WO2025201659A1

WO2025201659A1 - Combination therapy comprising therapeutic rna cancer vaccines and anti-ctla4 antibodies for cancer treatment

Info

Publication number: WO2025201659A1
Application number: PCT/EP2024/058693
Authority: WO
Inventors: Ugur Sahin; Lena KRANZ; Mathias VORMEHR
Original assignee: Biontech SE
Current assignee: Biontech SE
Priority date: 2024-03-28
Filing date: 2024-03-28
Publication date: 2025-10-02
Anticipated expiration: 2026-09-28
Also published as: WO2025202495A1

Abstract

The invention provides an RNA cancer vaccine and/or an anti-CTLA4 antibody for use in a method of treating cancer in a subject in need thereof, wherein the subject is administered a combination comprising the RNA cancer vaccine comprising at least one RNA and the anti- CTLA4 antibody. The invention further provides a compositions or kit of parts comprising the RNA cancer vaccine comprising the at least one RNA and the anti-CTLA4 antibody. The invention further provides methods of treating cancer in a subject in need thereof, wherein the method comprises administering to the subject the RNA cancer vaccine comprising the at least one RNA and the anti-CTLA4 antibody. The invention further provides a combination of the RNA cancer vaccine comprising at least one RNA and the anti-CTLA4 antibody.

Description

COMBINATION THERAPY COMPRISING THERAPEUTIC RNA CANCER

VACCINES AND ANTI-CTLA4 ANTIBODIES FOR CANCER TREATMENT

FIELD OF THE INVENTION

The present invention relates to methods for treating cancer in a subject using a combination of an RNA cancer vaccine and an anti-CTLA4 antibody. The invention further provides a composition or kit of parts and a combination of the RNA cancer vaccine and the anti-CTLA4 antibody.

BACKGROUND OF THE INVENTION

The immune system of humans and other mammals provides protection against infection and disease through mechanisms of innate and adaptive immunity. The evolutionary ancient innate immune system provides a rapid, i.e., within minutes, but non-specific immune response which relies on invariant receptors that recognize common molecular patterns associated with pathogens (antigens). In contrast, the immune response of the adaptive immune system is considerably slower, i.e., taking days to weeks, but involves highly specific antigen receptors on B cells (B lymphocytes) and T cells (T lymphocytes) for pathogen recognition. Like the innate immune system, the adaptive immune system also comprises a humoral immune and a cell-mediated immune response. The humoral response is primarily driven by antibodies produced by B cells, which are able to recognize and neutralize foreign target antigens. In contrast, the cell-mediated immune response involves the activation of macrophages, neutrophils, natural killer cells (NK), and antigen-specific cytotoxic T cells, and the release of various cytokines in response to the recognition of an antigen. Besides the protection against pathogens, the immune system plays a pivotal role in cancer prevention, development, and defense (Gonzalez et al., Genes Dev 2018, 32(19-20): 1267- 1284). Therefore, the field of cancer immunotherapy has attracted great attention from both the scientific and clinical communities over the past two decades. The overarching concept of cancer immunotherapy is to activate, induce and/or enhance a specific immune response in patients to control and/or eliminate the cancer disease. Cancer immunotherapies can generally be categorized into active and passive immunization strategies, depending on their ability to (re)-activate the immune system against cancer cells. Passive forms of cancer immunotherapy include tumor-targeting monoclonal antibodies (mAbs) and adoptively transferred T cells, while anti-cancer vaccines and immune checkpoint inhibitors are considered active forms of cancer immunotherapy (Galluzi et al., Oncotarget 2014, 5(24): 12472-12508).

Anti-cancer vaccines aim to elicit a tumor-specific immune response by active immunization, e.g., by inducing and expanding cancer antigen-specific T cells in patients, which are able to specifically recognize and kill malignant cells. The identification of a growing number of tumor-associated antigens (TAA) has led to a broad collection of suitable targets for immunotherapy using anti-cancer vaccination strategies. Specific TAAs can be delivered to the patients using different vaccination strategies, including proteins, peptides or immunizing vectors such as RNA, DNA or viral vectors that can be applied either directly in vivo or in vitro by pulsing of dendritic cells (DCs) following transfer into the patient. In particular, the field of RNA-based anti-cancer vaccines has developed rapidly in recent years (Liu et al., ACS Nano 2023, 17, 20: 19550-19580; Sahin et al., Nature 2020, 585: 107-112).

Therapeutic anti-cancer vaccines that stimulate the immune system against specific TAAs have shown promising results, but their efficacy has so far fallen short of expectations. Tumor heterogeneity, immunosuppressive tumor microenvironments, optimal TAA candidate identification, and immune response evaluation are still major challenges of anticancer vaccine development and application, resulting in their overall low efficacy in clinical trials (Fan et al., Sig Transduct Target Ther 2023, 8:450; Wang et al., Front Immunol 2023, 14: 1246682). Immunosuppressive tumor microenvironments are a particular challenge for RNA-based anti-cancer vaccines as the immune response elicited by the RNA cancer vaccines can be impeded by the inhibitory tumor microenvironment, e.g., by inhibiting the function and activation of immune cells and preventing T cell infiltration into tumor cells, leading to T cell exhaustion.

Overcoming the immunosuppressive tumor microenvironment is one of the major challenges of RNA anti-cancer vaccines.

Immune checkpoints play a pivotal role in the regulation of the immune response in tumor microenvironments. Immune checkpoints act as gatekeepers of the immune system modulating the nature, magnitude, and duration of the immune response and maintaining self-tolerance. One of the key inhibitory immune checkpoints is Cytotoxic T lymphocyte antigen-4 (CTLA4), also known as CD 152 (cluster of differentiation 152). The interaction between the B7.1 (CD80) (Freeman et al., J Immunol 1989, 143(8): 2714-22) and B7.2 (CD86) (Freeman et al., Science 1993, 262(5135): 909-11; Hathcock et al., Science 1993, 262(5135): 905-7; Wu et al., J Exp Med 1993, 178(5): 1789-93) ligands of antigen presenting cells and the CD28 and CTLA4 receptors (Leach et al., Science 1996, 271(5256): 1734-6; Linsley et al, J Exp Med 1991, 174(3): 561-9; Linsley et al., Proc Natl Acad Sci U S A 1990, 87(13): 5031-5) of T cells governs the activation or downregulation of T cells. CTLA4 is recognized as a key regulator of the adaptive immune response, having a central role in the maintenance of peripheral tolerance and in shaping the repertoire of emerging T cell responses and, is therefore a therapeutic target for the treatment of cancer and inflammation.

Treatment with anti-CTLA4 antibodies has been shown to be a powerful tool for enhancing anti-tumor immunity in preclinical models (Leach et al., Science 1996, 271(5256): 1734-6). Monotherapy with an antibody against CTLA4 promoted rejection of transplantable tumors of various origins. Based on promising preclinical tumor model studies, the clinical potential of antibodies against CTLA4 has been explored in different human malignancies. Unlike other checkpoint inhibitors such as anti-PD-l/PD-Ll antibodies, the anti-CTLA4 antibody, ipilimumab (YERVOY®), has gained market approval for one indication (melanoma) as a monotherapy. The toxicity profile significantly limits its dose and exposure that are required for achieving higher efficacy benefit. The less optimal dose may explain the consistently lower response rate than an anti-PD-1 antibody in head-to-head comparison studies in melanoma and its failure as a monotherapy in multiple Phase III clinical trials in other cancer indications. Despite its approval for multiple cancer indication as a combination therapy with nivolumab (Opdivo®, an anti-PD-1 antibody), the incidence of grade 3/4 immunotherapy-related adverse effects (irAEs) (e.g., up to 73-90% of patients with melanoma receiving ipilimumab/nivolumab as a neo-adjuvant therapy) remains high. CTLA4 remains a valid and attractive immunotherapy target, however, the less favorable safety profile significantly limits its clinical usage.

The molecular basis underlying irAEs and cancer immunotherapeutic effects (CITE) of anti-CTLA4 antibodies is traditionally viewed as antagonizing the endogenous function of CTLA4. In both mice and humans, genetic inactivation of CTLA4 caused severe autoimmune diseases. Therefore, an effective antagonist of CTLA4 is expected to likely induce autoimmune diseases. If inactivation of CTLA4 is necessary, then irAEs are expected to be a necessary price for cancer immunity.

Therefore, there is a desire to improve the therapeutic potential of anti-CTLA4 antibodies by increasing CITE efficacy while reducing the associated irAEs. In the past, different therapeutic immune check inhibitors were combined to enhance anti-tumor activity, particularly against poorly immunogenic tumors. However, this approach is associated with the risk of further increasing the autoimmune side effects further highlighting the need to selectively modulate cancer immunity without enhancing autoimmunity. There is therefore an unmet medical need for improved immunotherapeutic methods for treatment of cancer addressing the challenges posed by, e.g., immunosuppressive tumor microenvironments and autoimmune side effects.

SUMMARY OF THE INVENTION

Against the aforementioned background, it is therefore an object of the present invention to provide safe and effective immunotherapeutic treatment options for subjects afflicted with cancer. It is also an object of the present invention to provide therapeutic means to elicit a therapeutically effective immune response against cancer in a subject and to enhance said immune response. It is a further object of the present invention to provide effective therapeutic means for the treatment of cancer with reduced autoimmune side effects.

These objects are achieved by the invention set forth in the claims and embodiments explained in more detail below.

The invention concerns an RNA cancer vaccine for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering to the subject: a) the RNA cancer vaccine comprising at least one RNA; and b) an anti-CTLA4 antibody.

Main challenges of cancer immunotherapy are the low overall efficacy of immunotherapies and undesired side effects associated with the treatment, such as immune-related adverse events. In the research underlying the invention, the applicants surprisingly found that the combined use of the RNA cancer vaccine and the anti-CTLA4 antibody provided an unexpected synergistic effect in the treatment of cancer, while in parallel providing a safe treatment.

This surprising effect results in longer overall survival, longer progression-free survival, longer freedom from disease progression (stable disease state), reduction of tumor-related symptoms, and/or reduction of need for pain medications during and/or following the anticancer therapy (RNA cancer vaccine and anti-CTLA4 antibody combination therapy). The effect can be reflected in an improved quality of life, such as mobility, strength of appetite, and/or psychological status.

The invention further provides an anti-CTLA4 antibody for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering to the subject: a) the anti-CTLA4 antibody; and b) an RNA cancer vaccine comprising at least one RNA.

Furthermore, the invention concerns an RNA cancer vaccine and an anti-CTLA4 antibody for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering to the subject: a) the RNA cancer vaccine comprising at least one RNA; and b) the anti-CTLA4 antibody.

The invention also provides a composition or kit of parts comprising: a) an RNA cancer vaccine comprising at least one RNA; and b) an anti-CTLA4 antibody.

The invention further provides a combination of an RNA cancer vaccine comprising at least one RNA and an anti-CTLA4 antibody.

The invention further provides a method of treating cancer in a subject in need thereof, wherein the method comprises administering to the subject: a) an RNA cancer vaccine comprising at least one RNA; and b) an anti-CTLA4 antibody.

Advantageous embodiments of the invention are indicated in the dependent claims and in the following.

DETAILED DESCRIPTION

Although certain embodiments of the present invention are described in detail below, it is to be understood that this invention is not limited to the particular embodiments, methodologies, protocols and reagents described herein as these may vary within the scope set by the claims. It is also to be understood that terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which is defined by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

In the following description, certain elements of the present invention will be described. These elements may be discussed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples, features and particular embodiments should not be construed to limit the present invention to only the explicitly described embodiments or to the explicitly described combination of features. This description should be understood to disclose and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by this description unless the context indicates otherwise. The above objects are achieved by the following embodiments in accordance with the invention:

1. An RNA cancer vaccine for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering to the subject: a) the RNA cancer vaccine comprising at least one RNA; and b) an anti-CTLA4 antibody.

2. An anti-CTLA4 antibody for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering to the subject: a) the anti-CTLA4 antibody; and b) an RNA cancer vaccine comprising at least one RNA.

3. The RNA cancer vaccine for use according to embodiment 1 or the anti-CTLA4 antibody for use according to embodiment 2, wherein the method comprises administering an additional immunomodulatory agent to the subject.

4. The RNA cancer vaccine for use according to embodiment 1 or 3 or the anti-CTLA4 antibody for use according to embodiment 2 or 3, wherein the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the additional immunomodulatory agent are administered separately.

5. The RNA cancer vaccine for use according to embodiments 1, 3 or 4 or the anti-CTLA4 antibody for use according to embodiments 2-4, wherein the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the additional immunomodulatory agent are administered concurrently or consecutively. The RNA cancer vaccine for use according to embodiments 1 or 3-5 or the anti-CTLA4 antibody for use according to embodiments 2-5, wherein the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the additional immunomodulatory agent are administered intravenously. The RNA cancer vaccine for use according to embodiments 1 or 3-6 or the anti-CTLA4 antibody for use according to embodiments 2-6, wherein the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the additional immunomodulatory agent are administered via an intravenous injection or an intravenous infusion, preferably an intravenous infusion. The RNA cancer vaccine for use according to embodiments 1 or 3-7 or the anti-CTLA4 antibody for use according to embodiments 2-7, wherein the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the additional immunomodulatory agent are administered in a therapeutically effective amount. The RNA cancer vaccine for use according to embodiments 1 or 3-8 or the anti-CTLA4 antibody for use according to embodiments 2-8, wherein the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the additional immunomodulatory agent are formulated with one or more pharmaceutically acceptable carriers, diluents and/or excipients. The RNA cancer vaccine for use according to embodiments 1 or 3-9 or the anti-CTLA4 antibody for use according to embodiments 2-9, wherein the cancer comprises one or more solid tumors. The RNA cancer vaccine for use according to embodiments 1 or 3-10 or the anti- CTLA4 antibody for use according to embodiments 2-10, wherein the cancer is selected from the group consisting of melanoma, lung cancer, human papillomavirus (HPV)- induced cancer, breast cancer, hepatocellular carcinoma, ovarian cancer such as ovarian carcinoma, prostate cancer such as prostate carcinoma, Hodgkin's or non-Hodgkin's lymphoma, acute myelogenic Leukemia, chronic myelogenic Leukemia, acute lymphocytic Leukemia, chronic lymphocytic Leukemia, or renal cell carcinoma. The RNA cancer vaccine for use according to embodiments 1 or 3-11 or the anti- CTLA4 antibody for use according to embodiments 2-11, wherein the cancer is melanoma, ovarian cancer, or lung cancer. The RNA cancer vaccine for use according to embodiments 1 or 3-12 or the anti- CTLA4 antibody for use according to embodiments 2-12, wherein the cancer is nonsmall cell lung cancer (NSCLC). The RNA cancer vaccine for use according to embodiment 13 or the anti-CTLA4 antibody for use according to embodiment 13, wherein the NSCLC has a squamous histology. The RNA cancer vaccine for use according to embodiment 13 or the anti-CTLA4 antibody for use according to embodiment 13, wherein the NSCLC has a non-squamous histology. The RNA cancer vaccine for use according to embodiments 1 or 3-15 or the anti-CTLA4 antibody for use according to embodiments 2-15, wherein the at least one RNA encodes at least one of the following amino acid sequences:

(i) an amino acid sequence comprising claudin 6 (CLDN6), an immunogenic variant thereof, or an immunogenic fragment of the CLDN6 or the immunogenic variant thereof;

(ii) an amino acid sequence comprising Kita-kyushu lung cancer antigen 1 (KK- LC-1), an immunogenic variant thereof, or an immunogenic fragment of the KK-LC-1 or the immunogenic variant thereof; (iii) an amino acid sequence comprising Melanoma antigen A3 (MAGE-A3), an immunogenic variant thereof, or an immunogenic fragment of the MAGE- A3 or the immunogenic variant thereof;

(iv) an amino acid sequence comprising Melanoma antigen 4 (MAGE-A4), an immunogenic variant thereof, or an immunogenic fragment of the MAGE-A4 or the immunogenic variant thereof;

(v) an amino acid sequence comprising Preferentially Expressed Antigen In Melanoma (PRAME), an immunogenic variant thereof, or an immunogenic fragment of the PRAME or the immunogenic variant thereof; and

(vi) an amino acid sequence comprising Melanoma antigen Cl (MAGE-CI), an immunogenic variant thereof, or an immunogenic fragment of the MAGE-CI or the immunogenic variant thereof. The RNA cancer vaccine for use according to embodiment 16 or the anti-CTLA4 antibody for use according to embodiment 16, wherein a) the RNA encoding the amino acid sequence (i) comprises the nucleotide sequence of SEQ ID NO: 3 or 4, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 3 or 4; and/or b) the amino acid sequence (i) comprises the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 2. The RNA cancer vaccine for use according to embodiment 16 or 17 or the anti-CTLA4 antibody for use according to embodiment 16 or 17, wherein a) the RNA encoding the amino acid sequence (ii) comprises the nucleotide sequence of SEQ ID NO: 7 or 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 7 or 8; and/or b) the amino acid sequence (ii) comprises the amino acid sequence of SEQ ID NO: 5 or 6, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5 or 6. The RNA cancer vaccine for use according to embodiments 16-18 or the anti-CTLA4 antibody for use according to embodiments 16-18, wherein a) the RNA encoding the amino acid sequence (iii) comprises the nucleotide sequence of SEQ ID NO: 11 or 12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 11 or 12; and/or b) the amino acid sequence (iii) comprises the amino acid sequence of SEQ ID NO: 9 or 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 9 or 10. The RNA cancer vaccine for use according to embodiments 16-19 or the anti-CTLA4 antibody for use according to embodiments 16-19, wherein a) the RNA encoding the amino acid sequence (iv) comprises the nucleotide sequence of SEQ ID NO: 15 or 16, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15 or 16; and/or b) the amino acid sequence (iv) comprises the amino acid sequence of SEQ ID NO: 13 or 14, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 13 or 14. The RNA cancer vaccine for use according to embodiments 16-20 or the anti-CTLA4 antibody for use according to embodiments 16-20, wherein a) the RNA encoding the amino acid sequence (v) comprises the nucleotide sequence of SEQ ID NO: 19 or 20, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 19 or 20; and/or b) the amino acid sequence (v) comprises the amino acid sequence of SEQ ID NO: 17 or 18, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 17 or 18. The RNA cancer vaccine for use according to embodiments 16-21 or the anti-CTLA4 antibody for use according to embodiments 16-21, wherein a) the RNA encoding the amino acid sequence (vi) comprises the nucleotide sequence of SEQ ID NO: 23 or 24, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 23 or 24; and/or b) the amino acid sequence (vi) comprises the amino acid sequence of SEQ ID NO: 21 or 22, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 21 or 22. The RNA cancer vaccine for use according to embodiments 16-22 or the anti-CTLA4 antibody for use according to embodiments 16-22, wherein a) the RNA encoding the amino acid sequence (i) comprises the nucleotide sequence of SEQ ID NO: 4; b) the RNA encoding the amino acid sequence (ii) comprises the nucleotide sequence of SEQ ID NO: 8; c) the RNA encoding the amino acid sequence (iii) comprises the nucleotide sequence of SEQ ID NO: 12; d) the RNA encoding the amino acid sequence (iv) comprises the nucleotide sequence of SEQ ID NO: 16; e) the RNA encoding the amino acid sequence (v) comprises the nucleotide sequence of SEQ ID NO: 20; and f) the RNA encoding the amino acid sequence (vi) comprises the nucleotide sequence of SEQ ID NO: 24. The RNA cancer vaccine for use according to embodiments 16-23 or the anti-CTLA4 antibody for use according to embodiments 16-23, wherein the RNA cancer vaccine comprises at least two RNAs, preferably encoding at least two of the amino acid sequences (i), (ii), (iii), (iv), (v), and (vi). The RNA cancer vaccine for use according to embodiments 16-24 or the anti-CTLA4 antibody for use according to embodiments 16-24, wherein the RNA cancer vaccine comprises six RNAs, preferably each encoding one of the amino acid sequences (i), (ii), (iii), (iv), (v), and (vi). The RNA cancer vaccine for use according to embodiments 16-25 or the anti-CTLA4 antibody for use according to embodiments 16-25, wherein at least one of the amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) comprises an amino acid sequence which breaks immunological tolerance and/or the at least one RNA is co-administered with RNA encoding an amino acid sequence which breaks immunological tolerance. The RNA cancer vaccine for use according to embodiment 26 or the anti-CTLA4 antibody for use according to embodiment 26, wherein each amino acid sequence (i), (ii), (iii), (iv), (v), or (vi) comprises an amino acid sequence which breaks immunological tolerance and/or each RNA is co-administered with RNA encoding an amino acid sequence which breaks immunological tolerance. The RNA cancer vaccine for use according to embodiment 26 or 27 or the anti-CTLA4 antibody for use according to embodiment 26 or 27, wherein the amino acid sequence which breaks immunological tolerance comprises helper epitopes, preferably tetanus toxoid-derived helper epitopes. The RNA cancer vaccine for use according to embodiments 26-28 or the anti-CTLA4 antibody for use according to embodiments 26-28, wherein a) the RNA encoding the amino acid sequence which breaks immunological tolerance comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 26; and/or b) the amino acid sequence which breaks immunological tolerance comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 25. The RNA cancer vaccine for use according to embodiments 16-29 or the anti-CTLA4 antibody for use according to embodiments 16-29, wherein at least one of the amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) 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, wherein the codon-optimization and/or the increase in the G/C content preferably does not change the sequence of the encoded amino acid sequence. The RNA cancer vaccine for use according to embodiment 30 or the anti-CTLA4 antibody for use according to embodiment 30, wherein each of the amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) 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, wherein the codon-optimization and/or the increase in the G/C content preferably does not change the sequence of the encoded amino acid sequence. The RNA cancer vaccine for use according to embodiments 1 or 3-31 or the anti- CTLA4 antibody for use according to embodiments 2-31, wherein the at least one RNA comprises a 5' cap, preferably selected from the group consisting of G[5’]ppp[5’]G, m7G[5’]ppp[5’]G, m₃ ²’²’⁷G[5’]ppp[5’]G, m₂ ⁷’³'-°G[5’]ppp[5’]G (3’-ARCA), m₂ ⁷’²'' °GppSpG (p-S-ARCA), m₂ ⁷’²’-°GppSpG most preferably m₂ ^7,2 '° GppSpG. The RNA cancer vaccine for use according to embodiment 32 or the anti-CTLA4 antibody for use according to embodiment 32, wherein each of the RNAs comprises a 5’ cap, preferably selected from the group consisting of G[5’]ppp[5’]G, m7G[5’]ppp[5’]G, m₃ ²’²’⁷G[5’]ppp[5’]G, m₂ ⁷’³’-°G[5’]ppp[5’]G (3’-ARCA), m₂ ⁷’²’' °GpppG (2’-ARCA), m₂ ^7,2 '° GppSpG, m₂ ⁷’²’'°GppSpG (p-S-ARCA), m₂ ⁷’²’-°GppSpG (P-S-ARCA), and m₂ ^{7 3} ''⁰Gppp(mi² '°)ApG, most preferably m₂ ⁷’² '° GppSpG. The RNA cancer vaccine for use according to embodiments 1 or 3-33 or the anti- CTLA4 antibody for use according to embodiments 2-33, wherein the at least one RNA comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 29. The RNA cancer vaccine for use according to embodiment 34 or the anti-CTLA4 antibody for use according to embodiment 34, wherein each of the RNAs comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 29. The RNA cancer vaccine for use according to embodiments 16-35 or the anti-CTLA4 antibody for use according to embodiments 16-35, wherein at least one of the amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) comprises an amino acid sequence enhancing antigen processing and/or presentation. The RNA cancer vaccine for use according to embodiment 36 or the anti-CTLA4 antibody for use according to embodiment 36, wherein each of the amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) comprises an amino acid sequence enhancing antigen processing and/or presentation. The RNA cancer vaccine for use according to embodiment 36 or 37 or the anti-CTLA4 antibody for use according to embodiment 36 or 37, wherein the amino acid sequence enhancing antigen processing and/or presentation comprises an amino acid sequence corresponding to the transmembrane and cytoplasmic domain of an MHC molecule, preferably an MHC class I molecule. The RNA cancer vaccine for use according to embodiments 36-38 or the anti-CTLA4 antibody for use according to embodiments 36-38, wherein a) the RNA encoding the amino acid sequence enhancing antigen processing and/or presentation comprises the nucleotide sequence of SEQ ID NO: 31, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 31; and/or b) the amino acid sequence enhancing antigen processing and/or presentation comprises the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 30. The RNA cancer vaccine for use according to embodiments 1 or 3-39 or the anti- CTLA4 antibody for use according to embodiments 2-39, wherein the at least one RNA comprises a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 32, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 32. The RNA cancer vaccine for use according to embodiment 40 or the anti-CTLA4 antibody for use according to embodiment 40, wherein each of the RNAs comprises a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 32, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 32. The RNA cancer vaccine for use according to embodiments 1 or 3-41 or the anti- CTLA4 antibody for use according to embodiments 2-41, wherein the at least one RNA comprises a poly-A sequence. The RNA cancer vaccine for use according to embodiment 42 or the anti-CTLA4 antibody for use according to embodiment 42, wherein each of the RNAs comprises a poly-A sequence. The RNA cancer vaccine for use according to embodiment 42 or 43 or the anti-CTLA4 antibody for use according to embodiment 42 or 43, wherein the poly-A sequence comprises at least 100 nucleotides. The RNA cancer vaccine for use according to embodiments 42-44 or the anti-CTLA4 antibody for use according to embodiments 42-44, wherein the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 33. The RNA cancer vaccine for use according to embodiments 1 or 3-45 or the anti- CTLA4 antibody for use according to embodiments 2-45, wherein the RNA is formulated as a liquid, a solid, or a combination thereof. The RNA cancer vaccine for use according to embodiments 1 or 3-46 or the anti- CTLA4 antibody for use according to embodiments 2-46, wherein the RNA is formulated for injection. The RNA cancer vaccine for use according to embodiments 1 or 3-47 or the anti- CTLA4 antibody for use according to embodiments 2-47, wherein the RNA is formulated for intravenous administration. The RNA cancer vaccine for use according to embodiments 1 or 3-48 or the anti- CTLA4 antibody for use according to embodiments 2-48, wherein the RNA is formulated or is to be formulated as lipoplex particles. The RNA cancer vaccine for use according to embodiment 49 or the anti-CTLA4 antibody for use according to embodiment 49, wherein the RNA lipoplex particles are obtainable by mixing the RNA with liposomes. The RNA cancer vaccine for use according to embodiments 16-50 or the anti-CTLA4 antibody for use according to embodiments 16-50, wherein the at least one RNA encoding an amino acid sequence (i), (ii), (iii), (iv), (v), or (vi) is co-formulated or is to be co-formulated as lipoplex particles with RNA encoding an amino acid sequence which breaks immunological tolerance. The RNA cancer vaccine for use according to embodiment 51 or the anti-CTLA4 antibody for use according to embodiment 51, wherein each of the RNAs encoding an amino acid sequence (i), (ii), (iii), (iv), (v), or (vi) is co-formulated or is to be coformulated as lipoplex particles with RNA encoding an amino acid sequence which breaks immunological tolerance. The RNA cancer vaccine for use according to embodiments 1 or 3-52 or the anti- CTLA4 antibody for use according to embodiments 2-52, wherein the anti-CTLA4 antibody comprises: a) a heavy chain variable region comprising (1) a complementarity-determining region 1 (HCDR1) whose amino acid sequence is set forth in SEQ ID NO: 39, (2) a complementarity-determining region 2 (HCDR2) whose amino acid sequence is set forth in SEQ ID NO: 41, 42 or 43, and (3) a complementarity-determining region 3 (HCDR3) whose amino acid sequence is set forth in SEQ ID NO: 47; and b) a light chain variable region comprising: (1) a complementarity-determining region 1 (LCDR1) whose amino acid sequence is set forth in SEQ ID NO: 40, (2) a complementarity-determining region 2 (LCDR2) whose amino acid sequence is set forth in SEQ ID NO: 44, 45, or 46, and (3) a complementarity-determining region 3 (LCDR3) whose amino acid sequence is set forth in SEQ ID NO: 48. The RNA cancer vaccine for use according to embodiment 53 or the anti-CTLA4 antibody for use according to embodiment 53, wherein the anti-CTLA4 antibody comprises: a) a heavy chain variable region comprising (1) a HCDR1 whose amino acid sequence is set forth in SEQ ID NO: 39, (2) a HCDR2 whose amino acid sequence is set forth in SEQ ID NO: 41, and (3) a HCDR3 whose amino acid sequence is set forth in SEQ ID NO: 47; and b) a light chain variable region comprising: (1) a LCDR1 whose amino acid sequence is set forth in SEQ ID NO: 40, (2) a LCDR2 whose amino acid sequence is set forth in SEQ ID NO: 45, and (3) a LCDR3 whose amino acid sequence is set forth in SEQ ID NO: 48. The RNA cancer vaccine for use according to embodiment 53 or the anti-CTLA4 antibody for use according to embodiment 53, wherein the anti-CTLA4 antibody comprises: a) a heavy chain variable region comprising (1) HCDR1 whose amino acid sequence is set forth in SEQ ID NO: 39, (2) a HCDR2 whose amino acid sequence is set forth in SEQ ID NO: 43, and (3) a HCDR3 whose amino acid sequence is set forth in SEQ ID NO: 47; and b) a light chain variable region comprising: (1) a LCDR1 whose amino acid sequence is set forth in SEQ ID NO: 40, (2) a LCDR2 whose amino acid sequence is set forth in SEQ ID NO: 45, and (3) a LCDR3 whose amino acid sequence is set forth in SEQ ID NO: 48. The RNA cancer vaccine for use according to embodiment 53 or the anti-CTLA4 antibody for use according to embodiment 53, wherein the anti-CTLA4 antibody comprises: a) a heavy chain variable region comprising (1) a HCDR1 whose amino acid sequence is set forth in SEQ ID NO: 39, (2) a HCDR2 whose amino acid sequence is set forth in SEQ ID NO: 43, and (3) a HCDR3 whose amino acid sequence is set forth in SEQ ID NO: 47; and b) a light chain variable region comprising: (1) a LCDR1 whose amino acid sequence is set forth in SEQ ID NO: 40, (2) a LCDR2 whose amino acid sequence is set forth in SEQ ID NO: 46, and (3) a LCDR3 whose amino acid sequence is set forth in SEQ ID NO: 48. The RNA cancer vaccine for use according to embodiments 53-56 or the anti-CTLA4 antibody for use according to embodiments 53-56, wherein the anti-CTLA4 antibody comprises: a) A heavy chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 49, 50, or 51; and b) a light chain variable domain whose amino acid sequence is set forth in SEQ ID NO:

52, 43, or 54. The RNA cancer vaccine for use according to embodiment 57 or the anti-CTLA4 antibody for use according to embodiment 57, wherein the anti-CTLA4 antibody comprises: a) a heavy chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 49; and b) a light chain variable domain whose amino acid sequence is set forth in SEQ ID NO:

53. The RNA cancer vaccine for use according to embodiment 57 or the anti-CTLA4 antibody for use according to embodiment 57, wherein the anti-CTLA4 antibody comprises: a) a heavy chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 51; and b) a light chain variable domain whose amino acid sequence is set forth in SEQ ID NO:

54. The RNA cancer vaccine for use embodiments 1, 3-60 or the anti-CTLA4 antibody for use according to embodiments 2-60, wherein the anti-CTLA4 antibody comprises a heavy chain comprising a Fc region of a human Ig antibody, preferably comprising the amino acid sequence of SEQ ID NO: 55 or 56. The RNA cancer vaccine for use according to embodiments 53-61 or the anti-CTLA4 antibody for use according to embodiments 53-61, wherein the anti-CTLA4 antibody comprises: a) a heavy chain whose amino acid sequence is set forth in SEQ ID NO: 57, 59, or 61; and b) a light chain whose amino acid sequence is set forth in SEQ ID NO: 63, 65, or 67. The RNA cancer vaccine for use according to embodiment 62 or the anti-CTLA4 antibody for use according to embodiment 62, wherein the anti-CTLA4 antibody comprises: a) a heavy chain whose amino acid sequence is set forth in SEQ ID NO: 57; and b) a light chain whose amino acid sequence is set forth in SEQ ID NO: 65. The RNA cancer vaccine for use according to embodiment 62 or the anti-CTLA4 antibody for use according to embodiment 62, wherein the anti-CTLA4 antibody comprises: a) a heavy chain whose amino acid sequence is set forth in SEQ ID NO: 61; and b) a light chain whose amino acid sequence is set forth in SEQ ID NO: 65. The RNA cancer vaccine for use according to embodiment 62 or the anti-CTLA4 antibody for use according to embodiment 62, wherein the anti-CTLA4 antibody comprises: a) a heavy chain whose amino acid sequence is set forth in SEQ ID NO: 61; and b) a light chain whose amino acid sequence is set forth in SEQ ID NO: 67. The RNA cancer vaccine for use according to embodiments 1, 3-65 or the anti-CTLA4 antibody for use according to embodiments 2-65, wherein the anti-CTLA4 antibody is capable of binding to human CTLA4. The RNA cancer vaccine for use according to embodiments 1, 3-66 or the anti-CTLA4 antibody for use according to embodiments 2-66, wherein the anti CTLA4 antibody is a humanized anti-CTLA4 antibody. The RNA cancer vaccine for use according to embodiments 1, 3-60, 66-67 or the anti- CTLA4 antibody for use according to embodiments 2-60, 66-67, wherein the anti- CTLA4 antibody is an antigen-binding fragment or a variant thereof. The RNA cancer vaccine for use according to embodiment 68 or the anti-CTLA4 antibody for use according to embodiment 68, wherein the antigen binding fragment or variant thereof is selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a scFV, a diabody, a triabody, a minibody, and a single-domain antibody (sdAB), and variants thereof. The RNA cancer vaccine for use according to embodiments 1, 3-69 or the anti-CTLA4 antibody for use according to embodiments 2-69, wherein the anti-CTLA4 antibody is encoded by one or more RNAs. An RNA cancer vaccine and an anti-CTLA4 antibody for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering to the subject: a) the RNA cancer vaccine comprising at least one RNA; and b) the anti-CTLA4 antibody. A composition or kit of parts comprising: a) an RNA cancer vaccine comprising at least one RNA; and b) an anti-CTLA4 antibody. The composition or kit of parts according to embodiment 72, further comprising an additional immunomodulatory agent. The composition or kit of parts according to embodiment 72 or 73, comprising a therapeutically effective amount of the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the additional immunomodulatory agent. The composition or kit of parts according to embodiments 72-74, comprising one or more pharmaceutically acceptable carriers, diluents and/or excipients. The composition or kit of parts according to embodiments 72-75, comprising one or more containers comprising the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the additional immunomodulatory agent. 77. The composition or kit of parts according to embodiments 72-76, wherein the kit comprises individual containers each individually comprising the RNA cancer vaccine, the anti-CTLA4 antibody, and optionally the additional immunomodulatory agent.

78. The composition or kit of parts according to embodiments 72-76, wherein the kit comprises individual containers comprising combinations of the RNA cancer vaccine, the anti-CTLA4 antibody, and optionally the additional immunomodulatory agent.

79. A method of treating cancer in a subject in need thereof, wherein the method comprises administering to the subject: a) an RNA cancer vaccine comprising at least one RNA; and b) an anti-CTLA4 antibody.

80. A combination of an RNA cancer vaccine comprising at least one RNA and an anti- CTLA4 antibody.

Definitions

The terms indicated for explanation of the invention have the following meaning, unless otherwise indicated in the description or the embodiments. Additional definitions are set forth throughout the detailed description.

Terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the embodiments) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

The terms “about” or “approximately” as used herein denotes a range of ±10% of a reference value. For examples, “about 10” defines a range of 9 to 11. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” or “approximately” in that context. The term “plurality” refers to the state of being plural.

Unless expressly specified otherwise, the term “comprising” is used in the context of the present disclosure to indicate that further members may optionally be present in addition to the members of the list introduced by “comprising”. It is, however, contemplated as specific embodiments of the present invention that each time the term “comprising” is used, this shall also encompass the possibility of no further members being present, i.e., for the purpose of this embodiment “comprising” can be understood as having the meaning of “consisting of’.

Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G.W. Leuenberger, B. Nagel, and H. K51bl, Eds., (1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 4th Edition, M.R. Green, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012).

Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it was individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The terms “polynucleotide” and “nucleic acid” can be used interchangeably herein to refer to polymers of nucleotides. The term “polynucleotide” comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. In some embodiments, a polynucleotide is DNA. In some embodiments, a polynucleotide is RNA. A polynucleotide may be present as a mixture of DNA and RNA. A polynucleotide may be further present as a single-stranded or doublestranded and linear or covalently circularly closed molecule. A polynucleotide can be isolated. The term “isolated polynucleotide “ means, according to the present invention, that the polynucleotide (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.

The term “RNA” relates to a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, “ribonucleotide” refers to a nucleotide with a hydroxyl group at the 2'- position of a P-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of nonnucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present invention, these altered/modified nucleotides can be referred to as analogs of naturally occurring nucleotides, and the corresponding RNAs containing such altered/modified nucleotides (i.e., altered/modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains “a majority of ribonucleotide residues” if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, 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%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof). The term “RNA” further includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), selfamplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA). In some embodiments, “RNA” refers to mRNA. RNA as described herein may comprise in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

The term “mRNA” refers to messenger RNA that comprises a coding sequence encoding a polypeptide. An mRNA may further comprise non-coding sequences such as a 5 ’cap, a 5’UTR, a 3’UTR, a Kozak sequence, an FI element or a poly(A) tail or any combination thereof. According to the present disclosure, the term “mRNA” means “messenger-RNA” and includes a “transcript” which may be generated by using a DNA template. Generally, mRNA encodes a peptide or polypeptide. mRNA is single-stranded but may contain self- complementary sequences that allow parts of the mRNA to fold and pair with itself to form double helices. According to the present disclosure, “dsRNA” means double-stranded RNA and is RNA with two partially or completely complementary strands.

The term “3’UTR sequence” refers to a 3' untranslated region known to regulate mRNA- based processes, such as mRNA localization, mRNA stability, and translation. In addition, 3' UTRs can establish 3' UTR-mediated protein-protein interactions (PPIs), and thus can transmit genetic information encoded in 3' UTRs to proteins. This function has been shown to regulate diverse protein features, including protein complex formation or posttranslational modifications, but is also expected to alter protein conformations.

The term “5’ UTR sequence” refers to a 5 '-untranslated region which lies within the noncoding genome upstream of a coding sequence and plays an important role in regulating gene expression. Within 5'-UTR sequences may be numerous cis-regulatory elements present that can interact with the transcriptional machinery to regulate mRNA abundance. The 5 '-untranslated region may contain various RNA-based regulatory elements including the secondary structures, RNA-binding protein motifs, upstream open-reading frames (uORFs), internal ribosome entry sites, terminal oligo pyrimidine (TOP) tracts, and G- quadruplexes. These elements can alter the efficiency of mRNA translation; some can also affect mRNA transcript levels via changes in stability or degradation.

The term “5’cap” refers to a cap structure on the 5'-end of mRNAs, which is present in eukaryotic organisms. Naturally occurring Cap structures comprise a ribo-guanosine residue that is methylated at position N7 of the guanine base, abbreviated ^7mGppp. The presence of the ^7mGppp fragment on the 5'-end is essential for mRNA maturation, it protects the mRNAs from degradation by exonucleases, facilitates transport of mRNAs from the nucleus to the cytoplasm and plays a key role in assembly of the translation initiation complex. Exemplary 5 ’caps suitable for use in the polynucleotide (RNA) disclosed herein are described in the patent application WO 2017/053297 Al. In some embodiments, a 5’ cap may be or comprise a dinucleotide cap analog such as G[5’]ppp[5’]G, m7G[5’]ppp[5’]G, m₃ ²’²’⁷G[5’]ppp[5’]G, m₂ ⁷’³'-°G[5’]ppp[5’]G (3’- ARCA), m₂ ^7,2 '°GpppG (2’-ARCA), m₂ ⁷’²'-° GppSpG, m₂ ⁷’²'-°GppSpG (p-S- ARCA), m₂ ⁷’²’' °GppSpG (P-S-ARCA), and m2^{7 3} '°Gppp(mi² '°)ApG (CleanCap413). Various cap analogues are described herein and known in the art, e.g., commercially available.

The term “nucleoside” relates to nucleotide compounds without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine. The five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine. The five nucleosides are commonly abbreviated to their one letter codes U, A, T, C and G, respectively. However, thymidine is more commonly written as “dT” (“d” represents “deoxy”) as it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA). Conversely, uridine is found in RNA and not DNA. The remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G, whereas in DNA they would be represented as dA, dC and dG. A modified purine (A or G) or pyrimidine (C, T, or U) base moiety may be modified by one or more alkyl groups, e.g., one or more C1.4 alkyl groups, e.g., one or more methyl groups. Particular examples of modified purine or pyrimidine base moieties include N⁷-alkyl-guanine, N⁶-alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and N(l)-alkyl-uracil, such as N⁷-Ci-4 alkyl-guanine, N⁶-CI-4 alkyl-adenine, 5-C1-4 alkylcytosine, 5-C1-4 alkyl-uracil, and N(1)-CI-4 alkyl-uracil, preferably N⁷-methyl-guanine, N⁶- methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil, and N(l)-methyl-uracil.

The term “poly(A)-tail” is used interchangeably with the term “poly(A)-sequence” or “poly-A-sequence” within the meaning of the present invention and refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3 '-end of an RNA molecule. Poly-A-sequences are known to those of skill in the art and may follow the 3’-UTR in the RNAs described herein. An uninterrupted poly-A-sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly-A- sequence is typical. RNAs disclosed herein can have a poly-A-sequence attached to the free 3 ’-end of the RNA by a template independent RNA polymerase after transcription or a poly-A-sequence encoded by DNA and transcribed by a template dependent RNA polymerase. This process, called polyadenylation, adds a poly(A)-tail that is usually between 100 and 250 residues long. Poly(A) tails play an important role in the translation and stability of the mRNA. RNA having an unmasked poly-A sequence is translated more efficiently than RNA having a masked poly-A sequence. An unmasked poly-A sequence means that the poly-A sequence at the 3’ end of an RNA molecule ends with an A of the poly-A sequence and is not followed by nucleotides other than A located at the 3’ end, i.e., downstream, of the poly-A sequence. Furthermore, a long poly-A sequence of about 120 base pairs results in an optimal transcript stability and translation efficiency of RNA.

The term “Kozak sequence” refers to a sequence which typically extends from approximately position -6 to position +6, where +1 is assigned to the adenine of the START codon. The Kozak sequence is known to affect transcription initiation.

The term “FI element” refers to a sequence in the 3 ’-untranslated region known to improve mRNA stability and translation efficiency. The FI element can be positioned in the 3’UTR. Exemplary FI elements suitable for use in the polynucleotide (RNA) disclosed herein are descripted in the patent application WO 2017/059902 Al.

The term “in vitro transcription” or “IVT” means that the transcription (i.e., the generation of RNA) is conducted in a cell-free manner. I.e., IVT does not use living/cultured cells but rather the transcription machinery extracted from cells (e.g., cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)).

Herein, the term “DNA” relates to a nucleic acid molecule which is entirely or at least substantially composed of deoxyribonucleotide residues. In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, “deoxyribonucleotide” refers to a nucleotide which lacks a hydroxyl group at the 2’- position of a P-D-ribofuranosyl group. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of nonnucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present invention, these altered DNAs are considered analogs of naturally-occurring DNA. A molecule contains “a majority of deoxyribonucleotide residues” if the content of deoxyribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, 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%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof). DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA.

As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., mRNA) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or a single-stranded RNA (e.g., an mRNA) encodes a polypeptide if transcription and translation of mRNA corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of a single-stranded RNA encoding a target polypeptide agent refers to a coding strand, the nucleotide sequence of which is identical to the mRNA sequence of such a target polypeptide agent. In some embodiments, a coding region of a single-stranded RNA encoding a target polypeptide agent refers to a non-coding strand of such a target polypeptide agent, which may be used as a template for transcription of a gene or cDNA. As is understood in the art, the phrase “nucleic acid encoding a peptide or protein” means that the nucleic acid, if present in the appropriate environment, for example within a cell and/or in a cell-free translation system, can direct the assembly of amino acids to produce the peptide or protein via a process of translation.

The term “identity” or “sequence identity” refer to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules. “Sequence identity” between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences. The terms “% identical”, “% identity” or similar terms refer to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or “window of comparison”, in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sei. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLASTP, BLASTN and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). For example, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at the website blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST _SPEC=blast2seq&LINK_LOC=align2seq). The algorithm parameters used for BLASTN algorithm on the NCBI website may include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to O; (iv) Match/Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. The algorithm parameters used for BLASTP algorithm on the NCBI website may include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to O; (iv) Matrixset to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment. Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100. In some embodiments, the degree of identity is given for a region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid or amino acid sequence consists of 200 nucleotides or amino acids, the degree of identity is given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides or amino acids, in some embodiments in continuous nucleotides or amino acids. In some embodiments, the degree of identity is given for the entire length of the reference sequence. Nucleic acid sequences or amino acid sequences having a particular degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, may have at least one functional property of said given sequence, e.g., and in some instances, are functionally equivalent to said given sequence. One important property includes an immunogenic property, in particular when administered to a subject. In some embodiments, a nucleic acid sequence or amino acid sequence having a particular degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.

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 term “analogue” refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analogue” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, an analogue is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analogue is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. In some embodiments, an analogue is or can be generated through performance of a synthetic process different from that used to generate the reference substance.

The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids.

The term “recombinant” when used in the context of a polynucleotide means a polynucleotide having nucleotide sequences that are not naturally joined together and can be made by artificially combining two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. Recombinant polynucleotides include vectors comprising an amplified or assembled polynucleotide, which can be used to transform or transfect a suitable host cell. A host cell that comprises the recombinant polynucleotide is referred to as a “recombinant host cell.” The polynucleotide is then expressed in the recombinant host cell to produce a “recombinant polypeptide.” A recombinant polynucleotide can also comprise a non-coding function. The term “full length” with respect to a given polypeptide means the form of the polypeptide naturally translated from the coding DNA sequence, beginning with the ATG start codon, which encodes the first methionine in the amino acid sequence, and ending at the TGA, TAG, or TTA stop codon, or whichever stop codon employed by the organism.

The term “gene” refers to a DNA sequence in a chromosome that codes for a protein. In some embodiments, a gene includes coding sequence (i.e., sequence that encodes a particular protein); in some embodiments, a gene includes non-coding sequence. In some particular embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequences. In some embodiments, a gene may include one or more regulatory elements that, for example, may control or impact one or more aspects of gene expression (e.g., cell-type- specific expression, inducible expression, etc.).

The term “vaccine” refers to a composition that induces an immune response upon administration to a subject. In some embodiments, the induced immune response provides therapeutic immunity. An “RNA cancer vaccine” is a composition comprising at least one RNA, wherein the composition induces an immune response against cancer cells upon administration to a subject. The RNA cancer vaccine disclosed herein is a vaccine comprising at least one RNA for the treatment of cancer. The RNA may encode tumor- associated or tumor-specific antigens. By vaccinating a subject with an RNA cancer vaccine, the immune system can be trained to recognize and destroy cancer cells. In some embodiments, the RNA cancer vaccine is a therapeutic RNA cancer vaccine.

The terms “effective amount” or “therapeutically effective amount” refer to an amount of a given substance that is sufficient in quantity to produce a desired effect, including an improvement or remediation of the disease, disorder, or symptoms of the disease or condition.

The term “carrier” refers to a component which may be natural, synthetic, organic, inorganic in which the active ingredients of the invention are combined in order to facilitate, enhance or enable administration of the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the additional immunomodulatory agent. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to the subject. Suitable carriers include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxy ethylene/polyoxy-propylene copolymers. Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985). Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

The term “excipient” is a substance which may be present in a formulation of the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the additional immunomodulatory agent but is not an active ingredient. Examples of excipients, include without limitation carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.

The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” means solvents, dispersion media, coatings, antibacterial agents and antifungal agents, isotonic agents, and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. In certain embodiments, the pharmaceutically acceptable carrier or excipient is not naturally occurring. The term “pharmaceutically acceptable” refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.

The term “diluent” relates to a diluting and/or thinning agent. Moreover, the term “diluent” includes any one or more of fluid, liquid or solid suspension and/or mixing media. Nonlimiting examples of suitable diluents include ethanol, glycerol, and water. The term “subject” relates to a human of either gender (a male or a female). The subject may be of any age. In some embodiments, the subject is female. In some embodiments, the subject is male. In some embodiments, the subject is a subject having cancer, in particular a female subject having cancer and/or a male subject having cancer.

The term “treating” when used in the context of a disease or disease condition means ameliorating, improving or remedying a disease, disorder, or symptom of a disease or condition associated with the disease, or can mean completely or partially stopping, on a molecular level, the biochemical basis of the disease. It describes an act that leads to the elimination, reduction, alleviation, reversal, or prevention or delay of onset or recurrence of any symptom of a disease.

The term “pharmaceutical composition” relates to a composition comprising a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and/or excipients. Said pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease by administration of said pharmaceutical composition to a subject.

The term “polymer” relates to a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. The repeat units can all be identical, or in some cases, there can be more than one type of repeat unit present within the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties can also be present in the polymer, for example targeting moieties. If more than one type of repeat unit is present within the polymer, then the polymer is said to be a “copolymer”. It is to be understood that the polymer being employed herein can be a copolymer. The repeat units forming the copolymer can be arranged in any fashion. For example, the repeat units can be arranged in a random order, in an alternating order, or as a “block” copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks. The term “cancer” refers to a neoplasm or tumor resulting from abnormal uncontrolled growth of cells. The term “tumor” as it applies to a subject diagnosed with, or suspected of having, a cancer refers to a malignant or potentially malignant neoplasm or tissue mass of any size and includes primary tumors and secondary neoplasms. A solid tumor is an abnormal growth or mass of tissue that usually does not contain cysts or liquid areas. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors (National Cancer Institute, Dictionary of Cancer Terms).

The term “CTLA4” or “CTLA-4” relates to cytotoxic T lymphocyte antigen-4. “CTLA4” or “CTLA-4” are used interchangeably herein. CTLA4 is expressed on the surface of cells and, if it is located at the surface of said cells, is accessible to binding by CTLA4 specific antibodies. “Cell surface” is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules. For example, a transmembrane protein having one or more extracellular portions is considered as being expressed on the cell surface.

The term “immunomodulatory agent” relates to a natural or synthetic agent, substance, compound, or composition that modulates the immune system, i.e., that a specific immune response is induced, enhanced, attenuated and/or suppressed by the immunomodulatory agent. Immunomodulatory agents or “immunomodulators” are well known in the art (Bascones-Martinze et al., Med Oral Patol Oral Cir Bucal 2014, 19(1): e24-e31). Examples of immunomodulatory agents include, without limitation, immune checkpoint inhibitors, cytokines such as monokines, lymphokines, interleukins, and chemokines, monoclonal antibodies, growth factors, lipopolysaccharide (LPS), chaperone GP96, CpG oligodeoxynucleotides and anti -cancer vaccines. The immune checkpoint inhibitors may be selected from the group consisting of anti-PD-1, anti-B7-Hl, anti-B7-H4, anti-LIGHT, anti- LAG3, anti-TIM3, anti-TIM4, anti-OX40, anti-GITR, anti -B TLA, anti-CD27, and anti- ICOS antibodies. The anti-PD-1 antibodies may include cemiplimab (LIBTAYO, REGN2810), nivolumab (OPDIVO; BMS-936558), pembrolizumab (KEYTRUDA; MK- 3475), pidilizumab (CT-011), spartalizumab (PDR001), MEDI0680 (AMP-514), dostarlimab (TSR-042), cetrelimab (JNJ 63723283), toripalimab (JSOO1), AMP-224 (GSK- 2661380), PF-06801591, tislelizumab (BGB-A317), ABBV-181, BI 754091, or SHR-1210. The cytokines may include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF a, INF-y, GM-CSF, and LT-a.

The term “amino acid sequences which breaks immunological tolerance” relates to an amino acid sequence that supports to overcome self-tolerance mechanisms for efficient induction of immune responses to self-antigens by providing tumor-unspecific T-cell help during priming. The amino acid sequence which breaks immunological tolerance may comprise helper epitopes, preferably tetanus toxoid-derived helper epitopes, e.g., P2P16 amino acid sequences derived from the tetanus toxoid (TT) of Clostridium tetani. Helper epitopes can be selected to assist in or ensure binding to as many MHC class II alleles as possible. Reference is made to WO 2020/182869, which is incorporated herein in its entirety.

The term “immune cell” means any cell of hematopoietic lineage involved in regulating an immune response against an antigen (e.g., a bacterial or viral infection or an auto-antigen). In some embodiments, an immune cell is a leukocyte, such as a white blood cell. Immune cells include neutrophils, eosinophils, basophils, lymphocytes, and/or monocytes. Lymphocytes include T lymphocytes (T cells) and B lymphocytes (B cells). Immune cells can also be dendritic cells, natural killer (NK) cells, and/or a mast cell.

The term “antibody” refers to a molecule that possesses an antigen-binding site. The term encompasses functional antibody fragments, such as a Fab, a Fab’, a F(ab’)2, a scFV, a diabody, a triabody, a minibody and a single-domain antibody (sdAB). The antibody can comprise a “variable region”. The terms “variable region” and “variable domain” are used interchangeably herein. The term variable region is intended to distinguish such domain of the immunoglobulin from domains that are broadly shared by antibodies (such as an antibody Fc domain). The variable region comprises a “hypervariable region” whose residues are responsible for antigen binding. The hypervariable region comprises amino acid residues from a “complementarity determining region” or “CDR” (i.e., typically at approximately residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and at approximately residues 27-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain) and/or those residues from a “hypervariable loop” (i.e., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26- 32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain. The variable domains of the heavy and light chains each contain three CDRs, designated CDR1, CDR2 and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5^th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991; Kabat et al., J Biol Chem 1977, 252: 6609-6616; Kabat, Adv Prot Chem 1978, 32: 1-75), the Chothia numbering system (Chothia & Lesk, J Mol Biol 1987, 196: 901-917; Chothia et al., Nature 1989, 342: 878-883) or the IMGT numbering system (Lefranc et al., Dev Comparat Immunol 2003, 27: 55-77). For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between different numbering systems is well known to those skilled in the art (Lefranc et al., Dev Comparat Immunol 2003, 27: 55-77). “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region or CRR residues as herein defined. “Antibody” includes monoclonal antibodies, multi-specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies, single chain antibodies, disulfide- linked Fvs (sdFv), intrabodies, and anti -idiotypic (anti-Id) antibodies (including, e.g., anti- id and anti-anti-Id antibodies to antibodies disclosed herein). In particular, such antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, IgG2, IgGs, IgG4, IgAi and IgA2) or subclass. The term antibody further encompasses functional antibody fragments, such as a Fab, a Fab’, a F(ab’)2, a scFV, a diabody, a triabody, a minibody and a single-domain antibody (sdAB).

The term “antigen binding fragment” or “antibody binding portion” of an antibody refers to one or more portions of an antibody that contain the antibody’s CDRs and optionally the framework residues that comprise the antibody’s variable domain antigen recognition site, and exhibit an ability to immunospecifically bind an antigen. Examples of binding fragments encompassed within the term “antigen binding fragment” or “antigen binding portion” of an antibody include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH domains; (ii) F(ab’)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of the VH and CH domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments (Ward et al., 1989, Nature 341 :544-546), which consist of a VH domain; (vi) isolated complementarity determining regions (CDR), and (vii) combinations of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988, Science, 242:423-426; and Huston et al., 1988, Proc. Natl. Acad. Sci. USA, 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” or “antibody binding portion” of an antibody. A further example are binding-domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide that is fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. Binding-domain immunoglobulin fusion proteins are further disclosed in U.S. Patent Application Publication Nos. 2003/0118592 and 2003/0133939. These antibody fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. “Antigen binding fragment” or “antibody binding portion” may further include fusion proteins comprising the antibody’s variable region antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or receptor ligand, etc.). As used within the present invention, the term “fragment” refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

The term “human antibody” refers to an antibody that comprises human immunoglobulin protein sequences only. A human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, “mouse antibody” or “rat antibody” refer to an antibody that comprises only mouse or rat immunoglobulin sequences, respectively.

A “humanized antibody” is an immunoglobulin comprising a human framework region and one or more CDR’s from a non-human (usually a mouse or rat) immunoglobulin. The nonhuman immunoglobulin providing the CDR’s is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor”. Constant regions need not be present, but if they are, they preferably can be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDR’s, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody, because, e.g., the entire variable region of a chimeric antibody is non-human. One says that the donor antibody has been “humanized”, by the process of “humanization”, because the resultant humanized antibody is expected to bind to the same antigen as the donor antibody that provides the CDR’s. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or a non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non- human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin that immunospecifically binds to an Fc.gamma.RIIB polypeptide, that has been altered by the introduction of amino acid residue substitutions, deletions or additions (i.e., mutations).

An antibody that “specifically binds to” a specified target protein is an antibody that exhibits preferential binding to that target as compared to other proteins, but this specificity does not require absolute binding specificity. An antibody is considered “specific” for its intended target if its binding is determinative of the presence of the target protein in a sample, e.g., without producing undesired results such as false positives. Antibodies, or binding fragments thereof, useful in the present invention will bind to the target protein with an affinity that is at least two fold greater, preferably at least ten times greater, more preferably at least 20-times greater, and most preferably at least 100-times greater than the affinity with non-target proteins. As used herein, an antibody is said to bind specifically to a polypeptide comprising a given amino acid sequence, e.g., the amino acid sequence of a mature human CTLA4 molecule, if it binds to polypeptides comprising that sequence but preferably does not bind to proteins lacking that sequence.

The term “epitope” refers to the part of an antigen that as used herein, refers to an agent that elicits an immune response; and/or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody. For example, epitopes are the discrete, three-dimensional sites on an antigen, which are recognized by the immune system. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non- conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

The term “antigen” as used herein, refers to an agent that elicits an immune response; and/or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody. In some embodiments, an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies); in some embodiments, the antigen elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen). In some embodiments, antigen binds to an antibody and may or may not induce a particular physiological response in an organism. In general, an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments other than a biologic polymer [e.g., other than a nucleic acid or amino acid polymer) etc. In some embodiments, an antigen is or comprises a polypeptide. In some embodiments, an antigen is or comprises a glycan. Those of ordinary skill in the art will appreciate that, in general, an antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g., together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigen-containing source). In some embodiments, antigens utilized in accordance with the present invention are provided in a crude form. In some embodiments, an antigen is a recombinant antigen.

The RNA cancer vaccine and anti-CTLA4 antibody combination therapy of the invention

The present invention provides an RNA cancer vaccine for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering to the subject: a) the RNA cancer vaccine comprising at least one RNA; and b) an anti-CTLA4 antibody.

The invention further provides an anti-CTLA4 antibody for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering to the subject: c) the anti-CTLA4 antibody; and d) an RNA cancer vaccine comprising at least one RNA.

The invention further provides an RNA cancer vaccine and an anti-CTLA4 antibody for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering to the subject: e) the RNA cancer vaccine comprising at least one RNA; and f) the anti-CTLA4 antibody.

The RNA cancer vaccine comprises at least one RNA, wherein preferably the at least one RNA encodes at least one of the following amino acid sequences:

(ii) an amino acid sequence comprising Kita-kyushu lung cancer antigen 1 (KK- LC-1), an immunogenic variant thereof, or an immunogenic fragment of the KK-LC-1 or the immunogenic variant thereof;

(iii) an amino acid sequence comprising Melanoma antigen A3 (MAGE-A3), an immunogenic variant thereof, or an immunogenic fragment of the MAGE- A3 or the immunogenic variant thereof; (iv) an amino acid sequence comprising Melanoma antigen 4 (MAGE-A4), an immunogenic variant thereof, or an immunogenic fragment of the MAGE-A4 or the immunogenic variant thereof;

(vi) an amino acid sequence comprising Melanoma antigen Cl (MAGE-CI), an immunogenic variant thereof, or an immunogenic fragment of the MAGE-CI or the immunogenic variant thereof.

The RNA cancer vaccine of the present invention comprises RNA encoding preferably a set of tumor-associated antigen (TAA) amino acid sequences, immunogenic variants thereof, or immunogenic fragments of the TAAs, i.e., antigenic peptides or proteins. Each of the TAA amino acid sequences comprises an epitope of a tumor antigen for inducing an immune response against the tumor antigen in the subject. RNA encoding a single TAA or a plurality of TAAs is administered to provide, upon translation of the TAA-encoding polynucleotide by appropriate target cells, a single TAA or a plurality of TAAs for induction, i.e., stimulation, priming and/or expansion, of an immune response directed against the single TAA or the plurality of TAAs or the procession product thereof. In some preferred embodiments, the immune response which is to be induced according to the present disclosure is a T cell-mediated immune response. In some preferred embodiments, the immune response is an anti-cancer, in particular anti-lung cancer, anti-ovarian cancer, or anti-melanoma immune response. In some more preferred embodiments, the immune response is directed against non-small cell lung cancer (NSLC).

In some embodiments, the RNA cancer vaccine described herein comprise at least one RNA encoding at least one of the following TAAs: a claudin 6 (CLDN6) vaccine antigen (amino acid sequence (i)), a Kita-kyushu lung cancer antigen 1 (KK-LC-1) vaccine antigen (amino acid sequence (ii)), a Melanoma antigen A3 (MAGE- A3) vaccine antigen (amino acid sequence (iii)), a Melanoma antigen 4 (MAGE-A4) vaccine antigen (amino acid sequence (iv)), a Preferentially Expressed Antigen In Melanoma (PRAME) vaccine antigen (amino acid sequence (v)), and a Melanoma antigen Cl (MAGE-CI) vaccine antigen (amino acid sequence (vi)).

The human claudin 6 gene (CLDN6) is localized on chromosome 16 and contains two isoforms which encode a protein of 220 amino acids. CLDN6 is highly conserved among species, and belongs to the group of claudins which consists of at least 27 members. In general, claudins, including CLDN6, are important for epithelial barrier regulation and belong to the group of tight junction molecules. CLDN6 contains four transmembrane domains, two extracellular loops, intracellular N- and C-termini, and a PDZ-binding domain, and has been shown to play a role in maintaining permeability barriers and trans- epithelial resistance in epidermal cells. Additionally, CLDN6 appears to be required for normal blastocyst formation. In some embodiments, CLDN6 has the amino acid sequence according to SEQ ID NO: 1.

Kita-kyushu lung cancer antigen 1 (KK-LC-1), also cancer/testis antigen 83, CT83, cXorf61, is a protein and tumor antigen from the group of cancer/testis antigens. KK-LC-1 has a length of 113 amino acids. KK-LC-1 is rarely found as a tumor antigen in healthy cells (except in immune privileged spermatocytes), but is often expressed in various tumors, e.g., non-small cell lung cancer. In some embodiments, KK-LC-1 has the amino acid sequence according to SEQ ID NO: 5.

The human Melanoma antigen A3 (MAGE-A3) gene is a member of the melanoma- associated antigen gene family. The members of this family encode proteins with 50 to 80% sequence identity to each other. The MAGEA genes are clustered at chromosomal location Xq28. They have been implicated in some hereditary disorders, such as dyskeratosis congenita. The normal function of MAGE- A3 in healthy cells is unknown. In some embodiments, MAGE-A3 has the amino acid sequence according to SEQ ID NO: 9. The human Melanoma antigen 4 (MAGE-A4) gene is a member of the MAGEA gene family. The members of this family encode proteins with 50 to 80% sequence identity to each other. The MAGEA genes are clustered at chromosomal location Xq28. They have been implicated in some hereditary disorders, such as dyskeratosis congenita. In some embodiments, MAGE-A4 has the amino acid sequence according to SEQ ID NO: 13.

The human Preferentially Expressed In Melanoma (PRAME) gene is localized on chromosome 22 and contains eight isoforms out of which seven encode for an identical protein of 509 amino acids, while the eighth isoform lacks the first 16 amino acids. Localization studies using FLAG- or GFP -tagged PRAME suggest a nuclear localization of the protein. Furthermore, PRAME plays a critical role in apoptosis and cell proliferation. Further functional studies revealed that PRAME inhibits retinoic acid receptor signaling and thereby elicits its role in apoptosis and differentiation. PRAME belongs to a multigene family consisting of 32 PRAME-like genes and pseudogenes. The closest protein-coding relatives of PRAME exhibit 53% homology to the protein (using the BLASTP algorithm of NCBI). A detailed RT-qPCR-based analysis revealed a high expression of PRAME in testis, epididymis, and uterus. In some embodiments, PRAME has the amino acid sequence according to SEQ ID NO: 17.

Melanoma antigen Cl (MAGE-CI), also cancer/testis antigen 7 (CT7), is a human tumor antigen from the group of cancer/testis antigens. MAGE-CI has a length of 1,142 amino acids and a mass of 123,643 Da. It is phosphorylated on up to four serines, S63, S207, S382 and S1063. MAGE-CI has anti-apoptotic properties and binds to NY-ESO-1 (New York esophageal squamous cell carcinoma-1). It does not occur in healthy cells (except in immune-privileged spermatocytes), but is often expressed in tumors, e.g., multiple myelomas. There it is formed by malignant plasma cells. In some embodiments, MAGE-CI has the amino acid sequence according to SEQ ID NO: 21.

In some embodiments, the subject is a human. The RNA cancer vaccine described herein is combined with an anti-CTLA antibody described herein for the treatment of subjects or patients afflicted with cancer.

Cytotoxic T lymphocyte antigen-4 (CTLA4) is a regulator of adaptive immune responses, having a role in the maintenance of peripheral tolerance and in shaping the repertoire of emergent T cell responses. CTLA4 is expressed on the surface of cells and, if it is located at the surface of said cells, is accessible to binding by CTLA4 specific antibodies.

The present invention provides antibodies or antigen-binding fragments that immunospecifically bind to CTLA4, in particular human CTLA4, preferably expressed on the surface of a cell at an endogenous or transfected concentration. In some embodiments, the anti-CTLA4 antibody is capable of binding to human CTLA4. In some embodiments, the cell is a T cell. In some embodiments, the anti-CTLA4 antibody is a monoclonal antibody, a human antibody, a chimeric antibody, or a humanized antibody.

Without wishing to be bound by theory, CTLA4 is contemplated to be recycled between the cell surface and endosomes, where it is usually prevented from lysosomal degradation and can recycle back to the cell surface by binding to the lipopolysaccharide-responsive and beige-like anchor (LRBA) protein. The anti-CTLA4 antibodies disclosed herein have reduced autoimmune side effects when used to enhance immune responses and are particularly suitable for treating cancer in subjects, particularly lung and ovarian cancers and melanoma.

Surprisingly, the combination method of the present invention comprising administration of the RNA cancer vaccine and the anti-CTLA4 antibody shows an enhanced anti-tumoral efficacy compared to administration of either the RNA cancer vaccine or the anti-CTLA4 antibody alone. Furthermore, the combination of the RNA cancer vaccine and the anti- CTLA4 antibody shows good safety profiles in cancer subjects, in particular immune- related adverse events are reduced. Immune-related adverse events such as skin rash, hepatitis, colitis and endocrinopathies, particularly hypopituitarism are characteristic side effects of anti-cancer therapies based on immune checkpoint inhibitors such as anti-CTLA4 antibodies. It was therefore surprising that the combination therapy of the invention improved the efficiency of the anti-cancer treatment while in parallel reducing immune- related adverse events.

RNA cancer vaccine

In some preferred embodiments, the RNA cancer vaccine of the present disclosure comprises RNA encoding one or more tumor-associated antigen (TAA) amino acid sequences, immunogenic variants thereof, or immunogenic fragments of the TAAs, i.e., antigenic peptides or proteins. In some embodiments, the RNA cancer vaccine of the present disclosure comprises RNA encoding a set of tumor-associated antigen (TAA) amino acid sequences, immunogenic variants thereof, or immunogenic fragments of the TAAs, i.e., antigenic peptides or proteins. Each of the TAA amino acid sequences comprises an epitope of a tumor antigen for inducing an immune response against the tumor antigen in the subject.

In some embodiments, the RNA cancer vaccine described herein comprises at least one RNA encoding at least one of the following amino acid sequences:

In some embodiments, a) the RNA encoding the amino acid sequence (i) comprises the nucleotide sequence of SEQ ID NO: 3 or 4, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 3 or 4; and/or b) the amino acid sequence (i) comprises the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 2.

In some embodiments, a) the RNA encoding the amino acid sequence (i) comprises the nucleotide sequence of SEQ ID NO: 3 or 4, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 3 or 4; or b) the amino acid sequence (i) comprises the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 2.

In some embodiments, a) the RNA encoding the amino acid sequence (i) comprises the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 3; and/or b) the amino acid sequence (i) comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, a) the RNA encoding the amino acid sequence (i) comprises the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 3; or b) the amino acid sequence (i) comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, a) the RNA encoding the amino acid sequence (i) comprises the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 4; and/or b) the amino acid sequence (i) comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 2.

In some embodiments, a) the RNA encoding the amino acid sequence (i) comprises the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 4; or b) the amino acid sequence (i) comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 2.

In some embodiments, a) the RNA encoding the amino acid sequence (ii) comprises the nucleotide sequence of SEQ ID NO: 7 or 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 7 or 8; and/or b) the amino acid sequence (ii) comprises the amino acid sequence of SEQ ID NO: 5 or 6, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5 or 6.

In some embodiments, a) the RNA encoding the amino acid sequence (ii) comprises the nucleotide sequence of SEQ ID NO: 7 or 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 7 or 8; or b) the amino acid sequence (ii) comprises the amino acid sequence of SEQ ID NO: 5 or 6, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5 or 6.

In some embodiments, a) the RNA encoding the amino acid sequence (ii) comprises the nucleotide sequence of SEQ ID NO: 7, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 7; and/or b) the amino acid sequence (ii) comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, a) the RNA encoding the amino acid sequence (ii) comprises the nucleotide sequence of SEQ ID NO: 7, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 7; or b) the amino acid sequence (ii) comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5.

In some embodiments, a) the RNA encoding the amino acid sequence (ii) comprises the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8; and/or b) the amino acid sequence (ii) comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 6.

In some embodiments, a) the RNA encoding the amino acid sequence (ii) comprises the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8; or b) the amino acid sequence (ii) comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 6.

In some embodiments, a) the RNA encoding the amino acid sequence (iii) comprises the nucleotide sequence of

SEQ ID NO: 11 or 12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 11 or 12; and/or b) the amino acid sequence (iii) comprises the amino acid sequence of SEQ ID NO: 9 or

10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 9 or 10.

In some embodiments, a) the RNA encoding the amino acid sequence (iii) comprises the nucleotide sequence of SEQ ID NO: 11 or 12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 11 or 12; or b) the amino acid sequence (iii) comprises the amino acid sequence of SEQ ID NO: 9 or

In some embodiments, a) the RNA encoding the amino acid sequence (iii) comprises the nucleotide sequence of SEQ ID NO: 11, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 11; and/or b) the amino acid sequence (iii) comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 9.

In some embodiments, a) the RNA encoding the amino acid sequence (iii) comprises the nucleotide sequence of SEQ ID NO: 11, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 11; or b) the amino acid sequence (iii) comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, a) the RNA encoding the amino acid sequence (iii) comprises the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 12; and/or b) the amino acid sequence (iii) comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10.

In some embodiments, a) the RNA encoding the amino acid sequence (iii) comprises the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 12; or b) the amino acid sequence (iii) comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10.

In some embodiments, a) the RNA encoding the amino acid sequence (iv) comprises the nucleotide sequence of SEQ ID NO: 15 or 16, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15 or 16; and/or b) the amino acid sequence (iv) comprises the amino acid sequence of SEQ ID NO: 13 or 14, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 13 or 14.

In some embodiments, a) the RNA encoding the amino acid sequence (iv) comprises the nucleotide sequence of SEQ ID NO: 15 or 16, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15 or 16; or b) the amino acid sequence (iv) comprises the amino acid sequence of SEQ ID NO: 13 or 14, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 13 or 14.

In some embodiments, a) the RNA encoding the amino acid sequence (iv) comprises the nucleotide sequence of SEQ ID NO: 15, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15; and/or b) the amino acid sequence (iv) comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 13.

In some embodiments, a) the RNA encoding the amino acid sequence (iv) comprises the nucleotide sequence of SEQ ID NO: 15, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15; or b) the amino acid sequence (iv) comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 13.

In some embodiments, a) the RNA encoding the amino acid sequence (iv) comprises the nucleotide sequence of SEQ ID NO: 16, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 16; and/or b) the amino acid sequence (iv) comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 14.

In some embodiments, a) the RNA encoding the amino acid sequence (iv) comprises the nucleotide sequence of SEQ ID NO: 16, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 16; or b) the amino acid sequence (iv) comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 14.

In some embodiments, a) the RNA encoding the amino acid sequence (v) comprises the nucleotide sequence of SEQ ID NO: 19 or 20, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 19 or 20; and/or b) the amino acid sequence (v) comprises the amino acid sequence of SEQ ID NO: 17 or 18, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 17 or 18.

In some embodiments, a) the RNA encoding the amino acid sequence (v) comprises the nucleotide sequence of SEQ ID NO: 19 or 20, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 19 or 20; or b) the amino acid sequence (v) comprises the amino acid sequence of SEQ ID NO: 17 or

18, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 17 or 18. In some embodiments, a) the RNA encoding the amino acid sequence (v) comprises the nucleotide sequence of SEQ ID NO: 19, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 19; and/or b) the amino acid sequence (v) comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 17.

In some embodiments, a) the RNA encoding the amino acid sequence (v) comprises the nucleotide sequence of SEQ ID NO: 19, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 19; or b) the amino acid sequence (v) comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 17.

In some embodiments, a) the RNA encoding the amino acid sequence (v) comprises the nucleotide sequence of SEQ ID NO: 20, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 20; and/or b) the amino acid sequence (v) comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 18.

In some embodiments, a) the RNA encoding the amino acid sequence (v) comprises the nucleotide sequence of SEQ ID NO: 20, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 20; or b) the amino acid sequence (v) comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 18.

In some embodiments, a) the RNA encoding the amino acid sequence (vi) comprises the nucleotide sequence of SEQ ID NO: 23 or 24, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 23 or 24; and/or b) the amino acid sequence (vi) comprises the amino acid sequence of SEQ ID NO: 21 or 22, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 21 or 22.

In some embodiments, c) the RNA encoding the amino acid sequence (vi) comprises the nucleotide sequence of SEQ ID NO: 23 or 24, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 23 or 24; or d) the amino acid sequence (vi) comprises the amino acid sequence of SEQ ID NO: 21 or 22, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 21 or 22.

In some embodiments, a) the RNA encoding the amino acid sequence (vi) comprises the nucleotide sequence of SEQ ID NO: 23, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 23; and/or b) the amino acid sequence (vi) comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 21. In some embodiments, a) the RNA encoding the amino acid sequence (vi) comprises the nucleotide sequence of SEQ ID NO: 23, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 23; or b) the amino acid sequence (vi) comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 21.

In some embodiments, a) the RNA encoding the amino acid sequence (vi) comprises the nucleotide sequence of SEQ ID NO: 24, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 24; and/or b) the amino acid sequence (vi) comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 22.

In some embodiments, a) the RNA encoding the amino acid sequence (vi) comprises the nucleotide sequence of SEQ ID NO: 24, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 24; or b) the amino acid sequence (vi) comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 22.

In some preferred embodiments, a) the RNA encoding the amino acid sequence (i) comprises the nucleotide sequence of SEQ ID NO: 4; b) the RNA encoding the amino acid sequence (ii) comprises the nucleotide sequence of SEQ ID NO: 8; c) the RNA encoding the amino acid sequence (iii) comprises the nucleotide sequence of SEQ ID NO: 12; d) the RNA encoding the amino acid sequence (iv) comprises the nucleotide sequence of SEQ ID NO: 16; e) the RNA encoding the amino acid sequence (v) comprises the nucleotide sequence of SEQ ID NO: 20; and f) the RNA encoding the amino acid sequence (vi) comprises the nucleotide sequence of SEQ ID NO: 24.

In some preferred embodiments, a) the amino acid sequence (i) encoded by the at least on RNA comprises the amino acid sequence of SEQ ID NO: 2; b) the amino acid sequence (ii) encoded by the at least on RNA comprises the amino acid sequence of SEQ ID NO: 6; c) the amino acid sequence (iii) encoded by the at least on RNA comprises the amino acid sequence of SEQ ID NO: 10; d) the amino acid sequence (iv) encoded by the at least on RNA comprises the amino acid sequence of SEQ ID NO: 14; e) the amino acid sequence (v) encoded by the at least on RNA comprises the amino acid sequence of SEQ ID NO: 18; and f) the amino acid sequence (vi) encoded by the at least on RNA comprises the amino acid sequence of SEQ ID NO: 22.

In some preferred embodiments, a) the RNA encoding the amino acid sequence (i) set forth in SEQ ID NO: 2 comprises the nucleotide sequence of SEQ ID NO: 4; b) the RNA encoding the amino acid sequence (ii) set forth in SEQ ID NO: 6 comprises the nucleotide sequence of SEQ ID NO: 8; c) the RNA encoding the amino acid sequence (iii) set forth in SEQ ID NO: 10 comprises the nucleotide sequence of SEQ ID NO: 12; d) the RNA encoding the amino acid sequence (iv) set forth in SEQ ID NO: 14 comprises the nucleotide sequence of SEQ ID NO: 16; e) the RNA encoding the amino acid sequence (v) set forth in SEQ ID NO: 18 comprises the nucleotide sequence of SEQ ID NO: 20; and f) the RNA encoding the amino acid sequence (vi) set forth in SEQ ID NO: 22 comprises the nucleotide sequence of SEQ ID NO: 24.

In some embodiments, the RNA cancer vaccine comprises at least one RNA encoding only one of the amino acid sequences (i)-(vi). In some embodiments, the RNA cancer vaccine comprises at least one RNA encoding at least two of the amino acid sequences (i)-(vi). In some embodiments, the RNA cancer vaccine comprises at least one RNA encoding at least three of the amino acid sequences (i)-(vi). In some embodiments, the RNA cancer vaccine comprises at least one RNA encoding at least four of the amino acid sequences (i)-(vi). In some embodiments, the RNA cancer vaccine comprises at least one RNA encoding at least five of the amino acid sequences (i)-(vi). In some embodiments, the RNA cancer vaccine comprises at least one RNA encoding all of the amino acid sequences (i)-(vi).

In some embodiments, the RNA cancer vaccine comprises at least one RNA encoding the amino acid sequence (i), i.e., the amino acid sequence of claudin 6 (CLDN6), an immunogenic variant thereof, or an immunogenic fragment of the CLDN6 or the immunogenic variant thereof.

In some embodiments, the RNA cancer vaccine comprises one RNA encoding the amino acid sequence (i), i.e., the amino acid sequence of claudin 6 (CLDN6), an immunogenic variant thereof, or an immunogenic fragment of the CLDN6 or the immunogenic variant thereof. In some embodiments, the RNA cancer vaccine comprises at least two RNAs, preferably encoding at least two of the amino acid sequences (i), (ii), (iii), (iv), (v), and (vi). In some embodiments, the RNA cancer vaccine comprises at least three RNAs, preferably encoding at least three of the amino acid sequences (i), (ii), (iii), (iv), (v), and (vi). In some embodiments, the RNA cancer vaccine comprises at least four RNAs, preferably encoding at least four of the amino acid sequences (i), (ii), (iii), (iv), (v), and (vi). In some embodiments, the RNA cancer vaccine comprises at least five RNAs, preferably encoding at least five of the amino acid sequences (i), (ii), (iii), (iv), (v), and (vi). In some embodiments, the RNA cancer vaccine comprises at least six RNAs, preferably encoding all of the amino acid sequences (i), (ii), (iii), (iv), (v), and (vi). In some preferred embodiments, the RNA cancer vaccine comprises six RNAs, preferably each encoding one of the amino acid sequences (i), (ii), (iii), (iv), (v), and (vi).

For rendering RNA as described herein non-immunogenic, modified nucleosides may be incorporated into the RNA.

In some embodiments, one or more uridine in the RNA described herein is replaced by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine. In some embodiments, the modified uridine replacing uridine is pseudouridine (y), Nl-methyl-pseudouridine (ml y), or 5-methyl-uridine (m5U).

In some embodiments, the modified nucleoside replacing one or more, e.g., all, uridine in the RNA may be any one or more of 3 -methyl -uridine (m³U), 5-methoxy-uridine (mo⁵ U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s² U), 4-thio-uridine (s⁴ U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho⁵ U), 5-aminoallyl- uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (emo⁵ U), uridine 5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵ U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵ U), 5- carboxyhydroxymethyl-uridine methyl ester (mchm⁵ U), 5-methoxycarbonylmethyl-uridine (mcm⁵ U), 5-methoxycarbonylmethyl-2 -thio-uridine (mcm⁵s² U), 5-aminomethyl-2 -thiouridine (nm⁵ s² U), 5-methylaminomethyl-uridine (mnm⁵ U), 1-ethyl-pseudouridine, 5- methylaminomethyl-2-thio-uridine (mnm⁵s²U), 5-methylaminomethyl-2-seleno-uridine (mnm⁵ se² U), 5-carbamoylmethyl-uridine (ncm⁵ U), 5-carboxymethylaminomethyl-uridine (cmnm⁵ U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵ s² U), 5-propynyl- uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (rm⁵ U), 1-taurinom ethylpseudouridine, 5-taurinomethyl-2-thio-uridine(Tm⁵s²U), 1 -taurinomethyl-4-thio- pseudouridine, 5-methyl-2-thio-uridine (m⁵s² U), 1 -methyl -4-thio-pseudouri dine (m¹ s⁴ y),

4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m³ y), 2-thio-l -methylpseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵ D), 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2- methoxy-4-thio-uridine, 4-m ethoxy -pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl- methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp³ U), l-methyl-3-(3-amino- 3-carboxypropyl)pseudouridine (acp³ y), 5-(isopentenylaminomethyl)uridine (inm⁵U), 5- (isopentenylaminomethyl)-2-thio-uridine (inm⁵ s² U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m⁵Um), 2'-O-methyl-pseudouridine (y m), 2-thio-2'-O- methyl-uridine (s² Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm⁵Um),

5-carbamoylmethyl-2'-O-methyl-uridine (ncm⁵ Um), 5-carboxymethylaminomethyl-2'-O- methyl-uridine (cmnm⁵ Um), 3,2'-O-dimethyl-uridine (m³Um), 5- (isopentenylaminomethyl)-2'-O-methyl-uridine (inm⁵ Um), 1 -thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-3- [l-E-propenylamino)uridine, or any other modified uridine known in the art.

In some embodiments, at least one RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, at least one RNA comprises a modified nucleoside in place of each uridine. In some embodiments, each RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, each RNA comprises a modified nucleoside in place of each uridine.

In some embodiments, the modified nucleoside is independently selected from pseudouridine (y), Nl-methyl-pseudouridine (ml y), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (y). In some embodiments, the modified nucleoside comprises Nl-methyl-pseudouridine (ml y). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m5U). In some embodiments, at least one RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (y), Nl- methyl-pseudouridine (ml y), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (y) and Nl-methyl-pseudouridine (ml y). In some embodiments, the modified nucleosides comprise pseudouridine (y) and 5-methyl- uridine (m5U). In some embodiments, the modified nucleosides comprise Nl-methyl- pseudouridine (ml y) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (y), Nl-methyl-pseudouridine (ml y), and 5-methyl- uridine (m5U).

In some embodiments, the RNA comprises other modified nucleosides or comprises further modified nucleosides, e.g., modified cytidine. For example, in one embodiment, in the RNA 5-methylcytidine is substituted partially or completely, preferably completely, for cytidine. In some embodiments, the RNA comprises 5-methylcytidine and one or more selected from pseudouridine (y), Nl-methyl-pseudouridine (ml y), and 5-methyl-uridine (m5U). In some embodiments, the RNA comprises 5-methylcytidine and Nl-methyl- pseudouridine (ml y). In some embodiments, the RNA comprises 5-methylcytidine in place of each cytidine and Nl-methyl-pseudouridine (ml y) in place of each uridine.

Amino acid sequences derived from tetanus toxoid of Clostridium tetani can be employed to overcome self-tolerance mechanisms in order to efficiently mount an immune response to self-antigens by providing T cell help during priming. It is known that the tetanus toxoid heavy chain includes epitopes that can bind promiscuously to MHC class II alleles and induce CD4⁺ memory T cells in almost all tetanus vaccinated individuals. In addition, the combination of tetanus toxoid (TT) helper epitopes with TAAs is known to improve the immune stimulation compared to application of TAAs alone by providing CD4⁺-mediated T cell help during priming. To reduce the risk of stimulating CD8⁺ T cells with the tetanus sequences which might compete with the intended induction of TAA-specific T-cell response, not the whole fragment C of tetanus toxoid is used as it is known to contain CD8⁺ T cell epitopes. Two peptide sequences containing promiscuously binding helper epitopes were selected alternatively to ensure binding to as many MHC class II alleles as possible. Based on the data of the ex vivo studies the well-known epitopes p2 (QYIKANSKFIGITEL (SEQ ID NO 27); TT₈₃o-844) and pl6 (MTNSVDDALINSTKIYSYFPSVISKVNQGAQG (SEQ ID NO 28); TT578-609) were selected. The p2 epitope was already used for peptide vaccination in clinical trials to boost anti-melanoma activity.

Present non-clinical data (unpublished) showed that RNA cancer vaccines encoding both a TAA plus promiscuously binding tetanus toxoid sequences lead to enhanced CD8⁺ T-cell responses directed against the tumor antigen and improved break of immunological tolerance. Immunomonitoring data from patients vaccinated with vaccines including those sequences fused in frame with the TAA-specific sequences reveal that the tetanus sequences chosen are able to induce tetanus-specific T cell responses in almost all patients.

In some embodiments, at least one of the amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) comprises an amino acid sequence which breaks immunological tolerance and/or the at least one RNA is co-administered with RNA encoding an amino acid sequence which breaks immunological tolerance. The term “co-administered” or “co-administration” or the like as used herein refers to administration of two or more agents concurrently, simultaneously, or essentially at the same time, either as part of a single formulation or as multiple formulations that are administered by the same or different routes. “Essentially at the same time” as used herein means within about 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or 6 hours period of each other.

In some embodiments, each amino acid sequence (i), (ii), (iii), (iv), (v), or (vi) comprises an amino acid sequence which breaks immunological tolerance and/or each RNA is coadministered with RNA encoding an amino acid sequence which breaks immunological tolerance. In some embodiments, each amino acid sequence (i), (ii), (iii), (iv), (v), and (vi) comprises an amino acid sequence which breaks immunological tolerance and/or each RNA is co-administered with RNA encoding an amino acid sequence which breaks immunological tolerance.

In some preferred embodiments, the amino acid sequence which breaks immunological tolerance comprises helper epitopes. In some more preferred embodiments, the amino acid sequence which breaks immunological tolerance comprises tetanus toxoid-derived helper epitopes.

In some preferred embodiments, an amino acid sequence which breaks immunological tolerance is fused, either directly or through a linker, e.g., a linker having the amino acid sequence GGSGGGGSGG (SEQ ID NO: 36), to the antigenic peptide or protein, i.e., CLDN6 (SEQ ID NO: 1), KK-LC-1 (SEQ ID NO: 5), MAGE- A3 (SEQ ID NO: 9), MAGE-A4 (SEQ ID NO: 13), PRAME (SEQ ID NO: 17), or MAGE-CI (SEQ ID NO: 21), a variant thereof, or a fragment thereof.

Such amino acid sequences which break immunological tolerance are preferably located at the C-terminus of the antigenic peptide or protein (and optionally at the N-terminus of the amino acid sequence enhancing antigen processing and/or presentation, wherein the amino acid sequence which breaks immunological tolerance and the amino acid sequence enhancing antigen processing and/or presentation may be fused either directly or through a linker, e.g., a linker having the amino acid sequence GSSGGGGSPGGGSS (SEQ ID NO: 37)), without being limited thereto. Amino acid sequences which break immunological tolerance as defined herein preferably improve T cell responses. In some embodiments, the amino acid sequence which breaks immunological tolerance as defined herein includes, without being limited thereto, sequences derived from tetanus toxoid-derived helper sequences p2 and pl6 (P2P 16), in particular a sequence comprising the amino acid sequence of SEQ ID NO: 25 or a functional variant thereof.

In some particularly preferred embodiments, a) the RNA encoding the amino acid sequence which breaks immunological tolerance comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 26; and/or b) the amino acid sequence which breaks immunological tolerance comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 25.

In some embodiments, instead of using antigen RNAs fused with tetanus toxoid helper epitope, the TAA encoding RNAs are co-administered with a separate RNA coding for TT helper epitope during vaccination. Here, the TT helper epitope coding RNA may be added to each of the antigen-coding RNAs before preparation. Thereby, mixed lipoplex nanoparticles are formed comprising both, antigen and helper epitope coding RNA.

In some embodiments, at least one of the amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) 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. In some preferred embodiments, the codon-optimization and/or the increase in the G/C content does not change the sequence of the encoded amino acid sequence.

In some embodiments, each of the amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) 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. In some embodiments, each of the amino acid sequences (i), (ii), (iii), (iv), (v), and (vi) 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. In some preferred embodiments, the codon-optimization and/or the increase in the G/C content does not change the sequence of the encoded amino acid sequence.

In some embodiments, the RNA described herein comprises a 5'-cap structure. In some embodiments, the RNA cancer vaccine does not comprise RNA having uncapped 5'- triphosphates. In some embodiments, the RNA (in particular, mRNA) may comprise a conventional 5'-cap and/or a 5'-cap analog. The term “conventional 5'-cap” refers to a cap structure found on the 5'-end of an RNA molecule and generally comprises a guanosine 5'- triphosphate (Gppp) which is connected via its triphosphate moiety to the 5'-end of the next nucleotide of the RNA (i.e., the guanosine is connected via a 5' to 5' triphosphate linkage to the rest of the RNA). The guanosine may be methylated at position N⁷ (resulting in the cap structure m⁷Gppp). The term “5'-cap analog” includes a 5'-cap which is based on a conventional 5'-cap but which has been modified at either the 2'- or 3'-position of the m⁷guanosine structure in order to avoid an integration of the 5'-cap analog in the reverse orientation (such 5'-cap analogs are also called anti-reverse cap analogs (ARCAs)). Particularly preferred 5'-cap analogs are those having one or more substitutions at the bridging and non-bridging oxygen in the phosphate bridge, such as phosphorothioate modified 5'-cap analogs at the P-phosphate (such as m₂ ⁷’^{2 O}G(5')ppSp(5')G (referred to as beta-S-ARCA or P-S-ARCA)), as described in PCT/EP2019/056502 which disclosure is incorporated herein in its entirety. Providing an with a 5'-cap structure as described herein may be achieved by in vitro transcription of a DNA template in presence of a corresponding 5'-cap compound, wherein said 5'-cap structure is co-transcriptionally incorporated into the generated RNA strand, or the RNA may be generated, for example, by in vitro transcription, and the 5'-cap structure may be attached to the RNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.

In some embodiments, the RNA disclosed herein comprises a 5' cap. In some preferred embodiments, the 5' cap is selected from the group consisting of G[5’]ppp[5’]G, m7G[5’]ppp[5’]G, m₃ ²’²’⁷G[5’]ppp[5’]G, m₂ ⁷’³''⁰G[5’]ppp[5’]G (3’-ARCA), m₂ ⁷’²'-°GpppG (2’-ARCA), m₂ ⁷’²'⁰ GppSpG, m₂ ⁷’²'-°GppSpG (p-S- ARCA), m₂ ⁷’²’-°GppSpG (p-S-ARCA), and m₂ ^{7 3} '°Gppp(mi² '°)ApG. In some more preferred embodiments, the 5' cap is m₂ ^7,2 '° GppSpG.

In some embodiments, each of the RNAs disclosed herein comprises a 5’ cap. In some preferred embodiments, the 5’ cap is selected from the group consisting of G[5’]ppp[5’]G, m7G[5’]ppp[5’]G, m₃ ²’²’⁷G[5’]ppp[5’]G, m₂ ⁷’³’-°G[5’]ppp[5’]G (3’-ARCA), m₂ ⁷’²’-°GpppG (2’-ARCA), m₂ ⁷’² '⁰ GppSpG, m₂ ⁷’²’-°GppSpG (p-S- ARCA), m₂ ⁷’²’-°GppSpG (p-S-ARCA) and m₂ ^{7 3} '°Gppp(mi² '°)ApG. In some more preferred embodiments, the 5’ cap is m₂ ^7,2 '° GppSpG. In some embodiments, RNA described in present disclosure comprises a 5’-UTR and/or a 3’-UTR. The term “untranslated region” or “UTR” relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule. An UTR can be present 5' (upstream) of an open reading frame (5'-UTR) and/or 3' (downstream) of an open reading frame (3'-UTR). A 5'-UTR, if present, is located at the 5'-end, upstream of the start codon of a protein-encoding region. A 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. A 3'-UTR, if present, is located at the 3'-end, downstream of the termination codon of a protein-encoding region, but the term “3 '-UTR” does generally not include the poly-A sequence. Thus, the 3'-UTR is upstream of the poly-A sequence (if present), e.g., directly adjacent to the poly-A sequence. Incorporation of a 3'-UTR into the 3'-non translated region of an RNA molecule can result in an enhancement in translation efficiency. A synergistic effect may be achieved by incorporating two or more of such 3'- UTRs (which are preferably arranged in a head-to-tail orientation; cf., e.g., Holtkamp et al., Blood 108, 4009-4017 (2006)). The 3'-UTRs may be autologous or heterologous to the RNA (e.g., mRNA) into which they are introduced. In certain embodiments, the 3'-UTR is derived from a globin gene or mRNA, such as a gene or mRNA of alpha2 -globin, alpha 1- globin, or beta-globin, e.g., beta-globin, e.g., human beta-globin. For example, the RNA (e.g., mRNA) may be modified by the replacement of the existing 3'-UTR with or the insertion of one or more, e.g., two copies of a 3'-UTR derived from a globin gene, such as alpha2 -globin, alphal -globin, beta-globin, e.g., beta-globin, e.g., human beta-globin.

In some embodiments, the at least one RNA comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 29.

In some embodiments, each of the RNAs comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the at least one RNA comprises a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 32, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 32.

In some embodiments, each of the RNAs comprises a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 32, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 32.

In some embodiments, at least one of the amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) comprises an amino acid sequence enhancing antigen processing and/or presentation.

In some embodiments, each of the amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) comprises an amino acid sequence enhancing antigen processing and/or presentation. In some embodiments, each of the amino acid sequences (i), (ii), (iii), (iv), (v), and (vi) comprises an amino acid sequence enhancing antigen processing and/or presentation.

In some preferred embodiments, an amino acid sequence enhancing antigen processing and/or presentation is fused, either directly or through a linker to the antigen peptide or protein, i.e., MAGE-A3 (SEQ ID NO: 9), MAGE-A4 (SEQ ID NO: 13), PRAME (SEQ ID NO: 17), and MAGE-CI (SEQ ID NO: 21), a variant thereof, or a fragment thereof. Such amino acid sequences which enhance antigen processing and/or presentation are preferably located at the C-terminus of the antigenic peptide or protein (and optionally at the C- terminus of the amino acid sequence breaking immunological tolerance, wherein the amino acid sequence which breaks immunological tolerance and the amino acid sequence enhancing antigen processing and/or presentation may be fused either directly or through a linker, e.g., a linker having the amino acid sequence GSSGGGGSPGGGSS (SEQ ID NO: 37)), without being limited thereto. Amino acid sequences enhancing antigen processing and/or presentation as defined herein preferably improve antigen processing and presentation. In some preferred embodiments, the amino acid sequence enhancing antigen processing and/or presentation comprises an amino acid sequence corresponding to the transmembrane and cytoplasmic domain of an MHC molecule, preferably an MHC class I molecule. Within the meaning of the present disclosure, the transmembrane and cytoplasmic domain of an MHC class I molecule is also referred to as the MHC class I trafficking domain (MITD).In some more preferred embodiments, the amino acid sequence enhancing antigen processing and/or presentation as defined herein includes, without being limited thereto, sequences derived from the human MHC class I complex (HLA-B51, haplotype A2, B27/B51, Cw2/Cw3), in particular a sequence comprising the amino acid sequence of SEQ ID NO: 30 or a functional variant thereof.

In some particularly preferred embodiments, a) the RNA encoding the amino acid sequence enhancing antigen processing and/or presentation comprises the nucleotide sequence of SEQ ID NO: 31, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 31; and/or b) the amino acid sequence enhancing antigen processing and/or presentation comprises the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 30.

Such amino acid sequences enhancing antigen processing and/or presentation are preferably used in order to promote antigen processing and/or presentation of the encoded antigenic peptide or protein. More preferably, an amino acid sequence enhancing antigen processing and/or presentation as defined herein is fused to an encoded antigenic peptide or protein as defined herein. Accordingly, in particularly preferred embodiments, the RNA described herein comprises at least one coding region encoding an antigenic peptide or protein and an amino acid sequence enhancing antigen processing and/or presentation, said amino acid sequence enhancing antigen processing and/or presentation preferably being fused to the antigenic peptide or protein, more preferably to the C-terminus of the antigenic peptide or protein as described herein.

In some embodiments, the antigenic peptides or proteins described herein have their own amino acid sequences enhancing antigen processing and/or presentation and may not require the addition of an amino acid sequences enhancing antigen processing and/or presentation.

In some embodiments, at least one of the amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) comprises an amino acid sequence encoding for a secretory signal peptide.

In some embodiments, each of the amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) comprises an amino acid sequence encoding for a secretory signal peptide. In some embodiments, each of the amino acid sequences (i), (ii), (iii), (iv), (v), and (vi) comprises an amino acid sequence encoding for a secretory signal peptide.

In some preferred embodiments, a secretory signal peptide is fused, either directly or through a linker, e.g., a linker having the amino acid sequence GGSGGGGSGG (SEQ ID NO: 36), to the antigenic peptide or protein, e.g., MAGE- A3 (SEQ ID NO: 9), PRAME (SEQ ID NO: 17), or MAGE-CI (SEQ ID NO: 21), a variant thereof, or a fragment thereof. Such secretory signal peptides are sequences, which typically exhibit a length of about 15 to 30 amino acids and are preferably located at the N-terminus of the antigenic peptide or protein, without being limited thereto. Secretory signal peptides as defined herein preferably allow the transport of the antigenic peptide or protein as encoded by the RNA into a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartment. In some embodiments, the signal peptide sequence as defined herein includes, without being limited thereto, the signal peptide sequence derived from the sequence encoding the human MHC class I complex (HLA-B51, haplotype A2, B27/B51, Cw2/Cw3), and preferably corresponds to the 78 bp fragment coding for the secretory signal peptide, which guides translocation of the nascent polypeptide chain into the endoplasmatic reticulum, and includes, in particular a sequence comprising the amino acid sequence of SEQ ID NO: 34 or a functional variant thereof. In some particularly preferred embodiments, a) the RNA encoding the amino acid sequence of the secretory signal peptide comprises the nucleotide sequence of SEQ ID NO: 35, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 35; and/or b) the amino acid sequence of the secretory signal peptide comprises the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 34.

Such secretory signal peptides are preferably used in order to promote secretion of the encoded antigenic peptide or protein. More preferably, a secretory signal peptide as defined herein is fused to an encoded antigenic peptide or protein as defined herein. Accordingly, in particularly preferred embodiments, the RNA described herein comprises at least one coding region encoding an antigenic peptide or protein and a secretory signal peptide, said signal peptide preferably being fused to the antigenic peptide or protein, more preferably to the N-terminus of the antigenic peptide or protein as described herein.

In some embodiments, the antigenic peptides or proteins described herein have their own secretory signal peptides and may not require the addition of a secretory signal peptide.

In some embodiments, RNA described herein comprises a poly A sequence. In some embodiments, the at least one RNA comprises a poly-A sequence. In some embodiments, each of the RNAs comprises a poly-A sequence.

As used herein, the term “poly-A-sequence” or “poly-A sequence” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3 '-end of an RNA molecule. Poly-A-sequences or poly-A-sequences are known to those of skill in the art and may follow the 3’-UTR in the RNAs described herein. An uninterrupted poly-A-sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly-A-sequence is typical. RNAs disclosed herein can have a poly-A- sequence attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly-A-sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

It has been demonstrated that a poly-A-sequence of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5’) of the poly-A-sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

The poly-A-sequence may be of any length. In some embodiments, a poly-A-sequence comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, “essentially consists of’ means that most nucleotides in the poly-A-sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A-sequence are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, “consists of’ means that all nucleotides in the poly-A-sequence, i.e., 100% by number of nucleotides in the poly-A-sequence, are A nucleotides. The term “A nucleotide” or “A” refers to adenylate. In some embodiments, a poly-A-sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly-A-sequence (coding strand) is referred to as poly(A) cassette.

In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016/005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 Al may be used in the present disclosure. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coll and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. Consequently, in some embodiments, the poly-A- sequence contained in an RNA molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

In some embodiments, the poly-A-sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence of 10 nucleotides.

In some embodiments, no nucleotides other than A nucleotides flank a poly-A-sequence at its 3'-end, i.e., the poly-A-sequence is not masked or followed at its 3'-end by a nucleotide other than A.

In some embodiments, a poly-A-sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A-sequence may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A-sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A-sequence comprises at least 100 nucleotides. In some embodiments, the poly-A-sequence comprises about 150 nucleotides. In some embodiments, the poly-A-sequence comprises about 120 nucleotides.

In some preferred embodiments, the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the RNA of the present disclosure is formulated as a liquid, a solid, or a combination thereof. In some embodiments, the RNA cancer vaccine of the present disclosure is formulated for injection. In some embodiments, the RNA cancer vaccine of the present disclosure is formulated for intravenous administration.

In some embodiments, the RNA as described herein may be administered formulated as particles (RNA particles), e.g., protein and/or lipid particles. In some embodiments, the term “particle” relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term “particle” includes nanoparticles. An “RNA particle” can be used to deliver RNA to a target site of interest (e.g., cell, tissue, organ, and the like). As used in the present disclosure, “nanoparticle” refers to a particle comprising RNA and at least one cationic lipid and having an average diameter suitable for intravenous administration. An RNA particle may be formed from lipids comprising at least one cationic or cationically ionizable lipid. Without wishing to be bound by theory, it is believed that the cationic or cationically ionizable lipid combines together with the RNA to form aggregates, and this aggregation results in colloidally stable particles.

RNA particles described herein include lipoplex particle (LPX)-based and lipid nanoparticle (LNP)-based formulations.

In some preferred embodiments, the RNA is formulated or is to be formulated as lipoplex particles (LPX particles). LPX particles as described herein are obtainable by mixing two aqueous phases, namely a phase comprising RNA and a phase comprising a dispersion of lipids. In some embodiments, the lipid phase comprises liposomes. In some embodiments, the RNA lipoplex particles (RNA-LPX particles) are obtainable by mixing the RNA with liposomes.

In some embodiments, liposomes are self-closed unilamellar or multilamellar vesicular particles wherein the lamellae comprise lipid bilayers and the encapsulated lumen comprises an aqueous phase. A prerequisite for using liposomes for nanoparticle formation is that the lipids in the mixture as required are able to form lamellar (bilayer) phases in the applied aqueous environment.

In some embodiments, liposomes comprise unilamellar or multilamellar phospholipid bilayers enclosing an aqueous core (also referred to herein as an aqueous lumen). They may be prepared from materials possessing polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, cationic lipids employed in formulating liposomes designed for the delivery of RNA are amphiphilic in nature and consist of a positively charged (cationic) amine head group linked to a hydrocarbon chain or cholesterol derivative via glycerol.

In some embodiments, LPX particles are multilamellar liposome-based formulations that form upon electrostatic interaction of cationic liposomes with RNAs. In some embodiments, formed LPX particles possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact RNA-LPX particles.

In some embodiments, an LPX particle comprises an amphiphilic lipid, in particular cationic or cationically ionizable amphiphilic lipid, and RNA (especially mRNA) as described herein. In some embodiments, electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, in particular cationic or cationically ionizable amphiphilic lipids) and negatively charged RNA (especially mRNA) results in complexation and spontaneous formation of RNA-LPX particles. In some embodiments, an RNA-LPX particle (especially an mRNA-LPX particle) is a nanoparticle.

The RNA may be formulated in RNA-LPX particles to generate serum-stable formulations for systemic administration such as intravenous (IV) administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, ’’parenteral administration” refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In some embodiments, after intravenous administration of the RNA cancer vaccine, expression of the in spleen occurs. In some embodiments, after intravenous administration of the RNA cancer vaccine, expression of the RNA in antigen presenting cells, preferably professional antigen presenting cells occurs. In some embodiments, the antigen presenting cells are selected from the group consisting of dendritic cells, macrophages and B cells. In some preferred embodiments, the disclosure involves targeting the lymphatic system, in particular secondary lymphoid organs, more specifically spleen.

The RNA-LPX particles described herein are useful for delivery of the RNA to a target tissue such as lymphoid organs and in particular APCs after systemic administration, in particular after intravenous administration. RNA-LPX may target antigen-presenting cells (APCs) in lymphoid organs which results in an efficient stimulation of the immune system.

Spleen targeting RNA-LPX particles are described in WO 2013/143683 and are herein incorporated by reference. It has been found that RNA-LPX particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen presenting cells, in particular dendritic cells. Accordingly, following administration of the RNA-LPX particles, RNA accumulation and/or RNA expression in the spleen occurs. Thus, RNA-LPX particles disclosed herein may be used for expressing RNA in the spleen. In some embodiments, no or essentially no RNA accumulation and/or RNA expression in the lung and/or liver occurs after administration of the RNA-LPX particles. In some embodiments, after administration of the RNA-LPX particles, RNA accumulation and/or RNA expression in APCs, such as professional APCs in the spleen occurs. Thus, RNA- LPX particles of the disclosure may preferably be used for expressing RNA in such APCs. In some embodiments, the APCs are dendritic cells and/or macrophages.

In some embodiments, the RNA may be present in RNA-LNP nanoparticles for delivery of the RNA to a target tissue such as lymphoid organs and in particular APCs after parenteral administration, in particular after intravenous administration.

The term ’’antigen presenting cell” (APC) is a cell of a variety of cells capable of displaying, acquiring, and/or presenting at least one antigen or antigenic fragment on (or at) its cell surface. Antigen- presenting cells can be distinguished in professional antigen presenting cells and non-professional antigen presenting cells. The term ’’professional antigen presenting cells” relates to antigen presenting cells which constitutively express the Major Histocompatibility Complex class II (MHC class II) molecules required for interaction with naive T cells. If a T cell interacts with the MHC class II molecule complex on the membrane of the antigen presenting cell, the antigen presenting cell produces a costimulatory molecule inducing activation of the T cell. Professional antigen presenting cells comprise dendritic cells and macrophages.

The RNA-LPX particles may be prepared using liposomes that may be obtained by injecting a solution of the lipids in ethanol into water or a suitable aqueous phase. In some embodiments, the aqueous phase has an acidic pH. In some embodiments, the aqueous phase comprises acetic acid, e.g., in an amount of about 5 mM. In some embodiments, the liposomes and RNA-LPX particles comprise at least one cationic or cationically ionizable lipid and at least one additional lipid. As used herein, a “cationic lipid“ refers to a lipid having a net positive charge. As used herein, the term “cationically ionizable lipid” refers to a lipid or lipid-like material which has a net positive charge or is neutral, i.e., which is not permanently cationic. Thus, depending on the pH of the composition in which the cationically ionizable lipid is solved, the cationically ionizable lipid is either positively charged or neutral. For purposes of the present disclosure, cationically ionizable lipids are covered by the term “cationic lipid” unless contradicted by the circumstances.

In some embodiments, the lipid solutions, liposomes and RNA-LPX particles described herein include a cationic lipid. Cationic or cationically ionizable lipids bind negatively charged RNA by electrostatic interaction to the lipid matrix. Generally, cationic or cationically ionizable lipids possess a lipophilic moiety, such as a sterol, an acyl or diacyl chain, and the head group of the lipid typically carries the positive charge. Examples of cationic or cationically ionizable lipids suitable for the preparation of RNA-LPX particles as described herein include, but are not limited to N,N-dimethyl-2, 3 -di oleyloxypropylamine (DODMA), l,2-dioleoyl-3 -trimethylammonium propane (DOTAP), 1,2-di-O-octadecenyl- 3 -trimethylammonium propane (DOTMA), 3-(N — (N',N'-dimethylaminoethane)- carbamoyljcholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl- 3-dimethylammonium-propane (DODAP), l,2-diacyloxy-3 -dimethylammonium propanes, 1.2-dialkyloxy-3 -dimethylammonium propanes, dioctadecyldimethyl ammonium chloride (DODAC), l,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3- di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1 ,2-dimyristoyl-sn- glycero-3 -ethylphosphocholine (DMEPC), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy- N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2- dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-

9, 12-oc-tadecadienoxy)propane (CLinDMA), 2-[5 '-(cholest-5-en-3-beta-oxy)-3 oxapentoxy)-3 -dimethyl- 1 -(cis, ci s-9', 12'-octadecadienoxy)propane (CpLinDMA), N,N- dimethyl -3, 4-di oleyloxybenzylamine (DMOBA), l,2-N,N'-dioleylcarbamyl-3- dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), l,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2- Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA), 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-di oxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen- 19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2 -Hydroxy ethyl)-N, N-dimethyl-

2.3-bis(tetradecyloxy)-l-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N- dimethyl-2,3-bis(cis-9-tetradecenyloxy)-l-propanaminium bromide (GAP-DMORIE), (±)- N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-l-propanaminium bromide (GAP- DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (GAP -DMRIE), N-(2-Aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l- propanaminium bromide (PAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3- bis(oleoyloxy)propan-l-aminium (DOBAQ), 2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)- N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l -amine (Octyl- CLinDMA), l,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), l,2-dipalmitoyl-3- dimethylammonium-propane (DPDAP), Nl-[2-((lS)-l-[(3-aminopropyl)amino]-4-[di(3- amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2- dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2- hydroxyethyl )-N,N-dimethylpropan- l-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)propan-l-aminium bromide (DMORIE), di((Z)-non-2-en-l- yl) 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N- dimethyl-2,3-bis(dodecyloxy)propan-l -amine (DLDMA), N,N-dimethyl-2,3- bis(tetradecyloxy)propan- 1 -amine (DMDMA), Di((Z)-non-2-en- 1 -yl)-9-((4- (dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2- dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)- [2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipidoid 98N12-5), and l-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-l-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200).

In some embodiments, the cationic or cationically ionizable lipid comprises DOTMA, DOTAP, DODAC, and/or DOSPA. In some preferred embodiments, the cationic or cationically ionizable lipid comprises DOTMA and/or DOTAP. In some preferred embodiments, the cationic or cationically ionizable lipid comprises DOTMA and DOTAP.

An additional lipid may be incorporated to adjust the overall positive to negative charge ratio and physical stability of the RNA-LPX particles. In some embodiments, the RNA- LPX particles described herein comprise cationic or cationically ionizable lipids and one or more additional lipids.

In some embodiments, the one or more additional lipids comprised in the RNA-LPX particles described herein comprise one or more of the following: neutral lipids, steroids, and combinations thereof. In some embodiments, the one or more additional lipids comprise a neutral lipid which is a phospholipid. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines, cerebroside, ceramide, cephalin and sphingomyelins. Specific phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins. In particular, diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2- di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl -sn- glycero-3 -phosphocholine (Cl 6 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), 1,2- di-(9Z-octadecenoyl)-sn-glycero-3 -phosphocholine (DOPG), l,2-dipalmitoyl-sn-glycero-3- phospho-(l'-rac-glycerol) (DPPG), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), and N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM). In some preferred embodiments, the neutral lipid is DOPE. In some preferred embodiments, the neutral lipid is DOPC.

In some embodiments, the additional lipid comprises a phospholipid, cholesterol or a derivative thereof, or a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.

In some embodiments, the at additional lipid comprises DOPE, cholesterol and/or DOPC. In some embodiments, the additional lipid comprises DOPE, cholesterol and DOPC.

In some embodiments, the RNA-LPX particles include both a cationic or cationically ionizable lipid and an additional lipid. In some preferred embodiments, the cationic or cationically ionizable lipid comprises DOTMA and additional lipid comprises DOPE. In some preferred embodiments, the liposomes and RNA-LPX particles comprise DOTMA and DOPE.

Without wishing to be bound by theory, the amount of the cationic or cationically ionizable lipid compared to the amount of the additional lipid may affect important RNA-LPX particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the RNA. Accordingly, in some embodiments, the molar ratio of the cationic lipid to the additional lipid is from about 10:0 to about 1 :9, about 4: 1 to about 1 :2, or about 3 : 1 to about 1 : 1. In some preferred embodiments, the molar ratio may be about 3: 1, about 2.75: 1, about 2.5: 1, about 2.25: 1, about 2: 1, about 1.75: 1, about 1.5: 1, about 1.25: 1, or about 1 : 1. For example, the molar ratio of the cationic lipid to the additional lipid is about 2: 1. In one embodiment, at physiological pH, the charge ratio of positive charges to negative charges in the RNA-LPX particles is from about 1.6:2 to about 1 :2, or about 1.6:2 to about 1.1 :2. In specific embodiments, the charge ratio of positive charges to negative charges in the RNA-LPX particles at physiological pH is about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1 :2.0, or about 1 :2.0.

Liposomes may be used for preparing RNA-LPX particles by mixing the liposomes with RNA.

In general, a lipid nanoparticle (LNP) is obtainable from direct mixing of RNA in an aqueous phase with lipids in a phase comprising an organic solvent, such as ethanol. In that case, lipids or lipid mixtures can be used for particle formation, which do not form lamellar (bilayer) phases in water.

In some embodiments, the RNA is formulated or is to be formulated as lipid nanoparticles (LNP particles).

In some embodiments, LNPs comprise or consist of a cationic or cationically ionizable lipid and helper lipids such as phospholipids, cholesterol, and/or polymer-conjugated lipids (e.g., polyethylene glycol (PEG) lipids). In some embodiments, in the RNA-LNPs described herein the RNA (in particular, mRNA) is bound by cationically ionizable lipid that occupies the central core of the LNP. In some embodiments, polymer-conjugated lipid forms the surface of the LNP, along with phospholipids. In some embodiments, the surface comprises a bilayer. In some embodiments, cholesterol and cationically ionizable lipid in charged and uncharged forms can be distributed throughout the LNP. In some embodiments, RNA (e.g., mRNA) described herein may be noncovalently associated with a particle as described herein. In some embodiments, the RNA (especially mRNA) may be adhered to the outer surface of the particle (surface RNA (especially surface mRNA)) and/or may be contained in the particle (encapsulated RNA (especially encapsulated mRNA)).

In some embodiments, LNPs comprise four components: cationically ionizable lipids, neutral lipids such as phospholipids, a steroid such as cholesterol, and a polymer- conjugated lipid. In some embodiments, LNPs may be prepared by mixing lipids dissolved in ethanol rapidly with RNA in an aqueous buffer. While RNA particles described herein may comprise polymer-conjugated lipids such as PEG lipids, provided herein are also RNA particles which do not comprise PEG lipids, or do not comprise any polymer-conjugated lipids.

In some embodiments, the LNPs comprising RNA and at least one cationic or cationically ionizable lipid described herein are prepared by (a) preparing an RNA solution containing water and a buffering system; (b) preparing an ethanolic solution comprising the cationic or cationically ionizable lipid and, if present, one or more additional lipids; and (c) mixing the RNA solution prepared under (a) with the ethanolic solution prepared under (b), thereby preparing the formulation comprising LNPs. After step (c) one or more steps selected from diluting and filtrating, such as tangential flow filtrating, can follow.

Described herein are compositions comprising RNA (especially mRNA) and at least one cationic or cationically ionizable lipid which associates with the RNA to form RNA-LNP particles and formulations comprising such particles. The RNA-LNP particles may comprise RNA which is complexed in different forms by non-covalent interactions to the particle. The particles described herein are not viral particles, in particular infectious viral particles, i.e., they are not able to virally infect cells. Suitable cationic or cationically ionizable lipids are those that form RNA particles and are included by the term “particle forming components” or “particle forming agents”. The term “particle forming components” or “particle forming agents” relates to any components which associate with RNA to form RNA particles. Such components include any component which can be part of RNA particles.

In some embodiments, RNA-LNPs and RNA-LPX particles (especially mRNA-LNPs and mRNA-LPX particles) comprise more than one type of RNA molecules, wherein the molecular parameters of the RNA molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping, coding regions or other features.

In particulate formulation, it is possible that each RNA species is separately formulated as an individual particulate formulation. In that case, each individual particulate formulation will comprise one RNA species. The individual particulate formulations may be present as separate entities, e.g., in separate containers. Such formulations are obtainable by providing each RNA species separately (typically each in the form of an RNA-containing solution) together with a particle-forming agent, thereby allowing the formation of particles. Respective particles will contain exclusively the specific RNA species that is being provided when the particles are formed (individual particulate formulations). In some embodiments, LNP formulations comprises more than one individual particle formulation. Respective pharmaceutical compositions are referred to as mixed particulate formulations. Mixed particulate formulations according to the present disclosure are obtainable by forming, separately, individual particulate formulations, followed by a step of mixing of the individual particulate formulations. By the step of mixing, a formulation comprising a mixed population of RNA-containing particles is obtainable. Individual particulate populations may be together in one container, comprising a mixed population of individual particulate formulations. Alternatively, it is possible that all RNA species of the pharmaceutical composition are formulated together as a combined particulate formulation. Such formulations are obtainable by providing a combined formulation (typically combined solution) of all RNA species together with a particle-forming agent, thereby allowing the formation of particles. As opposed to a mixed particulate formulation, a combined particulate formulation will typically comprise particles which comprise more than one RNA species. In a combined particulate composition different RNA species are typically present together in a single particle.

The terms “lipid” and “lipid-like material” are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually insoluble or poorly soluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). The hydrophilic groups may comprise polar and/or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.

As used herein, the term “hydrophobic” refers to any a molecule, moiety or group which is substantially immiscible or insoluble in aqueous solution. The term hydrophobic group includes hydrocarbons having at least 6 carbon atoms. The monovalent radical of a hydrocarbon is referred to as hydrocarbyl herein. The hydrophobic group can have functional groups (e.g., ether, ester, halide, etc.) and atoms other than carbon and hydrogen as long as the group satisfies the condition of being substantially immiscible or insoluble in aqueous solution.

According to the disclosure, lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH. In some embodiments, the RNA compositions and formulations and RNA particles described herein comprise at least one cationic or cationically ionizable lipid as particle forming agent. Cationic or cationically ionizable lipids contemplated for use herein include any cationic or cationically ionizable lipids (including lipid-like materials) which are able to electrostatically bind nucleic acid. In some embodiments, cationic or cationically ionizable lipids contemplated for use herein can be associated with nucleic acid, e.g., by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

As used herein, a “cationic lipid” refers to a lipid or lipid-like material having a net positive charge. Cationic lipids bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.

In some embodiments, a cationic lipid has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.

As used herein, a “cationically ionizable lipid” refers to a lipid or lipid-like material which has a net positive charge or is neutral, i.e., which is not permanently cationic. Thus, depending on the pH of the composition in which the cationically ionizable lipid is solved, the cationically ionizable lipid is either positively charged or neutral. For purposes of the present disclosure, cationically ionizable lipids are covered by the term “cationic lipid” unless contradicted by the circumstances.

In some embodiments, the cationic or cationically ionizable lipid comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated, e.g., under physiological conditions. Examples of cationic or cationically ionizable lipids suitable for the preparation of RNA- LNPs as described herein include, but are not limited to N,N-dimethyl-2,3- di oleyloxypropylamine (DODMA), l,2-dioleoyl-3 -trimethylammonium propane (DOTAP), l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N — (N',N'- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); l,2-dioleoyl-3 -dimethylammonium -propane (DODAP), l,2-diacyloxy-3- dimethylammonium propanes, l,2-dialkyloxy-3 -dimethylammonium propanes, dioctadecyldimethyl ammonium chloride (DODAC), l,2-distearyloxy-N,N-dimethyl-3- aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3- trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy- N-[2(spermine carboxamide)ethyl]- N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5- en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9, 12-oc-tadecadienoxy)propane (CLinDMA), 2-[5 (cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9',12'- octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), l,2-N,N'-dioleylcarbamyl-3 -dimethylaminopropane (DOcarbDAP), 2,3- Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), l,2-N,N'-Dilinoleylcarbamyl-3- dimethylaminopropane (DLincarbD AP), 1 ,2-Dilinoleoylcarbamyl-3 -dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), 2,2- dilinoleyl-4-dimethylaminoethyl-[l,3]-di oxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4- (2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31- tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2 -Hydroxy ethyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (DMRIE), (±)-N-(3- aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-l-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-l- propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-l-propanaminium bromide (GAP -DMRIE), N-(2-Aminoethyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (PAE-DMRIE), N-(4- carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l-aminium (DOBAQ), 2-({8- [(3 P)-chol est-5 -en-3 -yloxy] octyl } oxy)-N,N -dimethyl-3 -[(9Z, 12Z)-octadeca-9, 12-dien- 1 - yloxy]propan-l -amine (Octyl-CLinDMA), l,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), l,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), Nl-[2-((lS)-l-[(3- aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4- di[oleyloxy]-benzamide (MVL5), l,2-dioleoyl-sn-glycero-3 -ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-l-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-l- aminium bromide (DMORIE), di((Z)-non-2-en-l-yl) 8,8'- ((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3- bis(dodecyloxy)propan- 1 -amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan- 1 - amine (DMDMA), Di((Z)-non-2-en-l-yl)-9-((4- (dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2- dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)- [2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipidoid 98N12-5), and l-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-l-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200).

In some embodiments, the cationic or cationically ionizable lipid is DOTMA. In some embodiments, the cationic or cationically ionizable lipid is DODMA.

DOTMA is a cationic lipid with a quaternary amine headgroup. The structure of DOTMA may be represented as follows:

DODMA is an ionizable cationic lipid with a tertiary amine headgroup. The structure of DODMA may be represented as follows:

In some embodiments, the cationic or cationically ionizable lipid may comprise from about 10 mol % to about 95 mol %, from about 20 mol % to about 95 mol %, from about 20 mol % to about 90 mol %, from about 30 mol % to about 90 mol %, from about 40 mol % to about 90 mol %, or from about 40 mol % to about 80 mol % of the total lipid present in the particle.

In some embodiments, the RNA-LNPs described herein also comprise lipids (including lipid-like materials) other than cationic or cationically ionizable lipids (also collectively referred to herein as cationic lipids), i.e., non-cationic lipids (including non-cationic or non- cationically ionizable lipids or lipid-like materials). Collectively, anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids. Optimizing the formulation of RNA particles (RNA-LNPs and RNA-LPX particles) by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to a cationic or cationically ionizable lipid may enhance particle stability and efficacy of RNA delivery.

One or more additional lipids may or may not affect the overall charge of the RNA particles. In some embodiments, the one or more additional lipids are a non-cationic lipid or lipid-like material. The non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids. As used herein, an “anionic lipid” refers to any lipid that is negatively charged at a selected pH. As used herein, a “neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH.

In some embodiments, the RNA compositions and formulations and RNA particles described herein comprise a cationic or cationically ionizable lipid and one or more additional lipids.

Without wishing to be bound by theory, the amount of the cationic or cationically ionizable lipid compared to the amount of the one or more additional lipids may affect important RNA particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the RNA. Accordingly, in some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1 :9, about 4: 1 to about 1 :2, about 4: 1 to about 1 : 1, about 3 : 1 to about 1 : 1, or about 3 : 1 to about 2: 1.

In some embodiments, the one or more additional lipids comprised in the RNA-LNPs described herein comprise one or more of the following: neutral lipids, steroids, and combinations thereof. In some embodiments, the one or more additional lipids comprise a neutral lipid which is a phospholipid. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins. Specific phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins. In particular, diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl- phosphatidylethanolamine (DPyPE), 1 ,2-di-(9Z-octadecenoyl)-sn-glycero-3 - phosphocholine (DOPG), l,2-dipalmitoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DPPG), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), and N-palmitoyl-D- erythro-sphingosylphosphorylcholine (SM). In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the neutral lipid is DOPE.

Thus, in some embodiments, the RNA-LNPs as described herein comprise (1) a cationic or cationically ionizable lipid, and a phospholipid such as DSPC or DOPE or (2) a cationic or cationically ionizable lipid and a phospholipid such as DSPC or DOPE and cholesterol.

In some embodiments, the RNA-LNP particles (especially the particles comprising mRNA) described herein comprise (1) DOTMA and DOPE, (2) DOTMA, DOPE and cholesterol, (3) DODMA and DOPE or (4) DODMA, DOPE and cholesterol.

In some embodiments, the vaccine RNA as described herein co-formulated or is to be coformulated as lipoplex particles with RNA encoding an amino acid sequence which breaks immunological tolerance.

In some preferred embodiments, the at least one RNA encoding an amino acid sequence (i), (ii), (iii), (iv), (v), or (vi) is co-formulated or is to be co-formulated as lipoplex particles with RNA encoding an amino acid sequence which breaks immunological tolerance.

In some preferred embodiments, each of the RNAs encoding an amino acid sequence (i),

(ii), (iii), (iv), (v), or (vi) is co-formulated or is to be co-formulated as lipoplex particles with RNA encoding an amino acid sequence which breaks immunological tolerance. In some preferred embodiments, each of the RNAs encoding an amino acid sequence (i), (ii),

(iii), (iv), (v), and (vi) is co-formulated or is to be co-formulated as lipoplex particles with RNA encoding an amino acid sequence which breaks immunological tolerance. In some more preferred embodiments, the RNA encoding an amino acid sequence (i), (ii),

(iii), (iv), (v), (vi), or (vi) is co-formulated or is to be co-formulated as lipoplex particles with the RNA encoding an amino acid sequence which breaks immunological tolerance at a ratio of about 4: 1 to about 16: 1, about 6: 1 to about 14: 1, about 8: 1 to about 12: 1, or about 10: 1. In some more preferred embodiments, the RNA encoding an amino acid sequence (i), (ii), (iii), (iv), (v), (vi), and (vi) is co-formulated or is to be co-formulated as lipoplex particles with the RNA encoding an amino acid sequence which breaks immunological tolerance at a ratio of about 4: 1 to about 16: 1, about 6: 1 to about 14: 1, about 8: 1 to about 12: 1, or about 10: 1.

In some embodiments, the vaccine RNA as described herein co-formulated or is to be coformulated as lipid nanoparticles with RNA encoding an amino acid sequence which breaks immunological tolerance.

In some preferred embodiments, the at least one RNA encoding an amino acid sequence (i), (ii), (iii), (iv), (v), or (vi) is co-formulated or is to be co-formulated as lipid nanoparticles with RNA encoding an amino acid sequence which breaks immunological tolerance.

In some embodiments, each of the RNAs encoding an amino acid sequence (i), (ii), (iii),

(iv), (v), or (vi) is co-formulated or is to be co-formulated as lipid nanoparticles with RNA encoding an amino acid sequence which breaks immunological tolerance. In some preferred embodiments, each of the RNAs encoding an amino acid sequence (i), (ii), (iii), (iv), (v), and (vi) is co-formulated or is to be co-formulated as lipid nanoparticles with RNA encoding an amino acid sequence which breaks immunological tolerance.

In some embodiments, the RNA encoding an amino acid sequence (i), (ii), (iii), (iv), (v), (vi), or (vi) is co-formulated or is to be co-formulated as lipid nanoparticles with the RNA encoding an amino acid sequence which breaks immunological tolerance at a ratio of about 4: 1 to about 16: 1, about 6: 1 to about 14: 1, about 8: 1 to about 12: 1, or about 10: 1. In some more preferred embodiments, the RNA encoding an amino acid sequence (i), (ii), (iii), (iv),

(v), (vi), and (vi) is co-formulated or is to be co-formulated as lipid nanoparticles with the RNA encoding an amino acid sequence which breaks immunological tolerance at a ratio of about 4: 1 to about 16: 1, about 6: 1 to about 14: 1, about 8: 1 to about 12: 1, or about 10: 1.

The anti-CTLA4 antibody

In some embodiments, the anti-CTLA4 antibody is capable of binding human CTLA4. The term “binding” preferably relates to a specific binding. An antibody usually is capable of binding to a predetermined target if it has a significant affinity for said predetermined target and binds to said predetermined target in standard assays. “Affinity” or “binding affinity” is often measured by equilibrium dissociation constant (KD). Preferably, the term “significant affinity” refers to the binding to a predetermined target with a dissociation constant (KD) of 10'⁵ M or lower, 10'⁶ M or lower, 10'⁷ M or lower, 10'⁸ M or lower, or 10'⁹ M or lower. An antibody is not (substantially) capable of binding to a target if it has no significant affinity for said target and does not bind significantly, in particular does not bind detectably, to said target in standard assays. For example, if the KD for binding of an antibody to the target to which the antibody is capable of binding is 10'⁹M, the KD for binding to a target for which the antibody has no significant affinity would be is at least around 10'⁸ M, 10'⁷ M, 10'⁶ M, 10'⁵ M, IO'⁴ M, 10'³ M, IO'² M, or 10'¹ M.

Binding of an antibody to a target can be determined experimentally using any suitable method; see, for example, Berzofsky et al., Antibody- Antigen Interactions In Fundamental Immunology, Paul, W. E., Ed., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, N Y (1992), and methods described herein. Affinities may be readily determined using conventional techniques, such as by equilibrium dialysis; by using the BIAcore 2000 instrument, using general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by another method known to the skilled artisan. The affinity data may be analyzed, for example, by the method of Scatchard et al. (Scatchard et al., Ann NY Acad ScL 1949, 51 :660). The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions, e.g., salt concentration, pH. Thus, measurements of affinity and other antigen-binding parameters, e.g., KD, IC50, are preferably made with standardized solutions of antibody and antigen, and a standardized buffer. For example, the affinity of an antibody can be evaluated by Octet. Multi -concentration kinetic experiments can be performed on the Octet Red96 system (ForteBio). Anti-hlgG Fc biosensors (ForteBio, #18-5064) can be hydrated in sample diluent (0.1% BSA in PBS and 0.02% Tween 20) and preconditioned in pH 1.7 glycine. The antigen can be diluted using a 7- point, 2-fold serial dilution starting at 600 nM with sample diluent. The antibody to be tested can be diluted to 10 pg/mL with sample diluent and then immobilized onto anti-hlgG Fc biosensors for 120 seconds. After baselines are established for 60 seconds in sample diluent, the biosensors can be moved to wells containing the antigen at a series of concentrations to measure the association. Association can be observed (e.g., for 120 seconds) and dissociation can be observed (e.g., for 180 seconds) for each protein of interest in the sample diluent. The binding affinities can be characterized by fitting the kinetic sensorgrams to a monovalent binding model (1 : 1 binding).

The anti-CTLA4 antibody disclosed herein is specific for CTLA4 if it is capable of binding to CTLA4 but is not (substantially) capable of binding to other targets.

The anti-CTLA4 antibody disclosed herein preferably does not inhibit binding of human CTLA4 to the B7.1 (CD80) and B7.2 (CD86) ligands of antigen presenting cells. In some embodiments, the level of B7.1 and B7.2 on immune cells following anti-CTLA4 treatment is used as a biomarker for measuring the biological activity of anti-CTLA4 antibodies in vivo and monitoring responses to anti-CTLA4 treatment by measuring the level B7.1 and/or B7.2 expression on immune cells, and comparing the level of expression before and after treatment. In some embodiments, the level of B7.1 and/or B7.2 expression is monitored over time during a course of therapy. The therapeutic effect of CTLA4 antibodies disclosed herein is preferably achieved through antibody-mediated depletion of Tregs specifically within tumor microenvironment. The anti-CTLA4 antibodies disclosed herein are preferably not capable of blocking B7-CTLA4 interactions under physiological conditions. A fundamental question for the generation of safe and effective anti-CTLA4 antibodies is whether cancer immunotherapeutic effects (CITE) and immunotherapy -related adverse effects (irAE) are intrinsically linked. The classical checkpoint blockade hypothesis stipulated that anti-CTLA4 antibodies promote cancer immunity by blocking a negative signal of B7-CTLA4 interactions to promote naive T cell activation in the lymphoid organ. According to this model, therapeutic antibodies are antagonists that functionally inactivate CTLA4-B7 interactions. Since genetic inactivation of CTLA4 expression leads to autoimmune diseases in mouse and human, it was assumed that the irAE would be a necessary price for CITE. However, there is no evidence to date that blocking CTLA-4 interaction with B7.1 and B7.2 is either necessary or sufficient for the CITE of anti-CTLA- 4 antibodies. Without wishing to be bound by theory, it is contemplated that selective depletion of Tregs in the tumor microenvironment constitutes the main mechanism of action of anti-CTLA4 antibodies. It is not relevant whether an antibody is capable of blocking B7-CTLA4 interactions under physiological conditions for the induction of CITE. In some embodiments, the anti-CTLA4 antibody disclosed herein can induce CITE without blocking B7-CTLA4 interactions.

In some embodiments, the anti-CTLA4 antibody is not ipilimumab (marketed as YERVOY®).

In particularly preferred embodiments, the antibody having the ability of binding to CTLA4 is described in International Patent Application Publication No., WO 2017/106372, which is incorporated herein in its entirety.

In some embodiments, the anti-CTLA4 antibody is an antigen-binding fragment or a variant thereof. In some preferred embodiments, the antigen binding fragment or variant thereof is selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a scFV, a diabody, a triabody, a minibody, and a single-domain antibody (sdAB), and variants thereof.

In some embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain variable region comprising (1) a complementarity-determining region 1 (HCDR1) whose amino acid sequence is set forth in SEQ ID NO: 39, (2) a complementarity-determining region 2 (HCDR2) whose amino acid sequence is set forth in SEQ ID NO: 41, 42, or 43, and (3) a complementarity-determining region 3 (HCDR3) whose amino acid sequence is set forth in SEQ ID NO: 47; and b) a light chain variable region comprising: (1) a complementarity-determining region 1 (LCDR1) whose amino acid sequence is set forth in SEQ ID NO: 40, (2) a complementarity-determining region 2 (LCDR2) whose amino acid sequence is set forth in SEQ ID NO: 44, 45, or 46, and (3) a complementarity-determining region 3 (LCDR3) whose amino acid sequence is set forth in SEQ ID NO: 48.

A CDR refers to one of three hypervariable regions (Hl, H2 or H3) within the nonframework region of the immunoglobulin (Ig or antibody) VH P-sheet framework, or one of three hypervariable regions (LI, L2 or L3) within the non-framework region of the antibody VL P-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. The CDRs of the VH P-sheet framework are denoted as HCDR1, HCDR2, and HCDR3. The CDRs of the VL P-sheet framework are denoted as LCDR1, LCDR2, and LCDR3. CDR regions are well known to those skilled in the art and have been defined by, for example, Rabat as the regions of most hypervariability within the antibody variable (V) domains (Kabat et al., J Biol Chem 1977, 252: 6609-6616; Kabat, Adv Prot Chem 1978, 32: 1-75). CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved P- sheet framework, and thus are able to adapt different conformations (Chothia and Lesk, J Mol Biol 1987, 196: 901-917). Both terminologies are well recognized in the art. The positions of CDRs within a canonical antibody variable domain have been determined by comparison of numerous structures (Al-Lazikani et al., J Mol Biol 1997, 273: 927-948; Morea et al., Methods 2000, 20: 267-279). Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al., J Mol Biol 1997, 273: 927-948). Such nomenclature is similarly well known to those skilled in the art.

The anti-CTLA4 antibody may comprise the CDRs listed in table 1.

Table 1: exemplary CDR sequences of the anti-CTLA4 antibody In some embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain variable region comprising (1) a HCDR1 whose amino acid sequence is set forth in SEQ ID NO: 39, (2) a HCDR2 whose amino acid sequence is set forth in SEQ ID NO: 41, and (3) a HCDR3 whose amino acid sequence is set forth in SEQ ID NO: 47; and b) a light chain variable region comprising: (1) a LCDR1 whose amino acid sequence is set forth in SEQ ID NO: 40, (2) a LCDR2 whose amino acid sequence is set forth in SEQ ID NO: 45, and (3) a LCDR3 whose amino acid sequence is set forth in SEQ ID NO: 48.

In some preferred embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain variable region comprising (1) HCDR1 whose amino acid sequence is set forth in SEQ ID NO: 39, (2) a HCDR2 whose amino acid sequence is set forth in SEQ ID NO: 43, and (3) a HCDR3 whose amino acid sequence is set forth in SEQ ID NO: 47; and b) a light chain variable region comprising: (1) a LCDR1 whose amino acid sequence is set forth in SEQ ID NO: 40, (2) a LCDR2 whose amino acid sequence is set forth in SEQ ID NO: 45, and (3) a LCDR3 whose amino acid sequence is set forth in SEQ ID NO: 48.

In some embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain variable region comprising (1) a HCDR1 whose amino acid sequence is set forth in SEQ ID NO: 39, (2) a HCDR2 whose amino acid sequence is set forth in SEQ ID NO: 43, and (3) a HCDR3 whose amino acid sequence is set forth in SEQ ID NO: 47; and b) a light chain variable region comprising: (1) a LCDR1 whose amino acid sequence is set forth in SEQ ID NO: 40, (2) a LCDR2 whose amino acid sequence is set forth in SEQ ID NO: 46, and (3) a LCDR3 whose amino acid sequence is set forth in SEQ ID NO: 48.

In some embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 49, 40, or 51; and b) a light chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 52, 53, or 54.

The anti-CTLA4 antibody may comprise the heavy and light chain variable domains listed in table 2. Table 2: exemplary heavy and light chain variable domain sequences of the anti- CTLA4 antibody

In some embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 49; and b) a light chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 53.

In some preferred embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 51; and b) a light chain variable domain whose amino acid sequence is set forth in SEQ ID NO:

53.

In some embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 51; and b) a light chain variable domain whose amino acid sequence is set forth in SEQ ID NO:

54.

In some embodiments, the antibody having the ability to bind to CTLA4 can be a polyclonal, monoclonal antibody or a chimeric antibody, optionally having an IgG or IgM isotype of any subclass, such as subclass I. Preferably, the anti-CTLA4 antibody is an IgG or IgM antibody, preferably an IgG antibody, more preferably an IgGl antibody. The term “monoclonal antibody” as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity. In some embodiments, the monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a non-human animal, e.g., mouse, fused to an immortalized cell.

In some embodiments, the anti-CTLA4 antibody comprises a heavy chain comprising a Fc region of a human Ig antibody. In some preferred embodiments, the Fc region of the human Ig antibody comprises the amino acid sequence of SEQ ID NO: 55 or 56. In some embodiments, the anti-CTLA4 antibody can be an anti-CTLA4 antibody with a mutated Fc region. Relative to the sequence of the IgGl backbone in SEQ ID NO: 55, the mutation may be M135Y, S137T, T139E, S181A, E216A, or K217A, or a combination thereof. These mutations are contemplated to lead to increased antibody dependent cellular cytotoxicity (ADCC) and increased half-life of the antibody in vivo. Preferably, the Fc region of the antibody may comprise all six mutations. In some particularly preferred embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 56.

In some embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain whose amino acid sequence is set forth in SEQ ID NO: 57, 59, or 61; and b) a light chain whose amino acid sequence is set forth in SEQ ID NO: 63, 65, or 67.

The anti-CTLA4 antibody may comprise the heavy and light chains listed in table 3.

Table 3: exemplary heavy and light chains of the anti-CTLA4 antibody In some embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain whose amino acid sequence is set forth in SEQ ID NO: 57; and b) a light chain whose amino acid sequence is set forth in SEQ ID NO: 65.

In some preferred embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain whose amino acid sequence is set forth in SEQ ID NO: 61; and b) a light chain whose amino acid sequence is set forth in SEQ ID NO: 65.

In some embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain whose amino acid sequence is set forth in SEQ ID NO: 61; and b) a light chain whose amino acid sequence is set forth in SEQ ID NO: 67.

In some embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain whose amino acid sequence is encoded by the nucleotide sequences set forth in SEQ ID NO: 58, 60, or 62, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence set forth in SEQ ID NO: 58, 60, or 62; and b) a light chain whose amino acid is encoded by the nucleotide sequences set forth in SEQ ID NO: 64, 66, or 68, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence set forth in SEQ ID NO: 64, 66, or 68.

In some embodiments, the tyrosine in the sequences set forth in SEQ ID NO: 85, 60, and 62 is uridine. Meaning, all tyrosines present in the sequences of SEQ ID NO: 85, 60, and 62 can be uridines.

In some embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain whose amino acid sequence is encoded by the nucleotide sequence set forth in SEQ ID NO: 58, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence set forth in SEQ ID NO: 58; and b) a light chain whose amino acid sequence is encoded by the nucleotide sequence set forth SEQ ID NO: 66, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence set forth in SEQ ID NO: 66.

In some embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain whose amino acid sequence is encoded by the nucleotide sequence set forth in SEQ ID NO: 62, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence set forth in SEQ ID NO: 62; and b) a light chain whose amino acid sequence is encoded by the nucleotide sequence set forth SEQ ID NO: 66, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence set forth in SEQ ID NO: 66.

In some embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain whose amino acid sequence is encoded by the nucleotide sequence set forth in SEQ ID NO: 62, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence set forth in SEQ ID NO: 62; and b) a light chain whose amino acid sequence is encoded by the nucleotide sequence set forth SEQ ID NO: 68, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence set forth in SEQ ID NO: 68. In some preferred embodiments, the anti CTLA4 antibody is a humanized anti-CTLA4 antibody. In some more preferred embodiments, the anti CTLA4 antibody is a humanized anti-CTLA4 IgGl monoclonal antibody.

In some embodiments, the anti-CTLA4 antibody is a pH-sensitive anti-CTLA4 antibody. For example, the pH-sensitive anti-CTLA4 antibody can dissociate from CTLA4 at pH 6.5 or below, more preferably pH 5.5 or below. In some preferred embodiments, binding to CTLA4 is reduced at an endosomal pH of 5.5 by more than 50% relative to binding at neutral pH (pH 7.0). Such a reduction may reach more than 75% at lysosomal pH 4.5 as compared to pH 7.0. The antibody- antigen complex preformed at pH 7.0 may dissociate under an acidic environment of pH 4.5-6.0. The reduction in binding may also be in comparison to a reference antibody which may be considerably less pH sensitive using the same standard. The reference antibody may be an antibody known in the art such as Ipilimumab or Tremelimumab. In the context of an engineered antibody, the changes may also be in comparison to a wild-type antibody which may be considerably less pH sensitive using the same standard. Without wishing to be bound by theory, it is contemplated that a pH-sensitive antibody is not only safer but also more effective in Treg depletion and tumor rejection than a pH-insensitive CTLA4 antibody (e.g., Ipilimumab). Meaning, Ipilimumab can bind to CTLA4 at a pH of 4-7 and no dissociation can be observed at pH 4-7. pH- insensitive antibodies can cause down-regulation of CTLA4 through lysosomal degradation. CTLA4 down-regulation can cause autoimmune diseases; while in the tumor CTLA4 down-regulation can reduce ADCC activity and thus anti-cancer efficacy. The sensitivity to pH can be measured by any method known to the skilled person. For example, human or monkey-CTLA4-Fc (0.5 pg/ml) can be coated on ELISA plates at 4°C overnight. Biotinylated anti-CTLA4 antibodies can be added at 1 pg/ml in 1% BSA PBS with pH 4.5- 7.0. Two hours later, antibodies binding with CTLA4 can be measured by using HRP- labeled streptavidin. A pH-sensitive anti-CTLA4 antibody can comprise, for example, the following CDR sequences: a) a heavy chain variable region comprising (1) a complementarity-determining region 1 (HCDR1) whose amino acid sequence is set forth in SEQ ID NO: 39, (2) a complementarity-determining region 2 (HCDR2) whose amino acid sequence is set forth in SEQ ID NO: 41-43, and (3) a complementarity-determining region 3 (HCDR3) whose amino acid sequence is set forth in SEQ ID NO: 47; and b) a light chain variable region comprising: (1) a complementarity-determining region 1 (LCDR1) whose amino acid sequence is set forth in SEQ ID NO: 40, (2) a complementarity-determining region 2 (LCDR2) whose amino acid sequence is set forth in SEQ ID NO: 44-46, and (3) a complementarity-determining region 3 (LCDR3) whose amino acid sequence is set forth in SEQ ID NO: 48.

Although the following provides considerations regarding the mechanism underlying the therapeutic efficacy of antibodies of the disclosure it is not to be considered as limiting to the invention in any way. In some embodiments, the antibodies described herein are contemplated to be a highly selective, humanized monoclonal immunoglobulin G1 (IgGl)- kappa isotype antibody against CTLA4 with a robust anti-tumor activity and lower autoimmune toxicity in comparison to ipilimumab. The disclosed antibodies preferably can dissociate from CTLA4 under low pH in endosomes to allow both CTLA4 and the antibody to escape from lysosomal degradation and recycle to the cell surface. Unlike ipilimumab that down-regulates CTLA4 expression on Treg cells, the antibodies can keep a high-level expression of CTLA4 on Treg cells through this recycling mechanism and makes Treg cells a better target for antibody-dependent cellular cytotoxicity, particularly in the tumor microenvironment (TME). The selective elimination of Treg cells in the tumor microenvironment and maintenance of CTLA4 expression in Treg cells in the peripheral tissues by the anti-CTLA4 antibody is contemplated to form the cellular and molecular basis for more potent tumor rejection and low toxicity. For example, the anti-CTLA4 antibody described herein dissociates from CTLA4 in endosomes, allows normal recycling of both antibodies and CTLA4, which lead to a much-reduced autoimmune toxicity. The preservation of the recycling of both CTLA4 and the anti-CTLA4 antibody facilitates more potent ADCC to eliminate Treg cells in the tumor microenvironment and induces strong CITE. ADCC preferably occurs when antibodies bind to antigens such as CTLA4 on Treg cells and the antibody Fc domains engage Fc receptors (FcR) on the surface of immune effector cells. In some embodiments, the anti-CTLA4 antibody is an antibody selected from the group consisting of (i) an antibody which is a chimerized or humanized form of the antibody defined by the sequence identifiers above, (ii) an antibody having the specificity of the antibody defined by the sequence identifiers above, and (iii) an antibody comprising the antigen binding portion or antigen binding site, in particular the variable region, of the antibody defined by the sequence identifiers above or variant thereof and preferably having the specificity of the antibody defined by the sequence identifiers above.

In some embodiments, the anti-CTLA4 antibody comprises one or more CDRs, a set of CDRs or a combination of sets of CDRs as described herein and comprises said CDRs together with their intervening framework regions. Construction of antibodies made by recombinant DNA techniques may result in the introduction of residues N- or C-terminal to the variable regions encoded by linkers introduced to facilitate cloning or other manipulation steps, including the introduction of linkers to join variable regions to further protein sequences including immunoglobulin heavy chains, other variable domains (for example in the production of diabodies) or protein labels. In one embodiment an antibody comprising one or more CDRs, a set of CDRs or a combination of sets of CDRs as described herein comprises said CDRs in a human antibody framework.

It will be appreciated by those skilled in the art that in particular the sequences of the CDR, hypervariable and variable regions can be modified without losing the ability to bind CTLA4. For example, CDR regions will be either identical or highly homologous to the regions of antibodies specified herein. By “highly homologous” it is contemplated that from 1 to 5, preferably from 1 to 4, such as 1 to 3 or 1 or 2 substitutions may be made in the CDRs. In addition, the hypervariable and variable regions may be modified so that they show substantial homology with the regions of antibodies specifically disclosed herein.

It will be appreciated by those skilled in the art that the anti-CTLA4 antibody can comprise variants of the sequence(s) disclosed herein without losing the ability to bind CTLA4. Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible.

Amino acid addition variants comprise amino- and/or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and/or to replacing amino acids with other ones having similar properties. Preferably, amino acid changes in protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.

Antibodies described herein and useful in the methods described herein can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 1975, 256: 495. Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibodies can be employed, e.g., viral or oncogenic transformation of B -lymphocytes or phage display techniques using libraries of antibody genes.

I l l In some embodiments, an animal system for preparing hybridomas that secrete monoclonal antibodies may be a murine system. Hybridoma production in the mouse is a very well- established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are the rat and the rabbit system (e.g., described in Spieker-Polet et al., Proc Natl Acad Sci U.S.A. 1995, 92: 9348; see also Rossi et al., Am J Clin Pathol 2005, 124: 295).

Yet another strategy for generating monoclonal antibodies is to directly isolate genes encoding antibodies from lymphocytes producing antibodies of defined specificity. For details of recombinant antibody engineering see also Welschof and Kraus, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Bemiy K.C. Lo Antibody Engineering ISBN 1 -58829- 092-1.

To generate antibodies, mice can be immunized with carrier-conjugated peptides derived from the antigen sequence, i.e., the sequence against which the antibodies are to be directed, an enriched preparation of recombinantly expressed antigen or fragments thereof and/or cells expressing the antigen, as described. Alternatively, mice can be immunized with nucleic acid encoding the antigen or fragments thereof. In the event that immunizations using a purified or enriched preparation of the antigen do not result in antibodies, mice can also be immunized with cells expressing the antigen, e.g., a cell line, to promote immune responses.

The immune response can be monitored over the course of the immunization protocol with plasma and serum samples being obtained by tail vein or retroorbital bleeds. Mice with sufficient titers of immunoglobulin can be used for fusions. Mice can be boosted intraperitonealy or intravenously with antigen expressing cells 3 days before sacrifice and removal of the spleen to increase the rate of specific antibody secreting hybridomas. To generate hybridomas producing monoclonal antibodies, splenocytes and lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can then be screened for the production of antigen-specific antibodies. Individual wells can then be screened by ELISA for antibody secreting hybridomas. By Immunofluorescence and FACS analysis using antigen expressing cells, antibodies with specificity for the antigen can be identified. The antibody secreting hybridomas can be re-plated, screened again, and if still positive for monoclonal antibodies can be subcloned by limiting dilution. The stable subclones can then be cultured in vitro to generate antibody in tissue culture medium for characterization. Antibodies also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as are well known in the art (Morrison, Science 1985, 229: 1202).

For example, in some embodiments, the gene(s) of interest, e.g., antibody genes, can be ligated into an expression vector such as a eukaryotic expression plasmid such as used by the GS gene expression system disclosed in International Patent Application Publication Nos. WO 87/04462 and WO 89/01036 and EP 338 841 A or other expression systems well known in the art. The purified plasmid with the cloned antibody genes can be introduced in eukaryotic host cells such as CHO cells, NS/0 cells, HEK293T cells or HEK293 cells or alternatively other eukaryotic cells like plant derived cells, fungal or yeast cells. The method used to introduce these genes can be methods described in the art such as electroporation, lipofectine, lipofectamine or others. After introduction of these antibody genes in the host cells, cells expressing the antibody can be identified and selected. These cells represent the transfectomas which can then be amplified for their expression level and upscaled to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and/or cells.

Alternatively, the cloned antibody genes can be expressed in other expression systems, including prokaryotic cells, such as microorganisms, e.g., E. coli. Furthermore, the antibodies can be produced in transgenic non-human animals, such as in milk from sheep and rabbits or in eggs from hens, or in transgenic plants; see e.g., Verma, R., et al., J Immunol Meth 1998,216: 165-181; Pollock, et al., J Immunol Meth 1999, 231 : 147-157; and Fischer, R., et al., Biol Chem 1999, 380: 825-839.

Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann et al., Nature 1998, 332:323-327; Jones et al., Nature 1986, 321 :522-525; and Queen et al., Proc Natl Acad Sci USA 1989, 86: 10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V (D) J joining during B cell maturation. Germline gene sequences will also differ from the sequences of a high affinity secondary repertoire antibody at individual evenly across the variable region.

The ability of antibodies to bind an antigen can be determined using standard binding assays (e.g., ELISA, Western Blot, Immunofluorescence and flow cytometric analysis).

To purify antibodies, selected hybridomas can be grown in two-liter spinner- flasks for monoclonal antibody purification. Alternatively, antibodies can be produced in dialysisbased bioreactors. Supernatants can be filtered and, if necessary, concentrated before affinity chromatography with protein G-sepharose or protein A-sepharose. Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using 1.43 extinction coefficient. The monoclonal antibodies can be ali quoted and stored at -80°C. To determine if selected monoclonal antibodies bind to unique epitopes and/or to characterize one or more binding properties, site-directed or multi-site directed mutagenesis can be used.

To determine the isotype of antibodies, isotype ELISAs with various commercial kits (e.g., Zymed, Roche Diagnostics) can be performed. Wells of microtiter plates can be coated with anti-mouse Ig. After blocking, the plates are reacted with monoclonal antibodies or purified isotype controls, at ambient temperature for two hours. The wells can then be reacted with either mouse IgGl, IgG2a, IgG2b or IgG3, IgA or mouse IgM-specific peroxidase- conjugated probes. After washing, the plates can be developed with ABTS substrate (1 mg/ml) and analyzed at OD of 405-650. Alternatively, the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche, Cat. No. 1493027) may be used as described by the manufacturer.

In order to demonstrate presence of antibodies in sera of immunized mice or binding of monoclonal antibodies to living cells expressing antigen, flow cytometry can be used. Cell lines expressing naturally or after transfection antigen and negative controls lacking antigen expression (grown under standard growth conditions) can be mixed with various concentrations of monoclonal antibodies in hybridoma supernatants or in PBS containing 1 % FBS, and can be incubated at 4 °C for 30 min. After washing, the APC- or Alexa647- labeled anti IgG antibody can bind to antigen-bound monoclonal antibody under the same conditions as the primary antibody staining. The samples can be analyzed by flow cytometry with a FACS instrument using light and side scatter properties to gate on single, living cells. In order to distinguish antigen-specific monoclonal antibodies from nonspecific binders in a single measurement, the method of co-transfection can be employed. Cells transiently transfected with plasmids encoding antigen and a fluorescent marker can be stained as described above. Transfected cells can be detected in a different fluorescence channel than antibody-stained cells. As the majority of transfected cells express both transgenes, antigen- specific monoclonal antibodies bind preferentially to fluorescence marker expressing cells, whereas non-specific antibodies bind in a comparable ratio to nontransfected cells. An alternative assay using fluorescence microscopy may be used in addition to or instead of the flow cytometry assay. Cells can be stained exactly as described above and examined by fluorescence microscopy.

In order to demonstrate presence of antibodies in sera of immunized mice or binding of monoclonal antibodies to living cells expressing antigen, immunofluorescence microscopy analysis can be used. For example, cell lines expressing either spontaneously or after transfection antigen and negative controls lacking antigen expression are grown in chamber slides under standard growth conditions in DMEM/F12 medium, supplemented with 10 % fetal calf serum (FCS), 2 raM L-glutamine, 100 lU/ml penicillin and 100 pg/ml streptomycin. Cells can then be fixed with methanol or paraformaldehyde or left untreated. Cells can then be reacted with monoclonal antibodies against the antigen for 30 min. at 25°C. After washing, cells can be reacted with an Alexa555-labelled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. Cells can then be examined by fluorescence microscopy.

Cell extracts from cells expressing antigen and appropriate negative controls can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes, blocked, and probed with the monoclonal antibodies to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and developed with ECL substrate.

Antibodies can be further tested for reactivity with antigen by Immunohistochemistry in a manner well known to the skilled person, e.g., using paraformaldehyde or acetone fixed cryosections or paraffin embedded tissue sections fixed with paraformaldehyde from noncancer tissue or cancer tissue samples obtained from patients during routine surgical procedures or from mice carrying xenografted tumors inoculated with cell lines expressing spontaneously or after transfection antigen. For immunostaining, antibodies reactive to antigen can be incubated followed by horseradish-peroxidase conjugated goat anti-mouse or goat anti-rabbit antibodies (DAKO) according to the vendors instructions. The testing of monoclonal antibody activity in vitro will provide an initial screening prior to testing in vivo models.

In some embodiments, the anti-CTLA4 antibody is encoded by one or more RNAs. In some preferred embodiments, an antigen-binding fragment of the anti-CTLA4 antibody or a variant thereof is encoded by one or more RNAs. In some preferred embodiments, the antigen-binding fragment of the anti-CTLA4 antibody or a variant thereof encoded by the one or more RNAs is selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a scFV, a diabody, a triabody, a minibody, and a single-domain antibody (sdAB), and variants thereof.

In some embodiments, the anti-CTLA4 antibody comprising: a) a heavy chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 49-51; and b) a light chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 52-54; is encoded by one or more RNAs.

In some embodiments, the anti-CTLA4 antibody comprising: a) a heavy chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 49; and b) a light chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 53; is encoded by one or more RNAs.

In some preferred embodiments, the anti-CTLA4 antibody comprising: a) a heavy chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 51; and b) a light chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 53; is encoded by one or more RNAs.

In some embodiments, the anti-CTLA4 antibody comprising: a) a heavy chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 51; and b) a light chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 54; is encoded by one or more RNAs.

In some embodiments, the heavy chain variable domains whose amino acid sequences are set forth in SEQ ID NO: 49-51 and the light chain variable domains whose amino acid sequences are set forth in SEQ ID NO: 52-54, are each individually encoded by one RNA.

In some embodiments, the at least one RNA encoding an amino acid sequence (i), (ii), (iii), (iv), (v), or (vi) is co-formulated or is to be co-formulated as lipoplex particles with RNA encoding an amino acid sequence which breaks immunological tolerance and with RNA encoding the anti-CTLA4 antibody.

In some preferred embodiments, the at least one RNA encoding an amino acid sequence (i), (ii), (iii), (iv), (v), or (vi) is co-formulated or is to be co-formulated as lipoplex particles with RNA encoding an amino acid sequence which breaks immunological tolerance and with RNA encoding an antigen-binding fragment of the anti-CTLA4 antibody or a variant thereof.

In some embodiments, each of the RNAs encoding an amino acid sequence (i), (ii), (iii), (iv), (v), or (vi) is co-formulated or is to be co-formulated as lipoplex particles with RNA encoding an amino acid sequence which breaks immunological tolerance and with RNA encoding the anti-CTLA4 antibody. In some embodiments, each of the RNAs encoding an amino acid sequence (i), (ii), (iii), (iv), (v), and (vi) is co-formulated or is to be co- formulated as lipoplex particles with RNA encoding an amino acid sequence which breaks immunological tolerance and with RNA encoding the anti-CTLA4 antibody.

In some preferred embodiments, each of the RNAs encoding an amino acid sequence (i), (ii), (iii), (iv), (v), or (vi) is co-formulated or is to be co-formulated as lipoplex particles with RNA encoding an amino acid sequence which breaks immunological tolerance and with RNA encoding an antigen-binding fragment of the anti-CTLA4 antibody or a variant thereof. In some preferred embodiments, each of the RNAs encoding an amino acid sequence (i), (ii), (iii), (iv), (v), and (vi) is co-formulated or is to be co-formulated as lipoplex particles with RNA encoding an amino acid sequence which breaks immunological tolerance and with RNA encoding an antigen-binding fragment of the anti- CTLA4 antibody or a variant thereof.

Treatment

In some embodiments, an additional immunomodulatory agent is administered to the subject. Meaning the additional immunomodulatory agent is administered together with the RNA cancer vaccine comprising the at least one RNA and the anti-CTLA4 antibody. Immunomodulatory agents are natural or synthetic agents, substances, compounds, or compositions that modulate the immune system. Thus, immunomodulatory agents induce, enhance, attenuate and/or suppress a specific immune response in a subject. Immunomodulatory agents or immunomodulators are well known in the art (Bascones- Martinze et al., Med Oral Patol Oral Cir Bucal 2014, 19(1): e24-e31). In some embodiments, the immunomodulatory agent is selected from the group consisting of immune checkpoint inhibitors, cytokines such as monokines, lymphokines, interleukins, and chemokines, monoclonal antibodies, growth factors, lipopolysaccharide (LPS), chaperone GP96, CpG oligodeoxynucleotides, and anti-cancer vaccines. In some embodiments the cytokines are selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INFa, INF-y, GM-CSF, and LT-a. In some embodiments, the additional immunomodulatory agent is an immune checkpoint inhibitor. In some preferred embodiments, the immune checkpoint inhibitor is selected from the group consisting of anti-PD-1, anti-B7-Hl, anti-B7-H4, anti-LIGHT, anti-LAG3, anti- TIM3, anti-TIM4, anti-OX40, anti-GITR, anti-BTLA, anti-CD27, and/or anti-ICOS antibodies. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody. For example, the anti-PD-1 antibody can be selected from the group consisting of cemiplimab (LIBTAYO, REGN2810), nivolumab (OPDIVO; BMS-936558), pembrolizumab (KEYTRUDA; MK-3475), pidilizumab (CT-011), spartalizumab (PDR001), MEDI0680 (AMP-514), dostarlimab (TSR-042), cetrelimab (JNJ 63723283), toripalimab (JSOO1), AMP-224 (GSK-2661380), PF-06801591, tislelizumab (BGB-A317), ABBV-181, BI 754091, or SHR-1210.

In some embodiments, an additional chemotherapeutic agent is administered to the subject. Meaning the chemotherapeutic agent is administered together with the anti-CTLA4 antibody and the RNA cancer vaccine comprising the at least one RNA.

In some embodiments, the invention relates to an RNA cancer vaccine comprising: a) at least one RNA, wherein the at least one RNA encodes the following amino acid sequences:

(ii) an amino acid sequence comprising Kita-kyushu lung cancer antigen 1 (KK-LC-1), an immunogenic variant thereof, or an immunogenic fragment of the KK-LC-1 or the immunogenic variant thereof;

(iii)an amino acid sequence comprising Melanoma antigen A3 (MAGE-A3), an immunogenic variant thereof, or an immunogenic fragment of the MAGE- A3 or the immunogenic variant thereof;

(iv)an amino acid sequence comprising Melanoma antigen 4 (MAGE-A4), an immunogenic variant thereof, or an immunogenic fragment of the MAGE-A4 or the immunogenic variant thereof; (v) an amino acid sequence comprising Preferentially Expressed Antigen In Melanoma (PRAME), an immunogenic variant thereof, or an immunogenic fragment of the PRAME or the immunogenic variant thereof; and

(vi)an amino acid sequence comprising Melanoma antigen Cl (MAGE-CI), an immunogenic variant thereof, or an immunogenic fragment of the MAGE-CI or the immunogenic variant thereof. b) a further therapeutic agent selected from an immune checkpoint inhibitor, a chemotherapeutic agent, or a combination thereof.

Chemotherapy is a type of cancer treatment that uses one or more anti-cancer drugs (chemotherapeutic agents), usually as part of a standardized chemotherapy regimen. The term chemotherapy has come to connote non-specific usage of intracellular poisons to inhibit mitosis. The connotation excludes more selective agents that block extracellular signals (signal transduction). The development of therapies with specific molecular or genetic targets, which inhibit growth-promoting signals from classic endocrine hormones (primarily estrogens for breast cancer and androgens for prostate cancer) are now called hormonal therapies. By contrast, other inhibitions of growth-signals like those associated with receptor tyrosine kinases are referred to as targeted therapy.

Importantly, the use of drugs (whether chemotherapy, hormonal therapy or targeted therapy) constitutes systemic therapy for cancer in that they are introduced into the blood stream and are therefore in principle able to address cancer at any anatomic location in the body. Systemic therapy is often used in conjunction with other modalities that constitute local therapy (i.e., treatments whose efficacy is confined to the anatomic area where they are applied) for cancer such as radiation therapy, surgery or hyperthermia therapy. Traditional chemotherapeutic agents are cytotoxic by means of interfering with cell division (mitosis) but cancer cells vary widely in their susceptibility to these agents. To a large extent, chemotherapy can be thought of as a way to damage or stress cells, which may then lead to cell death if apoptosis is initiated. Chemotherapeutic agents include alkylating agents, antimetabolites, anti -microtubule agents, topoisomerase inhibitors, and cytotoxic antibiotics.

Alkylating agents have the ability to alkylate many molecules, including proteins, RNA and DNA. The subtypes of alkylating agents are the nitrogen mustards, nitrosoureas, tetrazines, aziridines, cisplatins and derivatives, and non-classical alkylating agents. Nitrogen mustards include mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide and busulfan. Nitrosoureas include N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU) and semustine (MeCCNU), fotemustine and streptozotocin. Tetrazines include dacarbazine, mitozolomide and temozolomide. Aziridines include thiotepa, mytomycin and diaziquone (AZQ). Cisplatin and derivatives include cisplatin, carboplatin and oxaliplatin. They impair cell function by forming covalent bonds with the amino, carboxyl, sulfhydryl, and phosphate groups in biologically important molecules. Non-classical alkylating agents include procarbazine and hexamethylmelamine. In one particularly preferred embodiment, the alkylating agent is cyclophosphamide.

Anti-metabolites are a group of molecules that impede DNA and RNA synthesis. Many of them have a similar structure to the building blocks of DNA and RNA. Anti -metabolites resemble either nucleobases or nucleosides, but have altered chemical groups. These drugs exert their effect by either blocking the enzymes required for DNA synthesis or becoming incorporated into DNA or RNA. Subtypes of the anti-metabolites are the anti-folates, fluoropyrimidines, deoxynucleoside analogues, and thiopurines. The anti-folates include methotrexate and pemetrexed. The fluoropyrimidines include fluorouracil and capecitabine. The deoxynucleoside analogues include cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, and pentostatin. The thiopurines include thioguanine and mercaptopurine.

Anti -microtubule agents block cell division by preventing microtubule function. The vinca alkaloids prevent the formation of the microtubules, whereas the taxanes prevent the microtubule disassembly. Vinca alkaloids include vinorelbine, vindesine, and vinflunine. Taxanes include docetaxel (Taxotere) and paclitaxel (Taxol). Topoisomerase inhibitors are drugs that affect the activity of two enzymes: topoisomerase I and topoisomerase II and include irinotecan, topotecan, camptothecin, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin.

The cytotoxic antibiotics are a varied group of drugs that have various mechanisms of action. The common theme that they share in their chemotherapy indication is that they interrupt cell division. The most important subgroup is the anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, and aclarubicin) and the bleomycins; other prominent examples include mitomycin C, mitoxantrone, and actinomycin.

In some embodiments, a chemotherapeutic agent as described herein comprises a taxane such as docetaxel and/or paclitaxel, a folate antimetabolite such as pemetrexed, a deoxynucleoside analogue such as gemcitabine, a vinca alkaloid such as vinorelbine, a platinum compound such as cisplatin and/or carboplatin, or a combination thereof. In some embodiments, a chemotherapeutic agent as described herein comprises a taxane such as docetaxel and/or paditaxel, a folate antimetabolite such as pemetrexed, a platinum compound such as cisplatin and/or carboplatin, or a combination thereof.

Taxanes are a class of diterpene compounds that were first derived from natural sources such as plants of the genus Taxus, but some have been synthesized artificially. The principal mechanism of action of the taxane class of drugs is the disruption of microtubule function, thereby inhibiting the process of cell division. Taxanes indude docetaxel (Taxotere) and paditaxel (Taxol).

Folate antimetabolites (antifolates) are class of antimetabolites that antagonize the actions of folic acid (vitamin B9). Folie acid's primary function in the body is as a cofactor to various methyltransferases involved in serine, methionine, thymidine and purine biosynthesis. Consequently, antifolates inhibit cell division, DNA/RNA synthesis and repair and protein synthesis. The majority of antifolates work by inhibiting dihydrofolate reductase (DHFR). Pemetrexed is a folate antimetabolite which inhibits three enzymes used in purine and pyrimidine synthesis, thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). By inhibiting the formation of precursor purine and pyrimidine nucleotides, pemetrexed prevents the formation of DNA and RNA, which are required for the growth and survival of both normal cells and cancer cells. In some embodiments, the term “pemetrexed” refers to the compound N-[4-2-(2-Amino-4, 7-dihydro-4-oxo-lH-pyrrolo[2,3-d]pyrimidin-5- y l)ethy 1 ]b enzoy 1 ] -1 -glutami c aci d .

As used herein, the term “platinum compound” refers to compounds containing platinum in their structure such as platinum complexes. In some embodiments, this term refers to such compounds as used in platinum-based chemotherapy. In some embodiments, this term includes compounds such as cisplatin, carboplatin, and oxaliplatin. In some embodiments, a platinum compound is cisplatin and/or carboplatin.

In some embodiments, the term “oxaliplatin” refers to the compound [(1R,2R)- cyclohexane-l,2-diamine](ethanedioato-O,O')platinum(II). Oxaliplatin for injection is also marketed under the trade name Eloxatine.

In some embodiments, the chemotherapeutic agent comprises docetaxel. In these embodiments, the lung cancer may be second line or higher non-small-cell lung cancer (NSCLC). In some embodiments, the chemotherapeutic agent comprises docetaxel and is used in combination with ramucirumab. In these embodiments, the lung cancer may be of any histologic subtype. In some embodiments, the chemotherapeutic agent comprises docetaxel and is used in combination with nintedanib. In these embodiments, the lung cancer may be an adenocarcinoma. In some embodiments, the chemotherapeutic agent comprises paclitaxel. In some embodiments, the chemotherapeutic agent comprises paclitaxel and is used in combination with a platinum compound such as cisplatin and/or carboplatin. In some embodiments, the chemotherapeutic agent comprises pemetrexed. In some embodiments, the chemotherapeutic agent comprises pemetrexed and is used in combination with a platinum compound such as cisplatin and/or carboplatin. In some embodiments, the chemotherapeutic agent comprises cisplatin. In some embodiments, the chemotherapeutic agent comprises carboplatin. In some embodiments, the chemotherapeutic agent comprises a combination of paclitaxel and cisplatin and/or carboplatin (e.g., a combination of paclitaxel and cisplatin, a combination of paclitaxel and carboplatin, or a combination of paclitaxel, cisplatin, and carboplatin). In these embodiments, the lung cancer may be squamous carcinoma. In some embodiments, the chemotherapeutic agent comprises a combination of pemetrexed and cisplatin and/or carboplatin (e.g., a combination of pemetrexed and cisplatin, a combination of pemetrexed and carboplatin, or a combination of pemetrexed, cisplatin, and carboplatin). In these embodiments, the lung cancer may be non-squamous carcinoma.

Ramucirumab (LY3009806, IMC-1121B, trade name Cyramza) is a fully human monoclonal antibody (IgGl) developed for the treatment of solid tumors. Ramucirumab is a direct VEGFR2 antagonist, that binds with high affinity to the extracellular domain of VEGFR2 and blocks the binding of natural VEGFR ligands (VEGF-A, VEGF-C and VEGF-D). Binding of ramucirumab to VEGFR2 leads to inhibition of VEGF -mediated tumor angiogenesis.

Nintedanib, sold under the brand names Ofev and Vargatef, is an oral medication used for the treatment of idiopathic pulmonary fibrosis and along with other medications for some types of non-small-cell lung cancer. Nintedanib competitively inhibits both nonreceptor tyrosine kinases (nRTKs) and receptor tyrosine kinases (RTKs). nRTK targets of nintedanib include Lek, Lyn, and Src. RTK targets of nintedanib include platelet-derived growth factor receptor (PDGFR) a and 13; fibroblast growth factor receptor (FGFR) 1, 2, and 3; vascular endothelial growth factor receptor (VEGFR) 1, 2, and 3; and FLT3.

In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional immunomodulatory agent are administered separately. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and the optional additional immunomodulatory agent are administered separately. In some preferred embodiments, the RNA cancer vaccine and the anti-CTLA4 antibody are administered separately. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional immunomodulatory agent are administered concurrently or consecutively. In some embodiments, the RNA cancer vaccine and the anti-CTLA4 antibody are administered concurrently, for example by simultaneous (same day) administration. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and the optional additional immunomodulatory agent are administered concurrently. In some embodiments, the RNA cancer vaccine and the anti-CTLA4 antibody are administered consecutively, for example by administration on consecutive days. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and the optional additional immunomodulatory agent are administered consecutively.

In some embodiments, the RNAs of the anti-cancer vaccine encoding the TAA amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) are administered separately. Meaning, each RNA can be administered using a separate composition. In some embodiments, the separate RNA compositions of the anti-cancer vaccine encoding the TAA amino acid sequences (i), (ii), (iii), (iv), (v), and (vi) are administered concurrently or consecutively. In some embodiments, the RNAs of the anti-cancer vaccine encoding the TAA amino acid sequences (i), (ii), (iii), (iv), (v), and (vi) are administered concurrently to the subject, for example by simultaneous administration, i.e., administration via a single composition comprising all RNAs of the anti-cancer vaccine. In some embodiments, the RNAs of the anti -cancer vaccine encoding the TAA amino acid sequences (i), (ii), (iii), (iv), (v), and (vi) are administered consecutively, for example by sequential administration of the individual RNAs of the anti-cancer vaccine. The term “sequential administration” as used herein, relates to separate administration of the RNAs of the anti-cancer vaccine to the subject, wherein the individual RNAs are administered essentially at the same time. Essentially at the same time” as used herein with respect to sequential administration means within about 10 seconds, 15 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 1 hour period of each other. Sequential administration as used herein does not mean administration on separate days. In some embodiments, six RNAs of the anti-cancer vaccine, each encoding one of the TAA amino acid sequences (i), (ii), (iii), (iv), (v), and (vi), are administered concurrently.

In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional chemotherapeutic agent are administered separately. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and the optional additional chemotherapeutic agent are administered separately.

In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional chemotherapeutic agent are administered concurrently or consecutively. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and the optional additional chemotherapeutic agent are administered concurrently. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and the optional additional chemotherapeutic agent are administered consecutively, for example by administration on consecutive days.

The RNA cancer vaccine, the anti-CTLA4 antibody, the optional additional immunomodulatory agent, and/or the optional additional chemotherapeutic agent provided herein can be administered via any suitable enteral route or parenteral route of administration. The term “enteral route” of administration refers to the administration via any part of the gastrointestinal tract. Examples of enteral routes include oral, mucosal, buccal, and rectal route, or intragastric route. “Parenteral route” of administration refers to a route of administration other than enteral route. Examples of parenteral routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, intratumour, intravesical, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, transtracheal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal, subcutaneous, or topical administration.

The RNA cancer vaccine, the anti-CTLA4 antibody, the optional additional immunomodulatory agent, and/or the optional additional chemotherapeutic agent of the disclosure can be administered using any suitable method, such as by oral ingestion, nasogastric tube, gastrostomy tube, injection, infusion, implantable infusion pump, and osmotic pump. The suitable route and method of administration may vary depending on a number of factors such as the specific therapeutic agent being used, the rate of absorption desired, specific formulation or dosage form used, type or severity of the disorder being treated, the specific site of action, and conditions of the subject, and can be readily selected by a person skilled in the art. Administration can be systemic or local.

In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional immunomodulatory agent may be administered intravenously, intraarterially, subcutaneously, intradermally, dermally, intranodally, or intramuscularly. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional immunomodulatory agent may be administered intramuscularly. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional immunomodulatory agent are formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional immunomodulatory agent are formulated for systemic administration. In some embodiments, the systemic administration is by intravenous administration. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional immunomodulatory agent are formulated for intramuscular administration. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional immunomodulatory agent are formulated for intravenous administration.

In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional immunomodulatory agent are administered intravenously. In some preferred embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional immunomodulatory agent are administered via an intravenous injection or an intravenous infusion. In some more preferred embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional immunomodulatory agent are administered via an intravenous infusion. For example, the RNA cancer vaccine, the anti- CTLA4 antibody, and the optional additional immunomodulatory agent are preferably administered via an intravenous infusion. For example, the RNA cancer vaccine and the anti-CTLA4 antibody are preferably administered via an intravenous infusion.

In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional chemotherapeutic agent may be administered intravenously, intraarterially, subcutaneously, intradermally, dermally, intranodally, or intramuscularly. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional chemotherapeutic agent may be administered intramuscularly. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional chemotherapeutic agent are formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional chemotherapeutic agent are formulated for systemic administration. In some embodiments, the systemic administration is by intravenous administration. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional chemotherapeutic agent are formulated for intramuscular administration. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional chemotherapeutic agent are formulated for intravenous administration.

In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional chemotherapeutic agent are administered intravenously. In some preferred embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional chemotherapeutic agent are administered via an intravenous injection or an intravenous infusion. In some more preferred embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional chemotherapeutic agent are administered via an intravenous infusion. For example, the RNA cancer vaccine, the anti- CTLA4 antibody, and the optional additional chemotherapeutic agent are preferably administered via an intravenous infusion. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, the optional additional immunomodulatory agent, and/or the optional additional chemotherapeutic agent are administered in a therapeutically effective amount. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional immunomodulatory agent are administered in a therapeutically effective amount. For example, the RNA cancer vaccine and the anti-CTLA4 antibody are administered in a therapeutically effective amount. For example, the RNA cancer vaccine, the anti-CTLA4 antibody, and the optional additional immunomodulatory agent are administered in a therapeutically effective amount. For example, the RNA cancer vaccine, the anti-CTLA4 antibody, the optional additional immunomodulatory agent, and the optional additional chemotherapeutic agent are administered in a therapeutically effective amount.

In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, the optional additional immunomodulatory agent, and/or the optional additional chemotherapeutic agent are formulated with one or more pharmaceutically acceptable carriers, diluents and/or excipients. In some embodiments, the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional immunomodulatory agent are formulated with one or more pharmaceutically acceptable carriers, diluents and/or excipients.

Suitable carriers include, for example, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxy ethylene/polyoxy-propylene copolymers. For parenteral administration suitable carriers are preferably isotonic to the blood of the recipient. Non-limiting examples of suitable diluents include ethanol, glycerol, and water. Examples of excipients, include without limitation carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.

For example, the RNA cancer vaccine and the anti-CTLA4 antibody are formulated with one or more pharmaceutically acceptable carriers, diluents and excipients. For example, the RNA cancer vaccine, the anti-CTLA4 antibody, and the optional additional immunomodulatory agent are formulated with one or more pharmaceutically acceptable carriers, diluents and excipients. For example, the RNA cancer vaccine, the anti-CTLA4 antibody, the optional additional immunomodulatory agent, and the optional additional chemotherapeutic agent are formulated with one or more pharmaceutically acceptable carriers, diluents and excipients.

In some embodiments, the cancer to be treated by the combination of the present disclosure comprising the RNA cancer vaccine as disclosed herein and the anti-CTLA4 antibody as disclosed herein comprises one or more solid tumors.

Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularly, examples of such cancers include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, carcinoma of the sexual and reproductive organs, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumors, glioma, meningioma, and pituitary adenoma.

In some embodiments, the cancer to be treated by the combination of the RNA cancer vaccine as disclosed herein and the anti-CTLA4 antibody as disclosed herein, is selected from the group consisting of melanoma, lung cancer, human papillomavirus (HPV)-induced cancer, breast cancer, hepatocellular carcinoma, ovarian cancer such as ovarian carcinoma, prostate cancer such as prostate carcinoma, Hodgkin's or non-Hodgkin's lymphoma, acute myelogenic Leukemia, chronic myelogenic leukemia, acute lymphocytic Leukemia, chronic lymphocytic Leukemia, or renal cell carcinoma. In some embodiments, the HPV-induced cancer is selected from the group consisting of anal cancer, cervical cancer, oropharyngeal cancer, penile cancer, vaginal cancer, and vulvar cancer. In some preferred embodiments, the cancer is melanoma, ovarian cancer, or lung cancer. In some preferred embodiments, the cancer is melanoma. In some preferred embodiments, the cancer is ovarian cancer. In some preferred embodiments, the cancer is lung cancer. In some preferred embodiments, the cancer is triple negative breast cancer (TNBC).

In some more preferred embodiments, the cancer is non-small cell lung cancer (NSCLC). In some more preferred embodiments, the NSCLC has a squamous histology. In some more preferred embodiments, the NSLCL has a non-squamous histology.

In some embodiments, the cancer to be treated by the combination of the RNA cancer vaccine as disclosed herein and the anti-CTLA4 antibody as disclosed herein, expresses at least one of the following amino acid sequences:

(iii) an amino acid sequence comprising Melanoma antigen A3 (MAGE-A3), an immunogenic variant thereof, or an immunogenic fragment of the MAGE- A3 or the immunogenic variant thereof;

(v) an amino acid sequence comprising Preferentially Expressed Antigen In Melanoma (PRAME), an immunogenic variant thereof, or an immunogenic fragment of the PRAME or the immunogenic variant thereof; and (vi) an amino acid sequence comprising Melanoma antigen Cl (MAGE-CI), an immunogenic variant thereof, or an immunogenic fragment of the MAGE-CI or the immunogenic variant thereof.

The cancer to be treated or the TAAs of the cancer to be treated by the combination of the RNA cancer vaccine as disclosed herein and the anti-CTLA4 antibody as disclosed herein can be identified as described in the following. In an initial target discovery approach, RNA sequencing data of cancerous and healthy tissues can be explored in order to select for the most frequently and tumor-specifically expressed target genes. These targets can preferably be expressed in a significant number of tumors, weakly expressed or absent in essential organs like brain and heart, and lower expressed compared to tumors or absent in other human tissues except of reproductive or gynecological tissues. Selection and filtering of genes based on above-mentioned criteria aims at enlarging the probability that the target can induce immunogenicity (not recognized as self-antigen) with a limited toxicity (not represented in essential organs). Targets may be evaluated for the main cancer subtypes of e.g., non-small cell lung cancer, lung adenocarcinoma and squamous cell carcinoma, and finally selected to address disease subtypes.

All in silico analyses may be performed using publicly available (GTEx, Genotype-Tissue Expression project (Nature Genetics 2013, 45: 580-585) and TCGA, The Cancer Genome Atlas (Nature 2012, 489: 519-525); Campbell et al., Nature Genetics 2016, 48: 607-616; Nature 2014, 511 : 543-550) and proprietary RNA-Seq gene expression data. RNA reads can be aligned to the hgl9 reference genome and transcriptome, and gene expression can be determined by comparison with UCSC known genes transcript and exon coordinates, followed by normalization to RPKM units (Mortazavi et al., Nature Methods 2008, 5: 621- 628; Langmead et al., Genome Biology 2009, 10: R25). Targets can be selected by comparing expression in tumor and normal tissues, and to achieve a high coverage across the tumor cohorts. Target-expressing tumors can be defined by expression value > 1 rpkm. For qRT-PCR analysis using the Fluidigm Biomark™ Platform fresh frozen primary lung cancer tissue samples may be used (e.g., 164 primary lung cancer tissue samples). For example, 91 fresh frozen normal tissue samples from 43 different tissue types can be used in total for qRT-PCR analysis. RNA can be isolated from tissues using the Qiagen RNeasy Lipid Tissue Mini Kit according to the manufacturer's instructions. RNA can be converted to cDNA by first strand cDNA synthesis using the TAKARA - PrimeScript™ RT Reagent Kit with gDNA Eraser according to the manufacturer's instructions. qRT-PCR analysis using the Fluidigm detection system may be performed according to the manufacturer's instructions. After normalization to housekeeping genes such as HPRT1, HMBS, and TBP, relative RNA expression can be quantified using AACt calculation. For example, a calibrator of 18.2 corresponding to 30 (maximal number of cycles used in the PCR) minus the mean of the HPRT1 housekeeping gene value of the normal tissue samples can be used in this analysis. Suitable primers for this analysis are listed in Table 4. Technical replicates, including different cDNA syntheses can be summarized by using the median expression values. Relative expression of the gene of interest in normal tissue reveals the median expression value, if more than one tissue sample of the same tissue type is analyzed. Target-expressing tumors can be defined by specific cutoffs dependent on the expression intensities in critical normal tissues (Table 4). Normalized expression values are given in arbitrary units (a.u.).

Table 4: Oligonucleotides suitable for qRT-PCR analysis

* Primers for MAGEA3 also detect MAGEA5 and MAGEA6 transcripts because of a very high sequence homology.

New York esophageal squamous cell carcinoma-1 (NY-ESO-1), also referred to as cancer/testis antigen 1, LAGE2 or LAGE2B is a protein that in humans is encoded by the CTAG1B gene. CTAG1B is located on the long arm of chromosome X (Xq28). The gene encodes a 180-amino acid polypeptide, expressed from 18 weeks during embryonic development until birth in human fetal testis. It is also strongly expressed in spermatogonia and in primary spermatocytes of adult testis, but not in post-meiotic cells or testicular somatic cells. NY-ESO-1 belongs to the family of Cancer Testis Antigens (CTA) that are expressed in a variety of malignant tumors at the mRNA and protein levels, but also restricted to testicular germ cells in normal adult tissues.

Expression analysis of NSCLC target genes in tumor and normal Tissue may be performed using public and in-house generated RNA-Seq gene expression data from normal tissue samples and non-small-cell lung carcinoma (NSCLC) samples including lung adenocarcinoma (LUAD) and squamous cell lung carcinoma (LUSC) samples to generate expression heatmaps. For example, RNA-Seq gene expression data from 3809 normal tissue samples and 881 NSCLC samples including 466 LUAD and 415 LUSC samples may be used.

In order to calculate the percentage of cancer patients that can be potentially addressed by a vaccine approach, tumor percentage can be calculated for individual targets, as well as for cumulative coverage in target combinations.

Expression analysis of cancer target genes in tumor and normal tissue can be performed using qRT-PCR data. In order to confirm the expression of targets in cancer and normal tissue using an independent method and patient cohort, qRT-PCR analyses may be performed using the Fluidigm Biomark™ platform. RNA expression intensities of cancer and normal tissue can be used to generate expression heatmaps. For example, RNA expression intensities of 164 NSCLC and other lung tumors, and 43 normal tissue sites may be used to generate expression heatmaps.

In order to calculate the percentage of cancer patients that can be potentially addressed by a vaccine approach tumor percentage can be calculated for individual targets, as well as for cumulative coverage in target combinations.

Composition or kit of parts

The invention further provides a composition or kit of parts comprising: a) an RNA cancer vaccine comprising at least one RNA; and b) an anti-CTLA4 antibody.

The embodiments disclosed herein for the method of treatment comprising the RNA cancer vaccine in combination with the anti-CTLA4 antibody can be used in the composition or kit of parts as disclosed herein. Features described herein in more detail in connection with the “the RNA cancer vaccine for use in a method of treating, the anti-CTLA4 antibody for use in a method of treating, or the RNA cancer vaccine and the anti-CTLA4 antibody for use in a method of treating” embodiments equally apply to the corresponding composition or kit of parts embodiments.

In some embodiments, the composition or kit of parts further comprises an additional immunomodulatory agent.

In some embodiments, the composition or kit of parts further comprises an additional chemotherapeutic agent.

In some embodiments, the composition or kit of parts comprises a therapeutically effective amount of the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional immunomodulatory agent. In some embodiments, the composition or kit of parts comprises a therapeutically effective amount of the RNA cancer vaccine, the anti-CTLA4 antibody, the optional additional immunomodulatory agent, and/or the optional additional chemotherapeutic agent.

In some embodiments, the composition or kit of parts comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.

In some embodiments, the composition or kit of parts comprises one or more containers comprising the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional immunomodulatory agent. In some embodiments, the kit of parts comprises one or more containers comprising the RNA cancer vaccine, the anti-CTLA4 antibody, the optional additional immunomodulatory agent, and/or the optional additional chemotherapeutic agent.

In some embodiments, the kit of parts comprises individual containers each individually comprising the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional immunomodulatory agent. In some embodiments, the kit of parts comprises individual containers each individually comprising the RNA cancer vaccine, the anti- CTLA4 antibody, the optional additional immunomodulatory agent, and/or the optional additional chemotherapeutic agent.

In some embodiments, the kit of parts comprises individual containers comprising combinations of the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the optional additional immunomodulatory agent. In some embodiments, the kit of parts comprises individual containers comprising combinations of the RNA cancer vaccine, the anti-CTLA4 antibody, the optional additional immunomodulatory agent, and/or the optional additional chemotherapeutic agent.

In some embodiments, the kit of parts comprises individual containers each individually comprising RNA encoding one of the TAA amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) or comprising an RNA-LPX formulation comprising RNA encoding one of the TAA aminos acid sequences (i), (ii), (iii), (iv), (v), or (vi).

In some embodiments, the kit of parts comprises individual containers each individually comprising an RNA-LNP formulation comprising RNA encoding one of the TAA aminos acid sequences (i), (ii), (iii), (iv), (v), or (vi).

The embodiments disclosed herein for the method of treatment comprising the RNA cancer vaccine in combination with the anti-CTLA4 antibody can be used in the combination as disclosed herein. Features described herein in more detail in connection with the “the RNA cancer vaccine for use in a method of treating, the anti-CTLA4 antibody for use in a method of treating, or the RNA cancer vaccine and the anti-CTLA4 antibody for use in a method of treating” embodiments equally apply to the corresponding combination embodiments. Method of treatment

The embodiments disclosed herein for the method of treatment comprising the RNA cancer vaccine in combination with the anti-CTLA4 antibody can be used in the method of treating cancer in a subject in need thereof as disclosed herein. Features described herein in more detail in connection with the “the RNA cancer vaccine for use in a method of treating, the anti-CTLA4 antibody for use in a method of treating, or the RNA cancer vaccine and the anti-CTLA4 antibody for use in a method of treating” embodiments equally apply to the corresponding method of treating embodiments disclosed herein.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing file is named 240169WO_Sequence Listing.xml and 132 KB in size.

SEQ ID NOs: 1 and 2 are exemplary amino acid sequences of the CLDN6 TAA. SEQ ID NO: 2 comprises a P2P16 sequence.

SEQ ID NOs: 3 and 4 are exemplary polynucleotide sequences of the CLDN6 TAA. SEQ ID NO: 4 comprises 5' UTR, 3' UTR, Poly A, and P2P 16 sequences.

SEQ ID NOs: 5 and 6 are exemplary amino acid sequences of the KK-LC-1 TAA. SEQ ID NO: 6 comprises a P2P16 sequence. SEQ ID NOs: 7 and 8 are exemplary polynucleotide sequences of the KK-LC-1 TAA. SEQ ID NO: 8 comprises 5' UTR, 3' UTR, Poly A, and P2P 16 sequences.

SEQ ID NOs: 9 and 10 are exemplary amino acid sequences of the MAGE-A3 TAA. SEQ ID NO: 10 comprises P2P16, MITD, and secretory signal peptide sequences.

SEQ ID NOs: 11 and 12 are exemplary polynucleotide sequences of the MAGE-A3 TAA. SEQ ID NO: 12 comprises 5' UTR, 3' UTR, Poly A, P2P16, MITD, and secretory signal peptide sequences.

SEQ ID NOs: 13 and 14 are exemplary amino acid sequences of the MAGE-A4 TAA. SEQ ID NO: 14 comprises P2P16 and MITD sequences.

SEQ ID NOs: 15 and 16 are exemplary polynucleotide sequences of the MAGE-A4 TAA. SEQ ID NO: 16 comprises 5' UTR, 3' UTR, Poly A, P2P16, and MITD sequences.

SEQ ID NOs: 17 and 18 are exemplary amino acid sequences of the PRAME TAA. SEQ ID NO: 18 comprises P2P16, MITD, and secretory signal peptide sequences.

SEQ ID NOs: 19 and 20 are exemplary polynucleotide sequences of the PRAME TAA. SEQ ID NO: 20 comprises 5' UTR, 3' UTR, Poly A, P2P16, MITD, and secretory signal peptide sequences.

SEQ ID NOs: 21 and 22 are exemplary amino acid sequences of the MAGE-CI TAA. SEQ ID NO: 22 comprises P2P16, MITD, and secretory signal peptide sequences.

SEQ ID NOs: 23 and 24 are exemplary polynucleotide sequences of the MAGE-CI TAA. SEQ ID NO: 24 comprises 5' UTR, 3' UTR, Poly A, P2P16, MITD, and secretory signal peptide sequences.

SEQ ID NO: 25 is an exemplary amino acid sequence of the tetanus toxoid-derived P2P16 sequence.

SEQ ID NO: 26 is an exemplary polynucleotide sequence of the tetanus toxoid-derived P2P 16 sequence. SEQ ID NO: 27 is an exemplary amino acid sequence of the tetanus toxoid epitope P2.

SEQ ID NO: 28 is an exemplary amino acid sequence of the tetanus toxoid epitope P16.

SEQ ID NO: 29 is an exemplary 5' UTR polynucleotide sequence.

SEQ ID NO: 30 is an exemplary amino acid sequence of the MHC class I trafficking domain (MITD) sequence.

SEQ ID NO: 31 is an exemplary polynucleotide sequence of the MHC class I trafficking domain (MITD) sequence.

SEQ ID NO: 32 is an exemplary 3' UTR polynucleotide sequence.

SEQ ID NO: 33 is an exemplary poly-A polynucleotide sequence.

SEQ ID NO: 34 is an exemplary amino acid sequence of the secretory signal peptide sequence.

SEQ ID NO: 35 is an exemplary polynucleotide sequence of the secretory signal peptide sequence.

SEQ ID NOs: 36-38 are exemplary amino acid sequences of polypeptide linkers.

SEQ ID NOs: 39-48 are exemplary amino acid sequences of heavy and light chain CDR1-3 sequences of the anti-CTLA4 antibody.

SEQ ID NOs: 49-54 are exemplary amino acid sequences of heavy and light chain variable domains of the anti CTLA4 antibody.

SEQ ID NO: 55 is an exemplary amino acid sequence of an unmutated Fc region suitable for the anti-CTLA4 antibody.

SEQ ID NO: 56 is an exemplary amino acid sequence of a mutated Fc region suitable for the anti-CTLA4 antibody. SEQ ID NOs: 57, 59, 61, 63, 65, and 67 are exemplary amino acid sequences of the full- length heavy and light chains of the anti-CTLA4 antibody.

SEQ ID NOs: 58, 60, 62, 64, 66, and 68 are exemplary polynucleotide sequences of the full-length heavy and light chains of the anti-CTLA4 antibody.

SEQ ID NOs: 69-88 are exemplary polynucleotide sequences of primers suitable for the qRT-PCR analysis of specific TAAs.

EXAMPLES

Example 1 : Design and preparation of MC38-RNA cancer vaccine and anti-CTLA4 antibody

Test items were liposomally formulated RNA (RNA-lipoplex [RNA-LPX]) cancer vaccines, designed to be administered intravenously (i.v.) and to target the RNA-encoded antigen specifically to resident dendritic cells (DCs) within lymphoid organs. These DCs translate the encoded antigen and present antigen-derived epitopes on MHC molecules for T cell priming.

In vitro transcription of vaccine RNA constructs was based on DNA plasmids. These plasmids encode a T7 promoter, a 5 '-untranslated region (UTR), a coding region, a 3’ UTR and a poly(A) tail. The coding region comprises the vaccine antigen(s) or epitope(s), which are flanked by a secretion signal for routing to the endoplasmic reticulum, and the transmembrane domain derived from mouse MHC class I (MITD), based on the human sequence described by Kreiter et al., for improved presentation of MHC class I and II epitopes (Kreiter et al., J Immunol 2008, 180: 309-318). RNAs were generated by in vitro transcription as described (Kreiter et al., Cancer Immunol Immunother 2007, 56: 1577— 1587), and capped with a P-S-ARCA cap (Kuhn et al., Gene Ther 2010, 17: 961-971).

The 5 - UTR is a derivative of the 5 -UTR of homo sapiens hemoglobin subunit alpha 1 (hAg), the 3’-UTR is the FI element (where F is a 136 nucleotide long 3'-UTR fragment of amino-terminal enhancer of split mRNA and I is a 142 nucleotide long fragment of mitochondrially encoded 12S RNA both identified in Homo sapiens; WO 2017/060314), and the poly(A) tail consists of 50 nucleotides, which is elongated by PCR to 120 nucleotides before in vitro transcription.

Two vaccine RNA constructs were used for the MC38-RNA cancer vaccine, each coding for 10 MC38-derived neoepitopes (decatopes), where each neoepitope consisted of 27 amino acids with the mutated amino acid in the center (position 14). Neoepitope sequences contained the following published neoepitopes: Irgq, Repsl, Adpgk, Aatf, Dpagtl, Cpnel, Medl2, Ccdc96, Atg9a, Actrlb, Car7, Rpll8, Zbtb40, Spirel, Hnmpl, N4bp212, Fam46b, and Mttp (Yadav et al., Nature 2014, 515: 572-576; Capietto et al., J Exp Med 2020, 217; Pollock et al., Mol Cell Proteomics 2021, 20: 100108). Neoepitope sequences on a decatope were separated by lOmer non-immunogenic glycine/ serine linkers (Kreiter et al., Nature 2015, 520: 692-696).

Non-antigen coding RNA (vaccine control) contains a 5’-UTR and a 3’-UTR as described above, a poly(A) tail of 100 nucleotides with a linker after 30 nucleotides, and a GS linker (GGSGGGGSGGGGSGGGGSGG; SEQ ID NO: 38) instead of the antigen coding sequence.

All RNAs were formulated with liposomes composed of DOTMA and DOPE to yield RNA-LPXs with a negative net charge (Kranz et al., Nature 2016, 534: 396-401).

In vitro transcription and formulation of all RNA constructs was carried out at BioNTech SE.

Another test item was a humanized monoclonal antibody against human CTLA-4, of human (h) IgGl isotype (denoted as PP4637 herein; Du et al., Cell Res. 2018, 28: 433- 447). An isotype control of hlgGl was used to control for the anti-CTLA4 antibody. Example 2: The anti-CTLA4 antibody improves the therapeutic activity of the MC38-RNA cancer vaccine

Checkpoint inhibitors have been used to block immune-regulatory mechanisms that usually prevent autoimmunity. The anti-CTLA4 antibody was designed to enhance the efficacy and selectivity of Treg depletion in the tumor microenvironment. RNA-LPX vaccines are designed to prime de novo or expand pre-existing tumor-specific T cells. It is assumed that the tumor-specific depletion of one of the major and oftentimes decisive drivers of T cell suppression, i.e., intratumoral Tregs, would greatly enhance the anti-tumor efficacy of RNA cancer vaccine-induced tumor-specific T cells.

The therapeutic activity of the MC38-RNA cancer vaccine, the anti-CTLA4 antibody and the combination of the MC38-RNA cancer vaccine with the anti-CTLA4 antibody was investigated in order to determine whether these two anti-cancer therapies would synergize and achieve superior antitumor activity.

C57BL/6 mice transgenic for human CTLA-4 (hCTLA-4tg; (n=13 per group) were inoculated subcutaneously (s.c.) with 5* 10⁵ MC38 colon carcinoma cells on day 0 and vaccinated intravenously (i.v.) four times weekly (day 9, 16, 23 and 30) with 40 pg of MC38-RNA cancer vaccine (vaccine) encoding 20 different neoepitopes inherent to MC38 tumor cells, distributed across two different RNA constructs encoding 10 epitopes each (20 pg per RNA construct). Mice were treated in addition with increasing doses of the anti- CTLA4 antibody (antibody) intraperitoneally (i.p.) concomitant to the vaccine but starting with the second cycle (day 16, 23 and 30; 100, 150, and 250 pg, respectively).

Monotherapy groups received either the MC38-RNA cancer vaccine or the anti-CTLA4 antibody. The control group received a non-antigen coding RNA cancer vaccine (vaccine control or MC38-RNA cancer vaccine control) and a hlgGl isotype control to control for the anti-CTLA4 antibody (antibody control or anti-CTLA4 antibody control). Anti-tumor activity was determined as tumor growth inhibition in the test groups compared to the control groups during an observation period of up to day 38 after tumor inoculation. Animals were attested complete responses when tumor sizes had decreased to 1 mm³ or less. The study design is depicted in Figure 1.

The control group did not exhibit any complete responses. Compared to the control group, vaccination with the MC38-RNA cancer vaccine clearly delayed tumor growth but was unable to achieve complete responses (Figure 2). Treatment with the anti-CTLA4 antibody delayed tumor growth as well, and resulted in two complete responses, decreasing the fraction of tumor-bearing mice to 85% so far. Combined treatment with the MC38-RNA cancer vaccine and the anti-CTLA4 antibody not only inhibited tumor growth much more efficiently, but also led to seven complete responses, resulting in a reduced fraction of tumor-bearing mice of only 46%. The combination of the anti-CTLA4 antibody and the MC38-RNA cancer vaccine provided a surprisingly strong synergistic effect that was clearly greater than the expected sum of tumor growth reduction of the monotherapies.

Example 3: Study design to assess the therapeutic activity of the RNA cancer vaccine in combination with the anti-CTLA4 antibody.

The therapeutic RNA cancer vaccine is based on the RNA-LPX platform as described herein. The RNA cancer vaccine comprises six different RNAs encoding the amino acid sequences of six human tumor-associated antigens (TAAs), i.e., CLDN6, KK-LC-1, MAGE- A3, MAGE-A4, PRAME, and MAGE-CI. The RNAs of the RNA cancer vaccine are based on six single-stranded, 5'-capped non-nucleoside-modified uridine-containing mRNAs produced by in vitro transcription from the corresponding DNA templates, each encoding one TAA. All RNAs were formulated with liposomes composed of DOTMA and DOPE to form RNA-LPXs with a negative net charge (Kranz et al., Nature 2016, 534: 396- 401). The RNA in each of the six RNA-LPXs is translated into the respective antigen protein upon entering APCs. Each RNA comprises the open reading frame encoding the TAA fused to CD4+ T helper cell epitopes derived from tetanus toxoid at the 3’ end and is flanked by sequence elements for improved vaccine antigen processing and presentation. The open reading frame is flanked by non-coding sequence elements at the 5’ and 3’ end. In order to investigate the combination of the RNA cancer vaccine and the anti-CTLA antibody, a mouse tumor model was designed and established that expresses all six human CLDN6, KK-LC-1, MAGE- A3, MAGE-A4, PRAME, and MAGE-CI antigens and enables the demonstration of therapeutic activity in a syngeneic setting (unlike, e.g., humanized, or immunocompromised mouse models). To this end, TC-1 mouse tumor cells were lentivirally transduced with human CLDN6, KK-LC-1, MAGE- A3, MAGE-A4, PRAME, and MAGE-CI antigens. Single clones were picked and antigen expression confirmed by digital droplet PCR and western blotting. Tumor growth of the selected TC-1 clone was confirmed in C57BL/6 mice, and the tumors were shown to respond to vaccination with a mixture of all six RNA-LPXs of the RNA cancer vaccine.

In the next step, the therapeutic effect of the combination of the RNA cancer vaccine of and the anti-CTLA4 antibody are investigated in hCTLA-4tg mice compared to either monotherapy. hCTLA-4tg mice are inoculated subcutaneously (s.c.) with TC-1 tumor cells expressing all six human CLDN6, KK-LC-1, MAGE- A3, MAGE-A4, PRAME, and MAGE-CI antigens on day 0 and vaccinated intravenously (i.v.) four times weekly (day x, x+7, x+14 and x+21) with the RNA cancer vaccine (vaccine). Mice are treated in addition with increasing doses of the anti-CTLA4 antibody (antibody) intraperitoneally (i.p.) concomitant to the vaccine, starting on the same day (day x, x+7, x+14 and x+21; concomitant) or starting with the second cycle (day x+7, x+14 and x+21; delayed). Monotherapy groups receive either the RNA cancer vaccine or the anti-CTLA4 antibody. The control group receives a non-coding RNA cancer vaccine (RNA cancer vaccine control or vaccine control) and an isotype to control for the anti-CTLA4 antibody (anti-CTLA4 antibody control or antibody control). Anti-tumor activity is determined as tumor growth inhibition in the test groups compared to the control groups. The study design is depicted in Figure 3. The combination of the anti- CTLA4 antibody and the RNA cancer vaccine shows a surprising synergistic effect that is greater than the expected sum of tumor growth reduction of the monotherapies. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 depicts the study design to assess the therapeutic activity of the MC38-RNA cancer vaccine, i.e., RNA cancer vaccine encoding 20 MC38-derived different neoepitopes (vaccine) in combination with the anti-CTLA4 antibody (antibody). hCTLA-4tg mice (n=13 per group) were inoculated subcutaneously with MC38 colon carcinoma cells on day 0 and vaccinated intravenously four times weekly (day 9, 16, 23 and 30) with 40 pg of MC38-RNA cancer vaccine encoding 20 different neoepitopes inherent to MC38 tumor cells, distributed across two different RNA constructs encoding 10 epitopes each (20 pg per RNA construct). Mice were treated in addition with increasing doses of the anti-CTLA4 antibody intraperitoneally (i.p.) concomitant to the vaccine but starting with the second cycle (day 16, 23 and 30; 100, 150, and 250 pg, respectively). Monotherapy groups received either the MC38-RNA cancer vaccine or the anti-CTLA4 antibody. The control group received a non-coding RNA cancer vaccine (MC38-RNA cancer vaccine control or vaccine control) and an isotype control (anti-CTLA4 antibody control or antibody control). Anti-tumor activity was determined as tumor growth inhibition in the test groups compared to the control groups during an observation period of up to day 38 after tumor inoculation.

Fig. 2 depicts the results of treatment of hCTLA-4tg mice with the combination comprising the MC38-RNA cancer vaccine and the anti-CTLA antibody. hCTLA-4tg mice were treated as described in Figure 1 and tumor growth was assessed over time, a, Individual tumor growth curves. Ratios represent the number of tumor-free mice (CR, complete responses) over the total number of mice per group, b, Mean tumor growth. Last observations were carried forward (LOCF) in each group as long as more than five mice in that group were still alive. Dotted lines indicate days of vaccination, c, Percent of tumor-bearing mice. The days after tumor inoculation on which a complete response was achieved marked an event. Animals were attested complete responses when tumor sizes had decreased to 1 mm³ or less (a, c).

Fig. 3 depicts the study design to assess the therapeutic activity of the RNA cancer vaccine (vaccine) of the present disclosure (i.e., RNA cancer vaccine comprising six different RNAs encoding the amino acid sequences of six human TAAs: CLDN6, KK-LC-1, MAGE- A3, MAGE-A4, PRAME, and MAGE-CI) in combination with the anti-CTLA4 antibody of the present disclosure (antibody). hCTLA-4tg mice are inoculated s.c. with TC- 1 tumor cells expressing all six human TAAs CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, PRAME, and MAGE-CI on day 0 and are vaccinated i.v. four times weekly (day x, x+7, x+14 and x+21) with the RNA cancer vaccine. Mice are treated in addition with increasing doses of the anti-CTLA4 antibody i.p. concomitant to the vaccine, starting on the same day (day x, x+7, x+14 and x+21; concomitant) or starting with the second cycle (day x+7, x+14 and x+21; delayed). Monotherapy groups receive either the RNA cancer vaccine or the anti-CTLA4 antibody. The control group receives a non-coding RNA cancer vaccine (RNA cancer vaccine control or vaccine control) and an isotype control to control for the anti- CTLA4 antibody (anti-CTLA4 antibody control or antibody control). Anti-tumor activity is determined as tumor growth inhibition in the test groups compared to the control groups.

Claims

3. The RNA cancer vaccine for use according to claim 1 or the anti-CTLA4 antibody for use according to claim 2, wherein the method comprises administering an additional immunomodulatory agent to the subject.

4. The RNA cancer vaccine for use according to claim 1 or 3 or the anti-CTLA4 antibody for use according to claim 2 or 3, wherein the RNA cancer vaccine, the anti- CTLA4 antibody, and/or the additional immunomodulatory agent are administered separately.

5. The RNA cancer vaccine for use according to claims 1, 3 or 4 or the anti-CTLA4 antibody for use according to claims 2-4, wherein the RNA cancer vaccine, the anti- CTLA4 antibody, and/or the additional immunomodulatory agent are administered concurrently or consecutively.

6. The RNA cancer vaccine for use according to claims 1 or 3-5 or the anti-CTLA4 antibody for use according to claims 2-5, wherein the RNA cancer vaccine, the anti- CTLA4 antibody, and/or the additional immunomodulatory agent are administered intravenously.

7. The RNA cancer vaccine for use according to claims 1 or 3-6 or the anti-CTLA4 antibody for use according to claims 2-6, wherein the RNA cancer vaccine, the anti- CTLA4 antibody, and/or the additional immunomodulatory agent are administered via an intravenous injection or an intravenous infusion, preferably an intravenous infusion.

8. The RNA cancer vaccine for use according to claims 1 or 3-7 or the anti-CTLA4 antibody for use according to claims 2-7, wherein the RNA cancer vaccine, the anti- CTLA4 antibody, and/or the additional immunomodulatory agent are administered in a therapeutically effective amount.

9. The RNA cancer vaccine for use according to claims 1 or 3-8 or the anti-CTLA4 antibody for use according to claims 2-8, wherein the RNA cancer vaccine, the anti- CTLA4 antibody, and/or the additional immunomodulatory agent are formulated with one or more pharmaceutically acceptable carriers, diluents and/or excipients.

10. The RNA cancer vaccine for use according to claims 1 or 3-9 or the anti-CTLA4 antibody for use according to claims 2-9, wherein the cancer comprises one or more solid tumors.

11. The RNA cancer vaccine for use according to claims 1 or 3-10 or the anti-CTLA4 antibody for use according to claims 2-10, wherein the cancer is selected from the group consisting of melanoma, lung cancer, human papillomavirus (HPV)-induced cancer, breast cancer, hepatocellular carcinoma, ovarian cancer such as ovarian carcinoma, prostate cancer such as prostate carcinoma, Hodgkin's or non-Hodgkin's lymphoma, acute myelogenic Leukemia, chronic myelogenic Leukemia, acute lymphocytic Leukemia, chronic lymphocytic Leukemia, or renal cell carcinoma.

12. The RNA cancer vaccine for use according to claims 1 or 3-11 or the anti-CTLA4 antibody for use according to claims 2-11, wherein the cancer is melanoma, ovarian cancer, or lung cancer.

13. The RNA cancer vaccine for use according to claims 1 or 3-12 or the anti-CTLA4 antibody for use according to claims 2-12, wherein the cancer is non-small cell lung cancer (NSCLC).

14. The RNA cancer vaccine for use according to claim 13 or the anti-CTLA4 antibody for use according to claim 13, wherein the NSCLC has a squamous histology.

15. The RNA cancer vaccine for use according to claim 13 or the anti-CTLA4 antibody for use according to claim 13, wherein the NSCLC has a non-squamous histology.

16. The RNA cancer vaccine for use according to claims 1 or 3-15 or the anti-CTLA4 antibody for use according to claims 2-15, wherein the at least one RNA encodes at least one of the following amino acid sequences:

(ii) an amino acid sequence comprising Kita-kyushu lung cancer antigen 1 (KK-LC- 1), an immunogenic variant thereof, or an immunogenic fragment of the KK-LC-1 or the immunogenic variant thereof;

17. The RNA cancer vaccine for use according to claim 16 or the anti-CTLA4 antibody for use according to claim 16, wherein a) the RNA encoding the amino acid sequence (i) comprises the nucleotide sequence of SEQ ID NO: 3 or 4, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 3 or 4; and/or b) the amino acid sequence (i) comprises the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 2.

18. The RNA cancer vaccine for use according to claim 16 or 17 or the anti-CTLA4 antibody for use according to claim 16 or 17, wherein a) the RNA encoding the amino acid sequence (ii) comprises the nucleotide sequence of SEQ ID NO: 7 or 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 7 or 8; and/or b) the amino acid sequence (ii) comprises the amino acid sequence of SEQ ID NO: 5 or 6, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5 or 6.

19. The RNA cancer vaccine for use according to claims 16-18 or the anti-CTLA4 antibody for use according to claims 16-18, wherein a) the RNA encoding the amino acid sequence (iii) comprises the nucleotide sequence of SEQ ID NO: 11 or 12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 11 or 12; and/or b) the amino acid sequence (iii) comprises the amino acid sequence of SEQ ID NO: 9 or 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 9 or 10.

20. The RNA cancer vaccine for use according to claims 16-19 or the anti-CTLA4 antibody for use according to claims 16-19, wherein a) the RNA encoding the amino acid sequence (iv) comprises the nucleotide sequence of SEQ ID NO: 15 or 16, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15 or 16; and/or b) the amino acid sequence (iv) comprises the amino acid sequence of SEQ ID NO: 13 or 14, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 13 or 14.

21. The RNA cancer vaccine for use according to claims 16-20 or the anti-CTLA4 antibody for use according to claims 16-20, wherein a) the RNA encoding the amino acid sequence (v) comprises the nucleotide sequence of SEQ ID NO: 19 or 20, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 19 or 20; and/or b) the amino acid sequence (v) comprises the amino acid sequence of SEQ ID NO: 17 or 18, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 17 or 18.

22. The RNA cancer vaccine for use according to claims 16-21 or the anti-CTLA4 antibody for use according to claims 16-21, wherein a) the RNA encoding the amino acid sequence (vi) comprises the nucleotide sequence of SEQ ID NO: 23 or 24, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 23 or 24; and/or b) the amino acid sequence (vi) comprises the amino acid sequence of SEQ ID NO: 21 or 22, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 21 or 22.

23. The RNA cancer vaccine for use according to claims 16-22 or the anti-CTLA4 antibody for use according to claims 16-22, wherein a) the RNA encoding the amino acid sequence (i) comprises the nucleotide sequence of SEQ ID NO: 4; b) the RNA encoding the amino acid sequence (ii) comprises the nucleotide sequence of SEQ ID NO: 8; c) the RNA encoding the amino acid sequence (iii) comprises the nucleotide sequence of SEQ ID NO: 12; d) the RNA encoding the amino acid sequence (iv) comprises the nucleotide sequence of SEQ ID NO: 16; e) the RNA encoding the amino acid sequence (v) comprises the nucleotide sequence of SEQ ID NO: 20; and f) the RNA encoding the amino acid sequence (vi) comprises the nucleotide sequence of SEQ ID NO: 24.

24. The RNA cancer vaccine for use according to claims 16-23 or the anti-CTLA4 antibody for use according to claims 16-23, wherein the RNA cancer vaccine comprises at least two RNAs, preferably encoding at least two of the amino acid sequences (i), (ii), (iii), (iv), (v), and (vi).

25. The RNA cancer vaccine for use according to claims 16-24 or the anti-CTLA4 antibody for use according to claims 16-24, wherein the RNA cancer vaccine comprises six RNAs, preferably each encoding one of the amino acid sequences (i), (ii), (iii), (iv), (v), and (vi).

26. The RNA cancer vaccine for use according to claims 16-25 or the anti-CTLA4 antibody for use according to claims 16-25, wherein at least one of the amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) comprises an amino acid sequence which breaks immunological tolerance and/or the at least one RNA is co-administered with RNA encoding an amino acid sequence which breaks immunological tolerance.

27. The RNA cancer vaccine for use according to claim 26 or the anti-CTLA4 antibody for use according to claim 26, wherein each amino acid sequence (i), (ii), (iii), (iv), (v), or (vi) comprises an amino acid sequence which breaks immunological tolerance and/or each RNA is co-administered with RNA encoding an amino acid sequence which breaks immunological tolerance.

28. The RNA cancer vaccine for use according to claim 26 or 27 or the anti-CTLA4 antibody for use according to claim 26 or 27, wherein the amino acid sequence which breaks immunological tolerance comprises helper epitopes, preferably tetanus toxoid- derived helper epitopes.

29. The RNA cancer vaccine for use according to claims 26-28 or the anti-CTLA4 antibody for use according to claims 26-28, wherein a) the RNA encoding the amino acid sequence which breaks immunological tolerance comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 26; and/or b) the amino acid sequence which breaks immunological tolerance comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 25.

30. The RNA cancer vaccine for use according to claims 16-29 or the anti-CTLA4 antibody for use according to claims 16-29, wherein at least one of the amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) 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, wherein the codon-optimization and/or the increase in the G/C content preferably does not change the sequence of the encoded amino acid sequence.

31. The RNA cancer vaccine for use according to claim 30 or the anti-CTLA4 antibody for use according to claim 30, wherein each of the amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) 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, wherein the codon-optimization and/or the increase in the G/C content preferably does not change the sequence of the encoded amino acid sequence.

32. The RNA cancer vaccine for use according to claims 1 or 3-31 or the anti-CTLA4 antibody for use according to claims 2-31, wherein the at least one RNA comprises a 5' cap, preferably selected from the group consisting of G[5’]ppp[5’]G, m7G[5’]ppp[5’]G, m₃ ²’²’⁷G[5’]ppp[5’]G, m₂ ⁷’³'-°G[5’]ppp[5’]G (3’-ARCA), m₂ ⁷’²'' °GpppG (2’-ARCA), m₂ ⁷’²'-° GppSpG, m₂ ⁷’²'-°GppSpG (p-S-ARCA), m₂ ⁷’²’-°GppSpG (P-S-ARCA), and m₂ ^{7 3} '°Gppp(m i² '°)ApG, most preferably m₂ ^7,2 '° GppSpG.

33. The RNA cancer vaccine for use according to claim 32 or the anti-CTLA4 antibody for use according to claim 32, wherein each of the RNAs comprises a 5’ cap, preferably selected from the group consisting of G[5’]ppp[5’]G, m7G[5’]ppp[5’]G, m₃ ²’²’⁷G[5’]ppp[5’]G, m₂ ⁷’³’-°G[5’]ppp[5’]G (3’-ARCA), m₂ ⁷’²’-°GpppG (2’-ARCA), m₂ ^7,2 -° GppSpG, m₂ ^7,2 '°GppSpG (p-S-ARCA), m₂ ⁷’²’-°GppSpG (p-S-ARCA), and m₂ ^{7 3} ''⁰Gppp(mi² '°)ApG, most preferably m₂ ⁷’² '⁰ GppSpG.

34. The RNA cancer vaccine for use according to claims 1 or 3-33 or the anti-CTLA4 antibody for use according to claims 2-33, wherein the at least one RNA comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 29.

35. The RNA cancer vaccine for use according to claim 34 or the anti-CTLA4 antibody for use according to claim 34, wherein each of the RNAs comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 29.

36. The RNA cancer vaccine for use according to claims 16-35 or the anti-CTLA4 antibody for use according to claims 16-35, wherein at least one of the amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) comprises an amino acid sequence enhancing antigen processing and/or presentation.

37. The RNA cancer vaccine for use according to claim 36 or the anti-CTLA4 antibody for use according to claim 36, wherein each of the amino acid sequences (i), (ii), (iii), (iv), (v), or (vi) comprises an amino acid sequence enhancing antigen processing and/or presentation.

38. The RNA cancer vaccine for use according to claim 36 or 37 or the anti-CTLA4 antibody for use according to claim 36 or 37, wherein the amino acid sequence enhancing antigen processing and/or presentation comprises an amino acid sequence corresponding to the transmembrane and cytoplasmic domain of an MHC molecule, preferably an MHC class I molecule.

39. The RNA cancer vaccine for use according to claims 36-38 or the anti-CTLA4 antibody for use according to claims 36-38, wherein a) the RNA encoding the amino acid sequence enhancing antigen processing and/or presentation comprises the nucleotide sequence of SEQ ID NO: 31, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 31; and/or b) the amino acid sequence enhancing antigen processing and/or presentation comprises the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 30.

40. The RNA cancer vaccine for use according to claims 1 or 3-39 or the anti-CTLA4 antibody for use according to claims 2-39, wherein the at least one RNA comprises a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 32, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 32.

41. The RNA cancer vaccine for use according to claim 40 or the anti-CTLA4 antibody for use according to claim 40, wherein each of the RNAs comprises a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 32, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 32.

42. The RNA cancer vaccine for use according to claims 1 or 3-41 or the anti-CTLA4 antibody for use according to claims 2-41, wherein the at least one RNA comprises a poly-A sequence.

43. The RNA cancer vaccine for use according to claim 42 or the anti-CTLA4 antibody for use according to claim 42, wherein each of the RNAs comprises a poly- A sequence.

44. The RNA cancer vaccine for use according to claim 42 or 43 or the anti-CTLA4 antibody for use according to claim 42 or 43, wherein the poly-A sequence comprises at least 100 nucleotides.

45. The RNA cancer vaccine for use according to claims 42-44 or the anti-CTLA4 antibody for use according to claims 42-44, wherein the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 33.

46. The RNA cancer vaccine for use according to claims 1 or 3-45 or the anti-CTLA4 antibody for use according to claims 2-45, wherein the RNA is formulated as a liquid, a solid, or a combination thereof.

47. The RNA cancer vaccine for use according to claims 1 or 3-46 or the anti-CTLA4 antibody for use according to claims 2-46, wherein the RNA is formulated for injection.

48. The RNA cancer vaccine for use according to claims 1 or 3-47 or the anti-CTLA4 antibody for use according to claims 2-47, wherein the RNA is formulated for intravenous administration.

49. The RNA cancer vaccine for use according to claims 1 or 3-48 or the anti-CTLA4 antibody for use according to claims 2-48, wherein the RNA is formulated or is to be formulated as lipoplex particles.

50. The RNA cancer vaccine for use according to claim 49 or the anti-CTLA4 antibody for use according to claim 49, wherein the RNA lipoplex particles are obtainable by mixing the RNA with liposomes.

51. The RNA cancer vaccine for use according to claims 16-50 or the anti-CTLA4 antibody for use according to claims 16-50, wherein the at least one RNA encoding an amino acid sequence (i), (ii), (iii), (iv), (v), or (vi) is co-formulated or is to be coformulated as lipoplex particles with RNA encoding an amino acid sequence which breaks immunological tolerance.

52. The RNA cancer vaccine for use according to claim 51 or the anti-CTLA4 antibody for use according to claim 51, wherein each of the RNAs encoding an amino acid sequence (i), (ii), (iii), (iv), (v), or (vi) is co-formulated or is to be co-formulated as lipoplex particles with RNA encoding an amino acid sequence which breaks immunological tolerance.

53. The RNA cancer vaccine for use according to claims 1 or 3-52 or the anti-CTLA4 antibody for use according to claims 2-52, wherein the anti-CTLA4 antibody comprises: a) a heavy chain variable region comprising (1) a complementarity-determining region 1 (HCDR1) whose amino acid sequence is set forth in SEQ ID NO: 39, (2) a complementarity-determining region 2 (HCDR2) whose amino acid sequence is set forth in SEQ ID NO: 41, 42 or 43, and (3) a complementarity-determining region 3 (HCDR3) whose amino acid sequence is set forth in SEQ ID NO: 47; and b) a light chain variable region comprising: (1) a complementarity-determining region 1 (LCDR1) whose amino acid sequence is set forth in SEQ ID NO: 40, (2) a complementarity-determining region 2 (LCDR2) whose amino acid sequence is set forth in SEQ ID NO: 44, 45, or 46, and (3) a complementarity-determining region 3 (LCDR3) whose amino acid sequence is set forth in SEQ ID NO: 48.

54. The RNA cancer vaccine for use according to claim 53 or the anti-CTLA4 antibody for use according to claim 53, wherein the anti-CTLA4 antibody comprises: a) a heavy chain variable region comprising (1) a HCDR1 whose amino acid sequence is set forth in SEQ ID NO: 39, (2) a HCDR2 whose amino acid sequence is set forth in SEQ ID NO: 41, and (3) a HCDR3 whose amino acid sequence is set forth in SEQ ID NO: 47; and b) a light chain variable region comprising: (1) a LCDR1 whose amino acid sequence is set forth in SEQ ID NO: 40, (2) a LCDR2 whose amino acid sequence is set forth in SEQ ID NO: 45, and (3) a LCDR3 whose amino acid sequence is set forth in SEQ ID NO: 48.

55. The RNA cancer vaccine for use according to claim 53 or the anti-CTLA4 antibody for use according to claim 53, wherein the anti-CTLA4 antibody comprises: a) a heavy chain variable region comprising (1) HCDR1 whose amino acid sequence is set forth in SEQ ID NO: 39, (2) a HCDR2 whose amino acid sequence is set forth in SEQ ID NO: 43, and (3) a HCDR3 whose amino acid sequence is set forth in SEQ ID NO: 47; and b) a light chain variable region comprising: (1) a LCDR1 whose amino acid sequence is set forth in SEQ ID NO: 40, (2) a LCDR2 whose amino acid sequence is set forth in SEQ ID NO: 45, and (3) a LCDR3 whose amino acid sequence is set forth in SEQ ID NO: 48.

56. The RNA cancer vaccine for use according to claim 53 or the anti-CTLA4 antibody for use according to claim 53, wherein the anti-CTLA4 antibody comprises: a) a heavy chain variable region comprising (1) a HCDR1 whose amino acid sequence is set forth in SEQ ID NO: 39, (2) a HCDR2 whose amino acid sequence is set forth in SEQ ID NO: 43, and (3) a HCDR3 whose amino acid sequence is set forth in SEQ ID NO: 47; and b) a light chain variable region comprising: (1) a LCDR1 whose amino acid sequence is set forth in SEQ ID NO: 40, (2) a LCDR2 whose amino acid sequence is set forth in SEQ ID NO: 46, and (3) a LCDR3 whose amino acid sequence is set forth in SEQ ID NO: 48.

57. The RNA cancer vaccine for use according to claims 53-56 or the anti-CTLA4 antibody for use according to claims 53-56, wherein the anti-CTLA4 antibody comprises: a) a heavy chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 49, 50, or 51; and b) a light chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 52, 43, or 54.

58. The RNA cancer vaccine for use according to claim 57 or the anti-CTLA4 antibody for use according to claim 57, wherein the anti-CTLA4 antibody comprises: a) a heavy chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 49; and b) a light chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 53.

59. The RNA cancer vaccine for use according to claim 57 or the anti-CTLA4 antibody for use according to claim 57, wherein the anti-CTLA4 antibody comprises: a) a heavy chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 51; and b) a light chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 53.

60. The RNA cancer vaccine for use according to claim 57 or the anti-CTLA4 antibody for use according to claim 57, wherein the anti-CTLA4 antibody comprises: a) a heavy chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 51; and b) a light chain variable domain whose amino acid sequence is set forth in SEQ ID NO: 54.

61. The RNA cancer vaccine for use claims 1, 3-60 or the anti-CTLA4 antibody for use according to claims 2-60, wherein the anti-CTLA4 antibody comprises a heavy chain comprising a Fc region of a human Ig antibody, preferably comprising the amino acid sequence of SEQ ID NO: 55 or 56.

62. The RNA cancer vaccine for use according to claims 53-61 or the anti-CTLA4 antibody for use according to claims 53-61, wherein the anti-CTLA4 antibody comprises: a) a heavy chain whose amino acid sequence is set forth in SEQ ID NO: 57, 59, or 61; and b) a light chain whose amino acid sequence is set forth in SEQ ID NO: 63, 65, or 67.

63. The RNA cancer vaccine for use according to claim 62 or the anti-CTLA4 antibody for use according to claim 62, wherein the anti-CTLA4 antibody comprises: a) a heavy chain whose amino acid sequence is set forth in SEQ ID NO: 57; and b) a light chain whose amino acid sequence is set forth in SEQ ID NO: 65.

64. The RNA cancer vaccine for use according to claim 62 or the anti-CTLA4 antibody for use according to claim 62, wherein the anti-CTLA4 antibody comprises: a) a heavy chain whose amino acid sequence is set forth in SEQ ID NO: 61; and b) a light chain whose amino acid sequence is set forth in SEQ ID NO: 65.

65. The RNA cancer vaccine for use according to claim 62 or the anti-CTLA4 antibody for use according to claim 62, wherein the anti-CTLA4 antibody comprises: a) a heavy chain whose amino acid sequence is set forth in SEQ ID NO: 61; and b) a light chain whose amino acid sequence is set forth in SEQ ID NO: 67.

66. The RNA cancer vaccine for use according to claims 1, 3-65 or the anti-CTLA4 antibody for use according to claims 2-65, wherein the anti-CTLA4 antibody is capable of binding to human CTLA4.

67. The RNA cancer vaccine for use according to claims 1, 3-66 or the anti-CTLA4 antibody for use according to claims 2-66, wherein the anti CTLA4 antibody is a humanized anti-CTLA4 antibody.

68. The RNA cancer vaccine for use according to claims 1, 3-60, 66-67 or the anti-CTLA4 antibody for use according to claims 2-60, 66-67, wherein the anti-CTLA4 antibody is an antigen-binding fragment or a variant thereof.

69. The RNA cancer vaccine for use according to claim 68 or the anti-CTLA4 antibody for use according to claim 68, wherein the antigen binding fragment or variant thereof is selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a scFV, a diabody, a triabody, a minibody, and a single-domain antibody (sdAB), and variants thereof.

70. The RNA cancer vaccine for use according to claims 1, 3-69 or the anti-CTLA4 antibody for use according to claims 2-69, wherein the anti-CTLA4 antibody is encoded by one or more RNAs.

71. An RNA cancer vaccine and an anti-CTLA4 antibody for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering to the subject: a) the RNA cancer vaccine comprising at least one RNA; and b) the anti-CTLA4 antibody.

72. A composition or kit of parts comprising: a) an RNA cancer vaccine comprising at least one RNA; and b) an anti-CTLA4 antibody.

73. The composition or kit of parts according to claim 72, further comprising an additional immunomodulatory agent.

74. The composition or kit of parts according to claim 72 or 73, comprising a therapeutically effective amount of the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the additional immunomodulatory agent.

75. The composition or kit of parts according to claims 72-74, comprising one or more pharmaceutically acceptable carriers, diluents and/or excipients.

76. The composition or kit of parts according to claims 72-75, comprising one or more containers comprising the RNA cancer vaccine, the anti-CTLA4 antibody, and/or the additional immunomodulatory agent.

77. The composition or kit of parts according to claims 72-76, wherein the kit comprises individual containers each individually comprising the RNA cancer vaccine, the anti- CTLA4 antibody, and optionally the additional immunomodulatory agent.

78. The composition or kit of parts according to claims 72-76, wherein the kit comprises individual containers comprising combinations of the RNA cancer vaccine, the anti- CTLA4 antibody, and optionally the additional immunomodulatory agent.