino acid sequence selected from the group comprising of SEQ ID NO: 1 to SEQ ID NO: 18 or SEQ ID NO: 56; or a fragment thereof.

[0102] The invention furthermore provides a nucleic acid molecule encoding one or more of the proteins or fragments of the invention as provided in Tables 1-6. More in particular the nucleic acid molecule comprises or consists of the nucleic acid sequence selected from the group comprising of SEQ ID NO: 19 to SEQ ID NO: 36, SEQ ID NO: 54-55 or SEQ ID NO: 57, or a fragment thereof. In a further embodiment of the present invention, the amino acid sequence of the polypeptide of the invention has at least 85% sequence identity such as 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 (e.g. SEQ ID NO: 1-18, or SEQ ID NO: 56) or as represented by the respective accession numbers.

[0103] In a yet further embodiment of the present invention, the nucleic acid sequence of the nucleic acid molecule of the invention has at least 70% sequence identity such as at least 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity, preferably at least 95% sequence identity, more preferably at least 99%, most preferably 100% sequence identity with the nucleic acid sequence as provided herein (e.g. SEQ ID NO: 19-36 or SEQ ID NO: 54-55 or SEQ ID NO: 57) or as represented by the respective accession numbers.

[0104] 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.

[0105] 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 protein 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:

[0106] Class Amino acid examples

[0107] Nonpolar Ala, Vai, Leu, Pro, Met, Phe, Trp, He Uncharged polar Gly, Ser, Thr, Cys, Tyr, Asn, Gin

[0108] Acidic Asp, Gly

[0109] Basic Lys, Arg, His

[0110] The differing amino acids can be conservative substitutions, and / or are located outside of immunodominant epitope(s) of the antigen fragment.

[0111] 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 antigens of the invention. For example, provided herein is any protein fragment (meaning a polypeptide sequence which is 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.

[0112] 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 and / or 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 850, 8 to 700, 8 to 600, 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 molecule. 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, at least 100 amino acids or more, at least 110 amino acids or more, at least 120 amino acids or more, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more, at least 190 amino acids or more, at least 200 amino acids or more, at least 210 amino acids or more, at least 220 amino acids or more, at least 230 amino acids or more, at least 240 amino acids or more, at least 250 amino acids or more, or at least 260 amino acids or more of a protein sequence disclosed herein. In some embodiments, the present invention provides a vaccine or a pharmaceutical composition comprising immunogenic fragments of the antigens selected from the list: OIP5, PBK, CEP55, XAGE1 B, TTK or IGF2BP3 or any combination thereof. In a particular embodiment, said immunogenic fragments include or are characterized by an amino acid sequence set forth as any one of SEQ ID NO: 37 to SEQ ID NO: 49, or a sequence having at least 85% sequence identity thereto. In another embodiment, a vaccine or a pharmaceutical composition is provided comprising one or more nucleic acid molecules, in particular mRNA, preferably incorporated in or associated with a carrier, such as an antigen presenting cell, wherein the nucleic acid molecule encodes one or more of said immunogenic fragments of said antigens, or including or encoding at least the amino acid sequence set forth as any one of SEQ ID NO: 37 to SEQ ID NO: 49, or a sequence having at least 85%, at least 90% or at least 95% sequence identity thereto.

[0113] “Fragment” as used herein with respect to nucleic acid sequences means a nucleic acid sequence or a portion thereof, that encodes a polypeptide capable of eliciting an immune response in a mammal that cross reacts with an antigen disclosed herein. The fragments can be DNA / RNA fragments selected from at least one of the various nucleotide sequences that encode protein fragments provided herein.

[0114] In some embodiments, fragments can comprise at least 20 nucleotides or more, at least 30 nucleotides or more, at least 40 nucleotides or more, at least 50 nucleotides or more, at least 60 nucleotides or more, at least 70 nucleotides or more, at least 80 nucleotides or more, at least 90 nucleotides or more, at least 100 nucleotides or more, at least 150 nucleotides or more, at least 200 nucleotides or more, at least 250 nucleotides or more, at least 300 nucleotides or more, at least 350 nucleotides or more, at least 400 nucleotides or more, at least 450 nucleotides or more, at least 500 nucleotides or more, at least 550 nucleotides or more, at least 600 nucleotides or more, at least 650 nucleotides or more, at least 700 nucleotides or more, at least 750 nucleotides or more of at least one of the nucleic acid sequences defined herein.

[0115] The antigen or fragment of the invention may also be referred to as isolated antigen or fragment. The term “isolated” is used to indicate that a cell, protein or nucleic acid is separated from its native environment. Isolated proteins and nucleic acids may be substantially pure, i.e. essentially free of other substances with which they may be 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.

[0116] The protein or peptides described herein can be chemically or recombinantly produced. Proteins or peptides can be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteins or peptides from natural sources, in vitro translation, or the chemical synthesis of proteins or peptides. Peptides can be readily synthesized chemically utilizing reagents that are free of contaminating bacterial or animal substances. For example, peptides can be prepared by (1) parallel solid-phase synthesis on multi-channel instruments using uniform synthesis and cleavage conditions; (2) purification over a P-HPLC column with column stripping; and rewashing, but not replacement, between peptides; followed by (3) analysis with a limited set of the most informative assays.

[0117] Alternatively, a nucleic acid (e.g., a polynucleotide) encoding a peptide or protein of the invention can be used to produce the protein or peptide in vitro. The polynucleotide can be, e.g., DNA, cDNA, PNA, CNA, RNA, mRNA either single-and / or double-stranded, or native or stabilized forms of polynucleotides. In one embodiment in vitro translation is used to produce the protein or peptide of the invention. Many exemplary systems for recombinant protein production exist that one skilled in the art could utilize. An expression vector capable of expressing a polypeptide can also be prepared. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression, if necessary, the DNA can be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host (e.g., bacteria), although such controls are generally available in the expression vector. The vector is then introduced into the host cell for cloning using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). One further aspect of the invention provides for a host cell or carrier comprising the nucleic acid or the vector as described herein. Representative host cells that may be used include bacterial cells, yeast cells, plant cells and animal cells (e.g. insect cells and mammalian cells, (e.g. derived from Chinese hamster (e.g. CHO), and human cell lines, such as HeLa). Introduction of a vector in a host cell can be affected by, e.g., calcium phosphate transfection, virus infection, DEAE-dextran-mediated transfection, lipofectamine transfection or electroporation, and any person skilled in the art can select and use an introduction method suitable for the expression vector and host cell used.

[0118] In one embodiment, the pharmaceutical composition of the invention comprises a recombinant protein antigen selected from the group consisting of OIP5, PBK, CEP55, XAGE1 b, TTK and IGF2BP3.

[0119] 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.

[0120] The present invention further relates to a nucleic acid molecule, encoding the proteins or peptides 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.

[0121] 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 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.

[0122] A “nucleic acid” can be double-stranded, partly double stranded, or single-stranded. Where singlestranded, 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 proteins or polypeptides.

[0123] Vector and cells

[0124] 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.

[0125] 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.

[0126] 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.).

[0127] In one embodiment, the pharmaceutical composition of the invention comprises a nucleic acid molecule encoding the antigen selected from the group consisting of OIP5, PBK, CEP55, XAGE1 B, TTK and IGF2BP3, or encoding the immunogenic fragment thereof.

[0128] 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.

[0129] In a particular embodiment of the present invention, the nucleic acid molecule provided herein may be delivered naked to the subject or cell. The term “naked” as used herein refers to nucleic acids that are substantially free of other macromolecules, such as lipids, polymers, and proteins. In some embodiments, a naked nucleic acid molecule such as an RNA, in particular mRNA, may not be formulated with other macromolecules to improve cellular uptake. Accordingly, the naked nucleic acid as taught herein may not be encapsulated in or associated with a delivery vehicle.

[0130] In another embodiment, the nucleic acid of the invention 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 pressure-based systems.

[0131] 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 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). In a particular embodiment, the carrier is a nanoparticle, more particular a lipid nanoparticle (LNP) or polymeric nanoparticle. 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 exogenous mRNA refers to mRNA introduced into cells / carriers from an external source, 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 polypeptides of the invention in an effective level that exceeds expression by promoter leakage.

[0132] In a particular embodiment, the carrier is an antigen presenting cell, more in particular a dendritic cell (DC), a B-cell, a dendritic cell-line, a B-cell line, a macrophage, and a leukocyte with antigen-presenting properties; most in particular a dendritic cell (DC) or 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 cDCs (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.

[0133] 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) or part thereof, a 3’ UTR, and optionally a poly(A) sequence.

[0134] In one aspect, the RNA is a synthetic RNA, including (chemical) modifications, 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 RNA 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 RNA 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 vaccine should direct the expression of the protein of interest and generate an immune response against the antigen or epitope(s) therein.

[0135] 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.

[0136] 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.

[0137] 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 favorable codons forthe 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 and / or U nucleotides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and / or U or contain a lower content of A and / or U nucleotides. For example, the G / C 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 G / C content of the coding region of the wild type RNA.

[0138] The term “antigen” or “immunostimulatory protein or peptide” 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 present invention is based at least in part on the understanding that nucleic acid-encoded antigens, when expressed from DNA / RNA / mRNA administered to a cell or subject, in particular encompassed by a carrier, 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 and / or neutralizing antibodies, can trigger B- and / or T-cell responses specific to the expressed antigen, and ultimately can cause a therapeutic and / or a protective (prophylactic) response against (subsequent) encounter with the antigen.

[0139] The vaccine technology described herein features nucleic acids, particularly messenger RNA (mRNA) designed to encode an antigen of interest or an immunogenic fragment thereof. The pharmaceutical compositions of the present disclosure comprise one or more mRNAs having an open reading frame (ORF) encoding one or more antigens selected from the group consisting of OIP5, PBK, CEP55, XAGE1 B, TTK and IGF2BP3. 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 or TGA, or UAA, UAG or UGA). An ORF typically encodes a protein. Some short open reading frames (sORFs), also known as small open reading frames (smORFs), usually < 100 codons in length, can produce functional peptides. 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 alpha-globins 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. Accordingly, in one embodiment, the RNA molecule is flanked by a 5’ and 3’ untranslated region (UTR) designed to optimize protein translation. In a further embodiment, the 3' end of the RNA molecule is composed of a poly(A) tail, a chain of adenosine residues, which has several critical roles in both mRNA stability and translation. 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.

[0140] In specific embodiments, the RNA molecule is a mRNA comprising at least, from 5’ to 3’,

[0141] (i) a 5’-cap or cap analog,

[0142] (ii) a 5-untranslated region (UTR), (iii) an open reading frame (ORF) including coding sequences as described herein and, optionally intron (non-coding) sequences,

[0143] (iv) a 3’ UTR, and

[0144] (v) optionally, a poly(A) sequence.

[0145] 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.

[0146] In a further embodiment, the nucleic acid molecule is an mRNA molecule 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% sequence identity to the nucleotide sequence of SEQ ID NO: 19 to SEQ ID NO: 24. Examples of codon-optimized sequences as used in the present invention are represented by SEQ ID NO: 25 to SEQ ID NO:30.

[0147] Hence, in another embodiment, the nucleic acid molecule is an optimized mRNA molecule 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% sequence identity to the nucleotide sequence of SEQ ID NO: 25 to SEQ ID NO: 30.

[0148] The RNA (including mRNA) of the present disclosure encoding the antigen can be delivered directly to a desired subject (with or without a carrier as disclosed herein before), or can be delivered ex vivo to cells obtained or derived from a subject, and the cells can be (re-)implanted into another or the same subject. In a particular embodiment of the invention, the cells are antigen presenting cells (APCs), more specific APCs selected from the group consisting of: a dendritic cell (DC) or a B-cell, a dendritic cellline, a B-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 particular embodiment, the APC is a DC, a B-cell or a combination thereof.

[0149] 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:

[0150] Antigen capture: APCs capture antigens from their surrounding environment. Dendritic cells, for example, are specialized in capturing antigens at sites of infection or inflammation.

[0151] Antigen processing: Once captured, APCs process antigens by breaking them down into smaller fragments. This process typically occurs within cellular compartments called lysosomes.

[0152] 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.

[0153] 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.

[0154] 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 mRNA molecules encoding one or more polypeptides selected from OIP5, XAGEI b, CEP55, PBK, TTK and IGF2BP3, or immunogenic fragments thereof.

[0155] In a specific embodiment, the present invention provides a pharmaceutical composition comprising at least:

[0156] (a) one antigen comprising or consisting of an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to the amino acid sequence selected from the group comprising SEQ ID NO: 6-8;

[0157] (b) an immunogenic fragment of the antigen of (a) wherein the immunogenic fragment comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to the amino acid sequence selected from the group comprising of SEQ ID NO: 42-43; or

[0158] (c) a nucleic acid molecule encoding the antigen of (a) or the immunogenic fragment of (b), in particular a nucleic acid molecule comprising or consisting of a nucleic acid sequence having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to the nucleic acid sequence selected from the group comprising SEQ ID: 21 , 27, or 33.

[0159] Said pharmaceutical compositions are particularly useful for patients with a CEP55-positively identified tumor and which are HLA-A2 positive.

[0160] In a specific embodiment, the present invention provides a pharmaceutical composition comprising at least:

[0161] (a) one antigen comprising or consisting of an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to the amino acid sequence selected from the group comprising SEQ ID NO: 17-18;

[0162] (b) an immunogenic fragment of the antigen of (a) wherein the immunogenic fragment comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to the amino acid sequence selected from the group comprising of SEQ ID NO: 46-49; or

[0163] (c) a nucleic acid molecule encoding the antigen of (a) or the immunogenic fragment of (b), in particular a nucleic acid molecule comprising or consisting of a nucleic acid sequence having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to the nucleic acid sequence selected from the group comprising SEQ ID: 24, 30 or 36.

[0164] Said pharmaceutical compositions are particularly useful for patients with a IGF2BP3-positively identified tumor and which are HLA-A2 positive.

[0165] Combinations

[0166] In a second aspect of the invention, it can be of particular advantage to combine one or more (i.e. 1 , 2, 3, 4, 5 or 6) of the mRNA encoded OIP5, PBK, CEP55, XAGE1 B, TTK and IGF2BP3 such as for example but not limited to CEP55 combined with IGF2BP3 and / or other immunostimulatory polypeptides.

[0167] In yet another aspect of the invention, it can be of particular advantage to combine one or more (i.e. 1 , 2, 3, 4, 5 or 6) of the mRNA encoded OIP5, PBK, CEP55, XAGE1 B, TTK and IGF2BP3 with other mRNA encoded antigens, in particular antigens and / or antigens expressed in certain cancer types (tumor antigens). Examples of suitable tumor antigens are CLDN6, KK-LC-1 , MAGE-A3, MAGE-A4, PRAME, MAGE-CI, HORMAD1 , or NY-ESO-1 .

[0168] Where a combination is used, the invention provides that these are either used as a combination of polypeptides or mRNA molecules encoding these antigens, either as one naked mRNA or introduced in the same carrier, e.g. DC or nanoparticle, or a combination of mRNA molecules encoding these antigens, yet introduced as separate naked mRNAs or in different carriers, e.g. DCs or nanoparticles. The antigens or mRNAs may then be combined and administered as a single vaccine or pharmaceutical composition (e.g., comprising multiple RNA encoding multiple antigens) or may be administered separately.

[0169] Accordingly, the present invention also provides a kit, combination or composition comprising a carrier comprising or consisting of an mRNA encoding for two or more polypeptides of the invention selected from the group consisting of OIP5, IGF2BP3, CEP55, PBK, TTK, and XAGE1 B, and one or more carriers each independently comprising a mRNA molecule encoding a (immunostimulatory) polypeptide or antigen, in particular OIP5, IGF2BP3, CEP55, PBK, TTK, and XAGE1 B 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 or a B-cell.

[0170] Additionally, the invention provides a combination comprising or consisting of two or more (two, three, four) of the polypeptides IGF2BP3, CEP55, PBK and XAGE1 B, in particular a combination of two or more mRNA molecules encoding for these resp. polypeptides, more in particular wherein said polypeptide is characterized by SEQ ID NO: 3-10, 17 or 18, or a sequence having at least 85%, at least 90%, or at least 95% sequence identity thereto. In another embodiment, the invention provides a combination or pharmaceutical composition comprising at least the polypeptides IGF2BP3 and CEP55, in particular a combination or pharmaceutical composition comprising or consisting of the polypeptides IGF2BP3, CEP55, and XAGE-1 B, in particular a combination or pharmaceutical composition comprising or consisting of the polypeptides IGF2BP3, CEP55, and PBK more in particular a combination or pharmaceutical composition comprising or consisting of the polypeptides IGF2BP3, CEP55, XAGE-1 B and PBK; or a combination or pharmaceutical composition comprising or consisting of the polypeptides IGF2BP3, CEP55 and TTK, in particular a combination or pharmaceutical composition comprising or consisting of the polypeptides IGF2BP3, CEP55 and MAGEA3, more in particular a combination of pharmaceutical composition comprising or consisting of the polypeptides IGF2BP3, CEP55, TTK and MAGEA3.

[0171] Different protein-coding RNA splice variants have been found for MAGEA3:

[0172] Transcript ID Name bp protein CCDS UniProt

[0173] Match

[0174] ENST00000370278.4 MAGEA3-201 1682 314 aa CCDS76045 P43357

[0175] ENST00000933889.1 MAGEA3-204 1914 314 aa CCDS76045

[0176] ENST00000598245.2 MAGEA3-203 1762 314 aa CCDS76045 P43357

[0177] ENST00000417212.5 MAGEA3-202 853 198 aa E7EMU0

[0178] The longest amino acid sequence is generated from splice variant MAGEA3-201 and MAGEA3-203 (P43357) represented herein by SEQ ID NO:50. The amino acid sequence for MAGEA3-202 (E7EMU0) is represented by SEQ ID NO:51. The RNA sequence corresponding to MAGEA3-201 , MAGEA3-203 and MAGEA3-204 is represented by SEQ ID NO:52.

[0179] More specific, the invention provides a combination of two or more mRNA molecules encoding for one or more of the polypeptides PBK, CEP55, XAGE1 B, TTK, IGF2BP3, MAGEA3 more in particular wherein said mRNA is characterized by respectively SEQ ID NO: 20 or 26 or 32, SEQ ID NO: 21 or 27 or 33, SEQ ID NO: 22 or 28 or 34, SEQ ID NO: 23 or 29 or 35, SEQ ID NO: 24 or 30 or 36; and SEQ ID NO: 52 or 53, or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. Preferred mRNA sequences are selected from SEQ ID NO: 20-24, and 52. Said combination can be particularly useful for use in the treatment lung adenocarcinoma (LUAD), more particular for use in the treatment of lung squamous cell carcinoma (LUSC) or lung non-squamous cell carcinoma, in particular wherein said mRNAs are incorporated in or associated with a carrier, in particular a DC or a nanoparticle such as an LNP. In a further specific embodiment, herein provided is a vaccine or pharmaceutical composition comprising two or more polypeptides or mRNA molecules encoding for at least IGF2BP3, CEP55, XAGE1 B, optionally in combination with the polypeptides or mRNAs encoding for either one of PBK, OIP5, HORMAD1 (UniProt Q86X24-1 ; Acc. No. NM_032132.5; including splice variants) or any combination thereof, in particular wherein said mRNAs are incorporated in or associated with a carrier, in particular a DC or nanoparticle such as an LNP or polymeric nanoparticle. Furthermore, the invention provides a combination comprising or consisting of two or more (two, three, four) of the polypeptides CEP55, TTK, IGF2BP3, and MAGEA3, in particular a combination of two or more mRNA molecules encoding for these resp. polypeptides, more in particular wherein said polypeptide is characterized by SEQ ID NO: 6-8, SEQ ID NO: 11-16, SEQ ID NO: 17-18, and SEQ ID NO: 50-51 (or having Accession number NM_005362.3), or a sequence having at least 85%, at least 90%, or at least 95% sequence identity thereto. Said combination can be particularly useful for use in the treatment of lung squamous cell carcinoma (LUSC) or lung non-squamous cell carcinoma.

[0180] Accordingly, and in a particular embodiment, herein provided is a vaccine or pharmaceutical composition comprising two or more mRNA molecules encoding for at least CEP55 in combination with the same or a separate mRNA encoding for either one of IGF2BP3, OIP5, PBK, TTK, MAGEA3, or XAGE1 B, or any combination thereof; or a vaccine or pharmaceutical composition comprising two or more mRNA molecules encoding for at least IGF2BP3 in combination with the same or a separate mRNA encoding for either one of CEP55, OIP5, PBK, TTK, MAGEA3 or XAGE1 B, or any combination thereof.

[0181] In a preferred embodiment, provided herein is a vaccine or pharmaceutical composition comprising two or more mRNA molecules encoding for at least CEP55 and IG2BP3. In another embodiment, said mRNAs are incorporated in or associated with a carrier, in particular a DC, a B-cell or nanoparticle. In particular, said composition may comprise the same or separate mRNAs encoding for at least CEP55 in combination with IGF2BP3; alternatively CEP55 / IGF2BP3 in combination with OIP5; alternatively CEP55 / IGF2BP3 in combination with PBK; alternatively CEP55 / IGF2BP3 in combination with TTK; alternatively CEP55 / IGF2BP3 in combination with XAGE1 B; alternatively CEP55 / IGF2BP3 in combination with MAGEA3. In particular any combination is provided herein of CEP55 / IGF2BP3 in combination with one or more selected from: OIP5, PBK, TTK, MAGEA3 or XAGEI .

[0182] For example, Vaccine-001 as described is provided herein comprising 4 mRNA-encoded antigens for non-squamous NSCLC: XAGE-1 b, CEP55, PBK and IGF2BP3, in particular wherein said mixture comprises 4 equal fractions of mRNA-modified autologous monocyte-derived dendritic cells (moDCs). Also as an example, Vaccine-002 as described herein is provided comprising 4 mRNA-encoded antigens for squamous NSCLC: MAGE-A3, CEP55, TTK and IGF2BP3, in particular wherein said mixture comprises 4 equal fractions of mRNA-moDCs.

[0183] Therefore, in one embodiment, the present invention provides a combination or pharmaceutical composition comprising one or more mRNA molecules encoding at least the polypeptides XAGE-1 b, CEP55, PBK and IGF2BP3, in particular wherein the one or more mRNA molecules encoding the amino acids have a nucleic acid sequence having at least 85%, or at least 90%, in particular at least 95% sequence identity to the nucleic acid sequence as set forth in respectively SEQ ID NO: 22 (XAGE-1 b), SEQ ID NO: 21 (CEP55), SEQ ID NO: 26 (PBK) and SEQ ID NO: 24 (IGF2BP3). Such a combination or pharmaceutical composition is particularly suitable for use in the treatment with of non-squamous NSCLC. Therefore, in one embodiment, the present invention provides a combination or pharmaceutical composition comprising one or more mRNA molecules encoding at least the polypeptides MAGE-A3, CEP55, TTK and IGF2BP3, in particular wherein the one or more mRNA molecules encoding the amino acids have a nucleic acid sequence having at least 85%, or at least 90%, in particular at least 95% sequence identity to the nucleic acid sequence as set forth in respectively SEQ ID NO: 52 (MAGE-A3), SEQ ID NO: 21 (CEP55), SEQ ID NO: 23 (TTK) and SEQ ID NO: 24 (IGF2BP3). Such a combination or pharmaceutical composition is particularly suitable for use in the treatment with of squamous NSCLC.

[0184] The antigens and combinations of the present invention can induce an effective immune response which can be further strengthened by combination of the vaccine platform (polypeptide or nucleic acid vaccine, or mRNA loaded carrier as provided herein) with state-of-the art immunotherapies (e.g. checkpoint inhibitors) or chemotherapy. In a specific embodiment, the one or more vaccines of the invention are 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. Approved anti-CTLA4 antibodies are known under the name "ipilimumab" (BMS), and tremelimumab (AstraZeneca).

[0185] Another important immune-checkpoint receptor as defined herein is programmed cell death protein 1 (PD-1), 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-PD-1 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).

[0186] Hence, the invention also provides a combination therapy of (i) a carrier comprising one or more mRNA molecules encoding two or more of the antigens CEP55, IGF2BP3, OIP5, PBK, XAGE1 B, or TTK; or (ii) one or more mRNA molecules encoding one or more of the antigens CEP55, IGF2BP3, OIP5, PBK, XAGE1 B, or TTK and one or more mRNA molecules encoding at least one of MAGEA3, CLDN6, KK- LC-1 , PRAME, MAGE-CI, HORMAD1 , or NY-ESO-1 ; or the combination of (i) or (ii) together with a checkpoint inhibitor (such as a PD-1-, PD-L1-, PD-L2- or CTLA-4 inhibitor) and / or chemotherapeutic agent.

[0187] 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.

[0188] 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.

[0189] Examples of chemotherapeutic agents are cisplatin, carboplatin, pemetrexed, vinorelbine, gemcitabine, paclitaxel (Taxol), and docetaxel (Taxotere). 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).

[0190] Immunogenic compositions

[0191] In another aspect, the present invention provides a pharmaceutical composition, e.g. an immunogenic composition, or kit containing an antigen or RNA molecule as described herein, and one or more pharmaceutically acceptable excipients. In one embodiment, the pharmaceutical composition is a vaccine, more 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 specifically in cancer immunotherapy.

[0192] 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:

[0193] 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.

[0194] 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.

[0195] Maturation of dendritic cells: the dendritic cells are matured or activated in the laboratory ortargeted in vivo 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.

[0196] - 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.

[0197] 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.

[0198] 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, B-cell or nanoparticle, or the composition or the combination as defined herein, and at least one pharmaceutically acceptable excipient.

[0199] 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. 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.

[0200] Another approach encompasses direct parenteral injection of mRNA with or without a carrier / delivery system. The carrier can be a liposome, a lipid micro- or nanoparticle, a (biodegradable) polymeric micro- or nanoparticle, an exosome, a cationic nano-emulsion, a cationic peptide or polymer, polyplexes or viral-based delivery systems, wherein the mRNA molecule is encapsulated, entrapped or complexed.

[0201] 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.

[0202] 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).

[0203] Uses

[0204] The present invention provides the antigen, the nucleic acid molecule, vector or host cell as disclosed herein, optionally in combination with a carrier (incorporated in or associated with) as provided herein, 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 provide 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.

[0205] The present invention further provides the antigen, the nucleic acid molecule, vector, host cell, the composition, or the combination, 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.

[0206] 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.

[0207] - 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.

[0208] - 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.

[0209] In particular, a satisfactory cancer vaccine requires induction of a type 1 polarized effector T-cell response with sufficient affinity to cancer antigen-derived epitopes. 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+ cell carries cytotoxic granules wherein perforin and granzymes are stored. Upon specific recognition of antigen- derived epitopes on MHC class I molecules on the cancer cell surface, degranulation of the CD8 T-cell occurs with release of perforin which creates pores in the cancer target cell membrane. This allows granzyme to penetrate into the target cell cytoplasm where it enzymatically cleaves caspases, hereby triggering an apoptotic cascade ending in cancer cell death. In the present examples, cancer antigenspecific tumor cell killing by vaccine-primed CD8 T-cells is demonstrated using an in vitro tumor cell killing assay as well as in vivo by measuring growth of a tumor xenograft.

[0210] 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 antigen 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 one or more mRNA molecules encoding for OIP5, PBK, CEP55, XAGE1 B, TTK, IGF2BP3, or any combination thereof, or the amino acid sequence as set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 18 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, either or not 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.

[0211] The present invention further provides the antigen, the nucleic acid molecule, vector, host cell, composition, or the combination, as defined herein, for use in preventing tumor progression or for use in stabilizing tumor growth or inhibiting / reducing tumor size and / or growth 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 Tumours) 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.

[0212] The present invention further provides the antigen, the nucleic acid molecule, vector, host cell, the composition, or the combination, as defined herein, for use in therapy, in particular immunotherapy in a subject in need thereof. 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.

[0213] For example, OIP5, PBK, CEP55, XAGE1 B, TTK and / or IGF2BP3 based vaccines can be used to elicit an immune response against one or more of OIP5, PBK, CEP55, XAGE1 B, TTK and IGF2BP3 proteins expressed by 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 as provided in the present disclosure.

[0214] The present invention further provides the antigen, the nucleic acid molecule, vector, host cell, the composition, or the combination, as defined herein, for use in the treatment of cancer, in particular wherein cancer cells express OIP5, PBK, CEP55, XAGE1 B, TTK and IGF2BP3, and optionally another immunostimulatory polypeptide, such as CLDN6, KK-LC-1 , MAGE-A3, MAGE-A4, PRAME, MAGE-CI, HORMAD1 , or NY-ESO-1 ; in particular wherein said cancer is lung cancer, more in particular NSCLC (non-small cell lung cancer), e.g., advanced or metastasized non-small cell lung cancer, such as non- squamous and squamous cell carcinoma. In one embodiment, the cancer is unresectable Stage III or metastatic Stage IV NSCLC.

[0215] 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. The antigens used in the present invention are expressed in a large set of different cancers. Examples are given below and are not limitative.

[0216] Examples of cancers expressing OIP5 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), breast cancer, glioblastoma, hepatocellular carcinoma, colorectal cancer and bladder cancer.

[0217] Examples of cancers expressing PBK (also known as TOPK) 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) and prostate cancer.

[0218] Examples of cancers expressing CEP55 are lung cancer, breast cancer, colon cancer, liver cancer and oral cavity squamous cell carcinoma.

[0219] Examples of cancers expressing XAGEI b are lung cancer, particularly adenocarcinoma, melanoma, ovarian cancer and gastric cancer.

[0220] Examples of cancers expressing TTK 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), thyroid papillary cancer, hepatocellular cancer, pancreatic ductal adenocarcinoma, glioma and gastric cancer.

[0221] Examples of cancers expressing IGF2BP3 are non-small cell lung cancer (NSCLC) of both non- squamous and squamous cell histological subtypes, glioblastoma multiforma, head and neck squamous cell carcinoma, nasopharyngeal carcinoma, uterine corpus endometrial carcinoma, colon adenocarcinoma, esophageal cancer, mesothelioma and melanoma.

[0222] 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).

[0223] In one embodiment, the cancer is found to express one or more antigens selected from the list OIP5, PBK, CEP55, XAGE1 B, TTK or IGF2BP3, or any combination thereof, when a threshold of 1 transcript per million is determined e.g. by using the method of the present examples.

[0224] 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.

[0225] The three main subtypes of non-small cell lung cancer are:

[0226] Non-Squamous Cell Carcinoma (Adenocarcinoma): this is the most common subtype of NSCLC, often found in the outer regions of the lungs. Adenocarcinoma can also occur 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.

[0227] 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.

[0228] 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 (in the shape of immune checkpoint blockade). 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.

[0229] The present invention also provides a method for reducing the symptoms of or for the treatment of cancer, in particular wherein the cancer cells express the polypeptides of the invention, more in particular lung cancer, even more in particular NSCLC, said method comprising administering to a subject in need thereof the antigen, the nucleic acid molecule, carrier, vector, host cell, the composition, or the combination, as defined herein.

[0230] 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 antigen, the nucleic acid, carrier, vector, host cell, the composition, or the combination, as defined herein.

[0231] The invention further provides a method of inducing an immune response in a subject comprising: administering to a subject in need of such treatment, an RNA such as a mRNA, preferably incorporated in or associated with a carrier, such as an antigen presenting cell, wherein the RNA encodes one or more of the antigens selected from the list: OIP5, PBK, CEP55, XAGE1 B, TTK or IGF2BP3 or any combination thereof, more specific a polypeptide characterized by an amino acid sequence set forth as any one of SEQ ID NO:1 to SEQ ID NO:18 or SEQ ID NO: 56, or a sequence having at least 85% sequence identity thereto, or an immunogenic fragment thereof characterized by an amino acid sequence set forth as any one of SEQ ID NO: 37 to SEQ ID NO: 49, or a sequence having at least 85% sequence identity thereto, in an amount effective to induce an immune response in the subject.

[0232] 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:

[0233] (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;

[0234] (2) inhibiting the disease symptom, i.e., arresting its development; or

[0235] (3) relieving the disease symptom, i.e., causing regression of the disease or symptom, e.g. regression of a tumor or of tumor growth.

[0236] 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.

[0237] 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.

[0238] 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 RNA (including mRNA) molecules encoding one or more polypeptides in said antigen presenting cell, characterized in that amongst the polypeptide at least OIP5, PBK, CEP55, XAGE1 B, TTK or IGF2BP3, or any combination thereof, 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.

[0239] 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 RNA (including mRNA) molecule encoding OIP5, PBK, CEP55, XAGE1 B, TTK or IGF2BP3, or any combination thereof, is introduced in said dendritic cells.

[0240] Also provided are nucleic acid molecules as described herein, wherein the nucleic acid molecule is incorporated into a plasmid or vector. In some embodiments, the nucleic acid molecule may be operably linked to a regulatory element selected from a promoter and a poly-adenylation signal. In some embodiments, the promoter is a human cytomegalovirus immediate-early promoter (hCMV promoter). In still other embodiments, the poly-adenylation signal may be a bovine growth hormone polyadenylation signal (bGH poly A). In still further embodiments, the nucleic acid molecule may be incorporated into a viral vector. 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:

[0241] Viral Vectors:

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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. 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.

[0248] 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.

[0249] The present invention also provides a method for preparing an immunotherapy agent comprising the steps of obtaining a carrier and ex vivo loading or introducing in said carrier a nucleic acid as characterized in any one of Tables 1-6, and optionally transfer the nucleic acid-loaded carrier into a suitable buffer for storage or administration.

[0250] More specific, 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 of one or more mRNA molecules encoding OIP5, PBK, CEP55, XAGE1 B, TTK or IGF2BP3, in particular encoding the amino acid sequence having at least 85%, or at least 90%, in particular at least 95% sequence identity to the amino acid sequence selected from the group comprising of: SEQ ID NO: 1 to SEQ ID NO: 18 or SEQ ID NO: 56, or an immunogenic fragment thereof; c) harvesting the obtained RNA-loaded carrier. Optionally, the RNA- loaded carrier is transferred into a suitable buffer for storage or administration.

[0251] In another further embodiment, the method further includes ex vivo modifying another or the same carrier by introduction of one or more nucleic acids, such as mRNA molecules, encoding antigens and / or antigens expressed in certain cancer types (tumor antigens) selected from the list comprising: CLDN6, KK-LC-1 , MAGE- A3, MAGE-A4, PRAME, MAGE-CI, HORMAD1 , and NY-ESO-1. In another further embodiment, the carrier is an antigen presenting cell or a nanoparticle, in particular a dendritic cell, a B cell or a (lipid or polymeric) nanoparticle.

[0252] The invention thus also provides a method for preparing an immunotherapy agent comprising the steps of: a) obtaining or culturing antigen presenting cells, in particular dendritic cells; b) ex vivo modifying said antigen presenting cells of step a) comprising the introduction of an RNA encoding OIP5, PBK, CEP55, XAGE1 B, TTK or IGF2BP3 of the invention, in particular encoding an amino acid sequence having at least 85%, at least 90% or at least 95% sequence identity to the amino acid sequence selected from the group comprising of: SEQ ID NO: 1 to SEQ ID NO: 18 or SEQ ID NO: 56; c) harvesting the obtained antigen presenting cells.

[0253] In a particular embodiment, the antigen presenting cells of step a) are dendritic cells or B-cells, more in particular mature dendritic cells. 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 a nucleic acid (DNA or RNA) encoding OIP5, PBK, CEP55, XAGE1 B, TTK or IGF2BP3, including splice variants or fragments as specified herein.

[0254] Administration

[0255] The antigen, nucleic acid molecule, vector, host cell, carrier, composition, or combination of the invention 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.

[0256] In a particular embodiment, the composition, e.g. the dendritic cell-based vaccine, can be administered with physiologically acceptable excipients, buffers, diluents, adjuvants, immunomodulators, etc. In a particular embodiment, 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 OIP5, PBK, CEP55, XAGE1 B, TTK or IGF2BP3 included in the vaccine (i.e. immunomonitoring), along with the clinical evolution using measurable parameters (survival metrics, radiological tumor burden by regular or immune-related RECIST criteria, tumor markers, circulating tumor cells, plasma circulating tumor DNA, radiomics or other surrogate markers of disease load or disease activity).

[0257] It is well known to those skilled in the art that there is no evidence for a preferred dose of the vaccine be administered to achieve a specific level of biological and / or clinical effect. Likewise, no clear doselimiting toxicity (DLT) has been observed and accordingly no maximal tolerated dose (MTD) has been observed. In case the vaccine comprises DCs (such as e.g. monocyte-derived DCs), 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.

[0258] The vaccine may be administered as part of a combinatorial regimen, i.e. administered before, during or as an adjuvant / consolidation therapy in the setting of the following cancer treatments alone or combined: surgery, radiotherapy, chemotherapy, antibody-based targeted therapy (including antibodies to cell surface antigens, immune checkpoint inhibitors, and / or angiogenic factors), small molecule-based targeted therapy (eg kinase inhibitors), metabolic checkpoint inhibitors (including inhibitors of indoelamine-deoxygenase, adenosine production or adenosine receptor, Arginase-1), antibody-drug conjugates, bispecific T-cell engagers (BiTEs), chimeric antigen receptor (CAR) T-cells (CAR-T), TCR- transgenic T-cells, NK-cell based therapy, chimeric antigen receptor macrophages (CAR-Mac).

[0259] 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.

[0260] 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.

[0261] EXAMPLES

[0262] Materials and Methods

[0263] Tumor cell lines

[0264] H-1650 and H-2087 were obtained from American Type Culture Collection (ATCC). Full cell line authentication was performed by genomics (Eurofins Scientific, Luxembourg). Cells were expanded according to recommended culture conditions, kept free of mycoplasma contamination and stored in cryopreservation medium at -150°C until further use.

[0265] RNA sequencing on tumor cells

[0266] RNAseq: Triplicate samples of the human tumor cell line H-1650 were provided for RNAseq. For each sample, a sequencing library was constructed using the QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina and the UMI Second Strand Synthesis Module for QuantSeq FWD (Illumina, Read 1). This incorporates a unique molecular identifier (UMI) in the first 6 nucleotides of each read, allowing to identify PCR duplicates and eliminate amplification bias. The libraries were sequenced as single-end 76 on a NextSeq device.

[0267] Read preparation: UMI were removed using UMI-tools (v1 .1 .2), resulting in raw reads with a remaining length of 66 nt (removed 6 nt for the UMI + 4 nt spacer). Raw sequencing reads were inspected using FastQC (v0.11 .9) fortheir quality and length. Putative contaminations were checked using FastQ Screen (vO.15.1) and a set of genomes from common lab organisms. Read quality based on phred scores is good. Contamination screening with FastQScreen shows that up to 97% of the reads map on the human genome. Limited cross- mapping (i.e. reads mapping to multiple genomes) to other organisms is normal and caused by homology between organisms. Adaptor trimming was done using cutadapt (v3.7) with added filtering of reads containing ambiguities or not passing the phred score threshold of 20. The quality of the remaining read pairs was checked using FastQC as before.

[0268] Gene expression quantification: For each sample, trimmed reads were aligned on the latest human reference genome (GRCh38, ENSEMBL release 107) using the splice-aware STAR (v2.7.10a) mapper. UMI-based removal of PCR duplicates from the mapped reads was done with UMI-tools (v1.1.2). Feature counting at the gene level was done using rsem-calculate-expression (RSEM v1.3.3). Next, data was generated using preseq (v2.0.1) followed by rarefaction plot visualisation using python (v3.6) in order to verify library saturation and sequencing depth. In addition, a principal component analysis (PCA) plot using the rlog-transformed expression counts of the samples was generated. Finally, expression levels of each gene of interest were plotted as normalized counts per million (cpm).

[0269] HLA-peptidomics on tumor cells

[0270] (i) Generation of immunoaffinity columns for MHC Class I pull down

[0271] W6 / 32 antibody was purified from hybridoma cell (HB-95™, ATCC) supernatant as recommended by the cell line provider. To generate immunoaffinity columns, 2 mL of resuspended protein A sepharose 4B beads were washed with 50 mM TRIS , 150 mM NaCI at pH 8.0 before 3 mg of purified W6 / 32 antibody was added and allowed to bind at room temperature for 1 h in a rolling tube. Antibody-bound sepharose beads were then washed with 0.2 M Sodium tetraborate decahydrate at pH 9.0. Antibodybound beads were cross linked with 20 mM dimethylpimelimidate (DMP) dissolved in tetraborate decahydrate pH 9.0 at a ratio of 3 mg antibody per 5 mL of 20 mM DMP solution. Cross-linking occurred for 45 min at room temperature in a rolling tube. Cross linked-beads were washed and incubated for 2 h at room temperature with 50 mM TRIS, 150 mM NaCI at pH 8.0 supplemented with 0.2 M ethanol amine pH 8.0 to quench the crosslinking reaction. Beads were resuspended in solution containing 50 mM TRIS, 150 mM NaCI and 0.02% (m / v) sodium azide at pH 8.0 and kept at 4°C prior use. Antibody binding to beads and cross-linking were monitored by silver staining according to manufacturer protocol.

[0272] (ii) Isolation and purification of immunopeptides

[0273] Cells were lysed by addition of a mild lysis buffer containing 1 % (m / v) octyl-p,D-glucopyranoside, 0.25% (m / v) sodium deoxycholate, 1x complete protease inhibitor cocktail, 1 mM phenylmethylsulfonyl fluoride, 0.2 mM and 1 mM ethylendiamine tetraacetic acid (EDTA), 50 mM TRIS and 150 mM NaCI at pH 8.0. Cell lysis was performed with 12 mL of ice cold lysis buffer on 600 million cells at a ratio of 1 mL per 50 x 106cells. Lysates were incubated for 10 min at +4°C under gentle agitation to alleviate protein extraction. Lysates were cleared by an initial centrifugation at 7,000 x g for 5 min at 4 °C, and supernatants were further cleared at 20,000 x g for 10 min at 4 °C. Prior to immunoprecipitation, W6 / 32 immunoaffinity columns were washed with 1 % acetic acid, followed by washes in 50 mM TRIS and 150 mM NaCI pH 8.0. Supernatants were added to the washed W6 / 32 immunoaffinity columns and incubated for 10 minutes or overnight (~16 h) while rolling at 30 rpm at 4 °C. Reusable Econo glass columns were used for the immunoprecipitation. After immunoprecipitation, beads were washed five times with 10 mL of 50 mM TRIS and 150 mM NaCI at pH 8.0 followed by three washes with ultrapure water (MS grade). MHC Class I molecules were eluted by applying 1 mL of 10% (v / v) acetic acid and incubated 10 minutes with gentle agitation every 2 minutes. Elution was repeated once for a total of two elution. The pH of the eluate was checked to be at 2.5 or below, prior to loading on preconditioned Sep- Pack C18 cartridge for peptide desalting. After loading, C18 cartridge were washed twice with 5 mL of ultrapure water (MS grade) 0.1 % (v / v) trifluoracetic acid (TFA). Next, MHC class I peptides were specifically eluted by applying twice with 1 ,000 pL of 25% acetonitrile (ACN) in 0.1 % TFA, followed by pooling of the eluates and complete drying in 2 mL protein LoBind tubes. Peptides were reconstituted in 50 pL of 2% ACN in 0.1 % TFA for 5 min in an ultrasonic bath prior injection for LC-MS analysis.

[0274] (Hi) LC-MS / MS analysis

[0275] Samples were injected twice either on an Evosep One LC system or on an Ultimate 3000 RSLC nanoLC system. A sample volume of 15 pL out of 50 pL was injected for both analyses. For trapping, peptides were loaded onto Evosep loading tips according to manufacturer’s protocol while, for the nanoLC system, peptides were loaded at 20 pL / min for 2 min in loading solvent A (0.1 % TFA in water / acetonitrile (ACN) (99.5:0.5, v / v) on a trapping column (5 mm x 300 pm internal diameter (I.D.), 5 pm beads. Both LC systems run over a reverse phase column (150 mm x 75 pm I.D., 1.7 pm beads, Aurora Gen3 Elite, lonopticks), heated to 45°C. The Evosep One LC system was operated with the 20 SPD Whisper method with a gradient consisting of 0.1 % FA in LC-MS-grade water as solvent A and 0.1 % FA in ACN as solvent B. The nanoLC system was run with a linear gradient from 0.5 to 37.5% MS solvent B (0.1 % FA in water / acetonitrile (2:8, v / v)) for 30 min, increasing MS solvent B to 55% after 38 min, increasing further MS solvent B to 70% after 40 min and finishing with a wash in 99.5% of MS solvent A for 5 min. The flow rate was started at 250 nL / min switching to 100 nL / min after 20 min and increased to 250 nL / min after 40 min.

[0276] Both LC systems were connected to a nanoelectrospray ion source (Captive spray source Bruker) and coupled to a timsTOF SCP instrument (Bruker). Eluting peptides were measured in positive polarity with a full-scan range of 100 m / z to 1700 m / z. The MS instrument was run in DDA-PASEF mode with a 10 PASEF / MSMS scan per topN acquisition method. Precursor signals intensity threshold was set at 500 arbitrary units. An adapted polygon was set in the m / z-IM plane to exclude adverse ions, but include single-charge precursors based on their expected position in an m / z-IM plane (IMS polygon filter Massmobility: 270.54-0.551 ; 400.74-0.850; 702.59-1.101 ; 704.02-1.723; 1 ,716-1.764 and NA-0.55, respectively). The mass spectrometer was not operated in sensitivity mode. Accumulation and ramp time was set a 100 ms. Precursors were isolated with a 2 Th window below m / z 700 and 3 Th above and actively excluded for 0.4 min when reaching a target intensity threshold of 20,000 arbitrary units. A range from 100 to 1700 m / z and 0.6 to 1.6 Vs cm-2 was covered with collision energy (1 / K0 [Vs cm-2] - Collision energy [eV]: 0.7-20; 1.06-30; 1.16-40; 1.34-40)).

[0277] (iv) Raw data processing and searches timsTOF Pro (.d) data by four search engines in parallel: MSFragger version 4.0 (PMID:28394336), Comet version 2023.01 rev. 2 (PMID:23148064), Sage version 0.14.7 (PMID:37819886), and ( / v) PEAKS Studio 11.5 (build 20231027) (PMID:22186715). Raw MS data was first pre-processed and searched by MSFragger, whereafter recalibrated mzML files (PMID:32792501) were used for Sage, Comet and PEAKS searches. Spectra were searched against a concatenated target-decoy (reverse) database comprised of UniProtKB reference proteomes for human (UP00005640) and MaxQuant contaminants (247 protein sequences). No static modifications were set, and variable modifications were Cys cysteinylation, Met oxidation, protein N-terminal acetylation and pyro-Glu formation from peptide N-terminal Asp and Glu. An unspecific peptide digestion option was used, restricting peptide length to 7-20 and mass 600-5000 Da. Mass tolerance was set to 15 ppm for MS1 and MS2 tolerance. In case of Sage, RT and IM model fitting was used for PSM scoring.

[0278] (v) Rescoring of PSMs by predicting fragment ion intensities, retention time and ion mobility features

[0279] MSFragger, Sage and PEAKS outputted each a Percolator input (.pin) file with search engine-derived scoring features for all PSMs. These were formatted by custom Python scripts to a tab-separated value (TSV) file used as input by MS2Rescore v3.0.0b5. For both Orbitrap and timsTOF Pro data, DeepLC version 2.2.32 (PMID:34711972) was used to score PSMs by generating features describing the deviations to the predicted peptide RT. In addition, MS2PIP Immuno-HCD (PMID:35803561) and timsTOF (PMID:38480730) models were used to predicted b / y-ion intensities for Orbitrap and timsTOF Pro data, respectively, used for generating correlation features for PSM rescoring. Lastly, peptide CSS values were predicted by IM2Deep (GitHub, Inc. 2024); and used for rescoring in case of timsTOF pro data. After extending the search engine scoring features with DeepLC, MS2PIP and possibly IM2Deep features, mokapot (PMID:33596079) was used for rescoring and peptides were filtered at a 1 % peptide FDR. In case of PEAKS, the built-in ‘Deep Learning Boost’ option was enabled, which similarly provides scoring features for RT, IM and fragment ion intensities.

[0280] (vi) Bioinformatics

[0281] Sequence logos were made using Logomaker. NetMHCpan-4.1 was used for binding prediction to mouse MHC class I alleles. Plots were made using built-in functions of seaborn and matplotlib.

[0282] Target antigen cloning and mRNA production

[0283] The sequence of the antigens of interest i.e. OIP5, PBK, CEP55, XAGE1 B, TTK and IGF2BP3 were selected via the Ensembl human genome browser. The splice variant resulting in the longest polypeptide was selected for each of the antigens, and for OIP5 also splice variant OIP5-202 (H0YKL4). The latter was used to demonstrate vaccine-induced antigen-specific cytotoxic T cell responses even when the vaccine consists of a selected sequence that does not encode the longest polypeptide of the antigen. 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 a vector backbone containing a T7 promotor 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.

[0284] 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.

[0285] The selected antigen sequences are: SEQ ID NO: 1 (OIP5-201), SEQ ID NO: 2 (OIP5-202); SEQ ID NO: 4 (PBK-203); SEQ ID NO: 6 (CEP55-201); SEQ ID NO: 9 (XAGE1 B-203); SEQ ID NO: 11 (TTK- 202); and SEQ ID NO: 17 (IGF2BP3-201).

[0286] Used mRNA constructs, which include sequences that regulate amongst others stability, location and translation efficiency, are as follows:

[0287] OIP5 represented by SEQ ID NO: 31 (OIP5-201) or SEQ ID NO: 55 (OIP5-202); PBK represented by SEQ ID NO: 32; CEP55 represented by SEQ ID NO: 33; XAGE1 B represented by SEQ ID NO: 34; TTK represented by SEQ ID NO: 35; and IGF2BP3 represented by SEQ ID NO: 36. Generation of lipid nanoparticles

[0288] 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.

[0289] 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.

[0290] Monocyte-derived Autologous Dendritic Cell culture

[0291] Monocyte-derived dendritic cells were generated according to a proprietary accelerated culture protocol as described (Brabants et al, 2018; WO2019 / 243537). 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.

[0292] PBLs were frozen in RPMI containing 20% Alburex20 (Human serum Albumin 20 g / l) and 10% dimethyl sulfoxide (DMSO).

[0293] 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) (Leukine sargramostim), 500 U / ml GMP-certified recombinant human interleukine-4 (hulL-4). On day 3 of the culture, 2,5 pg / ml synthetic TLR4 agonist Monophosphoryl lipid A (MPLA) and 1000 lU / ml pharmaceutical-grade IFN-y (Immukine) were added to the culture medium for another 24h. Mature DCs (mDCs) were harvested on day 4.

[0294] Isolation of B cells

[0295] 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. DC loading with mRNA formulated in lipid nanoparticles

[0296] 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.

[0297] Electroporation of DCs

[0298] Cells were harvested and washed twice with Opti-MEM prior to electroporation. For large scale electroporation, 50x106DCs were resuspended in 350 pl Opti-MEM and transferred to a 4 mm gap cuvette, whereupon 50 pl nuclease-free water containing the mRNA at a dosage of 1 pg / 106cells was added to the cell suspension. For small scale electroporation, 5x106DCs were resuspended in 170 pl Opti-MEM and transferred to a 4 mm gap cuvette, whereupon a mixture of 5 pl nuclease-free water containing the mRNA at a dosage of 1 pg / 106cells supplemented with 25 pl 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.

[0299] Four hours after electroporation, DC phenotype and electroporation efficiency were evaluated using flow cytometry and DCs were cryopreserved in cryopreservation medium.

[0300] Peptide pulsing of DCs

[0301] Mature DCs were harvested and resuspended at a density of 5x106DC / ml in serum-free GMP CellGro medium whereupon the OIP5 peptide VLADSVHLA was added at a final concentration of 5 |j.M. Cells were incubated for 2 hours at 37°C and 5% CO2, whereupon they were harvested, washed and cryopreserved in cryopreservation medium. Non-peptide pulsed mature DCs served a s a control.

[0302] Isolation of B cells

[0303] 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. B cells were electroporated prior to cryopreservation. Electroporation of B cells

[0304] Cells were harvested and washed twice with Opti-MEM prior to electroporation. To that end, 50x106B cells were resuspended in 350 pl Opti-MEM and transferred to a 4 mm gap cuvette, whereupon 50 pl 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.

[0305] Four hours after electroporation, B cell phenotype and electroporation efficiency were evaluated using flow cytometry and B cells were cryopreserved in cryopreservation medium.

[0306] Flow cytometry

[0307] 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.

[0308] Surface staining of PBMC, PBL, CD14+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.

[0309] 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).

[0310] Samples were acquired on a Fortessa LSR and analyzed using FlowJo software.

[0311] Shotgun mass-spectrometry on mRNA-electroporated dendritic cells

[0312] (i) LC-MS / MS

[0313] Peptides were re-dissolved in 20 pl loading solvent A (0.1 % trifluoroacetic acid in water / acetonitrile (ACN) (99.5:0.5, v / v)) of which 1 pl was injected for LC-MS / MS analysis on an Ultimate 3000 ProFlow nanoLC system in-line connected to an Orbitrap Fusion Lumos mass spectrometer. Trapping was performed at 20 pl / min for 2 min in loading solvent A on a 5 mm trapping column (300 pm internal diameter (I.D.), 5 pm beads). The peptides were separated on a 250 mm Aurora Ultimate, 1 ,7pm C18, 75 pm inner diameter kept at a constant temperature of 45°C in a butterfly heater (. Peptides were eluted by a non-linear gradient starting at 1 % MS solvent B reaching 26.4% MS solvent B (0.1 % FA in acetonitrile) in 75 min, 44% MS solvent B in 95 min, 56% MS solvent B in 100 minutes followed by a 5- minute wash at 56% MS solvent B and re-equilibration with MS solvent A (0.1 % FA in water), all at a flow rate of 250 nl / min. The mass spectrometer was operated in data-independent mode, automatically switching between MS and MS / MS acquisition. Full-scan MS spectra ranging from 390-910 m / z with a target value of 4E5, a maximum fill time of 50 ms and a resolution at of 60,000 were followed by 30 quadrupole isolations with a precursor isolation width of 10 m / z for HCD fragmentation at an NCE of 34% after filling the trap at a target value of 4E5 for maximum injection time of 54 ms. MS2 spectra were acquired at a resolution of 30,000 at 200 m / z in the Orbitrap analyser without multiplexing. The isolation intervals were set from 400 - 900 m / z with a width of 10 m / z using window placement optimization. The polydimethylcyclosiloxane background ion at 445.120028 Da was used for internal calibration (lock mass) and QCIoud has been used to control instrument longitudinal performance during the project (Chiva, 2018).

[0314] (ii) Data analysis

[0315] LC-MS / MS runs of all samples were searched separately using the DiaNN algorithm (version 1.8.1), library free. Spectra were searched against the human protein sequences in the Swiss-Prot database (database release version of 2022_01), containing 20,588 sequences. Enzyme specificity was set as C- terminal to arginine and lysine, also allowing cleavage at proline bonds with a maximum of two missed cleavages. Variable modifications were set to oxidation of methionine residues and acetylation of protein Ntermini. Mainly default settings were used, except for the addition of a 400-900 m / z precursor mass range filter.

[0316] Further data analysis of the shotgun results was performed with an in-house script in the R programming language, version 4.2.2. Protein expression matrices were prepared as follows: the DIA-NN main report output table was filtered at a precursor and protein global q-value cut-off of 1 % and only proteins identified by at least one proteotypic peptide were retained. After pivoting into a wide format, iBAQ intensity columns were then added to the matrix using the DIAgui’s R package getJBAQ function these iBAQ values were Iog2 transformed and a ranking number was assigned according to the relative abundance in the samples.

[0317] Mice

[0318] NOD. Cg-Prkdc80^ H2rgtm1Wjl / Sz mice (further referred to as “NSG mice”) were obtained from The Jackson Laboratory.

[0319] Animals 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. 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. In vitro cancer antigen-specific T-cell cytotoxicity study

[0320] The mRNA loaded DCs, mRNA-LNP loaded DC, peptide loaded DC or mRNA loaded B cells were 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 “in vitro vaccination” were performed by which mRNA loaded DCs, mRNA-LNP loaded DC, peptide 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.

[0321] In vivo therapeutic cancer vaccination study

[0322] 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.

[0323] 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.

[0324] 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.

[0325] The investigators were blinded for treatment randomization as well as tumor measurements and data analysis.

[0326] Tumor growth inhibition (TGI) is calculated using following formula:

[0327] %TGI = (1 -{Tt / TO I Ct / C0} 1 1 -{COZCt}) x 100

[0328] Where:

[0329] Tt = median tumor volume of treated at time t

[0330] TO = median tumor volume of treated at time 0

[0331] Ct = median tumor volume of control at time t

[0332] CO = median tumor volume of control at time 0

[0333] A TGI >50% is considered clinically significant.

[0334] Statistical analysis

[0335] Shapiro-Wilk Normality test was performed to determine Gaussian distribution (a = 0,05) of the data.

[0336] When data were normally distributed according to Shapiro-Wilk testing, unpaired two-tailed student t- test was performed. If data were not normally distributed according to Shapiro-Wilk testing, unpaired nonparametric Mann-Whitney 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.

[0337] Clinical trial

[0338] Two different therapeutic mRNA-modified DC vaccines were designed according to the cancer indication and differing by the set of target antigens used.

[0339] • Vaccine-001 includes 4 mRNA-encoded antigens for non-squamous NSCLC: XAGE-1 b, CEP55, PBK and IGF2BP3. Based on in silico prediction > 97% of the patients with non-squamous NSCLC should express at least 2 of the 4 targeted antigens in the tumor. Vaccine-001 consists of a mixture of 4 equal fractions of mRNA-modified autologous monocyte-derived dendritic cells (moDCs). Respective mRNA sequences encoding the antigens are represented by SEQ ID NO: 22 (XAGE- 1 b), SEQ ID NO: 21 (CEP55), SEQ ID NO: 26 (PBK) and SEQ ID NO: 24 (IGF2BP3).

[0340] • Vaccine-002 includes 4 mRNA-encoded antigens for squamous NSCLC: MAGE-A3, CEP55, TTK and IGF2BP3. Based on in silico prediction > 99% of the patients with squamous NSCLC should express at least 2 of the 4 targeted antigens in the tumor. Vaccine-002 consists of a mixture of 4 equal fractions of mRNA-modified autologous monocyte-derived dendritic cells (moDCs). Respective mRNA sequences encoding the antigens are represented by SEQ ID NO: 52 (MAGE- A3), SEQ ID NO: 21 (CEP55), SEQ ID NO: 23 (TTK) and SEQ ID NO: 24 (IGF2BP3).

[0341] For conciseness, Vaccine-001 and Vaccine-002 will be further referred to collectively as Vaccine-OOx. The trial is a prospective, open-label, parallel group phase 1 b umbrella study to evaluate the safety, immunogenicity and clinical efficacy of an autologous DC immunotherapy defined by histologic subtype (as described above, i.e. squamous or non-squamous NSCLC) in combination with anti-PD-1 treatment in patients with metastatic NSCLC. The study is performed according to a modular protocol allowing the activation of different parallel cohorts reflecting different unmet medical need scenarios. For each vaccine treatment arm (Vaccine-001 and Vaccine-002), the following cohorts are defined.

[0342] Cohort 1 includes patients with metastatic NSCLC that will start on an anti-PD-1 therapy-containing regimen per SOC.

[0343] • Cohort 1 A: pembrolizumab or cemiplimab monotherapy (PD-L1 TPS > 50%).

[0344] • Cohort 1 B: cisplatin / carboplatin-pemetrexed-pembrolizumab or cisplatin / carboplatin-pemetrexed- cemiplimab.

[0345] Cohort 2 includes patients with metastatic NSCLC that do not progress after completion of SOC platinum-based chemotherapy in combination with anti-PD-1 therapy. Vaccine-OOx is added to SOC anti-PD-1 treatment from the maintenance phase. Cohort 3 includes patients with metastatic NSCLC that progress on anti-PD-1 monotherapy or in the maintenance phase of an anti-PD-1 therapy-containing regimen per standard-of-care, and fulfilling the criteria of treatment beyond progression.

[0346] • Cohort 3A: progressing on anti-PD-1 monotherapy, e.g. pembrolizumab or cemiplimab (PD-L1 TPS > 50%, no previous platinum-based chemotherapy).

[0347] • Cohort 3B: progressing in the maintenance phase of an anti-PD-1 therapy-containing regimen, e.g. cisplatin / carboplatin-pemetrexed-pembrolizumab or cisplatin / carboplatin-pemetrexed-cemiplimab.

[0348] Cohort 4 includes patients with metastatic NSCLC that progress after a previous anti-PD-1 therapycontaining regimen that was discontinued after a maximum duration per SOC and are eligible for anti- PD-1 therapy. Vaccine-OOx is added to SOC anti-PD-1 treatment.

[0349] In each cohort, Vaccine-OOx is administered alongside anti-PD-1 therapy, with or without chemotherapy, to subjects who are either already receiving or are scheduled to receive treatment as per the standard of care (SOC). Subjects will not be randomized into cohorts and SOC treatment will not be altered.

[0350] Preparation of Vaccine-OOx

[0351] Vaccine-OOx is formulated as aliquots of 15 x 106cells in a cryopreservation medium consisting of Cryostore CS10 / Plasmalyte A / 5% human serum albumin, with the cryoprotectant DMSO at a final volume concentration of 5%. The number of aliquots necessary to obtain the required dose of 35-45 x 106viable cells per injection is calculated based on cell viability measurements of a post-thawing quality control sample. Thawed aliquots are combined into one and diluted 3-fold in Plasmalyte A with 5% HSA, pooled, diluted to a total volume 20 ml with Plasmalyte A with 5% HSA and dispensed in a syringe.

[0352] Vaccine-OOx contains mRNA-modified autologous cells prepared according to the methods disclosed herein. The mRNA is entirely contained into the cells and transiently expressed into protein.

[0353] Administration, dosage and dose frequency of Vaccine-OOx

[0354] The vaccine is administered intravenously. Participants receive IV infusions of 35 to 45 x 106viable cells every 3 weeks for up to 4 cycles depending on the number of doses produced from 1 apheresis, or until the occurrence of a grade > 3 treatment-related toxicity or disease progression.

[0355] In all cohorts, treatment with the DC-vaccine will be combined with anti-PD-1 therapy per SOC (e.g. pembrolizumab or cemiplimab) with or without chemotherapy. During the screening and pre-treatment phase, anti-PD-1 therapy will be continued per SOC. There is an option to delay the next anti-PD-1 dose for 1 week to allow the simultaneous administration of the study treatment. Vaccine-OOx will be given on the same day after the infusion of anti-PD-1 therapy. For patients also receiving chemotherapy, injections will be given at D15 of a standard 21 -day chemotherapy cycle.

[0356] Duration of treatment

[0357] Participants receive Vaccine-OOx for a maximum of 4 cycles (treatment duration 12 to 14 weeks from leukapheresis) depending on the number of doses produced from 1 apheresis, or until occurrence of a grade > 3 treatment-related toxicity or disease progression. Anti-PD-1 therapy and chemotherapy will be given according to SOC.

[0358] Results

[0359] The value of the proteins of the present invention as target antigens for cancer immunotherapy is demonstrated in the examples herein, generated using an mRNA-modified dendritic cell-based vaccine approach. The mRNA-DCs are used with the aim to induce antigen-specific tumor killing T-cell responses, with readouts from preclinical (1) in vitro and (2) in vivo experiments.

[0360] Example 1 : Expression of antigens and presentation of antigen-derived epitopes by the model target cell line H1650

[0361] H1650, a non-small cell lung cancer cell line, was selected based on described expression profile for the target antigens of the present invention, as documented in the CCLE database (DepMap, Broad (2024). DepMap 24Q2 Public. Figshare+. Dataset). In addition, H1650 express the MHC class I allele HLA-A2, allowing to set up partially HLA-matched experiments using immune cells derived from HLA- A2 anonymous blood donors (HLA-A2 is present in 30-50% of Caucasians). We first performed RNA NGS to verify expression pattern from the literature and could confirm abundant transcripts for each of the antigens to be evaluated (FIG. 1 ). Next, we used HLA-immunopeptidomics to verify whether antigen- derived epitopes were generated and presented on surface MHC molecules of the H1650 cells. For each of the candidate target antigens, we could detect at least one peptide bound to one of the three MHC alleles expressed by HLA-A*02:01 , as listed in Table 7 hereunder.

[0362] Table 7: Summary of immunopeptidomic analysis on H1650 cells, showing detection of HLA-A2- restricted peptides of varying binding affinities for each of the antigens of the invention.

[0363] Example 2: Functionality of antigen-encoding mRNA when electroporated into dendritic cells Monocyte-derived dendritic cells were electroporated as separate fractions with mRNA encoding one of the proteins of the present invention. Proteomic analysis of each separate fraction was performed using shotgun mass-spectrometry. Protein abundance was reported as ranked iBAQ values (i.e. total precursor intensities divided by the number of theoretically observable peptides for a given protein). Abundant protein expression was detected for each of the target antigens in DCs electroporated with the corresponding mRNA, confirming the functionality of the mRNA sequences (FIG.2).

[0364] Examples 3: Vaccine-induced antigen-specific cytotoxic T-cell responses: in vitro H1650 tumor killing effect using T cells primed against targets of the invention by autologous mRNA-loaded DCs.

[0365] T-cells from an HLA-A2+ donor were exposed to mRNA-loaded autologous DCs (mRNA prepared as specified in ‘Target antigen cloning and mRNA production') and subsequently co-incubated with the H1650 NSCLC cell line known to express said targets. mRNA encoding the splice variant of each of the described antigens translating in the longest amino acid sequence was selected.

[0366] 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 3) shows selective killing of tumor cells when T-cells were primed beforehand with DCs loaded with each of the target mRNAs respectively and separately OIP5 (panel A), PBK (panel B), CEP55 (panel C), XAGE1 B (panel D), TTK (panel E) and IGF2BP3 (panel F). 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 for each of the target mRNAs.

[0367] Example 4: Vaccine-induced antigen-specific cytotoxic T-cell responses: in vivo tumor killing effect

[0368] The capacity of an mRNA-DC vaccine targeting one of the antigens of the invention to induce anti- tumoral T-cell responses in vivo was assessed 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 preengrafted 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.

[0369] Immunodeficient NSG mice were engrafted with human HLA-A2 lymphocytes via systemic route, concurrent with subcutaneous implantation of a human tumor xenograft of the abovementioned HLA- A2-positive NSCLC cell line. Mice were treated with systemic injections of mRNA-DCs autologous to the engrafted lymphocytes, 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. The main endpoint was tumor growth inhibition (%TGI).

[0370] The data in FIG. 4 shows significant suppression of tumor growth in mice treated with DCs loaded with mRNA encoding one of each antigen from the invention respectively OIP5 (panel A), PBK (panel B), CEPP5 (panel C), XAGE1 B (panel D), TTK (panel E) and IGF2BP3 (panel F). The effect is highly antigen-specific as mice injected with DCs loaded with a control antigen (i.e. protein not expressed in the target tumor) showed no response. This is reflected in the TGI% indices as shown in Table 8.

[0371] Table 8: Tumor growth inhibition indices (TGI) related to example 4 for tumor-bearing cohorts treated with DCs loaded with mRNA encoding the indicated target antigens. A TGI above 50% is generally considered clinically significant.

[0372] Example 5: Vaccine-induced antigen-specific cytotoxic T cell responses: in vitro H2087 tumor killing effect using T cells primed against targets of the invention by autologous mRNA-loaded DCs. T cells from an HLA-A2+ donor were exposed to mRNA-loaded autologous DCs and subsequently co-incubated with the H2087 NSCLC cell line known to express said targets. 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. 5) show selective killing of tumor cells when T cells were primed beforehand with DCs loaded with each of the target mRNAs respectively OIP5 (panel A), PBK (panel B), CEPP5 (panel C), XAGE1 B (panel D), TTK (panel E) and IGF2BP3 (panel F). 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 for each of the target mRNAs.

[0373] Example 6: Vaccine-induced OIP5-specific cytotoxic T cell response: in vitro H1650 tumor killing effect using T cells primed against the OIP5 target of the invention by autologous OIP5 mRNA- LNP loaded DCs. 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 OIP5 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 OIP5 mRNA encodes the splice variant resulting in the longest polypeptide and as such is able to generate the maximum number of potentially generated OIP5 epitopes, represented by OIP5 splice variant 201. 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 OIP5, as documented in the CCLE public database (RPKM 41 .64) and subsequently confirmed in our RNAseq analysis (normalized cpm 64.51). 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. 6) show selective killing of tumor cells when T cells were primed beforehand with OIP5 mRNA-LNP loaded DCs. The effect is clearly above background cytotoxicity measured when T cells were primed with DCs loaded with mRNA-LNPs containing an irrelevant antigen, indicating an antigen-specific effect.

[0374] Example 7: Vaccine-induced OIP5-specific cytotoxic T cell response: in vitro H1650 tumor killing effect using T cells primed against the OIP5 target of the invention by autologous OIP5 mRNA loaded B cells. B cells generated from HLA-A2-positive anonymous healthy donor leukocytes according to the aforementioned method were loaded with OIP5 mRNA and used as antigen-presenting cells to prime autologous T cells. The OIP5 mRNA encodes OIP5 splice variant 201 . 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 OIP5, as documented in the CCLE public database (RPKM 41.64) and subsequently confirmed in our RNAseq analysis (normalized cpm 64.51). 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. 7) show selective killing of tumor cells when T cells were primed beforehand with OIP5 mRNA loaded B cells. The effect is clearly above background cytotoxicity measured when T cells were primed with B cells loaded with mRNA encoding an irrelevant antigen, indicating an antigen-specific effect.

[0375] Example 8: Vaccine-induced OIP5-specific cytotoxic T cell response: in vitro H1650 tumor killing effect using T cells primed against OIP5 by autologous DCs loaded with mRNA encoding OIP5. The capacity of T cells primed against OIP5 splice variant 202 to specifically induce cell death in OIP5- 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 OIP5 mRNA and used as antigen-presenting cells to prime autologous T cells. The OIP5 mRNA encodes the sequence represented by OIP5 splice variant 202, which is a splice variant that does not result in the longest possible OIP5 polypeptide. 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 OIP5, as documented in the CCLE public database (RPKM 41 .64) and subsequently confirmed in our RNAseq analysis (normalized cpm 64.51). 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. 8) show selective killing of tumor cells when T cells were primed beforehand with OIP5 splice variant 202 mRNA 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. These data additionally demonstrate induction antigen-specific antitumor effects, even when the used antigenic sequence does not cover the maximum number of potentially generated epitopes as would be the case if the splice variant resulting in the longest polypeptide was used.

[0376] Example 9: Vaccine-induced OIP5-specific cytotoxic T cell response: in vitro H1650 tumor killing effect using T cells primed against OIP5 by autologous DCs loaded with VLADSVHLA peptide. Using immunopeptidomic analysis on the H1650 NSCLC cell line, the HLA-A2+OIP5 peptide VLADSVHLA was identified. To demonstrate the capacity of T cells primed against a particular antigen derived peptide to specifically induce cell death in an OIP5-expressing cancer cell line, following experiment was performed. DCs generated from HLA-A2- positive anonymous healthy donor leukocytes according to the aforementioned method were loaded with the VLADSVHLA peptide (SEQ ID NO: 37) of OIP5 and used as antigen-presenting cells to prime autologous T cells. Non-peptide loaded DCs function as a negative control. 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 OIP5, as documented in the CCLE public database (RPKM 41 .64) and subsequently confirmed in our RNAseq analysis (normalized cpm 64.51). 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) show selective killing of tumor cells when T cells were primed beforehand with VLADSVHLA peptide loaded DCs. The effect is clearly above background cytotoxicity measured when T cells were primed with non-peptide loaded DCs loaded, indicating an antigen-specific effect.

[0377] REFERENCES

[0378] 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.

[0379] Cytotherapy. 2018 Sep;20(9):1 164-1181 .

[0380] Chiva C, Olivella R, Borras E, Espadas G, Pastor O, Sole A, et al. (2018) QCIoud: A cloud-based quality control system for mass spectrometry-based proteomics laboratories. PLoS ONE 13(1): e0189209. https: / / doi.Org / 10.1371 / journal.pone.0189209

[0381] Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold

[0382] Spring Harbor, N.Y

Claims

CLAIMS1. A pharmaceutical composition comprising:(a) at least one antigen, wherein the at least one antigen comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence selected from the group comprising SEQ ID NO: 6-8, SEQ ID NO: 17-18, SEQ ID NO: 1-2, SEQ ID NO: 3-5 or 56, SEQ ID NO: 9-10 or SEQ ID NO: 11-16;(b) an immunogenic fragment of the antigen of (a) wherein the immunogenic fragment comprises or consists of an amino acid sequence having 85% sequence identity to the amino acid sequence selected from the group comprising of SEQ ID NO: 37 to SEQ ID NO: 49; or(c) a nucleic acid molecule encoding the antigen of (a) or the immunogenic fragment of (b).

2. The pharmaceutical composition according to claim 1 , wherein said antigen (a), immunogenic fragment (b) or nucleic acid molecule (c) is incorporated in or associated with a carrier.

3. The pharmaceutical composition according to any one of claims 1 or 2, wherein said nucleic acid molecule is DNA or RNA, in particular mRNA.

4. The pharmaceutical composition according to claim 3, wherein said nucleic acid molecule comprises or consists of a nucleic acid sequence having 70% sequence identity to the nucleic acid sequence selected from the group comprising: SEQ ID NO: 19 to 36, SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 57.

5. The pharmaceutical composition according to any one of claims 2 to 4, wherein said carrier is selected from the group comprising liposomes, lipid micro- or nanoparticles, polymeric micro- or nanoparticles, exosomes, cationic nano-emulsions, cationic peptides, cationic polymers, antigen presenting cells (APCs), plasmids or viral vectors.

6. The pharmaceutical composition according to claim 5, wherein said antigen presenting cell (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; in particular a dendritic cell (DC).

7. The pharmaceutical composition according to any one of claims 3 to 6, wherein said RNA molecule is a splice variant encoding the longest polypeptide from the protein-coding gene, and wherein said RNA molecule is incorporated in a dendritic cell or nanoparticle.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the composition further comprises a pharmaceutically acceptable excipient.

9. The pharmaceutical composition of any one of claims 1 to 8, for use in human and / or veterinary medicine.

10. The pharmaceutical composition according to any one of claims 1 to 9, for use in stabilizing tumor growth or inducing tumor regression in a subject in need thereof.

11. The pharmaceutical composition according to any one of claims 1 to 9, for use in immunotherapy in a subject in need thereof.

12. The pharmaceutical composition according to any one of claims 1 to 9, for use in the treatment of cancer, in particular wherein cancer cells express one or more of the polypeptides OIP5, PBK, CEP55, XAGE1 B, TTK, or IGF2BP3; in particular wherein said cancer is lung cancer, more in particular NSCLC.

13. A method for preparing an immunotherapy agent comprising the steps of: a) obtaining a carrier; b) ex vivo modifying said carrier of step a) comprising the introduction of an RNA encoding an amino acid sequence having at least 85% sequence identity to the amino acid sequence selected from the group comprising of: SEQ ID NO: 1 to SEQ ID NO: 18 or SEQ ID NO: 56, or an immunogenic fragment thereof; c) harvesting the obtained RNA-loaded carrier; d) optionally formulating the RNA-loaded carrier of (c) to be suitable for use in immunotherapy.

14. The method according to claim 13, wherein in step b) RNA is introduced by transfection or transduction, more specifically by electroporation, viral transduction, photoporation, or lipofection.

15. The method according to claim 13 or 14, wherein the carrier is an antigen presenting cell or a nanoparticle.

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