Therapeutic RNA for ovarian cancer

Abstract

To provide a medical preparation for treatment of ovarian cancers.SOLUTION: In one aspect, a composition or a medical preparation comprising at least one RNA is provided, wherein the at least one RNA encodes 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 p53, an immunogenic variant thereof, or an immunogenic fragment of the p53 or the immunogenic variant thereof; and (iii) 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.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to the field of therapeutic RNA for treating ovarian cancer.Ovarian cancer refers to the cancerous growth that starts from ovary.It is the fifth most common cause of cancer death in women, and the tenth most common cancer in women in the United States.Ovarian cancer is the most deadly of gynecological cancers that affect uterus, cervix and ovary. [Background technology]

[0002] Disclosed herein is the composition, use and method for treating ovarian cancer.Disclosed herein is the therapeutic RNA that is administered to patients with ovarian cancer can reduce tumor size, prolong the time to progressive disease, and / or prevent tumor metastasis and / or recurrence, and ultimately prolong survival. Summary of the Invention [Means for solving the problem]

[0003] In one aspect, provided herein is a composition or pharmaceutical formulation comprising at least one RNA, wherein the at least one RNA has the following amino acid sequence: (i) an amino acid sequence comprising claudin 6 (CLDN6), an immunogenic variant thereof, or an immunogenic fragment of CLDN6 or an immunogenic variant thereof; (ii) an amino acid sequence comprising p53, an immunogenic variant thereof, or an immunogenic fragment of p53 or an immunogenic variant thereof; and (iii) an amino acid sequence comprising a preferentially expressed melanoma antigen (PRAME), an immunogenic variant thereof, or an immunogenic fragment of PRAME or an immunogenic variant thereof; Code the following.

[0004] In one embodiment, each of the amino acid sequences of (i), (ii), or (iii) is encoded by a separate RNA.

[0005] In one embodiment, (i) the RNA encoding the amino acid sequence of (i) comprises the nucleotide sequence of SEQ ID NO: 2 or 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: 2 or 3; and / or (ii) The amino acid sequence of (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.

[0006] In one embodiment, (i) The RNA encoding the amino acid sequence of (ii) comprises the nucleotide sequence of SEQ ID NO: 6 or 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: 6 or 7; and / or (ii) The amino acid sequence of (ii) comprises the amino acid sequence of SEQ ID NO: 4 or 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: 4 or 5.

[0007] In one embodiment, (i) The RNA encoding the amino acid sequence of (iii) comprises the nucleotide sequence of SEQ ID NO: 10 or 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: 10 or 11; and / or The amino acid sequence of (ii)(iii) comprises the amino acid sequence of SEQ ID NO: 8 or 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: 8 or 9.

[0008] In one embodiment, at least one amino acid sequence of (i), (ii), or (iii) comprises an amino acid sequence that disrupts immune tolerance. In one embodiment, each amino acid sequence of (i), (ii), or (iii) comprises an amino acid sequence that disrupts immune tolerance.

[0009] In one embodiment, at least one RNA is co-administered with (iv) an RNA encoding an amino acid sequence that disrupts immune tolerance. In one embodiment, each RNA is co-administered with (iv) an RNA encoding an amino acid sequence that disrupts immune tolerance.

[0010] In one embodiment, the amino acid sequence that breaks immune tolerance comprises a helper epitope, preferably a helper epitope derived from tetanus toxoid.

[0011] In one embodiment, (i) the RNA encoding the immune tolerance-disrupting amino acid sequence comprises the nucleotide sequence of SEQ ID NO: 14 or 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: 14 or 15; and / or (ii) The amino acid sequence that disrupts immune tolerance comprises the amino acid sequence of SEQ ID NO: 12 or 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: 12 or 13.

[0012] In one embodiment, at least one of the amino acid sequences of (i), (ii), (iii), or (iv) is encoded by a coding sequence that has been codon-optimized and / or has an increased G / C content compared to a wild-type coding sequence, wherein the codon-optimization and / or increased G / C content preferably does not alter the sequence of the encoded amino acid sequence. In one embodiment, each of the amino acid sequences of (i), (ii), (iii), or (iv) is encoded by a coding sequence that has been codon-optimized and / or has an increased G / C content compared to a wild-type coding sequence, wherein the codon-optimization and / or increased G / C content preferably does not alter the sequence of the encoded amino acid sequence.

[0013] In one embodiment, at least one RNA is a modified RNA, particularly a stabilized mRNA. In one embodiment, at least one RNA contains a modified nucleoside in place of at least one uridine. In one embodiment, at least one RNA contains a modified nucleoside in place of each uridine. In one embodiment, each RNA contains a modified nucleoside in place of at least one uridine. In one embodiment, each RNA contains a modified nucleoside in place of each uridine. In one embodiment, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U).

[0014] In one embodiment, at least one RNA is 5' capped m2 7,2'-O Gpp s In one embodiment, each RNA comprises a 5' cap m2 7,2'-O Gpp s Contains p(5')G.

[0015] In one embodiment, at least one RNA comprises a 5'-UTR comprising 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. In one embodiment, each RNA comprises a 5'-UTR comprising 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.

[0016] In one embodiment, at least one of the amino acid sequences of (i), (ii), (iii), or (iv) comprises an amino acid sequence that enhances antigen processing and / or antigen presentation. In one embodiment, each of the amino acid sequences of (i), (ii), (iii), or (iv) comprises an amino acid sequence that enhances antigen processing and / or antigen presentation. In one embodiment, the amino acid sequence that enhances antigen processing and / or antigen presentation comprises an amino acid sequence corresponding to the transmembrane and cytoplasmic domains of an MHC molecule, preferably an MHC class I molecule.

[0017] In one embodiment, (i) the RNA encoding the amino acid sequence that enhances antigen processing and / or antigen presentation 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 (ii) The amino acid sequence that enhances antigen processing and / or antigen presentation comprises the amino acid sequence of SEQ ID NO: 19 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: 19.

[0018] In one embodiment, the amino acid sequence that enhances antigen processing and / or antigen presentation further comprises an amino acid sequence encoding a secretory signal peptide.

[0019] In one embodiment, (i) the RNA encoding the secretory signal peptide comprises the nucleotide sequence of SEQ ID NO: 18 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: 18; and / or (ii) the secretory signal peptide 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.

[0020] In one embodiment, at least one RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 21 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: 21. In one embodiment, each RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 21 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: 21.

[0021] In one embodiment, at least one RNA comprises a polyA sequence. In one embodiment, each RNA comprises a polyA sequence. In one embodiment, the polyA sequence comprises at least 100 nucleotides. In one embodiment, the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 22.

[0022] In one embodiment, RNA is formulated as a liquid, as a solid, or a combination thereof.In one embodiment, RNA is formulated for injection.In one embodiment, RNA is formulated for intravenous administration.

[0023] In one embodiment, the RNA is or will be formulated as a lipoplex particle. In one embodiment, the RNA lipoplex particle is obtained by mixing the RNA with a liposome. In one embodiment, at least one RNA encoding the amino acid sequence of (i), (ii), and / or (iii) is or will be co-formulated as a lipoplex particle with an RNA encoding an amino acid sequence that disrupts immune tolerance. In one embodiment, each RNA encoding the amino acid sequence of (i), (ii), and / or (iii) is or will be co-formulated as a lipoplex particle with an RNA encoding an amino acid sequence that disrupts immune tolerance. In one embodiment, the RNA encoding the amino acid sequence of (i), (ii), and / or (iii) is or will be co-formulated as a lipoplex particle with the RNA encoding the amino acid sequence that disrupts immune 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.

[0024] In one embodiment, the composition or pharmaceutical formulation is a pharmaceutical composition, hi one embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0025] In one embodiment, the composition or pharmaceutical formulation is a kit. In one embodiment, the RNA and optionally the liposomes are in separate vials.

[0026] In one embodiment, the composition or pharmaceutical preparation further comprises instructions for using the RNA and optionally the liposomes to treat or prevent ovarian cancer.

[0027] In one aspect, provided herein are compositions or pharmaceutical formulations described herein for medical use. In one embodiment, the medical use includes therapeutic or prophylactic treatment of a disease or disorder. In one embodiment, the therapeutic or prophylactic treatment of a disease or disorder includes treating or preventing ovarian cancer. In one embodiment, the compositions or pharmaceutical formulations described herein are for administration to humans.

[0028] In one embodiment, the therapeutic or prophylactic treatment of a disease or disorder further comprises administering an additional therapy. In one embodiment, the additional therapy comprises one or more selected from the group consisting of: (i) surgery to remove, excise, or debulk a tumor, (ii) radiation therapy, and (iii) chemotherapy. In one embodiment, the additional therapy comprises administering an additional therapeutic agent. In one embodiment, the additional therapeutic agent comprises an anti-cancer therapeutic agent. In one embodiment, the additional therapeutic agent is a checkpoint modulator. In one embodiment, the checkpoint modulator is an anti-PD1 antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD1 antibody and an anti-CTLA-4 antibody.

[0029] In one embodiment, there is provided a method of treating ovarian cancer in a subject comprising administering to the subject at least one RNA, wherein the at least one RNA has the following amino acid sequence: (i) an amino acid sequence comprising claudin 6 (CLDN6), an immunogenic variant thereof, or an immunogenic fragment of CLDN6 or an immunogenic variant thereof; (ii) an amino acid sequence comprising p53, an immunogenic variant thereof, or an immunogenic fragment of p53 or an immunogenic variant thereof; and (iii) an amino acid sequence comprising a preferentially expressed melanoma antigen (PRAME), an immunogenic variant thereof, or an immunogenic fragment of PRAME or an immunogenic variant thereof; Code the following.

[0030] In one embodiment, each of the amino acid sequences of (i), (ii), or (iii) is encoded by a separate RNA.

[0031] In one embodiment, (i) the RNA encoding the amino acid sequence of (i) comprises the nucleotide sequence of SEQ ID NO: 2 or 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: 2 or 3; and / or (ii) The amino acid sequence of (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.

[0032] In one embodiment, (i) The RNA encoding the amino acid sequence of (ii) comprises the nucleotide sequence of SEQ ID NO: 6 or 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: 6 or 7; and / or (ii) The amino acid sequence of (ii) comprises the amino acid sequence of SEQ ID NO: 4 or 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: 4 or 5.

[0033] In one embodiment, (i) The RNA encoding the amino acid sequence of (iii) comprises the nucleotide sequence of SEQ ID NO: 10 or 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: 10 or 11; and / or The amino acid sequence of (ii)(iii) comprises the amino acid sequence of SEQ ID NO: 8 or 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: 8 or 9.

[0034] In one embodiment, at least one amino acid sequence of (i), (ii), or (iii) comprises an amino acid sequence that disrupts immune tolerance. In one embodiment, each amino acid sequence of (i), (ii), or (iii) comprises an amino acid sequence that disrupts immune tolerance.

[0035] In one embodiment, at least one RNA is co-administered with (iv) an RNA encoding an amino acid sequence that disrupts immune tolerance. In one embodiment, each RNA is co-administered with (iv) an RNA encoding an amino acid sequence that disrupts immune tolerance.

[0036] In one embodiment, the amino acid sequence that breaks immune tolerance comprises a helper epitope, preferably a helper epitope derived from tetanus toxoid.

[0037] In one embodiment, (i) the RNA encoding the immune tolerance-disrupting amino acid sequence comprises the nucleotide sequence of SEQ ID NO: 14 or 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: 14 or 15; and / or (ii) The amino acid sequence that disrupts immune tolerance comprises the amino acid sequence of SEQ ID NO: 12 or 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: 12 or 13.

[0038] In one embodiment, at least one of the amino acid sequences of (i), (ii), (iii), or (iv) is encoded by a coding sequence that has been codon-optimized and / or has an increased G / C content compared to a wild-type coding sequence, wherein the codon-optimization and / or increased G / C content preferably does not alter the sequence of the encoded amino acid sequence. In one embodiment, each of the amino acid sequences of (i), (ii), (iii), or (iv) is encoded by a coding sequence that has been codon-optimized and / or has an increased G / C content compared to a wild-type coding sequence, wherein the codon-optimization and / or increased G / C content preferably does not alter the sequence of the encoded amino acid sequence.

[0039] In one embodiment, at least one RNA is a modified RNA, particularly a stabilized mRNA. In one embodiment, at least one RNA contains a modified nucleoside in place of at least one uridine. In one embodiment, at least one RNA contains a modified nucleoside in place of each uridine. In one embodiment, each RNA contains a modified nucleoside in place of at least one uridine. In one embodiment, each RNA contains a modified nucleoside in place of each uridine. In one embodiment, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U).

[0040] In one embodiment, at least one RNA is 5' capped m2 7,2'-O Gpp s In one embodiment, each RNA comprises a 5' cap m2 7,2'-O Gpp s Contains p(5')G.

[0041] In one embodiment, at least one RNA comprises a 5'-UTR comprising 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. In one embodiment, each RNA comprises a 5'-UTR comprising 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.

[0042] In one embodiment, at least one of the amino acid sequences of (i), (ii), (iii), or (iv) comprises an amino acid sequence that enhances antigen processing and / or antigen presentation. In one embodiment, each of the amino acid sequences of (i), (ii), (iii), or (iv) comprises an amino acid sequence that enhances antigen processing and / or antigen presentation. In one embodiment, the amino acid sequence that enhances antigen processing and / or antigen presentation comprises an amino acid sequence corresponding to the transmembrane and cytoplasmic domains of an MHC molecule, preferably an MHC class I molecule.

[0043] In one embodiment, (i) the RNA encoding the amino acid sequence that enhances antigen processing and / or antigen presentation 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 (ii) The amino acid sequence that enhances antigen processing and / or antigen presentation comprises the amino acid sequence of SEQ ID NO: 19 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: 19.

[0044] In one embodiment, the amino acid sequence that enhances antigen processing and / or antigen presentation further comprises an amino acid sequence encoding a secretory signal peptide.

[0045] In one embodiment, (i) the RNA encoding the secretory signal peptide comprises the nucleotide sequence of SEQ ID NO: 18 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: 18; and / or (ii) the secretory signal peptide 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.

[0046] In one embodiment, at least one RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 21 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: 21. In one embodiment, each RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 21 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: 21.

[0047] In one embodiment, at least one RNA comprises a polyA sequence. In one embodiment, each RNA comprises a polyA sequence. In one embodiment, the polyA sequence comprises at least 100 nucleotides. In one embodiment, the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 22.

[0048] In one embodiment, the RNA is administered by injection, hi one embodiment, the RNA is administered by intravenous administration.

[0049] In one embodiment, the RNA is formulated as a lipoplex particle. In one embodiment, the RNA lipoplex particle is obtained by mixing RNA with liposomes.

[0050] In one embodiment, at least one RNA encoding the amino acid sequence of (i), (ii), and / or (iii) is co-formulated as a lipoplex particle with an RNA encoding an amino acid sequence that disrupts immune tolerance. In one embodiment, each RNA encoding the amino acid sequence of (i), (ii), and / or (iii) is co-formulated as a lipoplex particle with an RNA encoding an amino acid sequence that disrupts immune tolerance. In one embodiment, the RNA encoding the amino acid sequence of (i), (ii), and / or (iii) is co-formulated as a lipoplex particle with the RNA encoding the amino acid sequence that disrupts immune 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.

[0051] In one embodiment, the subject is a human.

[0052] In one embodiment, the methods described herein further comprise administering an additional therapy. In one embodiment, the additional therapy comprises one or more selected from the group consisting of: (i) surgery to remove, excise, or debulk the tumor; (ii) radiation therapy; and (iii) chemotherapy. In one embodiment, the additional therapy comprises administering an additional therapeutic agent. In one embodiment, the additional therapeutic agent comprises an anti-cancer therapeutic agent. In one embodiment, the additional therapeutic agent is a checkpoint modulator. In one embodiment, the checkpoint modulator is an anti-PD1 antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD1 antibody and an anti-CTLA-4 antibody.

[0053] In one aspect, the RNA described herein for use in the methods described herein, e.g., (i) RNA encoding an amino acid sequence comprising claudin 6 (CLDN6), an immunogenic variant thereof, or an immunogenic fragment of CLDN6 or an immunogenic variant thereof; (ii) RNA encoding an amino acid sequence comprising p53, an immunogenic variant thereof, or an immunogenic fragment of p53 or an immunogenic variant thereof; and / or (iii) RNA encoding an amino acid sequence comprising a preferentially expressed melanoma antigen (PRAME), an immunogenic variant thereof, or an immunogenic fragment of PRAME or its immunogenic variant. is provided herein. [Brief explanation of the drawings]

[0054] [Figure 1] General structure of RNAs RBL005.2, RBL008.1, RBL012.1, and RBLTet.1. Schematic of the general structure of all RNA vaccines, including the 5' cap, 5' and 3' untranslated regions (UTRs), coding sequence with N- and C-terminal fusion tags (sec and MITD, respectively), and the A30L70 poly(A) tail. Note that individual elements are not drawn to scale relative to the length of their respective sequences. [Figure 2] 5'-capping structure β-S-ARCA (D1) (m2 7,2'-OGppSpG). Differences between β-S-ARCA (D1) and the basic cap analog m7GpppG are shown in red: an -OCH3 group at the C2' position of the building block m7G, and the replacement of the non-bridging oxygen of the β-phosphate with sulfur. Due to the presence of a stereogenic P center (indicated by an asterisk), the phosphorothioate cap analog β-S-ARCA exists as two diastereomers. These are designated D1 and D2 based on their elution order in reversed-phase HPLC. [Figure 3] Vector map of the plasmid pST1-hAg-Kozak-CLDN6-2hBgUTR-A30L70 for the production of RBL005.2. The insert with labeled sequence elements is shown in different colors. Eam1104I indicates the recognition site for the restriction endonuclease used for linearization. The kanamycin resistance gene is shown in black. [Figure 4]Vector map of the plasmid pST1-hAg-Kozak-sec-GS-P53-GS-MITD-2hBgUTR-A30L70 for RBL008.1 production. The insert with labeled sequence elements is shown in different colors. Eam1104I indicates the recognition site for the restriction endonuclease used for linearization. The kanamycin resistance gene is shown in black. [Figure 5] Vector map of the plasmid pST1-hAg-Kozak-sec-GS-PRAME-GS-MITD-2hBgUTR-A30L70 for the production of RBL0012.1. The insert with labeled sequence elements is shown in different colors. Eam1104I indicates the recognition site for the restriction endonuclease used for linearization. The kanamycin resistance gene is shown in black. [Figure 6] Vector map of the plasmid pST2-hAg-Kozak-sec-GS-P2P16-GS-MITD-2hBgUTR-A30L70 for RBLTet.1 production. The insert with labeled sequence elements is shown in different colors. Eam1104I indicates the recognition site for the restriction endonuclease used for linearization. The kanamycin resistance gene is shown in black. [Figure 7] Chemical structures of selected cationic lipids and co-lipids tested during formulation development. [Figure 8] Organ selectivity of RNA-lipoplexes with different charge ratios. Positively charged luc-RNA lipoplexes exhibit high luciferase expression in the lung, whereas negatively charged RNA-lipoplexes exhibit high selectivity for luciferase expression in the spleen. [Figure 9] The biological activity of RNA-lipoplexes depends on the particle size and size of the liposomes used in their preparation. 20 μg of luc-RNA was condensed with small (198 nm) and large (381 nm) liposomes to reconstitute RNA-lipoplexes, which were then injected intravenously into BALB / c mice (n = 5). Luciferase expression in the spleen was analyzed 6 hours after luc-RNA (LIP) administration (mean ± SD). [Figure 10]Particle size of RNA-lipoplexes prepared according to the clinical formulation protocol. The particle size of RNA-lipoplexes prepared by different researchers in different laboratories using different RNA constructs was analyzed by PCS measurement. For experiments 3 and 10, two independent preparations were performed. [Figure 11] Size and polydispersity index of RNA-lipoplexes with different charge ratios. The particle size (z-average) and polydispersity index of RNA-lipoplexes with different charge ratios (DOTMA:RNA) were measured 10 min, 2 h, and 24 h after preparation. [Figure 12] Size and biological activity of RNA-lipoplexes with different charge ratios. (A) The particle size (z-average) and polydispersity index of RNA-lipoplexes with different charge ratios (DOTMA:RNA) were measured immediately (10 min) after preparation. (B) Luciferase expression in the spleen was analyzed 6 h after intravenous administration of luc-RNA (LIP) (20 μg RNA) to BALB / c mice (n = 4–5). [Figure 13] Localization of bioluminescence signal after intravenous administration of luciferase RNA (LIP). Bioluminescence imaging of in vivo (A) and ex vivo explanted spleen, liver, and lungs (B) 6 hours after intravenous injection of luc-RNA (LIP) (20 μg RNA (HED: 4.74 mg)) into BALB / c mice (n=3). One representative mouse is shown. [Figure 14] RNA (LIP) is selectively internalized by splenic APCs. BALB / c mice (n = 3) were intravenously injected with 40 μg (HED: 9.48 mg) of Cy5-RNA formulated in rhodamine-labeled liposomes. Uptake of Cy5-labeled RNA (bottom row) or rhodamine-labeled liposomes (top row) by splenic cell populations was assessed by flow cytometry 1 h after lipoplex injection. Representative dot plots are shown. [Figure 15]Induction of antigen-specific CD8+ T cell responses and development of T cell memory. C57BL / 6 mice (n=5) were immunized intravenously with SIINFEKL-RNA (LIP) (40 μg RNA) on days 0, 3, 8, and 15 (green). The frequency of antigen-specific CD8+ T cells was monitored in the blood by SIINFEKL-MHC tetramer staining (gray). After a booster injection of RNA (LIP) on day 57, memory recall responses were assessed on day 62. The graph shows the mean tetramer frequency ± SD. [Figure 16] A reduced vaccination schedule does not reduce the efficacy of antigen-specific T cell induction during the induction phase. C57BL / 6 mice (n = 3) were intravenously immunized with 40 or 10 μg of SIINFEKL-RNA (LIP) on days 1, 4, and 8 (groups 1 and 3) or days 1 and 8 (groups 2 and 4) (black bars). On day 13, blood was collected and the induction of antigen-specific CD8+ T cells was analyzed by SIINFEKL-MHC class I tetramer staining (red bars). The graph shows the mean tetramer frequency ± SD. [Figure 17]Isolation of RBL005.2-specific TCRs from in vitro primed CD8+ T cells. (A) In vitro priming of RBL005.2-specific T cells. CD8+ T cells from a healthy HLA-A*02-expressing donor were primed in vitro using autologous mDCs transfected with RBL005.2. After three rounds of stimulation, antigen-specific CD8+ T cells were detected and sorted by flow cytometry based on specific RBL005.291-99 / HLA-A2 dextramer binding. Cells were gated on single lymphocytes. Negative control: T cells primed against a control antigen (RBL001.2). (B) Specificity testing of TCRs isolated from RBL005.2-specific CD8+ T cells. CD8+ T cells from HLA-A*02-positive healthy donors were transfected with TCR-α / β chain RNA and tested for recognition of K562-A2 cells transfected with RBL005.2 or pulsed with the RBL005.2 overlapping 15-mer peptide (=RBL005.2 pool) or the HLA-A*02-binding peptide RBL005.291-99 by IFN-γ ELISPOT assay. Negative controls: control RNA (RBL003.2), an irrelevant control peptide pool (HIV-gag), an irrelevant 9-mer peptide (MAGE-A3112-120), and a positive control: Staphylococcal enterotoxin B (SEB). [Figure 18] IFN-γ secretion by antigen-specific CD8+ T cells after electroporation of RBL005.2 into human DCs. Antigen-specific CD8+ T cells were co-incubated with DCs transfected with different amounts of RBL005.2: 0.25, 1, 4, or 16 μg (HED: 0.6–3.8 mg). DCs transfected with effector-only or irrelevant antigen-encoding RNA served as negative controls. Bars represent the average of two donors and two biological replicates. [Figure 19]Vaccination with WAREHOUSE antigen RNA results in robust cytokine secretion. Splenocytes from intravenously vaccinated A2 / DR1 mice were restimulated for 20 hours with the corresponding HLA-A*0201-restricted peptides ALFGLLVYL (RBL005.291-99), p53 peptide pool (RBL008.1), ALQSLLQHL (RBL012.1422-430), or a mixture of p2 and p16 peptides (RBLTet.1). Effector function was measured using an IFN-γ ELISPOT assay. Symbols represent the mean values of triplicate wells from individual animals. Bars represent the median values across all animals per group. All groups were significantly different from controls (Mann-Whitney test, p<0.05). [Figure 20] Vaccination with RNA (LIP) encoding a target antigen mixed with tetanus helper epitopes results in the disruption of immune tolerance in a self-antigen environment. Splenocytes from RNA (LIP)-vaccinated C57BL / 6 mice were restimulated for 20 hours with the Tyrp1 MHC class I epitope (A) or p2 and p16 peptides (B). Effector function was measured using an IFN-γ ELISPOT assay. Symbols represent the mean values of triplicate wells from individual animals. Bars represent the mean values (±SEM) of all animals per group. *A single comparison with group 1 (Tyrp1 RNA alone) indicates a statistically significant difference for group 2 (Tyrp1 + 4:1 RBLTet.1) using the Mann-Whitney test (p=0.0159). [Figure 21] Transient increase in IFN-α after RNA(LIP) vaccination. (A) C57BL / 6 mice (n = 3) were injected with HA-RNA(LIP) (40 μg RNA (HED: 9.48 mg)), liposomes alone, or PBS as a control. Serum concentrations of IFN-α and TNF-α were assessed by ELISA 6 and 24 h after treatment (mean ± SD). (B) Naive or splenectomized C57BL / 6 mice (n = 2) were intravenously injected with HA-RNA(LIP) (40 μg RNA (HED: 9.48 mg)). Serum concentrations of IFN-α were assessed by ELISA 6 h after treatment (mean ± SD). [Figure 22] Cell activation by RNA (LIP) containing non-immunogenic RNA (ni-RNA) and the absence of IFN-α. C57BL / 6 mice (n=3) were injected with HA-RNA (LIP) (10 μg RNA (HED: 2.37 mg)) containing either immunogenic (unmodified) RNA, non-immunogenic (pseudouridine-modified HPLC-purified) ni-RNA, or PBS as a control. (A) Immune cell activation in the spleen was determined by FACS 24 hours after treatment. (B) Serum concentrations of IFN-α were assessed by ELISA 6 and 24 hours after treatment (mean ± SD). nd: not detected [Figure 23] The transient reduction in total white blood cell (WBC) counts and T lymphocyte subpopulations in peripheral blood upon RNA (LIP) administration was IFN-α dependent. Wild-type C57BL / 6 mice (n = 36) and IFNAR- / - mice (n = 12) were intravenously injected with equal amounts of a mixture of four ATM RNAs (LIP) (40 μg total RNA (HED: 9.48 mg)) or liposomes alone. WBC and lymphocyte counts were examined by FACS analysis at different intervals after injection. Data are presented as a percentage of cell counts from untreated control mice (% of untreated counts). Similar effects were observed for other lymphocyte populations, including B cells and NK cells. [Figure 24] Upregulation of liver enzymes and absence of IFN-α by RNA (LIP) containing non-immunogenic RNA (ni-RNA). C57BL / 6 male mice (n = 5) were injected with the indicated amounts of HA-RNA (LIP) containing either immunogenic (unmodified) RNA, non-immunogenic (pseudouridine-modified, HPLC-purified) ni-RNA, or NaCl as a control. (A) Liver enzyme parameters were determined 6, 24, and 120 hours after treatment (*, p < 0.05; ***, p < 0.001). (B) Serum concentrations of IFN-α were assessed by ELISA 6 hours after treatment (mean ± SD). [Figure 25]Mean levels of IFN-α (black bars) and IL-6 (gray bars) in animals in the high-dose group. Error bars indicate standard deviation. IL-6 induction was much stronger after the first dose (day 1) than after the fifth dose (day 22). [Figure 26] DOTMA accumulation in the spleen, liver, lungs, heart, lymph nodes, and bone marrow was measured over 28 days using three mice per time point. DOTMA accumulation was measured over 28 days using three mice per time point. The y-axis of DOTMA concentration in organs is shown with the same scaling as for the liver and spleen. The solid line is used to delineate the eyes. [Figure 27] DOTMA accumulation in fat pads, brain, and kidneys. DOTMA accumulation is measured over 28 days using three mice per time point. The y-axis of DOTMA concentration in organs is shown with the same scaling as for the liver and spleen. [Figure 28] DOTMA in the spleen and liver during and after the injection period (8 weekly injections). The blue bar indicates the cumulative infusion volume; the blue squares represent DOTMA findings always measured 1 week after the previous injection; the red line represents the results from a single exponential model curve of the data points after the last injection, using y = A*exp(-t / τ), where t is time in weeks. For the model curve, t = 9 weeks was selected. [Figure 29] Relative expression of the WH_ova1 target antigen at the mRNA level in ovarian tumor and normal tissue samples. Expression was assessed by quantitative real-time RT-PCR (Fluidgm screening platform) in up to 91 ovarian tumor and 51 normal tissue samples. Median expression of replicates was calculated, which corresponds to relative expression <5,000 au (detection limit), 5,000-30,000 au (low / intermediate), and >30,000 au (high). The term "% tumor expression" refers to the % of positive samples (>5,000 au). DETAILED DESCRIPTION OF THE INVENTION

[0055] Array Description The following table provides a list of the specific sequences referenced herein.

[0056] [Table 1] TIFF2025122023000002.tif223153TIFF2025122023000003.tif223153TIFF2025122023000004.tif223153TIFF2025122023000005.tif223153 TIFF2025122023000006.tif223153TIFF2025122023000007.tif223153TIFF2025122023000008.tif223153TIFF2025122023000009.tif223153

[0057] Although the present disclosure will be described in detail below, it should be understood that the disclosure is not limited to the specific methodology, protocols and reagents described herein, which may vary.It should also be understood that the terms used herein are only intended to describe specific embodiments and are not intended to limit the scope of the present disclosure, which is limited only by the scope of the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0058] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0059] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0060] The elements of the present disclosure are described below. Although these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create further embodiments. The various described examples and embodiments should not be construed as limiting the disclosure to only the embodiments explicitly described. This description should be understood to disclose and encompass embodiments combining the explicitly described embodiment with any number of the disclosed elements. Furthermore, any permutation and combination of all described elements should be considered disclosed by this description unless the context dictates otherwise.

[0061] The term "about" means approximately or near, and in the context of numerical values or ranges described herein, means, in one embodiment, ±20%, ±10%, ±5%, or ±3% of the recited or claimed numerical value or range.

[0062] As used in the context of describing the present disclosure (particularly in the context of the claims), the terms "a," "an," "the," and similar references should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The 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 herein as if it were individually listed herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better explain the disclosure and does not impose limitations on the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0063] Unless otherwise specified, the term "comprises" is used in the context of this document to indicate that additional members may optionally be present in addition to the members of the list introduced by "comprises." However, it is contemplated as a specific embodiment of the present disclosure that the term "comprises" encompasses the possibility that additional members are not present, i.e., for the purposes of this embodiment, "comprises" should be understood to have the meaning of "consisting of."

[0064] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure.

[0065] definition The following provides definitions that apply to all aspects of this disclosure. The following terms have the following meanings unless otherwise indicated: Undefined terms have their art-accepted meanings.

[0066] As used herein, terms such as "reduce" or "inhibit" refer to the ability to cause an overall decrease in levels, for example, by about 5% or more, about 10% or more, about 20% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar phrases includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0067] In one embodiment, terms such as "increase" or "enhancement" relate to an increase or enhancement of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, or at least about 100%.

[0068] As used herein, "physiological pH" refers to a pH of about 7.5.

[0069] The term "ionic strength" refers to the mathematical relationship between the number of different ionic species in a particular solution and their respective charges. Therefore, ionic strength, I, is expressed by the formula

number

[0070] According to the present disclosure, the term "ionic strength" in one embodiment relates to the presence of monovalent ions. With respect to the presence of divalent ions, particularly divalent cations, their concentration or effective concentration (presence of free ions) is, in one embodiment, sufficiently low to prevent RNA degradation due to the presence of a chelating agent. In one embodiment, the concentration or effective concentration of divalent ions is lower than the catalytic level for hydrolysis of phosphodiester bonds between RNA nucleotides. In one embodiment, the concentration of free divalent ions is 20 μM or less. In one embodiment, free divalent ions are absent or essentially absent.

[0071] The term "freezing" refers to the solidification of a liquid, usually by the removal of heat.

[0072] The term "lyophilize" or "freeze-drying" refers to the lyophilization of a substance by freezing the substance and then reducing the surrounding pressure to cause the freezing medium in the substance to sublimate directly from the solid phase to the gas phase.

[0073] The term "spray drying" refers to spray drying a substance by mixing a (heated) gas with an atomized (atomized) fluid in a vessel (spray dryer), where the solvent from the formed droplets evaporates, resulting in a dry powder.

[0074] The term "cryoprotectant" relates to a substance added to a formulation to protect the active ingredient during the freezing step.

[0075] The term "lyoprotectant" relates to a substance added to a formulation to protect the active ingredient during the drying step.

[0076] The term "reconstitute" relates to adding a solvent, such as water, to a dried product to return it to a liquid state, such as its original liquid state.

[0077] The term "recombinant" in the context of the present disclosure means "produced through genetic engineering." In one embodiment, a "recombinant entity" in the context of the present disclosure is not naturally occurring.

[0078] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that exists in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring. The term "found in nature" means "existing in nature", and includes known objects and objects that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from natural sources in the future.

[0079] In the context of the present disclosure, the term "particle" relates to a structured entity formed by molecules or molecular complexes. In one embodiment, the term "particle" relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure.

[0080] In the context of the present disclosure, the term "RNA lipoplex particle" refers to a particle comprising a lipid, particularly a cationic lipid, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA result in the complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes can generally be synthesized using a cationic lipid, such as DOTMA, and an additional lipid, such as DOPE. In one embodiment, the RNA lipoplex particle is a nanoparticle.

[0081] As used in this disclosure, "nanoparticle" refers to a particle comprising RNA and at least one cationic lipid and having an average diameter suitable for intravenous administration.

[0082] The term "mean diameter" refers to the average hydrodynamic diameter of particles measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, resulting in the so-called Z 平均, and the dimensionless polydispersity index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Here, the "average diameter", "diameter" or "size" of a particle is defined as this Z 平均 Used synonymously with the value of

[0083] The term "polydispersity index" is used herein as a measure of the size distribution of a collection of particles, e.g., nanoparticles. The polydispersity index is calculated based on dynamic light scattering measurements by so-called cumulant analysis.

[0084] The term "ethanol injection technique" refers to a process in which an ethanol solution containing lipids is rapidly injected through a needle into an aqueous solution. This action disperses the lipids throughout the solution and promotes lipid structure formation, e.g., lipid vesicle formation, such as liposome formation. Generally, the RNA lipoplex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, such a colloidal liposome dispersion is formed, in one embodiment, as follows: an ethanol solution containing lipids, such as a cationic lipid such as DOTMA and an additional lipid, is injected into an aqueous solution with stirring. In one embodiment, the RNA lipoplex particles described herein can be obtained without an extrusion step.

[0085] The term "extrusion" or "extrusion" refers to the creation of particles having a fixed cross-sectional profile. In particular, the term refers to the compaction of particles by forcing them through a filter with defined pores.

[0086] The ovaries are organs found in the female reproductive system that produce eggs. Once released, they travel down the fallopian tubes to the uterus, where they can be fertilized by sperm. Ovaries are found on the left and right sides of the body. They also secrete hormones that play a role in the menstrual cycle and fertility. The ovaries progress through many stages, beginning in the prenatal period and ending in menopause. Because they secrete various hormones, they are also endocrine glands. As used herein, "ovarian cancer" refers to cancer that forms in or on the ovaries. This results in abnormal cells that have the ability to invade or spread to other parts of the body. When this process begins, there may be no symptoms or only vague symptoms. Symptoms become more noticeable as the cancer progresses and may include abdominal bloating, pelvic pain, abdominal swelling, and loss of appetite, among others. Common areas where cancer can spread include the lining of the abdomen, lymph nodes, lungs, and liver.

[0087] The risk of ovarian cancer increases in women who ovulate more frequently during their lifetime. This includes women who have never had children, women who begin ovulating at a younger age, and women who reach menopause at an older age. Other risk factors include postmenopausal hormone therapy, fertility treatments, and obesity. Risk-reducing factors include hormonal contraception, tubal ligation, and breastfeeding. Approximately 10% of cases are associated with genetic risk; women with mutations in the BRCA1 or BRCA2 genes have an approximately 50% chance of developing the disease. Ovarian carcinoma is the most common type of ovarian cancer, accounting for over 95% of cases. There are five main subtypes of ovarian carcinoma, of which high-grade serous carcinoma (HGSC) is the most common. These tumors are thought to originate in the cells lining the ovaries, although some can form in the fallopian tubes. Less common types of ovarian cancer include germ cell tumors and sex cord-stromal tumors. A diagnosis of ovarian cancer is usually confirmed by biopsy of tissue removed during surgery. If detected and treated early, ovarian cancer is often curable. Treatment usually involves some combination of surgery, radiation therapy, and chemotherapy. Outcome depends on the extent of the disease, the subtype of cancer present, and other medical conditions.

[0088] As used herein, terms such as "co-administered" or "co-administration" refer to the administration of two or more agents concurrently, simultaneously, or essentially simultaneously, either as part of a single formulation or as multiple formulations administered by the same or different routes. As used herein, "essentially simultaneously" means within about 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or 6 hours of each other.

[0089] The present disclosure describes nucleic acid and amino acid sequences that have a degree of identity, respectively, to a given nucleic acid or amino acid sequence (a reference sequence).

[0090] "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences.

[0091] The term "% identical", "% identity" or similar terms is intended to refer to the percentage of nucleotides or amino acids that are identical in the optimal alignment between the sequences being compared. The percentage is purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the sequences being compared. The comparison of two sequences is usually carried out by comparing the sequences over a segment or "comparison window" after optimal alignment to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually, or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, or the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, or the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or with the aid of computer programs that use the above algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithms available at the United States National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) an expectation threshold set to 10; (ii) a word size set to 28; (iii) a maximum match within the query range set to 0; (iv) match / mismatch scores set to 1, -2; (v) a gap cost set to linear; and (vi) a filter for low-complexity regions being used. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) an expectation threshold set to 10; (ii) a word size set to 3; (iii) a maximum match within the query range set to 0; (iv) a matrix set to BLOSUM62; (v) gap costs set to presence: 11, extension: 1; and (vi) a conditional composition score matrix adjustment.

[0092] The percent identity is obtained by determining the number of identical positions where the compared sequences match, 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.

[0093] In some embodiments, the degree of identity is given for a region that is 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 sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 consecutive nucleotides in some embodiments. In some embodiments, the degree of identity is given for the entire length of the reference sequence.

[0094] A nucleic acid sequence or amino acid sequence that has a certain degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, can have at least one functional property of the given sequence, for example, in some cases, it is functionally equivalent to the given sequence.One important property includes immunogenicity, especially when administered to a subject.In some embodiments, a nucleic acid sequence or amino acid sequence that has a certain degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.

[0095] RNA In this disclosure, the term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. In preferred embodiments, RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal RNA nucleotides or to either or both ends of the RNA. It is also contemplated herein that the nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. In this disclosure, these modified RNAs are considered analogs of naturally occurring RNA.

[0096] In certain embodiments of the present disclosure, the RNA is messenger RNA (mRNA), which is related to RNA transcripts encoding peptides or proteins. As established in the art, mRNA generally comprises a 5' untranslated region (5'-UTR), a peptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, the mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid comprising deoxyribonucleotides.

[0097] In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0098] In one embodiment, the RNA can have modified nucleosides. In some embodiments, the RNA includes a modified nucleoside in place of at least one (e.g., all) uridines.

[0099] The term "uracil" as used herein refers to one of the nucleobases that can occur in RNA nucleic acids. The structure of uracil is: [ka] is.

[0100] As used herein, the term "uridine" refers to one of the nucleosides that can occur in RNA. The structure of uridine is: [ka] is.

[0101] UTP (uridine 5'-triphosphate) has the following structure: [ka] It has.

[0102] Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure: [ka] It has.

[0103] "Pseudouridine" is an example of a modified nucleoside that is an isomer of uridine in which uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0104] Another exemplary modified nucleoside is N1-methylpseudouridine (m1Ψ), which has the structure: [ka] It has.

[0105] N1-methyl pseudo-UTP has the following structure: [ka] It has.

[0106] Another exemplary modified nucleoside is 5-methyluridine (m5U), which has the structure: [ka] It has.

[0107] In some embodiments, one or more uridines in the RNA described herein are replaced with a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0108] In some embodiments, the modified uridine substituting for uridine is pseudouridine (ψ), N1-methylpseudouridine (m1ψ), or 5-methyluridine (m5U).

[0109] In some embodiments, the modified nucleoside that replaces one or more uridines in the RNA is 3-methyluridine (m 3 U), 5-methoxyuridine (mo 5 U), 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s 2 U), 4-thiouridine (s 4 U), 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine (ho 5 U), 5-aminoallyl uridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyluridine (cm 5 U), 1-carboxymethylpseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thiouridine (nm 5 s 2 U), 5-methylaminomethyluridine (mnm 5 U), 1-ethylpseudouridine, 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-selenouridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyluridine, 1-propynylpseudouridine, 5-taurinomethyluridine (τm 5 U), 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thiouridine (τm5s2U), 1-taurinomethyl-4-thiopseudouridine), 5-methyl-2-thiouridine (m 5 s 2 U), 1-methyl-4-thiopseudouridine (m 1 s 4 Ψ), 4-thio-1-methylpseudouridine, 3-methylpseudouridine (m 3 Ψ), 2-thio-1-methylpseudouridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deazapseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m 5 D), 2-thiodihydrouridine, 2-thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypseudouridine, 4-methoxy-2-thiopseudouridine, N1-methylpseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 Ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyluridine (m 5 Um), 2'-O-methylpseudouridine (Ψm), 2-thio-2'-O-methyluridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm 5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm 5 Um), 3,2'-O-dimethyluridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm 5 Um), 1-thiouridine, deoxythymidine, 2'-F-aruridine, 2'-F-uridine, 2'-OH-aruridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0110] 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.

[0111] In some embodiments, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ). In some embodiments, the modified nucleoside comprises N1-methylpseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises 5-methyluridine (m5U). In some embodiments, at least one RNA may comprise two or more modified nucleosides, wherein the modified nucleosides are independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (ψ) and N1-methylpseudouridine (m1ψ). In some embodiments, modified nucleosides include pseudouridine (ψ) and 5-methyluridine (m5U). In some embodiments, modified nucleosides include N1-methylpseudouridine (m1ψ) and 5-methyluridine (m5U). In some embodiments, modified nucleosides include pseudouridine (ψ), N1-methylpseudouridine (m1ψ) and 5-methyluridine (m5U).

[0112] In one embodiment, the RNA contains other modified nucleosides or further modified nucleosides, such as modified cytidine. For example, in one embodiment, cytidine is partially or completely substituted with 5-methylcytidine in the RNA, preferably completely substituted. In one embodiment, the RNA contains 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ) and 5-methyluridine (m5U). In one embodiment, the RNA contains 5-methylcytidine and N1-methylpseudouridine (m1ψ). In some embodiments, the RNA contains 5-methylcytidine in place of each cytidine and N1-methylpseudouridine (m1ψ) in place of each uridine.

[0113] In some embodiments, the RNA of the present disclosure includes a 5' cap. In one embodiment, the RNA of the present disclosure does not have an uncapped 5'-triphosphate. In one embodiment, the RNA may be modified with a 5' cap analog. The term "5' cap" refers to the structure found at the 5' end of an mRNA molecule and generally consists of a guanosine nucleotide linked to the mRNA by a 5'-5' triphosphate bond. In one embodiment, the guanosine is methylated at position 7. Providing an RNA with a 5' cap or 5' cap analog can be achieved by in vitro transcription, in which the 5' cap is co-transcriptionally expressed on the RNA strand, or can be attached to the RNA post-transcriptionally using a capping enzyme.

[0114] In some embodiments, the building block cap for RNA is m2 7,3'-O Gppp(m1 2'-O ) ApG (sometimes m2 7,3'O G(5')ppp(5')m 2'-O ApG), which has the following structure: [ka] It has.

[0115] The following are RNA and m2 7,3'O G(5')ppp(5')m 2'-O An exemplary Cap1 RNA containing ApG is: [ka]

[0116] Below is another exemplary Cap1 RNA (no cap analog): [ka]

[0117] In some embodiments, the RNA has, in one embodiment, the structure: [ka] The anti-reverse cap of the cap analogue (ARCA cap (m2 7,3'O G(5')ppp(5')G)) is modified with a "cap 0" structure.

[0118] The following are RNA and m2 7,3'O An exemplary Cap 0 RNA containing G(5')ppp(5')G is: [ka]

[0119] In some embodiments, the "cap 0" structure has the structure: [ka] Cap analogue β-S-ARCA (m2 7,2'O G(5')ppSp(5')G).

[0120] The following is β-S-ARCA(m2 7,2'O G(5')ppSp(5')G) and RNA. [ka]

[0121] A particularly preferred cap is the 5' cap m2 7,2'O In some embodiments, at least one RNA described herein comprises a 5' cap m2 7,2'O In some embodiments, each RNA described herein comprises a 5' cap m2 7,2'O Contains G(5')ppSp(5')G.

[0122] In some embodiments, an RNA according to the present disclosure comprises a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA molecule, such as an mRNA molecule. The untranslated region (UTR) can be located 5' (upstream) of the open reading frame (5'-UTR) and / or 3' (downstream) of the open reading frame (3'-UTR). If present, the 5'-UTR is located at the 5' end upstream of the start codon of the protein-coding region. If present, the 5'-UTR is downstream of the 5' cap (if present), e.g., directly adjacent to the 5' cap. If present, the 3'-UTR is located at the 3' end downstream of the stop codon of the protein-coding region, although the term "3'-UTR" preferably does not include a polyA sequence. Thus, the 3'-UTR is upstream of the polyA sequence (if present), e.g., directly adjacent to the polyA sequence.

[0123] A particularly preferred 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 16. A particularly preferred 3'-UTR comprises the nucleotide sequence of SEQ ID NO:21.

[0124] In some embodiments, at least one RNA comprises a 5'-UTR comprising 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. In some embodiments, each RNA comprises a 5'-UTR comprising 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.

[0125] In some embodiments, at least one RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO:21, 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:21. In some embodiments, each RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO:21, 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:21.

[0126] As used herein, the term "poly A tail" or "poly A sequence" refers to a continuous or intermittent sequence of adenylate residues typically located at the 3' end of an RNA molecule. Poly A tails or poly A sequences are known to those skilled in the art and may follow the 3'-UTR of the RNA described herein. A continuous poly A tail is characterized by consecutive adenylate residues. Continuous poly A tails are typical in nature. The RNAs disclosed herein may have a poly A tail attached to the free 3' end of the RNA by a template-independent RNA polymerase after transcription, or a poly A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0127] Poly(A) tails of approximately 120 A nucleotides have been demonstrated to have beneficial effects on the levels of RNA in transfected eukaryotic cells and on the levels of proteins translated from open reading frames located 5' upstream of the poly(A) tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0128] The poly-A tail can be of any length. In some embodiments, the poly-A tail comprises, consists essentially 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, particularly about 120 A nucleotides. In this context, "consisting essentially of" means that most of the nucleotides in the poly-A tail, 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% of the number of nucleotides in the poly-A tail, are A nucleotides, while allowing for the remaining nucleotides to be nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid). In this context, "consisting of" means that all nucleotides of the poly A tail, i.e., 100% of the number of nucleotides in the poly A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylic acid.

[0129] In some embodiments, the poly(A) tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template containing repeated dT nucleotides (deoxythymidylic acid) in the strand complementary to the coding strand. The DNA sequence encoding the poly(A) tail (coding strand) is referred to as a poly(A) cassette.

[0130] In some embodiments, the poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides but is interrupted by random sequences of four nucleotides (dA, dC, dG, and dT). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length. Such cassettes are disclosed in International Publication No. 2016 / 005324A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in International Publication No. 2016 / 005324A1 can be used in the present invention. Poly(A) cassettes consisting essentially of dA nucleotides but interrupted by random sequences in which the four nucleotides (dA, dC, dG, and dT) are evenly distributed and have a length of, for example, 5-50 nucleotides, exhibit sustained propagation of plasmid DNA in Escherichia coli (E. coli) at the DNA level and are still associated with beneficial properties related to supporting RNA stability and translation efficiency at the RNA level. Consequently, in some embodiments, the poly-A tails included in the RNA molecules described herein consist essentially of A nucleotides, but are interrupted by random sequences of four nucleotides (A, C, G, U). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length.

[0131] In some embodiments, no nucleotides other than A nucleotides are adjacent to the polyA tail at its 3' end, i.e., the polyA tail is not masked or followed by a nucleotide other than A at its 3' end.

[0132] In some embodiments, the polyA tail comprises the sequence of SEQ ID NO:22.

[0133] In some embodiments, at least one RNA comprises a poly-A tail. In some embodiments, each RNA comprises a poly-A tail. In some embodiments, the poly-A tail 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 tail may consist essentially 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 tail 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 tail may comprise the poly A tail set forth in SEQ ID NO: 22. In some embodiments, the poly A tail comprises at least 100 nucleotides. In some embodiments, the poly A tail comprises about 150 nucleotides. In some embodiments, the poly A tail comprises about 120 nucleotides.

[0134] In the context of the present disclosure, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA, which can then be translated into peptides or proteins.

[0135] With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes by which a chain of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.

[0136] In one embodiment, after administration of the RNA described herein, for example, formulated as an RNA lipoplex particle, at least a portion of the RNA is delivered to a target cell. In one embodiment, at least a portion of the RNA is delivered to the cytosol of the target cell. In one embodiment, the RNA is translated by the target cell to produce its encoded peptide or protein. In one embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an antigen-presenting cell, such as a professional antigen-presenting cell in the spleen. In one embodiment, the target cell is a dendritic cell or a macrophage. The RNA lipoplex particles described herein can be used to deliver RNA to such target cells. Thus, the present disclosure also relates to a method for delivering RNA to a target cell in a subject, comprising administering to the subject an RNA lipoplex particle described herein. In one embodiment, the RNA is delivered to the cytosol of the target cell. In one embodiment, the RNA is translated by the target cell to produce the peptide or protein encoded by the RNA.

[0137] According to the present disclosure, the term "RNA-encoded" means that the RNA, when present in the appropriate environment, such as within the cells of a target tissue, can direct the assembly of amino acids to produce the peptide or protein it encodes during the translation process. In one embodiment, the RNA can interact with the cellular translation machinery to enable translation of the peptide or protein. The cell can produce the encoded peptide or protein intracellularly (e.g., in the cytoplasm and / or nucleus), secrete the encoded peptide or protein, or produce it on its surface.

[0138] According to this disclosure, the term "peptide" includes oligopeptides and polypeptides and refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids joined together by peptide bonds. The term "protein" refers to large peptides, particularly peptides having at least about 151 amino acids, although the terms "peptide" and "protein" are generally used interchangeably herein.

[0139] The term "antigen" refers to an agent containing an epitope capable of generating an immune response. The term "antigen" includes, inter alia, proteins and peptides. In one embodiment, the antigen is presented by a cell of the immune system, such as an antigen-presenting cell, such as a dendritic cell or a macrophage. In one embodiment, the antigen, such as a T cell epitope, or its processing product is bound by a T cell or B cell receptor, or by an immunoglobulin molecule, such as an antibody. Thus, the antigen or its processing product can specifically react with an antibody or a T lymphocyte (T cell). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, and the epitope is derived from such an antigen.

[0140] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Thus, a disease-associated antigen or its epitope can be used for therapeutic purposes. A disease-associated antigen can be associated with cancer, typically a tumor.

[0141] The term "tumor antigen" refers to a component of a cancer cell that can originate from the cytoplasm, cell surface, and cell nucleus. In particular, the term refers to an antigen that is produced intracellularly or as a surface antigen on a tumor cell.

[0142] The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by a T cell, a B cell, or an antibody. An epitope of an antigen can include continuous or discontinuous portions of the antigen and can be about 5 to about 100 amino acids in length. In one embodiment, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0143] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of an MHC molecule. The terms "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or diseased cells during the immune response; they bind to peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the surface of cells and present both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. For class I MHC / peptide complexes, the binding peptide is typically about 8 to about 10 amino acids long, although longer or shorter peptides can also be effective. For class II MHC / peptide complexes, the binding peptide is typically about 10 to about 25 amino acids long, particularly about 13 to about 18 amino acids long, although longer and shorter peptides can also be effective.

[0144] In certain embodiments of the present disclosure, the RNA encodes at least one epitope, hi certain embodiments, the epitope is derived from a tumor antigen as described herein.

[0145] Administered RNA In some embodiments, the compositions or pharmaceutical preparations described herein comprise RNA encoding claudin 6 (CLDN6) protein, RNA encoding p53 protein, and RNA encoding preferentially expressed melanoma antigen (PRAME) protein. Similarly, the methods described herein comprise administration of RNA encoding claudin 6 (CLDN6) protein, RNA encoding p53 protein, and RNA encoding preferentially expressed melanoma antigen (PRAME) protein.

[0146] Molecular structure and function of CLDN6 (RBL005.2) The human claudin 6 gene (CLDN6) is located on chromosome 16 and contains two isoforms encoding a 220-amino acid protein. CLDN6 is highly conserved across species and belongs to a group of claudins consisting of at least 27 members. In general, claudins, including CLDN6, are important for regulating the epithelial barrier and belong to a 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 the permeability barrier and transepithelial electrical resistance in epidermal cells. Furthermore, CLDN6 appears to be required for normal blastocyst formation. Detailed RT-qPCR-based analysis revealed CLDN6 expression below the detection limit in all tissues examined (Figure 29).

[0147] Claudin 6 (CLDN6) proteins include amino acid sequences comprising CLDN6, an immunogenic variant thereof, or an immunogenic fragment of CLDN6 or an immunogenic variant thereof, and may have an amino acid sequence comprising 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. RNA encoding CLDN6 proteins may (i) comprise the nucleotide sequence of SEQ ID NO: 2 or 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: 2 or 3; and / or (ii) encode an amino acid sequence comprising 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.

[0148] Molecular structure and function of tumor protein p53 (RBL008.1) The p53 gene locus on chromosome 17p13.1 encodes a 53-kDa protein that is well conserved across species. The p53 protein is primarily localized in the nucleus, but depending on its ubiquitin modification and isoform, it can also be detected in the cytoplasm. p53 is a transcription factor involved in pleiotropic cellular functions, such as DNA repair, cell proliferation, and apoptosis, depending on the physiological context, cell type, and post-translational modifications, including ubiquitination, sumoylation, phosphorylation, neddylation, acetylation, and methylation. In healthy tissues, p53 expression is tightly regulated via ubiquitination and subsequent proteasomal degradation. However, upon DNA damage, the p53 protein is stabilized and prevents genomic instability by inducing the DNA damage response.

[0149] p53 is a well-known tumor suppressor gene that is found mutated or overexpressed in over 50% of all cancers. The p53 protein is expressed in many tissues (Figure 29) and has been intensively studied as an antigen target for cancer immunotherapy. p53-specific cytotoxic T lymphocytes (CTLs) and CD4 + Adoptive transfer of T helper cells eradicates p53-overexpressing tumors in mice. Furthermore, it has been described that p53 is subject to "dissociated immune tolerance," which involves efficient deletion of lymphocytes that recognize p53-derived peptides on MHC class I molecules but not on MHC class II molecules. Consequently, p53 qualifies as a universal antigen for the induction of antitumor T helper cell responses.

[0150] To date, there are at least three CD8 + and two CD4 + T cell epitopes have been identified. Furthermore, p53 autoantibodies and p53-specific CTLs have been detected in cancer patients, supporting the protein's potential to induce effective immune responses.

[0151] Several immunotherapeutic phase I and II trials using p53 as an antigen have been initiated, most of which have shown p53-specific vaccine-induced immune responses. These trials include viral vector and dendritic cell- and peptide-based vaccination strategies and have been conducted in various cancer entities. Some trials have shown robust p53-specific CD4 + T helper cell induction and CD8 + Although mobilization of cytotoxic T lymphocytes has been demonstrated, clear evidence of clinical efficacy is lacking.

[0152] The p53 protein comprises an amino acid sequence comprising p53, an immunogenic variant thereof, or an immunogenic fragment of p53 or an immunogenic variant thereof, and may have an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 4 or 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: 4 or 5. The RNA encoding the p53 protein may (i) comprise the nucleotide sequence of SEQ ID NO: 6 or 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: 6 or 7; and / or (ii) encode an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 4 or 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: 4 or 5.

[0153] Molecular structure and function of PRAME (RBL012.1) The PRAME gene, which is preferentially expressed in human melanoma, is located on chromosome 22 and contains eight isoforms, seven of which encode identical proteins of 509 amino acids, while the eighth isoform lacks the first 16 amino acids. Localization studies using FLAG-tagged or GFP-tagged PRAME suggest nuclear localization of the protein. Furthermore, PRAME plays an important role in apoptosis and cell proliferation. Further functional studies revealed that PRAME inhibits retinoic acid receptor signaling, thereby inducing its role in apoptosis and differentiation. PRAME belongs to a multigene family consisting of 32 PRAME-like genes and pseudogenes. The closest protein-coding relative of PRAME shows 53% homology to this protein (using the blastp command in the blast software package). Detailed RT-qPCR-based analysis revealed high expression of PRAME in the testis, epididymis, and uterus. Moderate PRAME expression was detected in the placenta, ovary, fallopian tube, and adrenal gland (Figure 29).

[0154] The melanoma preferentially expressed antigen (PRAME) protein comprises an amino acid sequence comprising PRAME, an immunogenic variant thereof, or an immunogenic fragment of PRAME or an immunogenic variant thereof, and may have an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 8 or 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: 8 or 9. The RNA encoding the PRAME protein may (i) comprise the nucleotide sequence of SEQ ID NO: 10 or 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: 10 or 11; and / or (ii) encode an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 8 or 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: 8 or 9.

[0155] Molecular structure and function of tetanus toxoid-derived helper sequences p2 and p16 (RBLTet.1) Amino acid sequences derived from tetanus toxoid of Clostridium tetani can be used to overcome self-tolerance mechanisms to efficiently initiate immune responses to self-antigens by providing T cell help during priming.

[0156] Tetanus toxoid heavy chains bind indiscriminately to MHC class II alleles and induce CD4 + Furthermore, the combination of tetanus toxoid (TT) helper epitopes with tumor-associated antigens induces CD4 T cell activation during priming. + It is known that tumor-associated antigens improve immune stimulation compared to the application of tumor-associated antigens alone by providing mediated T cell help. +To reduce the risk of stimulating T cells, the whole tetanus toxoid fragment C stimulates CD8 + The entire fragment C is not used because it is known to contain a T cell epitope. To ensure binding to as many MHC class II alleles as possible, two peptide sequences containing promiscuous helper epitopes were instead selected. Based on data from ex vivo studies, the known epitope p2 (QYIKANSKFIGITEL; TT 830-844 ) and p16(MTNSVDDALINSTKIYSYFPSVISKVNQGAQG;TT 578-609 ) was selected. The p2 epitope has already been used in peptide vaccination in clinical trials to boost anti-melanoma activity.

[0157] Current preclinical data (unpublished) indicate that an RNA vaccine encoding both tumor antigens and promiscuous tetanus toxoid sequences can stimulate CD8 activation against tumor antigens. + This has been shown to result in enhanced T cell responses and improved tolerance breaking. Immune monitoring data from patients vaccinated with vaccines containing sequences fused in-frame with tumor antigen-specific sequences reveals that selected tetanus sequences are able to induce tetanus-specific T cell responses in nearly all patients.

[0158] Instead of using a self-antigen RNA fused with a tetanus toxoid helper epitope, the WH_ova1 shared tumor antigen RNA can be co-administered with a separate RNA encoding a TT helper epitope (i.e., RBLTet.1) during vaccination. Here, the RNA encoding the TT helper epitope is added to each antigen-encoding RNA prior to preparation. In this way, mixed lipoplex nanoparticles containing both the antigen and the helper epitope-encoding RNA are formed to deliver both compounds to a given APC.

[0159] Thus, in some embodiments, the compositions described herein can include RNA encoding the tetanus toxoid-derived helper sequences p2 and p16 (P2P16). Similarly, the methods described herein can include administration of RNA encoding the tetanus toxoid-derived helper sequences p2 and p16 (P2P16).

[0160] Thus, a further aspect is (i) RNA encoding a vaccine antigen, and (ii) RNA encoding an amino acid sequence that disrupts immune tolerance The present invention relates to compositions, such as pharmaceutical compositions, comprising particles, such as lipoplex particles, comprising: Such compositions are useful in methods of inducing an immune response against vaccine antigens and, therefore, against disease-associated antigens.

[0161] A further aspect is a method of inducing an immune response, comprising: (i) RNA encoding a vaccine antigen, and (ii) RNA encoding an amino acid sequence that disrupts immune tolerance The present invention relates to methods comprising administering particles, such as lipoplex particles, comprising:

[0162] In one embodiment, the amino acid sequence that breaks immune tolerance comprises a helper epitope, preferably a helper epitope derived from tetanus toxoid.

[0163] In one embodiment, (i) the RNA encoding the immune tolerance-disrupting amino acid sequence comprises the nucleotide sequence of SEQ ID NO: 14 or 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: 14 or 15; and / or (ii) The amino acid sequence that disrupts immune tolerance comprises the amino acid sequence of SEQ ID NO: 12 or 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: 12 or 13.

[0164] In one embodiment, RNA encoding a vaccine antigen is co-formulated with RNA encoding an amino acid sequence that disrupts immune tolerance as a particle, such as a lipoplex particle. In one embodiment, RNA encoding a vaccine antigen is co-formulated with RNA encoding an amino acid sequence that disrupts immune 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 as a particle, such as a lipoplex particle.

[0165] The tetanus toxoid-derived helper sequences p2 and p16 (P2P16) protein comprises an amino acid sequence comprising P2 and P16, an immunogenic variant thereof, or an immunogenic fragment of P2 and P16 or an immunogenic variant thereof, and may have an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 12 or 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: 12 or 13. The RNA encoding the P2P16 protein may (i) comprise the nucleotide sequence of SEQ ID NO: 14 or 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: 14 or 15; and / or (ii) encode an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 12 or 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: 12 or 13.

[0166] As used herein, "variant" refers to an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid modification. The parent amino acid sequence can be a natural or wild-type (WT) amino acid sequence, or can be a modified version of the wild-type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid modifications compared to the parent, preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0167] As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally modified.

[0168] For purposes of this disclosure, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformational variants, isoform variants, allelic variants, species variants, and species homologs, particularly those occurring naturally.

[0169] Amino acid insertion variants include the insertion of one or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at a specific site in the amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example, 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, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may occur at any position in the protein. Amino acid deletion variants containing deletions at the N- and / or C-termini of a protein are also referred to as N- and / or C-terminal truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications at positions in the amino acid sequence that are not conserved between homologous proteins or peptides and / or substitutions of amino acids with other amino acids with similar properties are preferred. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of members of a family of amino acids whose side chains are related. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (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 together as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: Glycine, Alanine; valine, isoleucine, leucine; Aspartic acid, glutamic acid; Asparagine, glutamine; Serine, threonine; lysine, arginine; and Phenylalanine, tyrosine.

[0170] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given over an amino acid region that is 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 amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given over at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably consecutive amino acids. In a preferred embodiment, the degree of similarity or identity is given over the entire length of the reference amino acid sequence.

[0171] "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between these sequences.

[0172] An amino acid sequence (peptide, protein, or polypeptide) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the initial amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof.

[0173] The peptide and protein antigens described herein (CLDN6 protein, p53 protein, and PRAME protein), when provided to a subject by administration of RNA encoding the antigen, i.e., vaccine antigen, preferably result in the stimulation, priming, and / or expansion of T cells in the subject. The stimulated, primed, and / or expanded T cells are preferably directed against a target antigen, particularly a target antigen, i.e., a disease-associated antigen, expressed by diseased cells, tissues, and / or organs. Thus, the vaccine antigen may comprise a disease-associated antigen, or a fragment or variant thereof. In one embodiment, such a fragment or variant is immunologically equivalent to the disease-associated antigen. In the context of the present disclosure, the term "antigen fragment" or "antigen variant" refers to an agent that results in the stimulation, priming, and / or expansion of T cells, and the stimulated, primed, and / or expanded T cells target the disease-associated antigen, particularly when expressed on the surface of diseased cells, tissues, and / or organs. Thus, vaccine antigens administered according to the present disclosure may correspond to or comprise disease-associated antigens, may correspond to or comprise fragments of disease-associated antigens, or may correspond to or comprise antigens homologous to disease-associated antigens or their fragments. When vaccine antigens administered according to the present disclosure comprise fragments of disease-associated antigens or amino acid sequences homologous to fragments of disease-associated antigens, the fragments or amino acid sequences may comprise epitopes of disease-associated antigens or sequences homologous to epitopes of disease-associated antigens, where T cells bind to the epitopes. Thus, according to the present disclosure, antigens may comprise immunogenic fragments of disease-associated antigens or amino acid sequences homologous to immunogenic fragments of disease-associated antigens. "Immunogenic fragments of antigens" according to the present disclosure preferably refer to fragments of antigens capable of stimulating, priming, and / or expanding T cells. Vaccine antigens (like disease-associated antigens) preferably provide relevant epitopes for binding by T cells. It is also preferred that vaccine antigens (like disease-associated antigens) are expressed on the surface of cells, such as antigen-presenting cells, to provide relevant epitopes for binding by T cells.Vaccine antigens according to the present invention may be recombinant antigens.

[0174] The term "immunologically equivalent" means that an immunologically equivalent molecule, such as an immunologically equivalent amino acid sequence, exhibits the same or essentially the same immunological properties and / or exerts the same or essentially the same immunological effect, e.g., with respect to the type of immunological effect. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effect or properties of an antigen or antigen variant. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if, when exposed to T cells that bind to the reference amino acid sequence or cells that express the reference amino acid sequence, it induces an immune response, particularly the stimulation, priming, and / or expansion of T cells, with specificity that reacts with the reference amino acid sequence. Thus, a molecule that is immunologically equivalent to an antigen exhibits the same or essentially the same properties and / or exerts the same or essentially the same effect as the antigen targeted by the T cells with respect to the stimulation, priming, and / or expansion of T cells.

[0175] As used herein, "activation" or "stimulation" refers to a state of T cells that are sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells undergoing cell division.

[0176] The term "priming" refers to the process by which a T cell first contacts its specific antigen and triggers its differentiation into an effector T cell.

[0177] The term "clonal expansion" or "expansion" refers to the process of increasing a specific entity. In the context of the present disclosure, the term is preferably used in the context of an immunological response in which lymphocytes are stimulated by an antigen, proliferate, and the specific lymphocytes that recognize said antigen are amplified. Preferably, clonal expansion results in differentiation of lymphocytes.

[0178] Lipoplex particles In certain embodiments of the present disclosure, the RNA described herein can be present in RNA lipoplex particles. The RNA lipoplex particles and compositions comprising the RNA lipoplex particles described herein are useful for delivering RNA to target tissues after parenteral administration, particularly intravenous administration. The RNA lipoplex particles can be prepared using liposomes, which can be obtained by injecting an ethanolic solution of lipids into water or a suitable aqueous phase. In one embodiment, the aqueous phase has an acidic pH. In one embodiment, the aqueous phase contains acetic acid, for example, in an amount of about 5 mM. In one embodiment, the liposomes and RNA lipoplex particles contain at least one cationic lipid and at least one additional lipid. In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP). In one embodiment, at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol), and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In one embodiment, at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), and at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, liposomes and RNA lipoplex particles comprise 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). Liposomes can be used to prepare RNA lipoplex particles by mixing liposomes with RNA.

[0179] RNA lipoplex particles targeted to the spleen are described in International Publication No. 2013 / 143683, which is incorporated herein by reference. It has been discovered that RNA lipoplex particles with a net negative charge can be used to selectively target spleen tissue or cells, such as antigen-presenting cells, particularly dendritic cells. Thus, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occurs in the spleen. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and / or RNA expression occurs in the lung and / or liver. In one embodiment, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occurs in antigen-presenting cells, such as professional antigen-presenting cells in the spleen. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA in such antigen-presenting cells. In one embodiment, the antigen-presenting cells are dendritic cells and / or macrophages.

[0180] RNA lipoplex particle size In one embodiment, the RNA lipoplex particles described herein have an average diameter in the range of about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 nm to about 700 nm, about 400 nm to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In one embodiment, the RNA lipoplex particles have an average diameter in the range of about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter in the range of about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0181] In one embodiment, the RNA lipoplex particles described herein exhibit a polydispersity index of less than about 0.5, less than about 0.4, or less than about 0.3. By way of example, the RNA lipoplex particles may exhibit a polydispersity index ranging from about 0.1 to about 0.3.

[0182] lipids In one embodiment, the lipid solutions, liposomes, and RNA lipoplex particles described herein contain cationic lipids. As used herein, "cationic lipid" refers to a lipid with a net positive charge. Cationic lipids bind negatively charged RNA to the lipid matrix through electrostatic interactions. Generally, cationic lipids have a lipophilic moiety, such as a sterol, acyl, or diacyl chain, and the lipid head group typically carries a positive charge. Examples of cationic lipids include 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); 1,2-dioleoyl-3-dimethylammonium propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 2,3-di(tetradecoxy)propane. Cationic lipids include, but are not limited to, pyr-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA).Preferably, DOTMA, DOTAP, DODAC, and DOSPA are cationic lipids.In certain embodiments, cationic lipids are DOTMA and / or DOTAP.

[0183] Additional lipids may be incorporated to adjust the overall ratio of positive and negative charges and the physical stability of the RNA lipoplex particles. In certain embodiments, the additional lipid is a neutral lipid. As used herein, "neutral lipid" refers to a lipid with a zero net charge. Examples of neutral lipids include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, and cerebroside. In certain embodiments, the additional lipid is DOPE, cholesterol, and / or DOPC.

[0184] In certain embodiments, the RNA lipoplex particles comprise both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE. Without wishing to be bound by theory, the amount of at least one cationic lipid relative to the amount of the at least one additional lipid can affect important RNA lipoplex particle properties, such as charge, particle size, stability, tissue selectivity, and RNA bioactivity. Thus, in some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In certain embodiments, the molar ratio can 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. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.

[0185] charge ratio The charge of the RNA lipoplex particles of the present disclosure is the sum of the charge present on at least one cationic lipid and the charge present on the RNA. The charge ratio is the ratio of the positive charge present on at least one cationic lipid to the negative charge present on the RNA. The charge ratio of the positive charge present on at least one cationic lipid to the negative charge present on the RNA is calculated by the following formula: Charge ratio = [(cationic lipid concentration (mol)) * (total number of positive charges in cationic lipids)] / [(RNA concentration (mol)) * (total number of negative charges in the RNA)]. The concentration of RNA and the amount of at least one cationic lipid can be determined by one of skill in the art using routine methods.

[0186] In one embodiment, the charge ratio of positive to negative charges in the RNA lipoplex particles at physiological pH is about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2. In certain embodiments, the charge ratio of positive to negative charges in the RNA lipoplex 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.

[0187] It has been found that RNA lipoplex particles having such a charge ratio can be used to selectively target spleen tissue or spleen cells, such as antigen-presenting cells, particularly dendritic cells. Thus, in one embodiment, RNA accumulation and / or RNA expression occurs in the spleen after administration of the RNA lipoplex particles. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, RNA accumulation and / or RNA expression in the lung and / or liver does not occur at all or essentially does not occur after administration of the RNA lipoplex particles. In one embodiment, RNA accumulation and / or RNA expression occurs in antigen-presenting cells, such as professional antigen-presenting cells in the spleen after administration of the RNA lipoplex particles. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA in such antigen-presenting cells. In one embodiment, the antigen-presenting cells are dendritic cells and / or macrophages.

[0188] A. Salt and Ionic Strength According to the present disclosure, the compositions described herein may contain salts such as sodium chloride. Without wishing to be bound by theory, sodium chloride functions as an ionic osmotic agent for pretreating RNA before mixing with at least one cationic lipid. Certain embodiments contemplate organic or inorganic salts to replace sodium chloride in the present disclosure. Alternative salts include, but are not limited to, potassium chloride, dipotassium phosphate, monopotassium phosphate, potassium acetate, potassium bicarbonate, potassium sulfate, potassium acetate, disodium phosphate, monosodium phosphate, sodium acetate, sodium bicarbonate, sodium sulfate, sodium acetate, lithium chloride, magnesium chloride, magnesium phosphate, calcium chloride, and the sodium salt of ethylenediaminetetraacetic acid (EDTA).

[0189] Generally, compositions comprising the RNA lipoplex particles described herein preferably contain sodium chloride at a concentration ranging from 0 mM to about 500 mM, from about 5 mM to about 400 mM, or from about 10 mM to about 300 mM. In one embodiment, the composition comprising the RNA lipoplex particles contains an ionic strength corresponding to such a sodium chloride concentration.

[0190] B. Stabilizer The compositions described herein may include stabilizers to avoid substantial loss of product quality, particularly substantial loss of RNA activity, during storage, such as freezing, lyophilization, spray drying, or storage of frozen, lyophilized, or spray-dried compositions.

[0191] In one embodiment, the stabilizer is a carbohydrate. As used herein, the term "carbohydrate" refers to and includes monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides.

[0192] In an embodiment of the present disclosure, the stabilizer is mannose, glucose, sucrose, or trehalose.

[0193] According to the present disclosure, the RNA lipoplex particle compositions described herein have stabilizer concentrations suitable for the stability of the composition, particularly the stability of the RNA lipoplex particles and the stability of the RNA.

[0194] C. pH and Buffers According to the present disclosure, the RNA lipoplex particle compositions described herein have a pH suitable for the stability of the RNA lipoplex particles, particularly the stability of the RNA. In one embodiment, the RNA lipoplex particle compositions described herein have a pH of about 5.5 to about 7.5.

[0195] According to the present disclosure, a composition comprising a buffer is provided. Without wishing to be bound by theory, the use of a buffer maintains the pH of the composition during its manufacture, storage, and use. In a specific embodiment of the present disclosure, the buffer is sodium bicarbonate, monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate, [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), 2-(bis(2-hydroxyethyl)amino)acetic acid (bicine), 2-amino-2-(hydroxymethyl)propane-1,3-diol (tris), N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine), 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl] The buffer may be 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 1,4-piperazinediethanesulfonic acid (PIPES), dimethylarsinic acid, 2-morpholin-4-ylethanesulfonic acid (MES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), or phosphate-buffered saline (PBS). Other suitable buffers may be acetic acid in salt, citric acid in salt, boric acid in salt, and phosphoric acid in salt.

[0196] In one embodiment, the buffering agent is HEPES.

[0197] In one embodiment, the buffering agent has a concentration of about 2.5 mM to about 15 mM.

[0198] D. Chelating Agents Certain embodiments of the present disclosure contemplate the use of chelating agents. A chelating agent refers to a compound capable of forming at least two coordinate covalent bonds with a metal ion, thereby forming a stable, water-soluble complex. Without wishing to be bound by theory, chelating agents reduce the concentration of free divalent ions that may otherwise induce accelerated RNA degradation in the present disclosure. Examples of suitable chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), salts of EDTA, desferrioxamine B, deferoxamine, dithiocarbamate sodium, penicillamine, calcium pentetate, sodium pentetate, succinic acid, trientine, nitrilotriacetic acid, trans-diaminocyclohexanetetraacetic acid (DCTA), diethylenetriaminepentaacetic acid (DTPA), bis(aminoethyl)glycol ether-N,N,N',N'-tetraacetic acid, iminodiacetic acid, citric acid, tartaric acid, fumaric acid, or salts thereof. In certain embodiments, the chelating agent is EDTA or a salt of EDTA. In an exemplary embodiment, the chelating agent is disodium EDTA dihydrate.

[0199] In some embodiments, the EDTA is at a concentration of about 0.05 mM to about 5 mM.

[0200] E. Physical State of the Compositions of the Present Disclosure In embodiments, the compositions of the present disclosure are liquid or solid. Non-limiting examples of solids include frozen or lyophilized forms. In preferred embodiments, the compositions are liquid.

[0201] Pharmaceutical Compositions of the Present Disclosure The RNA described herein, for example, formulated as RNA lipoplex particles, is useful as, or for the preparation of, pharmaceutical compositions or medicaments for therapeutic or prophylactic treatments.

[0202] The compositions of the present disclosure may be administered in the form of any suitable pharmaceutical composition.

[0203] The term "pharmaceutical composition" relates to a formulation comprising a therapeutically active agent, preferably together with a pharmaceutically acceptable carrier, diluent and / or excipient. The pharmaceutical composition is useful for treating, preventing, or lessening the severity of a disease or disorder by administering the pharmaceutical composition to a subject. A pharmaceutical composition is also known in the art as a pharmaceutical formulation. In the context of the present disclosure, a pharmaceutical composition comprises an RNA as described herein, for example, formulated as an RNA lipoplex particle.

[0204] The pharmaceutical compositions of the present disclosure preferably contain one or more adjuvants or can be administered with one or more adjuvants. The term "adjuvant" refers to a compound that prolongs, enhances, or accelerates an immune response. Adjuvants include groups of compounds such as oil emulsions (e.g., Freund's adjuvant), inorganic compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immune-stimulating complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines such as monokines, lymphokines, interleukins, and chemokines. Chemokines can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INFα, INF-γ, GM-CSF, or LT-α. Further known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils such as Montanide® ISA 51. Other suitable adjuvants for use in the present disclosure include lipopeptides such as Pam3Cys.

[0205] Pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and a "pharmaceutically acceptable formulation."

[0206] The term "pharmaceutically acceptable" refers to the non-toxicity of a substance that does not interact with the action of the active ingredients of the pharmaceutical composition.

[0207] The term "pharmaceutically effective amount" refers to an amount that, alone or together with further doses, achieves the desired response or desired effect. In the case of the treatment of a specific disease, the desired response preferably relates to the inhibition of the course of the disease. This includes slowing the progression of the disease, particularly halting or reversing the progression of the disease. The desired response in the treatment of a disease can also be the delay or prevention of the onset of the disease or condition. The effective amount of the compositions described herein depends on the condition being treated, the severity of the disease, individual patient parameters including age, physiological condition, size and weight, the duration of treatment, the type of concomitant treatment (if any), the specific route of administration, and similar factors. Thus, the dosage of the compositions described herein can depend on such various parameters. If the patient's response is inadequate with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) can be used.

[0208] In some embodiments, an effective amount includes an amount sufficient to shrink a tumor / lesion. In some embodiments, an effective amount is an amount sufficient to reduce the rate of tumor growth (e.g., inhibit tumor growth). In some embodiments, an effective amount is an amount sufficient to delay tumor onset. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. In some embodiments, an effective amount is an amount sufficient to increase a subject's immune response against the tumor such that tumor growth and / or size and / or metastasis is reduced, delayed, ameliorated, and / or prevented. An effective amount can be administered in one or more administrations. In some embodiments, administration of an effective amount (e.g., of a composition comprising mRNA) may (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow to some extent, or stop cancer cell invasion into peripheral organs; (iv) inhibit (e.g., slow to some extent and / or block or prevent) metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor onset and / or recurrence; and / or (vii) alleviate to some extent one or more symptoms associated with cancer.

[0209] The pharmaceutical compositions of the present disclosure may include salts, buffering agents, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure include one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0210] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0211] The term "excipient" as used herein refers to a substance that may be present in the pharmaceutical compositions of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0212] The term "diluent" refers to an agent that is diluted and / or diluted. Furthermore, the term "diluent" includes any one or more of a fluid, liquid, or solid suspension and / or mixture medium. Examples of suitable diluents include ethanol, glycerol, and water.

[0213] The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, with which an active ingredient is combined to facilitate, enhance, or enable administration of a pharmaceutical composition. As used herein, a carrier may be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure comprises isotonic saline.

[0214] Pharmaceutically acceptable carriers, excipients, or diluents for pharmaceutical use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985).

[0215] Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0216] Routes of Administration of the Pharmaceutical Compositions of the Present Disclosure In one embodiment, the pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally, intranodally, or intramuscularly. In certain embodiments, the pharmaceutical compositions are formulated for local or systemic administration. Systemic administration may include enteral administration, including absorption via the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to administration by any means other than via the gastrointestinal tract, such as by intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for systemic administration. In another preferred embodiment, systemic administration is by intravenous administration.

[0217] Uses of the Pharmaceutical Compositions of the Present Disclosure For example, the RNA described herein, formulated as RNA lipoplex particles, can be used in the therapeutic or prophylactic treatment of diseases in which providing to a subject an amino acid sequence encoded by the RNA has a therapeutic or prophylactic effect.

[0218] The term "disease" refers to an abnormal condition affecting an individual's body. Disease is often interpreted as a medical condition associated with specific symptoms and signs. Diseases can be caused by external factors, such as infection, or by internal dysfunction, such as autoimmune disease. In humans, "disease" is often used more broadly to refer to conditions that cause pain, impairment, distress, social problems, or death in the affected individual or that cause similar problems in those who come into contact with the individual. In this broader sense, disease sometimes includes impairment, incapacity, disability, syndrome, infection, isolated symptoms, deviant behavior, and atypical changes in structure and function, although in other contexts and for other purposes, these may be considered distinct categories. Because suffering from and living with many illnesses can alter one's outlook on life and personality, illnesses typically affect individuals not only physically but also emotionally.

[0219] In the present context, the terms "treatment", "treating" or "therapeutic intervention" relate to the management and care of a subject with the aim of combating a condition, such as a disease or disorder. This term is intended to include the full range of treatments for a given condition from which a subject is suffering, such as the administration of therapeutically effective compounds to alleviate symptoms or complications, slow the progression of a disease, disorder or condition, relieve or reduce symptoms and complications, and / or cure or eliminate a disease, disorder or condition, as well as to prevent a condition, where prevention is to be understood as the management and care of an individual with the aim of combating a disease, condition or disorder and includes the administration of active compounds to prevent the onset of symptoms or complications.

[0220] The term "therapeutic treatment" relates to any treatment that improves the health status and / or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, halt or delay the onset of the disease in an individual, inhibit or delay the onset of the disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual who currently has or has previously had the disease.

[0221] The term "prophylactic treatment" or "preventative treatment" relates to any treatment intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventative treatment" are used interchangeably herein.

[0222] The terms "individual" and "subject" are used interchangeably herein. They refer to a human or another mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that may or may not have a disease or disorder (e.g., cancer), but that may or may not have the disease or disorder. In many embodiments, the individual is a human. Unless otherwise specified, the terms "individual" and "subject" do not denote a particular age and thus encompass adults, elderly people, children, and newborns. In embodiments of the present disclosure, an "individual" or "subject" is a "patient."

[0223] The term "patient" refers to an individual or subject for treatment, particularly an afflicted individual or subject.

[0224] In one embodiment of the present disclosure, the objective is to provide an immune response against cancer cells expressing one or more tumor antigens and to treat cancer diseases involving cells expressing one or more tumor antigens. In one embodiment, the cancer is ovarian cancer. In one embodiment, the tumor antigen is CLDN6, p53, and / or PRAME.

[0225] Pharmaceutical compositions comprising RNA can be administered to a subject to induce an immune response against one or more antigens or one or more epitopes encoded by the subject's RNA, which can be therapeutic or partially or completely protective.Those skilled in the art will understand that one of the principles of immunotherapy and vaccination is based on the fact that immunizing a subject with an immunologically related antigen or epitope for the disease to be treated generates an immune protective response against the disease.Therefore, the pharmaceutical compositions described herein can be applied to induce or enhance immune response.Therefore, the pharmaceutical compositions described herein are useful in the preventive and / or therapeutic treatment of diseases involving antigens or epitopes, particularly ovarian cancer.

[0226] As used herein, "immune response" refers to the integrated body response to an antigen or a cell expressing an antigen, and refers to a cellular immune response and / or a humoral immune response. Cellular immune responses include, but are not limited to, cellular responses directed against cells expressing an antigen and characterized by antigen presentation by class I or class II MHC molecules. Cellular responses involve T lymphocytes, which can be divided into helper T cells (also called CD4+ T cells), which play a central role by regulating the immune response, or killer cells (cytotoxic T cells, CD8+ T cells), which induce apoptosis in infected or cancer cells. + In one embodiment, administering a pharmaceutical composition of the present disclosure induces anti-tumor CD8 T cells against cancer cells that express one or more tumor antigens. + In certain embodiments, the tumor antigen is presented in association with a class I MHC molecule.

[0227] The present disclosure contemplates an immune response that may be protective, defensive, preventative, and / or therapeutic. As used herein, "inducing an immune response (or inducing)" may refer to the absence of an immune response to a particular antigen prior to induction, or to a basal level of immune response to a particular antigen prior to induction that is enhanced after induction. Thus, "inducing an immune response (or inducing)" includes "enhancing an immune response (or enhancing)."

[0228] The term "immunotherapy" relates to the treatment of a disease or condition by inducing or enhancing an immune response. The term "immunotherapy" includes antigen immunization or antigen vaccination.

[0229] The terms "immunization" or "vaccination" refer to the process of administering an antigen to an individual with the intent of inducing an immune response, for example, for therapeutic or prophylactic reasons.

[0230] In one embodiment, the present disclosure contemplates an embodiment in which the RNA lipoplex particles described herein are administered to target spleen tissue. The RNA encodes a peptide or protein containing an antigen or epitope, for example, as described herein. The RNA is taken up by antigen-presenting cells in the spleen, such as dendritic cells, to express the peptide or protein. Following optional processing and presentation by the antigen-presenting cells, an immune response against the antigen or epitope can be generated, resulting in preventive and / or therapeutic treatment of a disease involving the antigen or epitope. In one embodiment, the immune response induced by the RNA lipoplex particles described herein includes presentation of the antigen or fragment thereof, such as an epitope, by antigen-presenting cells, such as dendritic cells and / or macrophages, and activation of cytotoxic T cells through this presentation. For example, the peptide or protein encoded by the RNA, or its processing product, can be presented by major histocompatibility complex (MHC) proteins expressed on antigen-presenting cells. The MHC-peptide complex can then be recognized by immune cells, such as T cells or B cells, resulting in their activation.

[0231] Thus, in one embodiment, the RNA in the RNA lipoplex particles described herein is delivered to and / or expressed in the spleen after administration. In one embodiment, the RNA lipoplex particles are delivered to the spleen to activate antigen-presenting cells in the spleen. Thus, in one embodiment, administration of the RNA lipoplex particles results in delivery of the RNA to and / or expression of the RNA in the antigen-presenting cells. The antigen-presenting cells may be professional or non-professional antigen-presenting cells. The professional antigen-presenting cells may be dendritic cells and / or macrophages, and even more preferably splenic dendritic cells and / or splenic macrophages.

[0232] Thus, the present disclosure relates to RNA lipoplex particles or pharmaceutical compositions comprising RNA lipoplex particles as described herein for inducing or enhancing an immune response, preferably an immune response against ovarian cancer.

[0233] In one embodiment, systemic administration of the RNA lipoplex particles or pharmaceutical compositions comprising the RNA lipoplex particles described herein results in targeting and / or accumulation of the RNA lipoplex particles or RNA in the spleen, but not in the lungs and / or liver. In one embodiment, the RNA lipoplex particles release RNA in the spleen and / or enter cells in the spleen. In one embodiment, systemic administration of the RNA lipoplex particles or pharmaceutical compositions comprising the RNA lipoplex particles described herein delivers RNA to antigen-presenting cells in the spleen. In certain embodiments, the antigen-presenting cells in the spleen are dendritic cells or macrophages.

[0234] The term "macrophage" refers to a subgroup of phagocytes produced by differentiation of monocytes. Activated by inflammation, immune cytokines, or microbial products, macrophages nonspecifically engulf foreign pathogens within the macrophage and kill them through hydrolytic and oxidative attack, resulting in their degradation. Peptides from degraded proteins are displayed on the macrophage cell surface where they can be recognized by T cells and can directly interact with antibodies on the surface of B cells, leading to T and B cell activation and further stimulation of the immune response. Macrophages belong to a class of antigen-presenting cells. In one embodiment, the macrophages are splenic macrophages.

[0235] The term "dendritic cell" (DC) refers to another subtype of phagocyte belonging to the class of antigen-presenting cells. In one embodiment, dendritic cells are derived from hematopoietic bone marrow progenitors. These progenitors first transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation capacity. Immature dendritic cells constantly sample the surrounding environment for pathogens, such as viruses and bacteria. Upon contact with presentable antigens, they are activated to become mature dendritic cells and begin migrating to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, degrade their proteins into small fragments, and upon maturation, present these fragments on their cell surface using MHC molecules. At the same time, they upregulate cell surface receptors that function as coreceptors for T cell activation, such as CD80, CD86, and CD40, greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that directs dendritic cells to migrate through the bloodstream to the spleen or through the lymphatic system to lymph nodes. Here, they function as antigen-presenting cells, activating helper T cells, killer T cells, and B cells by presenting antigens together with non-antigen-specific costimulatory signals. Thus, dendritic cells can actively induce T cell or B cell-related immune responses. In one embodiment, the dendritic cells are splenic dendritic cells.

[0236] The term "antigen-presenting cell" (APC) refers to any of a variety of cells that can display, acquire, and / or present at least one antigen or antigenic fragment on (or at) their cell surface. Antigen-presenting cells can be distinguished into professional and non-professional antigen-presenting cells.

[0237] The term "professional antigen-presenting cells" refers to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules, which are necessary for interaction with naive T cells. When T cells interact with the MHC class II molecule complex on the membrane of the antigen-presenting cell, the antigen-presenting cell produces costimulatory molecules that induce T cell activation. Professional antigen-presenting cells include dendritic cells and macrophages.

[0238] The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules but do so upon stimulation with certain cytokines, such as interferon gamma. Exemplary non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.

[0239] "Antigen processing" refers to the breakdown of an antigen into processing products that are fragments of the antigen (e.g., breakdown of a protein into peptides), and the association (e.g., by binding) of one or more of these fragments with an MHC molecule for presentation to specific T cells by a cell, such as an antigen-presenting cell.

[0240] The term "antigen-associated disease" or "epitope-associated disease" refers to any disease in which an antigen or epitope is involved, for example, a disease characterized by the presence of an antigen or epitope. The antigen- or epitope-associated disease may be a cancer disease or simply cancer. As noted above, the antigen may be a disease-associated antigen, such as a tumor-associated antigen, and the epitope may be derived from such an antigen.

[0241] The term "cancer disease" or "cancer" refers to or describes a physiological condition in an individual that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specifically, examples of such cancer include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, 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, cancer of the genital and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumor, glioma, meningioma, and pituitary adenoma. One particular form of cancer that can be treated by the compositions and methods described herein is ovarian cancer. The term "cancer," according to the present disclosure, also includes cancer metastasis.

[0242] Combination strategies in cancer treatment can be desirable due to the resulting synergistic effects, which can be significantly more potent than the effects of monotherapy approaches. In one embodiment, the pharmaceutical composition is administered with an immunotherapeutic agent. As used herein, "immunotherapeutic agent" refers to any agent that can be involved in activating a specific immune response and / or immune effector function(s). The present disclosure contemplates the use of antibodies as immunotherapeutic agents. Without wishing to be bound by theory, antibodies can achieve therapeutic effects on cancer cells through various mechanisms, including inducing apoptosis, blocking components of signaling pathways, or inhibiting tumor cell proliferation. In certain embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies can induce cell death via antibody-dependent cell-mediated cytotoxicity (ADCC) or can bind to complement proteins, resulting in direct cytotoxicity, known as complement-dependent cytotoxicity (CDC). Non-limiting examples of anti-cancer antibodies and potential antibody targets (in parentheses) that may be used in combination with the present disclosure include abagovomab (CA-125), abciximab (CD41), adecatumumab (EpCAM), afutuzumab (CD20), alacizumab pegol (VEGFR2), altumomab pentetate (CEA), amatuximab (MORAb-009), anatumomab mafenatox (TAG-72), apolizumab (HLA-DR), arcitumomab (CEA), atezolizumab (PD-L1), bavituximab (phosphatidylserine), bectumomab (CD22), belimumab (BAFF), bevacizumab (VEGF-A), bivacizumab mertansine (CD44 v6), blinatumomab (CD19), brentuximab vedotin (CD30TNFRSF8), cantuzumab mertansine (mucin CanAg), cantuzumab mertansine (MUC1), capromab pendetide (prostate cancer cells), carlumab (CNT0888), catumaxomab (EpCAM, CD3), cetuximab (EGFR), sitatuzumab bogatox (EpCAM), cixutumumab (IGF-1 receptor), claudiximab (claudin), clivatuzumab tetraxetan (MUC1), conatumumab (TRAIL-R2), dacetuzumab (CD40), dalotuzumab (insulin -like growth factor I receptor), denosumab (RANKL), detumomab (B lymphoma cells), drozitumab (DR5), ecloneximab (GD3 ganglioside), edrecolomab (EpCAM), elotuzumab (SLAMF7), enavatuzumab (PDL192), ensituximab (NPC-1C), epratuzumab (CD22), ertumaxomab (HER2 / neu, CD3), etaracizumab (integrin ανβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), ficlatuzumab (SCH900105), figitumumab (IGF-1 receptor), framvotumab (glycoprotein 75), fresolimumab (TGF-β), galiximab (CD80), ganitumab (IGF-I), gemtuzumab ozogamicin (CD33), gevokizumab (IL-Ιβ), girentuximab (carbonic anhydrase 9 (CA-IX)), glembatumumab vedotin (GPNMB), ibritumomab tiuxetan (CD20), icrucumab (VEGFR-1), igovoma (CA-125), indatuximab ravtansine (SDC1), intetumumab (CD51), inotuzumab ozogamicin (CD22), ipilimumab (CD152), iratumumab (CD30), labetuzumab (CEA), lexatumumab (TRAIL-R2), ribivirumab (hepatitis B surface antigen), lintuzumab (CD33), lorvotuzumab mertansine (CD56), lucatumumab (CD40), rumiliximab (CD23), mapatumumab (TRAIL-R1), matuzumab (EGFR), mepolizumab (IL-5), milatuzumab (CD74), mitsumomab (GD3 ganglionic Osido), mogamulizumab (CCR4), moxetumomab pasudotox (CD22), nacolomab butafenatox (C242 antigen), naptumomab estafenatox (5T4), namatumab (RON), necitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4), ofatumumab (CD20), olaratumab (PDGF-Ra), onartuzumab (human scatter factor receptor kinase), oportuzumab monatox (EpCAM), oregovomab (CA-125), oxelumab (OX-40), panitumumab (EGFR), patritumab (HER3), pemtumomab (MUC1), pertuzumab (HER2 / neu), pintumomab (adenocarcinoma antigen), pritumumab (vimentin), racotumomab (N-glycolylneuraminic acid), radletumab (fibronectin extra domain B), rafivirumab (rabies virus glycoprotein), ramucirumab (VEGFR2), rilotumumab (HGF), rituximab (CD20), lobatumumab (IGF-1 receptor), samalizumab (CD200), sib Lotuzumab (FAP), siltuximab (IL-6), tabalumab (BAFF), tacatuzumab tetraxetan (α-fetoprotein), taplitumomab paptox (CD19), tenatumomab (tenascin-C), teprotumumab (CD221), ticilimumab (CTLA-4), tigatuzumab (TRAIL-R2), TNX-650 (IL-13), tositumomab (CD20), trastuzumab (HER2 / neu), TRBS07 (GD2), tremelimumab (CTLA-4), tucotuzumab celmoleukin (EpCAM), ublituximab (MS4A1), urelumab (4-1BB), boroximab (integrin α5β1), votumumab (tumor antigen CTAA 16.88), zalutumumab (EGFR), and zanolimumab (CD4).

[0243] In one embodiment, the immunotherapeutic agent is a PD-1 axis binding antagonist. PD-1 axis binding antagonists include, but are not limited to, PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists. Other names for "PD-1" include CD279 and SLEB2. Other names for "PD-L1" include B7-H1, B7-4, CD274, and B7-H. Other names for "PD-L2" include B7-DC, Btdc, and CD273. In some embodiments, the PD-1 binding antagonist is a molecule that inhibits PD-1 from binding to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PD-L1 and / or PD-L2. In another embodiment, the PD-L1 binding antagonist is a molecule that inhibits PD-L1 from binding to its binding partner. In certain embodiments, the PD-L1 binding partner is PD-1 and / or B7-1. In another embodiment, the PD-L2 binding antagonist is a molecule that inhibits PD-L2 from binding to its binding partner. In certain embodiments, the PD-L2 binding partner is PD-1. The PD-1 binding antagonist can be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). Examples of anti-PD-1 antibodies include, but are not limited to, MDX-1106 (nivolumab, OPDIVO), Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA), MEDI-0680 (AMP-514), PDR001, REGN2810, BGB-108, and BGB-A317.

[0244] In one embodiment, the PD-1 binding antagonist is an immunoadhesin comprising the extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region. In one embodiment, the PD-1 binding antagonist is AMP-224 (also known as B7-DCIg, which is PD-L2-Fc), a fused soluble receptor described in WO 2010 / 027827 and WO 2011 / 066342.

[0245] In one embodiment, the PD-1 binding antagonist is an anti-PD-L1 antibody, including but not limited to YW243.55.S70, MPDL3280A (atezolizumab), MEDI4736 (durvalumab), MDX-1105, and MSB0010718C (avelumab).

[0246] In one embodiment, the immunotherapeutic agent is a PD-1 binding antagonist. In another embodiment, the PD-1 binding antagonist is an anti-PD-L1 antibody. In an exemplary embodiment, the anti-PD-L1 antibody is atezolizumab.

[0247] Citation of documents and tests referenced herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements regarding the contents of these documents are based on information available to applicant and do not constitute an admission as to the accuracy of the contents of these documents.

[0248] The following description is presented to enable those skilled in the art to make and use various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Accordingly, the various embodiments are not intended to be limited to the examples described and shown herein, but are to be accorded the scope consistent with the appended claims. [Example]

[0249] Example 1 Intravenous vaccine for treating ovarian cancer - Patent Application 20070122999 The vaccines described herein are serum-stable RNA-lipoplexes (RNA (LIP) It consists of RNA separately complexed with liposomes to generate a tumor-associated antigen (TAA)-targeting RNA, which can be administered together with RNA encoding a helper epitope to boost the resulting immune response. (LIP) targets antigen-presenting cells (APCs) in lymphoid organs, resulting in efficient stimulation of the immune system.

[0250] The ovarian cancer (OC) vaccine consists of three different RNA cancer vaccines: RBL005.2, RBL008.1, and RBL012.1. Each RNA cancer vaccine consists of one RNA drug substance encoding the antigen claudin 6 (CLDN6), the universal tumor-associated antigen p53, and the "preferentially expressed melanoma antigen" (PRAME), respectively.

[0251] Targets were included (WH_ova1) based on the following criteria: Low or absent expression in toxicity-related organs assessed by quantitative real-time RT-PCR (RT-qPCR) (Figure 29). Expression in a significant proportion of tumors as assessed by quantitative real-time RT-PCR (RT-qPCR) (Figure 29). The ability to induce an antigen-specific immune response as evidenced by published literature and / or assessed by in vitro stimulation of human T cells with antigen-specific TCRs and / or in vivo priming of HLA-transgenic mice.

[0252] Furthermore, the suitability of p53, a well-known tumor suppressor gene found to be mutated or overexpressed in more than 50% of all cancers, as a target antigen was considered a universal tumor-associated antigen for ovarian tumors.

[0253] Thus, all of WH_ova1's RNA medicines may confer tumor-selective immune-mediated benefits to patients with a low risk of adverse reactions.

[0254] To boost the resulting immune response, each RNA is co-administered with an additional RNA (RBLTet.1) encoding the helper epitopes p2 and p16 (P2P16) derived from tetanus toxoid (TT).

[0255] RNA-lipoplex (RNA (LIP) ) can be prepared prior to administration according to established protocols. The RNA pharmaceutical can be provided in three RNA pharmaceutical vials. For each of the three RNA pharmaceuticals, one vial of RBLTet.1 can be additionally provided. Sterile isotonic NaCl solution (e.g., 40 mL, 0.9%) as the primary diluent and liposomes as the excipient can also be delivered. RNA (LIP) Specialized materials such as syringes and cannulas for preparation, as well as RNA (LIP) Further isotonic saline solution to allow further dilution of may be supplied as a clinical standard.

[0256] drug substance RBL005.2, β-S-ARCA(D1)-hAg-Kozak-CLDN6-2hBgUTR-A30L70 Encoded antigen: human claudin 6 (Gene ID(HG19):uc002csu.4) RBL008.1, β-S-ARCA(D1)-hAg-Kozak-sec-GS-p53-GS-MITD-2hBgUTR-A30L70 Encoded antigen: human p53 (Gene ID(HG18):uc002gij.2) RBL012.1, β-S-ARCA(D1)-hAg-Kozak-sec-GS-PRAME-GS-MITD-2hBgUTR-A30L70 Encoded antigen: human PRAME (Gene ID(HG19):uc002zwg.3) RBLTet.1, β-S-ARCA(D1)-hAg-Kozak-sec-GS-P2P16-GS-MITD-2hBgUTR-A30L70 Encoded antigens: Tetanus p2 and p16 (UniProtKB / Swiss-Prot identifier P04958)

[0257] The active ingredient of each drug substance is a single-stranded 5'-capped mRNA that is translated into its respective protein upon entry into antigen-presenting cells (APCs). Figure 1 illustrates the general structure of antigen-encoding RNAs, determined by the respective nucleotide sequences of linearized plasmid DNA used as templates for in vitro RNA transcription. In addition to the wild-type or codon-optimized sequence encoding the target protein, each RNA contains common structural elements optimized for maximum RNA potency with respect to stability and translation efficiency (5' cap, 5'-UTR, 3'-UTR, poly(A) tail; see below). Additionally, a sec (secretory signal peptide) and an MITD (MHC class I transport domain) are fused to the antigen-coding region so that each element is translated as an N- or C-terminal tag, respectively. Both fusion tags have been shown to improve antigen processing and presentation. For some antigens listed below, one or both fusion tags are not necessary and are therefore omitted.

[0258] mRNA cap β-S-ARCA (D1) (Figure 2) is utilized as a specific capping structure at the 5' end of the RNA drug substance.

[0259] mRNA sequence The general sequence elements of an mRNA as displayed in Figure 1 are shown below.

[0260] Codon-optimized sequences encoding the target proteins CLDN6, p53, PRAME, and P2P16 are used. In the case of P2P16, the two epitopes are fused together by a short linker peptide consisting primarily of the amino acids glycine (G) and serine (S), a peptide commonly used in fusion proteins.

[0261] hAg-Kozak: 5'-UTR sequence of human α-globin mRNA with an optimized "Kozak sequence" to increase translation efficiency.

[0262] sec / MITD: A fusion protein tag derived from the sequence encoding the human MHC class I complex (HLA-B51, haplotype A2, B27 / B51, Cw2 / Cw3) that has been shown to improve antigen processing and presentation. sec corresponds to a 78-bp fragment encoding the secretory signal peptide, which directs the translocation of the nascent polypeptide chain into the endoplasmic reticulum. MITD corresponds to the transmembrane and cytoplasmic domains of the MHC class I molecule, also known as the MHC class I transport domain. Note that CLDN6 has its own secretory signal peptide and transmembrane domain. Therefore, no fusion tag was added to this antigen.

[0263] GS / Linker: A sequence encoding a short linker peptide, consisting primarily of the amino acids glycine (G) and serine (S), commonly used in fusion proteins.

[0264] 2hBgUTR: two re-repeated 3'-UTRs of human β-globin mRNA placed between the coding sequence and the poly(A) tail to ensure higher maximal protein levels and long-term persistence of the mRNA.

[0265] A30L70: A 110-nucleotide-long poly(A) tail consisting of a stretch of 30 adenosine residues, followed by a 10-nucleotide linker sequence and another 70 adenosine residues. This poly(A) tail sequence was designed to increase RNA stability and translation efficiency in dendritic cells.

[0266] The complete nucleotide sequences of the four RNA drug substances RBL005.2, RBL008.1, RBL012.1 and RBLTet.1 are shown below:

[0267] Nucleotide sequence of RBL005.2. The nucleotide sequence is shown with individual sequence elements indicated in bold. Additionally, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (* = stop codon).

[0268] [Table 2] TIFF2025122023000026.tif192153

[0269] Nucleotide sequence of RBL008.1. The nucleotide sequence is shown with individual sequence elements indicated in bold. Additionally, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (* = stop codon).

[0270] [Table 3] TIFF2025122023000028.tif205153TIFF2025122023000029.tif202153TIFF2025122023000030.tif100153

[0271] Nucleotide sequence of RBL012.1. The nucleotide sequence is shown with individual sequence elements indicated in bold. Additionally, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (* = stop codon).

[0272] [Table 4] TIFF2025122023000032.tif207153TIFF2025122023000033.tif215153TIFF2025122023000034.tif213153

[0273] Nucleotide sequence of RBLTet.1. The nucleotide sequence is shown with individual sequence elements indicated in bold. Additionally, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (* = stop codon).

[0274] [Table 5] TIFF2025122023000036.tif137153

[0275] Individual plasmid DNAs for the production of RBL005.2 (pST1-hAg-Kozak-CLDN6-2hBgUTR-A30L70), RBL008.1 (pST1-hAg-Kozak-sec-GS-P53-GS-MITD-2hBgUTR-A30L70), RBL012.1 (pST1-hAg-Kozak-sec-GS-PRAME-GS-MITD-2hBgUTR-A30L70), and RBLTet.1 (pST2-hAg-Kozak-sec-GS-P2P16-GS-MITD-2hBgUTR-A30L70) were generated using a combination of gene synthesis and recombinant DNA techniques. In addition to the sequence encoding the transcribed region, the plasmid DNA contains a promoter for T7 RNA polymerase, a recognition sequence for the class IIs endonuclease used for linearization, a kanamycin resistance gene, and an origin of replication (ori).

[0276] Plasmid DNA pST1-hAg-Kozak-sec-GS-SIINFEKL-GS-MITD-2hBgUTR-A30L70 served as the DNA template for RBL008.1 and RBL012.1, respectively, and as the starting point for the generation of plasmid DNA pST2-hAg-Kozak-sec-GS-SIINFEKL-GS-MITD-2hBgUTR-A30L70 for RBL Tet.1. Plasmid DNA pST1-hAg-Kozak-2hBgUTR-A30L70 served as the starting point for the generation of RBL005.2. Because this antigen contains its own secretory signal peptide and transmembrane domain, no fusion tag had to be added.

[0277] The vector map is shown in Figures 3-6. Note that the plasmid DNA encoding RBLTet.1 contains an additional 800 base pair sequence inserted between the replication origin and the T7 promoter. This modification of the plasmid backbone is based on our observation that for short mRNAs (i.e., mRNAs with a total length of less than 1,200 nucleotides), the poly(A) tail-encoding region of the corresponding plasmid DNA is partially unstable when propagated in E. coli. Subsequently, we identified the distance between the replication origin (or a nearby sequence element) and the poly(dA:dT) sequence as a critical parameter for the stability of the DNA sequence encoding the poly(A) tail. Therefore, we inserted an 800 base pair sequence between the replication origin and the T7 RNA polymerase promoter to generate pST2 plasmid DNA for constructing a plasmid encoding RBLTet.1, thereby mimicking a longer RNA-coding sequence in terms of the distance between the upstream sequence element and the poly(dA:dT) sequence.

[0278] To obtain the starting material for RNA transcription, the circular plasmid DNA was linearized with a suitable restriction enzyme. Here, the enzyme Eam1104I (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) was chosen. This is because linearization with such a class IIs restriction endonuclease allows the transcription of RNA encoding a "free" poly(A) tail, i.e., without additional nucleotides at the 3' end. We were able to demonstrate that this resulted in higher protein expression.

[0279] RNA (LIP) The product can be prepared in a three-step procedure, including (i) addition of RBLTet.1 RNA, (ii) dilution of the RNA mixture with NaCl solution, and (iii) addition of liposomes to form an RNA-lipoplex. The synthetic cationic lipid DOTMA and the naturally occurring phospholipid DOPE can be used as lipids.

[0280] The intravenous product is a formulation with pharmaceutical and physiological properties that enable selective targeting of RNA to APCs, primarily located in the spleen. RNA-lipoplexes are formed by first condensing RNA in an appropriate ionic environment, followed by incubation with positively charged liposomes.

[0281] For RNA condensation, various monovalent and divalent ions, peptides, and buffers were applied at various concentrations. Monovalent ions such as sodium and ammonium were tested at concentrations up to 1.5 M. Divalent ions, especially Ca, were also tested. 2+ , Mg 2+ , Zn 2+ and Fe 2+ was tested at concentrations up to 50 mM. Additionally, various commercially available buffers were tested.

[0282] RNA (LIP) Liposomes containing cationic lipids and different co-lipids were extensively tested for formation. Liposomes with different charge, phase state, size, lamellae, and surface functionalization were investigated. Only lipid components available in GMP grade and previously tested in clinical trials or used in market-approved products were considered (Figure 7).

[0283] Using the liposome components described above, RNA-lipoplexes were constructed with different cationic lipid:RNA and different charge ratios, where the charge ratio was calculated from the number of positive charges from the lipid and the number of negative charges from the RNA nucleotides, i.e., the RNA phosphate groups. More specifically, the charge ratio was calculated as follows: We assumed that RNA consisted of nucleotides with an average molar mass of 330 Da, each carrying one negatively charged phosphate group. Therefore, a 1 mg / mL solution of RNA would occupy approximately 3 mM of negative charge. Meanwhile, we considered one positive charge per monovalent cationic lipid. For example, the cationic lipid DOTMA has a molar mass of 670 Da, and liposomes with a DOTMA concentration of 2 mg / mL were considered to have a positive charge concentration of 3 mM. Therefore, in this case, the (+:-) charge ratio was considered to be 1:1. The concentration of uncharged co-lipids, which were present in most cases, did not contribute to this calculation.

[0284] The chemical and physicochemical properties (i.e., chemical composition, particle size, and zeta potential) of the liposomes and RNA-lipoplexes formed based on this method were thoroughly investigated. For regular control of product quality, the chemical composition was determined by HPLC analysis, and the particle size was measured by photon correlation spectroscopy (PCS). The zeta potential was also measured by PCS. Furthermore, electron microscopy, small-angle X-ray scattering (SAXS), calorimetry, field-flow fractionation, analytical ultracentrifugation, and spectroscopic techniques were applied during formulation development. This procedure identified optimized formulations for further pharmaceutical development.

[0285] Appropriate liposome formulations were tested in vitro and in vivo. To optimize targeting to APCs, primarily located in the spleen, expression of luciferase as a reporter gene was monitored in vivo. We were able to demonstrate that colloidal, stable nanoparticle lipoplex formulations with distinct particle sizes can be formed with an appropriate charge ratio (excess negative or positive charge). Furthermore, negatively charged luciferase-RNA-lipoplex formulations have been shown in vivo to exhibit high selectivity for the spleen, which serves as a reservoir for professional APCs. By varying the charge ratio, the selectivity of luciferase expression in the spleen can be tailored as desired, as shown in Figure 8, which shows the organ selectivity of RNA-lipoplexes from the same liposomes with different cationic lipid-to-RNA ratios. The observation that negatively charged lipoplexes target splenic APCs was verified with multiple lipid compositions. Liposomes composed of the cationic lipid DOTMA and the helper phospholipid DOPE were identified as the most suitable in terms of particle properties for forming RNA-lipoplexes suitable for the intended splenic APC targeting. Optimized selectivity and efficacy for splenic targeting were observed with a slight excess of negative charge, resulting from excess RNA. RNA lipoplexes with slightly more positive charge, which showed comparable efficacy, were too colloidally unstable and had a high risk of aggregation and precipitation under these conditions, making them unsuitable for pharmaceutical development.

[0286] Furthermore, for a given RNA, we were able to show that the biological activity of the formulation increased with the particle size of the RNA lipoplex. More specifically, we were able to show that RNA-lipoplexes formed from larger liposomes (e.g., approximately 400 nm) themselves exhibited greater and higher biological activity than those prepared using smaller liposomes (e.g., approximately 200 nm) (Figure 9). Therefore, liposomes larger than 200 nm were used to prepare RNA. (LIP) Used for formation.

[0287] Based on the above findings, RNA (LIP)A robust and reproducible protocol for preparation was developed. By using specified components and a defined preparation protocol, RNA-lipoplexes are formed by self-assembly to achieve the intended physicochemical properties and biological activity. As an example, the particle sizes of RNA-lipoplexes from various independent preparations are shown in Figure 10. The limited spread in the resulting RNA-lipoplex particle sizes demonstrates the robustness of the reconstitution procedure.

[0288] RNA (LIP) To determine the limits and robustness of the preparation, particle sizes were measured for different charge ratios (mixing ratios between cationic lipids and nucleotides) ranging from 1.0:2.0 to 1.9:2.0. Figure 11 shows the results from size measurements of RNA lipoplexes after mixing liposomes and RNA at various ratios. (LIP) Particle size was measured at various time points after preparation. Ratios between 1.0:2.0 and 1.6:2.0 yielded comparable particle sizes that remained stable over time. At ratios of 1.7:2.0 and above, the size of the RNA-lipoplexes increased both initially and over time, with this finding being most pronounced after 24 hours.

[0289] Based on these data, a charge ratio of 1.0:2.0 to 1.6:2.0 was considered appropriate for obtaining acceptable particle characteristics of the RNA-lipoplex product. At higher ratios (1.7:2.0 or higher), particle size increased and product quality could deviate. While no changes in particle characteristics were observed toward lower charge ratios, lower ratios were not considered due to potentially lower activity in that range (data not shown). The experiment was repeated with a range of 1.1:2.0 to 1.6:2.0, and in addition to size measurements (Figure 12A), biological activity was examined (Figure 12B). Consistent with previous experiments, particle size remained virtually constant. The same was true for biological activity (luciferase expression). In summary, all tested RNA-lipoplexes delivered RNA to APCs without significant changes in physicochemical properties or biological performance. Therefore, the range of 1.1:2.0 to 1.6:2.0 appears to yield RNA lipoplexes of comparable quality.

[0290] Example 2 Non-clinical data In this example, RNA (LIP) We review the preclinical studies conducted to clarify the mechanism of action, pharmacodynamics, antitumor activity, pharmacokinetics, and potential toxicity of the vaccine. The most important findings are summarized in Table 1.

[0291] The first part of this section provides a brief overview of the scientific rationale and preparatory work for the development of the vaccine platform, including an overview of the target properties of the WAREHOUSE_ova1 target antigen as well as the tetanus toxoid-derived helper epitopes p2 and p16 (section 1).

[0292] In the following sections, we will discuss (i) the induction of antigen-specific T cells in vitro and in vivo, (ii) BioNTech's RNA (LIP) (iii) the transient immunomodulatory effects of vaccination; and (iii) RNA (LIP) RNA, including data on antigen-specific T cell stimulation and antitumor activity of vaccination (LIP) Primary pharmacodynamic studies of the drug are described (Section 2).

[0293] Secondary pharmacodynamic studies included RNA analysis of inflammatory cytokines. (LIP) The results of testing for mediated induction of ribosomal RNA are presented in Section 3. A pharmacodynamic non-GLP study in cynomolgus monkeys was performed to refine the analysis of cytokine kinetics and hematological changes observed in mice. (LIP) In vitro studies analyzing cytokine secretion from human and cynomolgus monkey blood cells after incubation with the preparations are also summarized in this section.

[0294] Respiratory and neurological safety pharmacology studies are summarized in Section 4.

[0295] A brief overview of in vivo biodistribution and metabolism is provided in Section 5.

[0296] A GLP-compliant repeat-dose toxicity study incorporating immunotoxicity testing was performed and is presented and discussed in Section 6.

[0297] [Table 6] TIFF2025122023000038.tif34153

[0298] Section 1: Scientific basis and preparatory work Sequence features that improve RNA translation and intracellular stability The RNA vaccine platform targets antigen-specific CD8 + and CD4 + They have been systematically developed and optimized over the past decade to aid in the safe and efficient induction of T cell responses.

[0299] The active ingredient (drug substance) is a single-stranded, capped messenger RNA (mRNA) that is translated into protein antigens upon entry into dendritic cells (DCs). Our Ribological® RNA vaccine format was optimized through the use of (i) modified cap analogs for stabilization of translationally active RNA, (ii) optimized 5'- and 3'-UTRs to increase stability and RNA translation, (iii) signal peptides and MITD sequences to improve MHC class I and II antigen processing, and (iv) extended free-end poly(A) tails to further enhance RNA stability and translation efficiency. Table 2 provides an overview of the various structural elements that have been subject to optimization and are currently undergoing clinical trials.

[0300] [Table 7]

[0301] Targeting antigen-encoding RNA to lymphocyte-resident antigen-presenting cells For systemic delivery of RNA to dendritic cells, individual RNA medicines from W_ova1 are formulated with liposomes to form RNA-lipoplexes (RNAs) that allow for intravenous administration. (LIP) ) and most importantly, RNA (LIP) The formulation has been engineered to protect the RNA from degradation by plasma RNases and optimized for selective delivery of the formulated RNA DP to antigen-presenting cells (APCs) residing primarily in the spleen (Figure 13) and other lymphoid organs (Figure 14), where selective uptake of RNA by dendritic cells and macrophages has been shown.

[0302] Once the RNA-lipoplex reaches APCs in the spleen, the mechanism of action is to deliver the intralymph node-applied RNA vaccine (RNA) formulated in Ringer's solution to the lymph nodes. (RIN) ) and RNA (LIP) Antigen-specific CD8 expression is further supported by the immune-stimulatory environment within the spleen induced by intravenous injection of the product. + and CD4 + It results in a potent induction of T cell responses as well as T cell memory.

[0303] Induction of antigen-specific CD8+ and CD4+ T cell responses and T cell memory RNA uptake and translation are prerequisites for peptide processing and presentation on APCs. (LIP) The efficacy of immunization was determined in study number STR-30207-021. For this purpose, naive C57BL / 6 mice were injected with liposomally formulated RNA encoding the immunodominant epitope of chicken ovalbumin (SIINFEKL). (LIP) The SIINFEKL-specific CD8 + Flow cytometric monitoring of T cells demonstrated a significant expansion of antigen-specific T cells after iv immunization (Figure 15).

[0304] After the end of the repeated immunization scheme, antigen-specific CD8 +A decrease in T cell frequency was observed. To assess whether memory T cells were formed during this period, SIINFEKL-RNA was administered 42 days after the last immunization. (LIP) Mice were restimulated with IgG, which led to a rapid expansion of antigen-specific memory T cells, detected at day 62, and RNA (LIP) Evidence was obtained for the formation of T cell memory upon immunization.

[0305] The schedules were further explored in Study No. STR-30207-015, which demonstrated that a reduced-intensity vaccination schedule in week 1, omitting the vaccination on day 4 (initially day 3), resulted in a similar size of antigen-specific immune response, and indicated that a vaccination schedule of six (rather than eight) vaccinations at initial one-week intervals and on days 1, 8, 15, 21, 29, and 43 appears sufficient for adequate induction of antigen-specific T cells.

[0306] Pharmacology RNA (LIP) The mechanism of action of vaccination relies on (i) the recruitment of antigen-specific T lymphocytes after presentation of peptides derived from RNA-encoded antigens by professional APCs, and (ii) TLR-mediated immunomodulatory effects, which result in cell activation and induction of inflammatory cytokines such as type I interferon, thereby enhancing the efficacy of vaccination. Intravenously injected RNA lipoplexes home to secondary lymphoid tissues, including the spleen, lymph nodes, and bone marrow, where they are rapidly taken up by professional APCs.

[0307] In Section 2, we will discuss (i) the activation and expansion of target antigen-specific T cells upon immunization with WAREHOUSE RNA encoding cancer antigens, and (ii) the RNA in the cell activation process accompanied by inflammatory cytokine induction. (LIP) associated induction, and (iii) WAREHOUSE antigen RNA (LIP) The cytocidal and antitumor effects of vaccination are reported.

[0308] RNA (LIP)Potential secondary effects of vaccine administration, e.g., RNA (LIP) Extensive in vitro and in vivo studies were performed to investigate the inflammatory cytokine induction and hematological changes caused by the intended immunomodulatory effects of .

[0309] Section 3 discusses a series of studies assessing the degree of cellular activation of human peripheral blood cells (PBMCs) and blood cells in heparinized whole blood. Furthermore, we demonstrate the extent of vaccination-induced cytokine induction and hematological changes in cynomolgus macaques treated with doses exceeding the highest intended clinical dose in humans. Finally, we perform a side-by-side comparison of in vitro cytokine induction in blood samples from human donors and cynomolgus macaques, and use the data generated in these studies to support ongoing clinical trials in malignant melanoma (RB_0003-01 / Lipo-MERIT) and RNAi. (LIP) Supported the definition of a safe starting dose for other trials investigating immunotherapy.

[0310] RNA (LIP) Section 3 provides an overview of nonclinical studies using human blood cells, mice, and cynomolgus monkeys as test systems to evaluate the secondary pharmacodynamics of .

[0311] The inventors take the position that the secondary pharmacodynamic effects observed with the liposomally formulated RNA in the study are not sequence dependent, and therefore the presented studies are equally applicable to the RNA pharmaceuticals used in this study.

[0312] [Table 8]

[0313] Section 2: Primary Pharmacodynamics RNA from WH_ova1 and other WAREHOUSE RNAs (LIP)To demonstrate the immunogenicity of the vaccination, several in vitro and in vivo experiments were performed. In vitro experiments were performed using selected WAREHOUSE RNA RBL005.2 and antigen-specific T cells from healthy volunteers restimulated with transfected or peptide-primed autologous dendritic cells. Human leukocyte antigen (HLA)-A * 0201 and (HLA)-DRB1 * RNAi assays were performed in vivo using A2 / DR1 mice expressing 01 and all WAREHOUSE RNAi. (LIP) The immunogenicity of the vaccination was demonstrated.

[0314] In vitro stimulation of antigen-specific T cells with selected warehouse antigens To exemplarily analyze the immunogenicity of RBL005.2 against WH_ova1 RNA in a human setting, we used autologous mature DCs (mDCs) transfected with research-grade antigen-encoding mRNA to stimulate CD8+ cells from healthy donors. + T cells were primed in vitro against RBL005.2 (Report_CG_14_001_B). After three weekly stimulations, antigen-specific CD8 + T cells were detected based on specific MHC-dextramers staining. As shown in Figure 17A, CD8 + 0.462% of T cells are HLA-A * 02 / RBL005.2 91-99 The CD8 T cells specifically bound to dextramer, but this was not the case for T cells primed against a control antigen. + RBL005.2-specific T cells were sorted in multiwell plates, and the corresponding TCR genes were cloned and validated by IFN-γ secretion assay. One TCR was shown to mediate specific recognition of K562-A2 cells transfected with RBL005.2 or pulsed with RBL005.2-derived peptides (Figure 17B).

[0315] Additionally, the ability of WAREHOUSE RNA drug RBL005.2 to generate surface-expressed MHC class I epitopes was exemplarily tested after electroporation of the RNA into human DCs. * 0201-restricted epitope ALFGLLVYL(CLDN6 91-99 Isolated human CD8 carrying the α and β chains of the T cell receptor (TCR) specific for + Antigen-specific cell activation was determined by co-incubation of T cells with transfected DCs for 24 hours. Antigen-specific cell activation was analyzed by IFN-γ Bio-plex bead assay (Bio-Rad Laboratories) of cell culture supernatants. To account for donor diversity, two different donors were used for each test article. ATM-quality RNA was used. Experiments were performed using up to 16 μg of RNA for electroporating DCs (Test Report Number: STR_21591_003).

[0316] Cells from both donors tested were able to produce IFN-γ after T cell stimulation with RBL005.2, and a clear dose-dependence could be demonstrated in both experiments (Figure 18).

[0317] In conclusion, RBL005.2 is translated and processed by human DCs, which then present MHC class I-restricted peptides and induce antigen-specific CD8 + It can efficiently induce effector cytokine secretion by T cells in a dose-dependent manner.

[0318] In vivo stimulation of antigen-specific T cells with WAREHOUSE RNA To obtain more information about the in vivo induction of antigen-specific T cells by RBL005.2, RBL008.1, RBL012.1, and RBLTet.1, we used CTM quality RNA. (LIP) The product was prepared. The liposome components used in the test were of ATM quality.

[0319] RNA (LIP)The product was purified by human leukocyte antigen (HLA)-A * 0201 and (HLA)-DRB1 * These were intravenously injected into transgenic mice engineered to express 01. These mice could be used to examine the priming and expansion of T cells specific for HLA-restricted epitopes in vivo. A2 / DR1 mice were vaccinated four to five times by injection of 30 μg of each antigen RNA complexed with liposomes (HED: 7.14 mg), followed by splenocyte isolation (5 days after the final vaccination). After restimulation with bone marrow-derived dendritic cells (BMDCs) electroporated with antigen-specific peptides or RNA, the priming efficiency of the test substances was evaluated by an IFN-γ ELISPOT assay.

[0320] For all antigens, RNA (LIP) Four vaccinations with the preparation primed specific T cell responses in all treated animals. T cells expressed the defined cognate HLA-A * After restimulation with BMDCs electroporated with 0201-restricted peptide or RNA, they were able to produce IFN-γ in an ELISPOT assay (Figure 19). All WAREHOUSE RNAs were able to induce strong immune responses in A2 / DR1 mice.

[0321] In summary, these studies demonstrate that the RNA encoding the WAREHOUSE antigen RNA (LIP) demonstrate the in vivo ability to induce de novo antigen-specific T cell responses in a human MHC background.

[0322] Improving immune responses using RBLTet.1 As known from the literature and our own preclinical data, the TT-derived helper epitopes p2 and p16 mediate autoantigen-specific CD8 +For the W_ova1 approach, p2 and p16 sequences were used as independent RNAs (i.e., RBLTet.1) formulated with each tumor antigen RNA to disrupt the tolerance mechanism for T cells. (LIP) Both RNAs are combined into one RNA (LIP) By formulating the product, tumor antigens and tetanus epitopes are presented by the same DCs and then CD4 + It is ensured that the T cells can help prime tumor-specific T cells.

[0323] To obtain information about the validity of this concept, we tested the in vivo induction of antigen-specific T cells against mouse self-antigens. For this purpose, C57BL / 6 mice were treated with RNA encoding mouse 5,6-dihydroxyindole-2-carboxylic acid oxidase (Tyrp1) alone (30 μg) and in combination with p2 and p16 as independent RNAs (RBLTet.1) at molar ratios of 4:1, 8:1, and 16:1 (3.1, 1.6, and 0.8 μg of RBLTet.1 RNA, respectively). (LIP) The mice were immunized with the vaccine. From previous studies, it is known that the p2 and p16 sequences can induce T cell responses in C57BL / 6 mice. (LIP) Animals were immunized three times by intravenous injection of 1000 mg of IFN-γ. As a primary endpoint, the priming efficiency of the test substances was assessed by an IFN-γ ELISPOT assay, using each peptide to detect specific T cell responses against the major Tyrp1 MHC class I epitope as well as the p2 and p16 epitopes. Immunization with the antigen resulted in approximately 360 mg of IFN-γ. + Spot / 5×10 5 The RNAi-containing IgG induced an immune response in splenocytes (Fig. 20A). (LIP) The addition of RBLTet.1 during the preparation improved the IFN-γ activity (average of 640, 670, and 605, respectively). + Spot / 5×10 5Responses to the p2 and p16 epitopes appeared to be dose-dependent, averaging 730, 490, and 220 IFN-γ responses, respectively. + Spot / 5×10 5 It was splenocytes.

[0324] These data suggest that co-formulation of RNA encoding a TAA with the helper epitope RNA RBLTet.1 can improve the immune response to the antigen even at low molar ratios.

[0325] In vivo antitumor activity of antigen-specific T cells induced by model antigen RNA Due to the known challenges of identifying mouse tumor models, further studies examining the WH_ova1 antigen were not performed due to the lack of mouse homologs of these antigens. Instead, tumor models suitable for the SIINFEKL epitope from ovalbumin, E6 / E7 antigens from human papillomavirus, and gp70 were developed as models of foreign and self-antigens for vaccination, respectively.

[0326] RNA (LIP) A summary of the in vivo antitumor effects induced by IL-1 is shown in Table 3. [Table 9]

[0327] Induction of cell activation processes In addition to its characteristic of encoding protein antigens, RNA IMP exerts immunomodulatory effects based on its ability to induce cell activation processes. From the literature and our own studies, there is good evidence that RNA is a ligand for human Toll-like receptors (TLRs) and can therefore induce immunomodulatory effects. When in vitro transcribed RNA is taken up by cells, recognition by TLRs occurs in the endosomal compartment, where these receptors are primarily localized. This initiates a cascade of signaling events, leading to the activation of our RNA. (LIP)This ultimately leads to DC activation and maturation, as demonstrated by the maturation of splenic DCs after intravenous vaccination in mice. A further consequence of these immunomodulatory effects is the subsequent activation of splenic T cells, B cells, NK cells, and macrophages, as well as the reversible induction of proinflammatory cytokines.

[0328] Most importantly, influenza hemagglutinin HA-RNA (LIP) Injection of a model antigen encoded by IFN-α showed a strong induction of IFN-α in mice (Fig. 21A), and its splenic origin was demonstrated in splenectomized mice (Fig. 21B). Notably, the induction of IFN-α was observed in RNA (LIP) Liposomes alone did not induce IFN-α in mice (Test Report STR-30207-005).

[0329] Interestingly, RNA (LIP) The activation of various immune cells in the spleen (Figure 22A) and systemic IFN-α (Figure 22B) observed with IFN-α was abolished when pseudouridine-modified HPLC-purified RNA (which was previously reported to be non-immunogenic) was used. These results support the conclusion that IFN-α activation is a potent anti-inflammatory agent. (LIP) provides further evidence that the immunostimulatory activity of is derived from its RNA component (Test Report STR-30207-019).

[0330] RNA (LIP) The transient cell activation and cytokines observed in vaccine-treated mice are consistent with our findings that RNA vaccines can bind to and trigger TLRs. Other researchers have also shown that RNA formulated as particles and in aqueous solutions can activate TLRs. TLR activation has been shown to induce lymphopenia and trigger type I interferon-dependent recirculation events of leukocytes. Consistent with this, activation of dendritic cells and other spleen cell populations was reported in our IMPD for RB_0003-01 / Lipo-MERIT trial, which also showed TLR7 activation. - / - Mouse or IFNAR- / - In mice, the effect was significantly impaired (Test Report No. STR-30207-005). Therefore, the IFNAR effect of four liposome-formulated RNAs was significantly impaired. - / - Tests in mice were performed using intravenous RNA (LIP) We demonstrated that the transient hematological changes observed after delivery were primarily mediated by downstream effects of IFN-α (FIG. 23).

[0331] Higher doses of RNA in mice (Section 6) and cynomolgus monkeys (Section 3) (LIP) Non-clinical in vivo studies using RNA lipoplexes revealed that treatment with RNA lipoplexes was associated with transient induction of inflammatory cytokines, transient hematological changes, and transient elevation of liver enzymes. The increase in liver enzymes was due to the RNA rather than a toxic reaction to synthetic lipids or nanoparticles in the liver. (LIP) Further non-clinical studies were carried out applying non-immunogenic RNA complexed with liposomes to assess whether this is a downstream effect of the desired immunomodulatory effects of RNA. To this end, the inventors conducted RNA-complexed with non-immunogenic RNA. (LIP) C57BL / 6 mice were immunized with IFN-α and the subsequent elevation of liver enzymes was assessed (FIG. 24).

[0332] RNA (LIP) Transient assessment of liver enzymes observed in the rats was performed using pseudouridine-modified HPLC-purified non-immunogenic RNA (ni-RNA). (LIP) The formation of ni-RNA significantly neutralized the immunogenicity compared to unmodified RNA (Figure 24A) by using ATM-grade liposomes (batch number: F12 / L2-ATM; EUFETS-13-45-01-F2). The high non-specific deviations of some liver enzyme parameters may be due to stress-related changes in male mice that were not related to the test substance (Test Report STR-30207-019). Furthermore, as can be seen in Figure 22B, the use of ni-RNA significantly neutralized the immunogenicity of RNA. (LIP) When the lipid component RNA was formed, no systemic IFN-α was observed (Figure 24B). (LIP)This provides further evidence that the RNA component, rather than the ATP, is responsible for the observed effects, which can be further confirmed with research-grade lipids (data not shown).

[0333] Section 3: Secondary Pharmacodynamics To investigate potential secondary effects of administering the RNA lipoplex vaccine, such as inflammatory cytokines and hematological changes induced by the intended immunomodulatory effects, extensive in vitro and in vivo studies were performed using human blood cells and cynomolgus monkeys as test systems.

[0334] In the following, RNA (LIP) The extent of vaccination-induced cytokine induction, hematological changes, complement activation, and clinical chemistry were investigated in cynomolgus monkeys treated with doses corresponding to the intended human dose. Additionally, the extent of cytokine release in human and cynomolgus monkey peripheral blood cells (PBMCs) and blood cells in heparinized whole blood in response to RNA lipoplex treatment was investigated in non-GLP and GLP studies.

[0335] Furthermore, as described below, a bioinformatics homology search between the RNA vaccine sequence and the human proteome was performed to exclude potential cross-reactivity of the induced T cells.

[0336] [Table 10]

[0337] In vitro activation of PBMCs and whole blood in healthy human donors and cynomolgus monkeys In addition to its ability to encode protein antigens, RNA possesses immunomodulatory properties derived from its ability to induce cell activation processes via TLR triggering. On the one hand, the immunomodulatory potential of RNA vaccines enhances the induction of antigen-specific T cell responses and should be considered as the primary pharmacodynamic effect. On the other hand, excessive or nonspecific activation of immune cells may result in undesirable secondary effects that should be addressed in preclinical trials.

[0338] To examine the extent of cellular activation of human blood cells, heparinized whole blood and PBMCs (isolated from heparinized whole blood) from four healthy donors were incubated in vitro with an equal mixture of ATM-quality liposomally formulated RBL001.1, RBL002.2, RBL003.1, and RBL004.1. Because TLR activation by RNA is not sequence-dependent, the study was not repeated with WH_ova1 WAREHOUSE RNA.

[0339] RNA for each of the four RNA drugs (LIP) Each RNA was prepared separately according to the clinical formulation protocol. For this first study (Study 1, STR-30207-013), a concentration range of 0.014 to 3.333 μg RNA / mL was selected, corresponding to a human dose of 0.07 mg to 16.65 mg total RNA (Table 4). The primary endpoint was cell activation, determined by secretion of cytokines (IP-10, IFN-α, IFN-γ, TNF-α, IL-1β, IL-2, IL-6, and IL-12) into cell culture medium (PBMCs) or plasma (whole blood) after 6 and 24 hours, respectively.

[0340] [Table 11]

[0341] PBMC RNA (LIP)After incubation with the mixture, there was detectable dose-dependent activation for all eight tested analytes, albeit at highly variable concentration levels. The cytokine response was dominated by five of the eight selected markers: IP-10, IFN-γ, TNF-α, IL-1β, and IL-6 (see Table 5 for a summary). IFN-α, IL-2, and IL-12 showed only slight induction at the highest dose levels tested.

[0342] Conversely, the secretion of IFN-γ, TNF-α, IL-1β, IL-2, and IL-12 was mediated by RNA (LIP) In this study, a dose-dependent increase in the secretion of IP-10 and IL-6 was observed after incubation with IFN-α, which was comparable to the diluent control, and the RNA (LIP) Only low levels of baseline secretion were observed that were not further elevated by incubation with (see Table 5 for summary).

[0343] In summary, the findings in PBMCs showed clear differences compared to whole blood, suggesting a higher sensitivity of the test system using PBMCs. Elevated cytokine levels were detected for all eight tested analytes in PBMCs, whereas cytokine detection was limited to IFN-α, IP-10, and IL-6 when whole blood samples were used as the test system.

[0344] [Table 12]

[0345] In vitro RNA (LIP)To further explore cytokine release in human cells in response to and compare and categorize the in vivo data from the mouse immunotoxicity study (see below) and the cynomolgus monkey study (see below), an additional GLP-compliant in vitro study was conducted at an external CRO (LPT No. 31031). The primary objectives of this study were (i) whether the findings in cynomolgus monkeys were comparable to humans, and (ii) which test system better reflected the cytokine response pattern observed in cynomolgus monkeys in vivo. Study LPT No. 31031 was designed as follows: In vitro induction of inflammatory cytokines in healthy human donors and cynomolgus monkeys was tested in two test systems: PBMC and whole blood. RNA was administered over the same dose range and dose tier as Study No. STR-30207-013. (LIP) The test article was again a mixture of separately prepared ATM-quality liposomally formulated RBL001.1, RBL002.2, RBL003.1, and RBL004.1 RNAs. As mentioned above, data generated using these IVT RNAs also account for WAREHOUSE RNAs, as TLR activation is RNA sequence-independent. In total, four individual samples of each species were analyzed. The primary endpoint was cell activation, determined by secretion of inflammatory cytokines into cell culture medium (PBMCs) or plasma (whole blood) after 6, 24, and 48 hours, respectively.

[0346] The cytokine responses observed are summarized in Table 6 for the whole blood test system and Table 7 for the PBMC test system, respectively.

[0347] [Table 13]

[0348] [Table 14]

[0349] Table 8 shows the results of whole blood analysis of 4 cynomolgus monkeys and 4 healthy donors with 6 different doses of RNA. (LIP) Data from LPT Study No. 31031 are shown, analyzed after 6 and 24 hours of incubation with IL-6. In Study No. STR-30207-013, the proinflammatory cytokines TNF-α, IL-6, and IFN-γ were primarily upregulated in human PBMCs, so analysis focused on these cytokines. As shown, the in vitro cytokine responses in the two species were highly comparable. For IL-6, a 122-fold induction was observed in cynomolgus monkeys and a 108-fold induction in healthy donors after 24 hours of incubation. At the highest dose level, only low levels of TNF-α were detectable in both species. Very low IFN-γ induction was observed only at the highest dose level in cynomolgus monkeys after 24 hours of incubation.

[0350] Most importantly, the strong test article-associated cytokine induction of these three proinflammatory cytokines was observed only at dose levels of 5,500 μg or higher, exceeding the highest intended dose level of 100 μg in patients and more than 100-fold higher than the planned dose for the initial vaccination cycle (=50 μg RNA). Notably, the results from healthy donors confirmed the findings from in vitro study STR-30207-013, and demonstrated that the cynomolgus monkey cytokine response pattern observed in the whole blood test system was significantly higher than that observed with RNA. (LIP) This was similar to findings from in vivo study LPT No. 29928, in which only IL-6 was detectable in cynomolgus monkeys treated with IL-6 (see below).

[0351] Table 9 shows the results of PBMCs from four cynomolgus monkeys and four healthy donors treated with six different doses of RNA. (LIP) Figure 1 shows data generated in LPT study No. 31031, analyzed after 6 and 24 hours of incubation with IL-6 and TNF-α. Induction of IL-6 and TNF-α was measured by: (i) the absolute amount of cytokine induced (less than two-fold difference between species), (ii) kinetics (early induction of IL-6 and TNF-α after 6 hours), and (iii) the RNA that resulted in cytokine induction.(LIP) The results were comparable in human and cynomolgus monkey PBMCs for the dose levels. (LIP) Only after stimulation with intermediate doses of RNA (LIP) IFN-γ was detected in PBMCs from both species treated with IFN-γ, but to a greater extent in humans. (LIP) The cytokine profile induced by IL-6 and TNF-α was comparable between species. (LIP) These results suggest that human PBMCs constitute a relevant species for assessing IFN-γ mediated cytokine induction and that human PBMCs constitute a more sensitive system for capturing IFN-γ induction.

[0352] [Table 15]

[0353] [Table 16]

[0354] Comparison of the results observed in the whole blood and PBMC test systems revealed that inflammatory cytokines in the PBMC test system were generally more widespread, reached higher absolute values, and started at lower dose levels compared to the whole blood test system.

[0355] In this most sensitive in vitro test system, a rapid increase in cytokine levels measured after 24 hours was observed over the dose range of 615 μg to 1,850 μg RNA for IL-6, 1,850 μg to 5,550 μg RNA for IFN-γ, and 5,550 μg to 16,650 μg RNA for TNF-α. Even for IL-6, the most sensitive cytokine marker in vitro, the intended starting dose of the first injection cycle of 50 μg was 12-37 times lower than the dose levels that showed the onset of potent in vitro cytokine induction.

[0356] In addition to the GLP study of LPT No. 31031, we conducted a non-GLP in vitro study (Report_RB_14_001_B) using a similar experimental setup, testing samples from three individuals per species, with similar observations and confirming the results from the GLP study (data not shown). Taking all three studies together, the findings (i) highlight the equivalence of both species, cynomolgus monkeys and humans, with respect to cell stimulation after incubation with the test substance. Furthermore, these observations (ii) demonstrate that the whole blood test system more closely reflects the in vivo situation than PBMCs. In the whole blood test system, cytokine induction was generally less pronounced and was observed only in the highest dose group, with the predominant induction of IL-6 resembling the findings in cynomolgus monkeys in the in vivo study (see below).

[0357] We observed more pronounced findings in PBMCs, which are considered an artificial but also more sensitive in vitro test system, and therefore incorporated results from this more sensitive test system into our strategy to define a safe initial starting dose.

[0358] In vivo studies of secondary pharmacology in cynomolgus monkeys RNA (LIP) To better understand the kinetics and secondary effects of CYP2000 and the correlation with cytokine expression, a non-GLP study was conducted in male cynomolgus monkeys (see Table 10 for treatment schedule and doses, and Table 11 for detailed study design and amounts of all formulation components). Groups 1-5 of animals (2 males per dose) were treated with a schedule similar to that planned for patients, i.e., 4 RNAi. (LIP) The vaccine (ATM quality) and control solutions were administered sequentially as slow bolus injections (approximately 10 seconds) with a 30-minute interval between each injection (i.e., the final injection was administered 1.5 hours later). Because secondary effects are not sequence-dependent, the results of this study are based on the results of WAREHOUSE RNA. (LIP) This should also apply to injections.

[0359] Human equivalent doses (HEDs) up to 20 times the clinical dose were tested within the study (HED = animal dose divided by 3.1, as recommended by the FDA Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers). Additionally, animals in dose group 6 received a single dose of 4 x 88.6 μg RNA on day 1, followed by a single dose of 4 x 3.6 μg RNA on day 22.

[0360] [Table 17]

[0361] [Table 18]

[0362] Clinical observations Overall, the treatment was very well tolerated, with no abnormal signs of intolerance noted in any of the animals regarding local and systemic tolerance observations (including behavior, appearance, feces, mortality, body weight, and food and water intake).

[0363] Cytokine analysis Cytokine release into plasma was measured for IFN-α, IFN-γ, TNF-α, IL-1β, IL-2, IL-6, IL-10, IL-12p70, and IP-10 after the first and fifth injections, before administration, and after completion of treatment (i.e., all four RNA (LIP) Two kinetics were studied at 0.5, 2, 5, 9, 24, and 48 hours (after completing the product injection cycle).

[0364] At the doses tested, only IL-6 showed a dose-dependent and test article-related induction. maxThe IL-6 induction reached levels of 1,071 pg / mL, returning to pre-dose levels 24 hours later (Figure 25). Animal 11 (Group 6) was an outlier showing a very strong response, with a peak IL-6 level of 1,071 pg / mL, approximately five-fold higher than other animals in the same dose group. Notably, IL-6 induction was much lower after the fifth treatment, suggesting an adaptive effect to IL-6 in monkeys.

[0365] IFN-α induction was observed only at very low levels in animals in the high-dose group 6, reaching a peak level after 5 hours and returning to pre-dose levels after 24 hours (Figure 25). In contrast to in vivo observations in cultured human cells and mice, no IP-10 induction was observed in monkeys. As reported by other researchers, IP-10 induction has been observed in monkeys after TLR-activating agonists, so the reason why IP-10 was not observed in this study remains unclear.

[0366] The other cytokines tested (IFN-γ, TNF-α, IL-1β, IL-2, IL-10, IL-12p70) were unchanged. Liposomes alone did not affect cytokine release.

[0367] hematology Standard hematology parameters were tested after the first and fifth injections, pre-dose, and 5, 9, 24, and 48 hours after completion of treatment (including an additional 2 hours after the fifth dose). Additionally, hematology was tested daily from study days 4 through 12, and 1 and 3 weeks after the last dose.

[0368] A transient decrease in lymphocytes and a transient increase in neutrophils were observed in a dose-dependent manner as test article-related findings. Lymphocytes declined very rapidly, up to 5-fold, in high-dose animals 5 hours after treatment completion (lowest dose of approximately 1,000 lymphocytes / μL in Group 6 animals). The effect was transient, with recovery occurring at approximately 48 hours. Notably, lymphocyte depletion was also observed to a lesser extent in animals in the liposome group, but not in the NaCl control group (Table 12). There was no adaptive effect, as observed for IL-6 induction.

[0369] An increase in neutrophils was also observed in the NaCl control group due to treatment, but significant differences were observed in groups 3 to 6 when compared to the control. The maximum effect was observed 10 hours after treatment and was 44%, 34%, 89%, and 91% compared to the control in groups 3, 4, 5, and 6, respectively.

[0370] Transient treatment-related effects (even in the NaCl group) were observed for eosinophils, leukocytes, and reticulocytes (probably due to constant blood sampling).

[0371] [Table 19]

[0372] Complement activation C3a was measured pre-dose, 0.5, 2, 5, 9, 24, and 48 hours after completion of the first and fifth treatments, and 1 and 3 weeks after the last dose. No test article-related changes were observed, and all values were considered to be within the normal range of biological variation.

[0373] clinical chemistry Standard parameters were tested pre-dose, 24 hours after each dose, and 4 days after the third and fourth doses, and 1 and 3 weeks after the final dose.

[0374] Test article-related effects were not evaluated on biochemical parameters of liposome-treated and test article-treated animals compared with control animals and / or background data available at the CRO conducting the study. The data show some variability, in part due to the small number of animals used per group.

[0375] No test article-related changes were observed in serum levels of bile acids, bilirubin, cholesterol, creatinine, glucose, phosphate, total protein, triglycerides, urea, calcium, chloride, potassium, and sodium, and serum proteins (albumin, globulin, and albumin / globulin ratio).

[0376] Serum enzyme activities of alanine aminotransferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH), α-amylase, creatine kinase (CK, including isoforms CK-BB, CK-MB, and CK-MM), γ-glutamyltransferase (γ-GT), and glutamate dehydrogenase (GLDH) were considered within the range of normal biological variation.

[0377] On study day 23, elevated values were observed for LDH, α-amylase, and CK enzyme activities in animal number 11, which was treated with 4 × 3.5 μg RNA / animal on study day 22. However, these changes were not related to the test article and are believed to be related to the stress of the monkeys being restrained in the infusion chair.

[0378] Although not considered test article-related, minor changes in CK were evaluated in more detail. Differential analysis of the CK isoenzymes CK-BB, CK-MB, and CK-MM revealed that increases in CK activity observed in individual animals from Groups 4, 5, or 6 compared with control animals on study days 9, 16, or 23 were primarily due to increases in the CK-MM fraction. In general, no increases were observed for CK-BB and CK-MB, thus confirming that the overall increases in CK levels were stress-related.

[0379] Cardiovascular testing ECG and blood pressure measurements showed no effects on the cardiovascular system.

[0380] Sequence homology screening between WAREHOUSE RNA and the human proteome Three of the four mRNA sequences used in the W_ova1 approach are fused in-frame to up to two adjacent glycine / serine (GS)-rich linker sequences, an MITD region, and a secretion signal region. These fusion junctions, if homologous to human proteins, could generate novel antigenic fusion proteins or peptides that could potentially trigger unwanted autoimmune responses. Therefore, we determined whether the junctions associated with the linker and enhancer sequences, antigens, and transmembrane domains share sequence homology with known human proteins by blastp-based homology searches against established human protein databases.

[0381] The fusion protein sequence to be analyzed was decomposed into smaller peptide sequences by using a sliding window with a length of 9–15 and a step size of 1 amino acid residue. All resulting peptides were compared to a reference database using the blastp command in the blast software package (e-value cutoff 10, no gaps).

[0382] For 100% homologous peptide subsequences, no significant alignment to human protein sequences could be found.

[0383] Section 4: Safety Pharmacology The ICH guideline S7A describes a core battery of tests that should be performed on pharmaceuticals prior to human exposure, including assessment of respiratory, central nervous system (CNS), and cardiovascular function. (RIN) and RNA (LIP) The safety pharmacology of was studied as an integral part of six GLP toxicology studies described in Section 6.

[0384] Potential effects on CNS and respiratory system function were evaluated in a pivotal repeated-dose toxicity study, which revealed no test article-related effects in the animals.

[0385] RNA for the cardiovascular system (LIP) A risk analysis was conducted regarding the potential impact of the vaccine. RNA distributed throughout the body is degraded in the circulation, and RNA (LIP) RNA formulated as a soluble form is cleared from the blood within minutes and is distributed primarily to the spleen and liver as shown in biodistribution studies (see below). (LIP) There is no suggestion that RNA accumulates in the cardiovascular system. (LIP) Potential systemic side effects of vaccination are expected to be related to a transient increase in IFN-α, but are not expected to lead to cardiovascular side effects, as has been demonstrated in thousands of patients who have received IFN-α. As a result, GLP cardiovascular safety pharmacology studies according to ICH S7A / B were not performed. However, RNA (LIP) Supportive ECG and blood pressure data are available from a non-GLP pharmacology study in cynomolgus monkeys treated with RNA (LIP) Addresses the assessment of cardiovascular function after treatment with the vaccine.

[0386] In summary, no test article or treatment-related changes in the respiratory, nervous, and cardiovascular systems were observed in any of the dose groups tested in mice (respiratory system and CNS function) and cynomolgus monkeys (cardiovascular function).

[0387] respiratory safety Respiratory safety was included in GLP-compliant repeated-dose toxicity studies in mice using WAREHOUSE RNA (LPT Nos. 28864 and 30283). For example, plethysmography was performed on 4 animals / sex / group treated with either control buffer, low dose, or high dose (5 μg and 50 μg RNA formulated in 9 μg and 90 μg liposomes, respectively) of WAREHOUSE RNA. (LIP)The study was conducted in a study using carbamyl-β-methylcholine chloride (LPT No. 30283). A positive control of animals treated with 30 mg of carbamyl-β-methylcholine chloride (bethanechol) / kg body weight was also included. Plethysmography was performed 1 day after the fourth through seventh doses. Testing included assessment of respiratory rate, tidal volume, minute volume, inspiratory time, expiratory time, peak expiratory and inspiratory flow rates, expiratory time, and airway resistance index. None of the pulmonary parameters tested showed test article-related changes in treated animals compared with the control group. Only animals in the positive control group showed the expected changes.

[0388] CNS safety CNS safety was included in GLP-compliant repeated-dose toxicity studies in mice using WAREHOUSE RNA (LPT Nos. 28864 and 30283). (LIP Test using ) For LPT No. 30283, five animals per sex were tested for observational screening approximately 24 hours after the fifth dose of control buffer, low-dose, and high-dose (5 μg and 50 μg RNA formulated in 9 μg and 90 μg liposomes, respectively). The following tests were included in the observational screening: righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, piloerection, diarrhea, pupil size, pupillary response, lacrimation, gait disturbance, stereotypy, toe pinch, tail pinch, wire manipulation, hind limb splay, positional passivity, tremor, positive geotropism, limb rotation, and auditory function. Additionally, functional tests were included to assess grip strength and locomotor activity.

[0389] Neurological screening revealed no test article-related effects in the mice that could be attributed to neurological toxicity. These findings were confirmed by the results of GLP-compliant repeat-dose toxicity study LPT No. 28864, conducted for the RB_0003-01 / Lipo-MERIT study using different RNAs, as reported in detail in the respective IMPDs.

[0390] cardiovascular safety RNA is degraded within seconds in the circulation,(LIP) Since there is no indication that thiazol-2000 accumulates in the cardiovascular system, no cardiovascular safety studies were conducted according to ICH S7.

[0391] However, the RNA targeting melanoma-associated antigens used in the RB_0003-01 / Lipo-MERIT trial (LIP) Supporting data are available from a non-GLP pharmacology study in cynomolgus monkeys with CI 1.25. In this study, 12 cynomolgus monkeys were treated in 6 groups (see Table 11 for study design), and ECG and blood pressure measurements were performed after the 4th dose at three time points: pre-dose, 5 hours after completion of dosing, and 24 hours after dosing.

[0392] RNA (LIP) Treatment with CYNOMOLGUS monkeys was well tolerated (no clinical findings were observed). None of the measured parameters (blood pressure, heart rate, QTc value, QT interval, P segment, PQ, QRS) showed any effects related to the test substance. Furthermore, serum levels of CK-MB and troponin I were measured to exclude the possibility of necrotic damage to myocardial tissue. All measured parameters were negative, indicating no adverse effects of RNA on the cardiovascular system at the dose levels tested in the study. (LIP) This confirmed that there was no toxic effect of

[0393] Discussion and Conclusions In mouse and human in vitro test systems, RNA (LIP) The mechanism of action and primary pharmacodynamics of RNA were extensively studied. (LIP) This indicates that the vaccine primarily targets the spleen after iv administration. (LIP) Vaccines induce a dual effect: induction of antigen-specific T cell responses and cell activation processes, and immune modulation following TLR triggering.

[0394] The data generated confirm that all antigen RNA lead constructs applied in vivo, including RBLTet.1, which encodes a tetanus toxoid helper epitope, induce antigen-specific T cell responses. Furthermore, we demonstrated that the concept of co-administering RBLTet.1 RNA with tumor antigen-encoding RNA improved immune responses to tumor antigens in mouse models.

[0395] Two representative WAREHOUSE RNAs (RBL001.2 and RBL007.1) demonstrated potent antigen-specific in vivo cytotoxicity upon liposomal formulation and intravenous vaccination. (LIP) The vaccine was shown to induce antitumor effects in vivo when applied to prophylactic and therapeutic mouse tumor models targeting either xenogeneic model antigens or endogenous gp70 antigen in BALB / c mice.

[0396] RNA (LIP) The functional properties of the formulation are (i) RNA protection in serum and (ii) efficient in vivo targeting of APCs capable of presenting antigenic peptides and activated after TLR7 triggering. The immunomodulatory activity of the RNA resulted in dose-dependent cytokine induction in human samples, mice, and cynomolgus monkeys, all of which showed varying degrees of IFN-α, IP-10, and IL-6 induction depending on the species tested or the test system applied. Given good evidence from our own RNA studies and the literature, we investigated the efficacy of RNA in PBMCs. (LIP) Independent of these expectations, the modest induction of IFN-α and induction of the chemokine IP-10 (CXCL10) likely reflects the onset of an intended pharmacological effect rather than an undesired immunotoxicological event.

[0397] Interestingly, RNA (LIP) When non-immunogenic RNA was used to form the RNA, activation of various immune cells in the spleen and RNA (LIP) Both the systemic IFN-α induction observed after treatment was abolished, and RNA(LIP) This indicates that the RNA component, rather than the lipid component, of α-glucan is responsible for the observed effects.

[0398] Data generated in mice showed that splenocytes express TLR7 - / - The TLR7-dependent cytokine response was reduced in mice, indicating that TLR7 is the primary source of IFN-α secretion. The observed transient and fully reversible cytokine response is considered to be the intended pharmacodynamic effect contributing to the efficient induction of vaccine-induced antitumor T cell responses. Favorable immunological properties were observed in mice and cynomolgus monkeys after administration of RNAi. (LIP) This was linked to the vaccine being well tolerated.

[0399] Additionally, RNA has been shown to be highly efficacious in several in vitro and in vivo studies using human, cynomolgus monkey, and mouse test systems. (LIP) Secondary effects of vaccine treatment were also examined. (LIP) This was particularly focused on because the immunomodulatory effects of the vaccine were more potent than those observed with unformulated RNA vaccines administered to lymph nodes, which only resulted in local cell activation and cytokine induction.

[0400] Experiments were performed using whole blood samples and PBMCs from human and cynomolgus donors, and RNA was analyzed. (LIP) This ruled out nonspecific or uncontrolled activation of human immune cells by the vaccine, but still showed modest cytokine induction, as expected. In these experiments, human cells and cynomolgus monkeys were treated with doses that covered at least the highest intended clinical dose cohort.

[0401] Although differences in cytokine levels were observed between donors, different in vitro test systems (cultured PBMCs vs. whole blood), or between species, the observed cytokine patterns and the transient nature of the cytokine responses were similar across all studies, with few exceptions, such as the lack of IP-10 induction in cynomolgus monkeys. Human PBMCs showed IP-10 induction and low IL-6 and IFN-α responses, and these levels were even lower when examined in whole blood. Cynomolgus monkeys showed very low IFN-α responses and did not show IP-10 induction or a more pronounced IL-6 response at the dose levels tested. Mice showed robust responses in IFN-α, IP-10, and IL-6, but at doses approximately 10-fold higher (based on dose per kg body weight) than those tested in monkeys. The differences in cytokine expression between mice and monkeys may be explained, on the one hand, by the different doses tested. On the other hand, mice have different activity towards TLR7 / 8, which may also be a plausible explanation for the different cytokine expression patterns.

[0402] The cytokine response pattern observed in cynomolgus monkeys in vivo was better reflected by the whole blood test system compared to the PBMC test system, where broader and higher cytokine responses were observed at lower dose levels. Nevertheless, the more sensitive PBMC findings were incorporated into the strategy for defining a safe starting dose for patients. Side-by-side comparison of cytokine secretion in human and cynomolgus monkey whole blood demonstrated that both species exhibited RNA (LIP) The results showed that cynomolgus monkeys were highly comparable in terms of inflammatory cytokine induction during treatment, and that the RNA levels of the patients were significantly higher. (LIP) These results suggest that this animal model is suitable for predicting secondary pharmacodynamic effects that may occur after vaccination with HIV.

[0403] RNA (LIP)In addition to cell activation processes and cytokine induction after exposure to riboflavin, we evaluated hematological changes in mice and cynomolgus monkeys. Here, transient lymphopenia was observed equally in mice and monkeys at all dose levels. Overall, RNA (LIP) Monkeys treated with α-TNF-α show responses in cytokine profiles and hematological parameters similar to those observed in monkeys treated with other TLR agonists. (LIP) This is consistent with the assumption that the primary activation process for cytokine expression by wild-type TLR7 occurs via TLR stimulation. - / - and IFNAR - / - Extensive pharmacodynamic studies in mice demonstrated that hematological findings were consistent with RNA (LIP) This suggests that this is a secondary effect of the induced cytokines. (LIP) It has been shown that unformulated and naked RNA can activate TLRs. TLR activation has been shown to induce lymphopenia and B cell accumulation in the spleen. Supportively, histopathology data generated in toxicity studies showed transient lymphoid hyperplasia in the spleen but not in other organs or tissues. This is consistent with the observed lymphopenia in the blood, and the RNA (LIP) It also highlights the intended targeting of, and subsequent attraction of effector cells to lymphoid organs.

[0404] The safety pharmacology tests conducted were (LIP) Neurological screening revealed no test article-related effects in mice in any of the studies performed. None of the lung parameters tested were significantly different from RNA. (LIP) There were no signs of cardiovascular effects in cynomolgus monkeys. Overall, RNA (LIP) shows a very good overall safety profile in terms of safety pharmacology parameters.

[0405] Section 5: Pharmacokinetics Although pharmacokinetic studies are not typically performed during cancer vaccine development, in vivo studies were performed to determine the biodistribution of intravenously injected RNA-lipoplexes and the presence or persistence of residual plasmid loads due to impurities in the drug product.

[0406] In vitro transcribed RNA consists of ribonucleotides and is therefore structurally identical to the RNA synthesized by human cells, except for the 5' cap structure. Therefore, RNA undergoes the same degradation process as natural mRNA. Especially in the extracellular space and serum, abundant RNases lead to rapid degradation of RNA.

[0407] The distribution / distribution and potential accumulation of RNA in the spleen, liver, and lung were investigated in pharmacokinetic studies, as outlined below. (LIP) Potential plasmid DNA impurities were quantified in gonads from mice treated with .

[0408] The biodistribution and persistence of the synthetic cationic lipid DOTMA have been investigated in initial exploratory in vivo studies. The fully synthetic DOPE cannot be distinguished from the body's own natural phospholipid DOPE, and therefore we refrain from further investigation of the biodistribution and accumulation of this lipid.

[0409] [Table 20]

[0410] Biodistribution RNA RNA was collected by organ sampling during the GLP repeat-dose toxicity study (LPT No. 28864) conducted for clinical trial RB_0003-01 / Lipo-MERIT. (LIP)The biodistribution of was investigated in detail in mice. Under non-GLP conditions, organs were analyzed for the sum of all IVT-RNA using a quantitative RT-PCR method developed at IMGM Laboratories GmbH, Martinsried, Germany (Study ID: RS297). In summary, RNA was very rapidly eliminated from the blood with an estimated half-life of approximately 5 minutes. After 48 hours and 7 days, respectively, RNA was detectable only at marginal levels in the blood and organs, suggesting that it was rapidly degraded and did not persist.

[0411] Residual Plasmid Impurities RNA (LIP) The biodistribution of residual plasmid impurities from vaccination was investigated using samples from a GLP repeat-dose toxicity study (LPT No. 28864). A GLP-compliant method was developed at BioNTech IMFS GmbH, Idar-Oberstein, Germany, to analyze residual plasmid impurities in organ samples. All test samples were below or slightly above the lower limit of detection (LLOD), suggesting that plasmid DNA did not accumulate or persist in the gonads (Study ID: 36X130313).

[0412] DOTMA RNA (LIP) The biodistribution of the two synthetic lipids used in the formulation can provide insight into the physical distribution of the lipoplex carrier particles over time. The synthetic cationic lipid DOTMA was chosen for biodistribution studies because it is not a naturally occurring molecule and therefore can be easily detected in the background of the biological matrix.

[0413] In the first exploratory study of DOTMA biodistribution, mice were administered intravenously with RNA (LIP)Lipids were extracted from blood collected after injection and from seven selected organs. An equal mixture of ATM-quality liposome-formulated IVT-RNA was used. This initial study included five mice: one was left untreated, two received a single injection of 60 μg RNA, and two received two injections of 60 μg RNA each, separated by a 20-day interval. All mice were sacrificed 24 hours after the last injection. DOTMA was quantified by LC / MS. The purpose of the experiment was to test the general feasibility of the extraction and quantification protocol and to characterize the RNA. (LIP) The aim was to obtain the first hints regarding the biodistribution of DOTMA after vaccination.

[0414] DOTMA could be clearly determined from all organs examined, and significant differences between findings in different organs could be observed. Consistent with the proposed mechanism of action, the highest DOTMA findings were in the spleen.

[0415] Based on these initial results, RNA (LIP) A single-dose study of DOTMA was conducted (Report_BN_14_004). DOTMA concentrations in selected organs were assessed over a period of up to 28 days (days 0, 1, 4, 7, 14, 21, and 28). In this study, 200 μL of RNA containing 20 μg of RNA and 26 μg of DOTMA was administered. (LIP) (In the initial study, 60 μg was administered per injection). The DOTMA concentration in the administered product was 195 μM. Three mice were studied per time point. The results for all seven time points are shown in Figures 26 and 27.

[0416] As can be seen, DOTMA was found primarily in the spleen and liver, which showed slightly different accumulation kinetics. In all other organs / tissues investigated (lungs, heart, kidneys, lymph nodes, fat pads, bone marrow, and brain), the findings were 10-50 times lower (Figures 26 and 27). From the liver and spleen data, we were able to estimate the pharmacokinetics of DOTMA: maximum concentrations were detected several days after administration. Within 20 days, DOTMA concentrations had decreased to approximately 50% of their maximum value. These findings support the assumption that DOTMA is eliminated from organs within acceptable timescales and that there is no indication of a risk of persistent accumulation in any organ.

[0417] Subsequent studies assessed DOTMA concentrations in selected organs using eight weekly RNA samples containing 20 μg of RNA (RBL005.2) and 26 μg of DOTMA, respectively. (LIP) The initial RNA was evaluated before (control group), during, and after injection (Report_RB_15_004_V02). (LIP) Organs were sampled from the mice 1 hour after administration, and then every other week after the previous application. After completing eight application cycles, mice were sacrificed after an additional 3, 6, 9, 12, and 15 weeks to examine organ DOTMA clearance. RNA (LIP) Repeated administration of the test article and organ sampling were performed in-house, while extraction and quantification of DOTMA from donated organ samples was performed by Charles River Laboratories Edinburgh Ltd. (Study No. 322915). The results were in good agreement with previous studies performed by the inventors: again, the highest DOTMA concentrations were observed in the spleen as the primary target organ, followed by the liver (Figure 28). In all other organs, approximately 5% or less of the concentrations present in the spleen samples were found (data not shown). As can be seen, DOTMA concentrations were significantly higher in the RNA samples than in the control group. (LIP) It increased with increasing injection frequency and then decreased continuously during the recovery period after the last application. The terminal half-lives of DOTMA in plasma (7.07 weeks), spleen (6.76 weeks), and liver (6.57 weeks) were comparable to those obtained from animals during the recovery period after the eighth dose.

[0418] In summary, DOTMA as an indicator of lipid vesicles is essential for the ivRNA (LIP) After administration, DOTMA is rapidly delivered to the spleen (and other organs) (in less than 1 hour). In addition to the spleen, DOTMA accumulates primarily in the liver, but RNA translation is not observed. In absolute terms, the amount of DOTMA found in these two organs approached the total cumulative amount of DOTMA infused in absolute terms, while the amount of DOTMA in all other organ samples was negligible.

[0419] Repeat RNA as assessed from animals in the recovery group (LIP) After application, accumulated DOTMA is cleared from organs with kinetics that can be reasonably described by first-order decay with an approximate half-life of 6-7 weeks. Such clearance kinetics is also consistent with findings from repeated applications, where transient accumulation was observed.

[0420] Taken together, these findings support the assumption that DOTMA is cleared from organs within an acceptable time frame and that the potential risk of persistent lipid accumulation in plasma, liver, spleen, lungs, heart, brain, kidneys, uterus, lymph nodes, and bone marrow is fairly low.

[0421] metabolism RNA The metabolic pathway of RNA is well understood: RNA is first deadenylated, then decapped, and further degraded to nucleoside monophosphates by RNases. Most of the enzymes involved are well described. Only the β-S-ARCA (D1) cap in our RNA is a natural cap in mRNA. 7 Unlike GpppG, the β-S-ARCA(D1) cap must nevertheless be cleaved by the cleavage enzyme Dcp2.

[0422] Further biotransformation studies were not considered to add further relevant information and were not performed.

[0423] DOTMA / DOPE RNA(LIP) The lipids used for this formulation were the naturally occurring phospholipid DOPE and the synthetic cationic lipid DOTMA. DOPE is metabolized similarly to the body's own DOPE. While detailed insights into DOTMA metabolism are not yet available in the literature, cationic DOTMA, as an ether lipid, is expected to be metabolized at a slower rate than the phospholipid DOPE. DOTMA has already been safely applied in clinical settings at doses up to 2.4 mg. Furthermore, the applied dose of DOTMA in this study was relatively low compared to other liposomal products containing similar cationic lipids.

[0424] discharge No specific studies were performed. Shedding studies for RNA vaccines are not considered to add value to the nonclinical data package.

[0425] Pharmacokinetic Drug Interactions RNA (LIP) No pharmacokinetic interaction studies are planned for the vaccine.

[0426] Other pharmacokinetic studies In accordance with the ICH Guideline M3(R2) on "Non-clinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorisation for Pharmaceuticals", further information on distribution and metabolism in test species will be generated by the inventors before exposing or treating a larger number of human subjects for extended periods (late stage clinical development / pre-Phase III).

[0427] Discussion and Conclusions In vitro transcribed RNA, consisting of ribonucleotides, has the same structure as the RNA produced by human cells, with only the 5' cap structure differing. Therefore, IVT-RNA undergoes the same degradation process as natural mRNA. Especially in the extracellular space and serum, abundant RNases lead to rapid degradation of RNA.

[0428] As shown by in vitro experiments described in the IMPD of our clinical trial RB_0001-01 / MERIT, intracellular RNA is degraded within hours (t 1 / 2 = approx. 6 hours).

[0429] RNA (LIP) The results of the biodistribution test of RNA (LIP) The results show high levels of RNA in the blood immediately after injection of C. The RNA was rapidly cleared from the blood and was subsequently found, albeit at much lower levels, in the spleen and liver, with only trace amounts found in the lungs. Because RNA distributed to the liver may result in transient immune activation via TLR triggering, liver enzymes will be closely monitored in patients after the first injection and throughout the study. After 48 hours and 7 days, only residual amounts of RNA were found in the blood and organs, suggesting that RNA did not accumulate or persist in any organs. C after the first and eighth injections max Comparison of levels also showed no cumulative effect.

[0430] In the gonads, either no plasmid DNA was detected or the samples were slightly above the LLOD, suggesting that there is a small risk of integration of plasmid residues, e.g., the kanamycin resistance gene, into the germ cell genome.

[0431] The biodistribution of DOTMA has been shown to be primarily found in the spleen and liver, with the spleen being the primary site of RNA (LIP) The main target organ for vaccination is RNA (LIP)Significantly lower exposure in plasma and other tissues was observed after a single dose and eight repeated doses of DOTMA. DOTMA was eliminated from plasma, spleen, and liver with similar terminal half-lives of approximately 6–7 weeks.

[0432] Section 6: Toxicology The toxicity program for our RNA vaccine platform includes RNA vaccines across a range of doses. (LIP) This included several pharmacological studies to test the vaccination, a repeat-dose toxicity study including local tolerance and safety pharmacology parameters, and an immunotoxicity investigation. The studies were performed under GLP conditions at an external CRO (LPT, Hamburg, Germany) using RNA and liposome batches comparable to the clinical trial material in terms of manufacturing process and analytical quality control.

[0433] GLP-compliant testing involves the use of RNA encoding six different WAREHOUSE antigens. (LIP) and a 6-week repeated-dose toxicity study by intravenous administration to C57BL / 6 mice of RBL008.1 encoding p53 and RBLTet.1 RNA encoding tetanus helper toxoid (LPT No. 30283).

[0434] Additionally, a complementary GLP-compliant 6-week repeat-dose toxicity study was conducted using the Ribological® RNA vaccine platform, which targets multiple melanoma-specific antigens (LPT No. 28864). While a variety of RNA sequences were tested, the toxicity data are also relevant to WAREHOUSE RNA applications, and the same types of liposomes were used to deliver RNA. (LIP) This adds important information: the toxicity profiles of the formulated RNAs in both studies are likely to be the same or at least comparable due to the fact that potential side effects are related to the intrinsic molecular properties of liposome-formulated RNA, which are independent of RNA sequence and length.

[0435] Additionally, an additional 4-week repeat-dose toxicity study was conducted to evaluate the comparability of the liposomes used in the 6-week repeat-dose toxicity study with a pH-matched liposome formulation in which the buffer conditions were slightly adjusted for long-term stability reasons (LPT No. 30586).

[0436] [Table 21]

[0437] Selection of related species WAREHOUSE RNA based on the following main reasons: (LIP) The mouse is considered a relevant species for testing potentially toxic direct effects of vaccines. The mouse as a model system provides all the relevant features of innate and adaptive immunity that are relevant to characterizing the direct toxic effects of WAREHOUSE RNA. Mice are CD4 + / CD8 + They exhibit all the expected primary and secondary pharmacological effects, from induction of T cell responses to immunomodulatory effects that enhance immune responses and result in subsequent TLR triggering, cell activation, and cytokine secretion. The mouse system includes a wealth of available tools and techniques for investigating biological effects (e.g., availability of transgenic mouse models, MHC tetramers, antibodies, etc.), far exceeding experimental possibilities in other species, allowing for a deeper analysis of any unexpected events. The on-target effect of vaccines cannot be fully investigated in animal species. Therefore, the use of other animal species does not provide additional information and, as a result, the use of higher mammals should not be considered.

[0438] Single-Dose Toxicology Dose-ranging studies are typically performed to justify doses for pivotal toxicity studies and to obtain initial information on target organ and symptomatic toxicity. (LIP)Several pharmacological studies were carried out to examine the effects of RNA (LIP) Administration of 100 mg ...

[0439] Furthermore, previous toxicity studies by the inventors have shown that intravenously administered naked RNA is very well tolerated in mice, even at high doses. Liposomes containing either DOTMA or DOPE as the synthetic lipid component have been tested in numerous clinical trials, and several approved liposomal pharmaceuticals have proven very well tolerated. Some liposomal formulations have even been applied to reduce drug-specific toxicity, such as the nephrotoxicity or hepatotoxicity of nucleic acids at high doses, or the toxicity of small molecules such as doxorubicin or clofazimine. Therefore, from data generated by a series of in-house studies and literature research, the inventors conclude that: A tolerated dose in mice that provides a sufficient safety margin for the first dose in humans can be estimated from the pharmacological studies performed. A single dose is not sufficient to induce a significant immune response. The maximum immune response was observed after at least three applications. RNA vaccines and lipoplex formulations are generally well tolerated.

[0440] Based on these conclusions, it was decided not to conduct a single-dose toxicity study, but to directly conduct a repeat-dose toxicity study.

[0441] Repeated Dose Toxicology RNA from the Ribological® RNA Vaccine Platform (LIP) The product is RNA (LIP) The product was analyzed for safety and toxicity in several GLP-compliant repeat-dose toxicity studies involving iv injection. Table 13 shows the RNA (LIP) Provides an overview of GLP repeat-dose toxicity studies supporting Phase I clinical trials with vaccines.

[0442] [Table 22]

[0443] ATM preparations The composition, formulation, and specifications of the animal test materials were designed to be as close as possible to those of the drug product intended for human use. The batches of test articles were RNA for Tests LPT No. 28864, LPT No. 30283, and LPT No. 30586, respectively. (LIP) Used in product preparation.

[0444] For the following reasons, RNA (LIP) I had to make some small changes to the preparation process: To obtain a higher dose that increases the likelihood of capturing potential dose-dependent toxic effects and thereby meets the criteria for toxicity studies outlined in the guidelines ICH S6 or M3(R2). To prevent administration of more than the maximum feasible volume in mice, which is a volume of 250 μL in a slow bolus injection. Higher injection volumes were not ethically recommended and risked losing the mouse during injection.

[0445] The differences from the clinical protocol are: Patients were treated with various WAREHOUSE RNA (LIP) This was not possible in mice due to volume limitations. (LIP) were prepared individually, then mixed, and all four RNA-lipoplexes were injected simultaneously in a total volume of 250 μL. RNA for patient treatment (LIP) For the formulation of RNAi, 150 mM NaCl is used. To obtain higher doses in toxicity studies, higher NaCl solutions are used. (LIP) It had to be used for formation. RNA for patient treatment (LIP)A concentration of 0.5 mg / mL of RNA drug is used for the preparation of the product. To obtain a higher dose in toxicity tests, a higher concentration of RNA, i.e., 1 mg / mL, is used for the RNA drug of test LPT No. 28864. (LIP) It had to be used in the preparation of the product.

[0446] It is the inventors' position that the above modifications to the formulation protocol at ATM will have no or little effect on the outcome or conduct of the study.

[0447] Study design See Table 13 for study design. Data from standard toxicity studies were evaluated for signs of potential immunotoxicity in accordance with ICH S6 and S8. The following investigations were performed in accordance with FDA, ICH, and CHMP guidance documents: mortality, histopathology (especially spleen), gross pathology and organ weights, clinical observations, ophthalmology, local tolerance, injection site reactions, body weight, food consumption, standard hematology parameters, as well as clinical chemistry and cytokines (IL-1β, IL-2, IL-6, IL-10, IL-12, TNF-α, INF-α, INF-γ, and IP-10).

[0448] Safety pharmacology studies were included to test for the respiratory and central nervous systems as outlined in Section 4.

[0449] result Toxicity evaluation in a 6-week repeated dose toxicity study using our vaccine platform was performed using RNA (LIP) The results revealed only minor effects that could be primarily attributed to the predicted pharmacological mechanism of action of RNA (Table 14). (LIP) The desired immunomodulatory effect of IFN-α is TLR activation and cytokine release (see sections 2 and 3 for details). Induction of IFN-α in mice leads to secondary effects such as leukopenia, decreased platelets, and increased liver parameters such as ALAT, effects commonly described in patients treated with IFN-α.

[0450] Consistent with this, test article-related changes in treated animals were primarily transient (Table 14). Furthermore, transient activation of the cytokines IP-10, IFN-α, IL-6, and IFN-γ was observed. All inductions returned to normal levels after 24 hours (with the exception of IP-10 levels, which remained slightly above normal).

[0451] Hematological findings in mice included primarily lymphopenia with small, reversible decreases in total white blood cells, neutrophils, and reticulocytes, as well as thrombocytopenia in all treatment groups. These findings were fully reversible. Lymphoid hyperplasia observed in the spleen by histopathology was completely resolved, outlining the desired effect and intended targeting of the test substance and lymphocytes to the spleen.

[0452] The observed mild changes in liver parameters such as GLDH, LDH, ALAT, and ASAT mainly affected the high-dose group and were not observed in animals in the recovery group, suggesting that the effects were fully reversed within at least 3 weeks. No hepatotoxicity was detected by histopathology.

[0453] Because there were no findings in the low-dose groups of Study LPT No. 30283, a dose of 5 μg total RNA per animal (i.e., approximately 0.2 mg / kg body weight in mice) met the no-observed-adverse-effect level.

[0454] To address the change in liposome buffer composition, an additional 4-week repeated-dose toxicity study was conducted (LPT No. 30586). The data demonstrate that the new liposome type is fully equivalent to those used in the main study in terms of the parameters measured.

[0455] [Table 23] TIFF2025122023000056.tif221153TIFF2025122023000057.tif139153

[0456] Genotoxicity RNA (LIP) The components of the product (lipids and RNA) are not considered to have genotoxic potential. There are no impurities or components of the delivery system that justify genotoxicity testing. No genotoxicity testing is planned in accordance with the recommendations set out in the ICH Guideline on Preclinical Safety Evaluation of Pharmaceuticals Derived from Biotechnology S6(R1) (June 2011).

[0457] Carcinogenicity The RNA itself and the lipids used as vehicles have no carcinogenic or tumorigenic potential. In accordance with ICH S1A, if there is no cause for concern from laboratory and toxicology testing and the drug is not expected to be used chronically, long-term carcinogenicity testing is not required.

[0458] Reproductive and Developmental Toxicity Macroscopic and microscopic evaluation of male and female reproductive tissues was performed using RNA (LIP) No findings were observed in these studies, and therefore, (LIP) No specific reproductive and developmental toxicity studies will be performed prior to the initiation of Phase I trials with the vaccine. Direct cytotoxic effects on reproductive tissues are unlikely to occur with RNAi, as supported by experience from other cancer vaccines that have not shown reproductive and developmental effects. (LIP) Not expected. Reproductive effects cannot be excluded; women of childbearing potential should use effective contraception during treatment. No further long-term or reproductive toxicity studies are planned at this time.

[0459] Local tolerance Local tolerance testing was evaluated in a GLP repeat-dose toxicity study for i.v. injection, as recommended by ICH. No signs of local intolerance were observed during the study.

[0460] Other toxicity studies antigenic Due to the rapid extracellular degradation of in vitro transcribed RNA within seconds to minutes, the formation of anti-drug antibodies (ADAs) is not expected. Therefore, no additional immunogenicity testing for antibody induction is planned.

[0461] immunotoxicity WAREHOUSE RNA (LIP) Because the product is intended to activate the immune system, particular attention was paid to immunotoxicity parameters to rule out unintended activation or suppression. Immunotoxicology tests were performed in both 6-week repeated-dose toxicity studies (LPT Nos. 28864 and 30283). In addition to monitoring serum cytokine levels, the following relevant parameters were considered to evaluate immunotoxicity: body weight, body temperature, lymphoid organ weight, lymphoid organ gross and histopathology, absolute and relative differential blood counts, total serum protein, albumin / immunoglobulin ratio, bone marrow myeloid / erythrocyte ratio, and coagulation parameters.

[0462] hematology Decreases in lymphocytes, white blood cell counts (primarily due to lymphocyte depletion), and platelets were observed in all treatment groups on Study Day 44, approximately 24 hours after the eighth injection in both studies. All effects were fully reversible after two weeks. Results are shown in Tables 15 and 16 for Studies LPT Nos. 28864 and 30283, respectively.

[0463] [Table 24]

[0464] [Table 25]

[0465] Cytokine measurements In the repeat-dose toxicity study, we analyzed the excretion of the following cytokines, known to be sensitive indicators of immune activation or TLR7 signaling: IL-1β, IL-2, IL-6, IL-10, IL-12p70, TNF-α, IFN-γ, and IP-10. In toxicity study LPT No. 28864, mice showed a dose-dependent and test article-related increase in the cytokine IP-10. IP-10 levels transiently increased and were highest 6 hours after the fourth injection. After 24 hours, levels were still significantly higher than control levels but had already returned to near-normal levels. Compared to the control group, IP-10 showed a maximum induction of 28-fold and 16-fold (males and females, respectively) at 6 hours. Significant increases were also observed for TNF-α (females only, groups 2, 3, and 4), IL-10 (females only, groups 3 and 4), IL-6 (males, group 4), and IFN-γ (males, group 4). The maximal induction was 3-fold for TNF-α, 4-fold for IL-10, 7-fold and 8-fold for IL-6, and 6-fold and 2-fold for IFN-γ. All effects were fully reversible after 24 hours (except for TNF-α levels in females in Group 4). The results are summarized in Table 17.

[0466] [Table 26]

[0467] Serum samples were collected 6 and 24 hours after the fifth injection for cytokine measurements in Study LPT No. 30283. Increased serum levels of IL-2, IL-6, IP-10, IFN-α, and IFN-γ were observed (Table 18). A clear dose-dependent induction was observed for IL-6. For IFN-γ, induction was observed after high-dose treatment (statistically significant at p<0.01) and was more pronounced in male animals. For IFN-α, induction was observed after low-dose and high-dose treatment (statistically significant at p<0.01 or p<0.05) and was more pronounced in female animals in Group 5 (RNA Set 2, high dose). Induction of all of the above cytokines subsided by 24 hours post-dose.

[0468] A relatively low but dose-related induction of IL-2 was observed in male and female animals 6 and 24 hours after low- or high-dose treatment.

[0469] A significant dose-dependent effect was detected for IP-10 in both male and female animals at all dose levels compared to the control group 6 hours after high-dose treatment. At 24 hours post-administration, IP-10 levels were still elevated at all dose levels compared to the control group. This induction of IP-10 reflects the intended pharmacological effect and is not considered an undesired immunotoxic event.

[0470] [Table 27]

[0471] Cytokine measurements were also performed during a study comparing L1 and L2 liposomes (LPT No. 30586). Cytokines were analyzed 6 and 24 hours after the fourth immunization. No test article-related changes were observed between groups.

[0472] Discussion and Conclusions As shown for a number of antigen-encoding RNAs evaluated in three different repeat-dose toxicity studies (LPT studies Nos. 28864, 30283, and 30586), RNA (LIP) Treatment with is very well tolerated in mice. Overall, treatment with up to eight iv injections was well tolerated, even in animals at the higher doses. No premature deaths related to the test substance were observed in the toxicity studies. There were no findings in the lower dose groups in Test No. 30283, so the no-observed-toxicity level was reached at a dose of 5 μg total RNA per animal (i.e., approximately 0.2 mg / kg body weight in mice). Furthermore, RNA (LIP) Vaccination with the product was also well tolerated (no clinical findings) in a non-GLP pharmacology study in 12 cynomolgus monkeys.

[0473] RNA in repeated-dose toxicity studies in mice (LIP)Toxicity evaluation of revealed effects attributable to the test article, including transient induction of cytokines, hematological changes, and elevation of liver enzymes. The effects observed were primarily induction of the cytokines IP-10, IFN-α, IFN-γ, and IL-6 in the in vivo studies reported herein and in the in vitro studies described and discussed in Section 3.

[0474] Notably, none of the inflammatory cytokines, such as TNF-α, IFN-γ, or IL-2, were excessively upregulated in mice. However, in the cynomolgus monkey study, at least one animal showed a high transient induction of IL-6 (1,076 pg / mL). IL-6 induction was also observed in mice in a dose-dependent manner, but to a lesser extent. IL-6, along with other cytokines, will be closely monitored throughout clinical trials and directly analyzed in patients.

[0475] Effects such as lymphopenia and liver enzyme deregulation are also commonly observed as secondary effects caused by IFN-α secretion, which has been reported after treatment with plasmid lipoplexes and by TLR activation in mice and monkeys and has been commonly described in patients treated with recombinant IFN-α, which has been commercially available for many years for the treatment of several oncological and non-oncological diseases.

[0476] The observed changes in liver parameters in the high-dose group in mice suggest that the liver may be a target of toxicity at higher doses of liposome-formulated RNA. Changes included increased liver weight and increased plasma GLDH, LDH, ASAT, and ALAT levels. These changes were considered mild and were not observed in animals in the recovery group, suggesting complete recovery within at least 3 weeks. Furthermore, histopathology did not reveal any liver toxicity. In cynomolgus monkeys, biochemical parameters in liposome-treated and test article-treated animals compared with control animals were considered to be within the range of normal biological variation. The slight increases in CK observed in individual animals in groups 4, 5, or 6 compared with control animals on study days 9, 16, or 23 were primarily due to increases in the CK-MM fraction and were considered stress-related.

[0477] The mild elevation of liver parameters in mice was due to RNA production by liver target cells such as Kupffer cells. (LIP) This may be due to an immunomodulatory effect that may be triggered by phagocytosis of the IgG. In contrast to the effect observed in the mouse spleen (lymphoid hyperplasia), this does not result in leukocyte recruitment to the liver, suggesting that the desired pharmacological effects, such as TLR activation and lymphocyte trafficking, are restricted to lymphoid organs.

[0478] Complement activation has previously been reported for liposome-formulated materials. (LIP) Following vaccination, a slight increase in C5a levels was observed in female mice, but this was considered to be of low biological relevance. Furthermore, mice are not considered a good model for extrapolating complement action to humans.

[0479] Overall, all three RNA (LIP)The immunological responses observed in the repeated-dose toxicity studies (LPT Nos. 28864, 30283, and 30586) suggest a comprehensive picture based on increased spleen weight, cytokine / chemokine activation, and lymphocyte trafficking. This reflects the intended induction of pharmacological events and emphasizes the validity of mice as a valid test model for toxicity studies. In the bridging study LPT No. 30583 evaluating the toxicity of the pH-adapted L2 liposome formulation, no notable differences were observed between the two liposome formulations. Based on these findings, we will apply these pH-adapted L2 liposomes to clinical trials, based on the view that L2 liposomes have been shown to be more stable than L1 liposomes.

Claims

1. 1. A composition or pharmaceutical formulation comprising at least one RNA, wherein the at least one RNA has the following amino acid sequence: (i) Claudin 6 (CLDN6), an immunogenic variant thereof, or an amino acid sequence comprising an immunogenic fragment of CLDN6 or an immunogenic variant thereof; (ii) an amino acid sequence comprising p53, an immunogenic variant thereof, or an immunogenic fragment of p53 or an immunogenic variant thereof; and (iii) an amino acid sequence comprising a melanoma preferentially expressed antigen (PRAME), an immunogenic variant thereof, or an immunogenic fragment of PRAME or its immunogenic variant. A composition or pharmaceutical formulation encoding the

2. 2. The composition or pharmaceutical preparation of claim 1, wherein each of the amino acid sequences of (i), (ii), or (iii) is encoded by a separate RNA.

3. (i) the RNA encoding the amino acid sequence of (i) comprises the nucleotide sequence of SEQ ID NO: 2 or 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: 2 or 3; and / or (ii) the amino acid sequence of (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; 3. A composition or pharmaceutical formulation according to claim 1 or 2.

4. (i) the RNA encoding the amino acid sequence of (ii) comprises the nucleotide sequence of SEQ ID NO: 6 or 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: 6 or 7; and / or (ii) the amino acid sequence of (ii) comprises the amino acid sequence of SEQ ID NO: 4 or 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: 4 or 5; A composition or pharmaceutical formulation according to any one of claims 1 to 3.

5. (i) the RNA encoding the amino acid sequence of (iii) comprises the nucleotide sequence of SEQ ID NO: 10 or 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: 10 or 11; and / or (ii) the amino acid sequence of (iii) comprises the amino acid sequence of SEQ ID NO: 8 or 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: 8 or 9; A composition or pharmaceutical formulation according to any one of claims 1 to 4.

6. At least one RNA (iv) an amino acid sequence that disrupts immune tolerance The composition or pharmaceutical formulation of any one of claims 1 to 5, co-administered with RNA encoding the

7. Each RNA is (iv) an amino acid sequence that disrupts immune tolerance The composition or pharmaceutical formulation of any one of claims 1 to 6, co-administered with RNA encoding the

8. 8. The composition or pharmaceutical formulation of claim 6 or 7, wherein the immune tolerance-breaking amino acid sequence comprises a helper epitope, preferably a helper epitope derived from tetanus toxoid.

9. (i) the RNA encoding the immune tolerance-disrupting amino acid sequence comprises the nucleotide sequence of SEQ ID NO: 14 or 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: 14 or 15; and / or (ii) the immune tolerance-disrupting amino acid sequence comprises the amino acid sequence of SEQ ID NO: 12 or 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: 12 or 13; A composition or pharmaceutical formulation according to any one of claims 6 to 8.

10. 10. The composition or pharmaceutical formulation of any one of claims 1 to 9, wherein at least one of the amino acid sequences of (i), (ii), (iii) or (iv) is encoded by a coding sequence that has been codon-optimized and / or has an increased G / C content compared to a wild-type coding sequence, and wherein the codon-optimization and / or increased G / C content preferably does not change the sequence of the encoded amino acid sequence.

11. 11. The composition or pharmaceutical formulation of any one of claims 1 to 10, wherein each of the amino acid sequences of (i), (ii), (iii) or (iv) is encoded by a coding sequence that has been codon-optimized and / or has an increased G / C content compared to a wild-type coding sequence, and wherein the codon-optimization and / or increased G / C content preferably does not alter the sequence of the encoded amino acid sequence.

12. At least one RNA has a 5' cap ２ ７，２'－Ｏ Gpp ｓ The composition or pharmaceutical preparation according to any one of claims 1 to 11, comprising p(5')G.

13. Each RNA is 5' capped ２ ７，２'－Ｏ Gpp ｓ The composition or pharmaceutical preparation of any one of claims 1 to 12, comprising p(5')G.

14. 14. The composition or pharmaceutical formulation of any one of claims 1 to 13, wherein at least one RNA comprises a 5'UTR comprising 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.

15. 15. The composition or pharmaceutical formulation of any one of claims 1 to 14, wherein each RNA comprises a 5'UTR comprising 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.

16. 16. The composition or pharmaceutical formulation of any one of claims 1 to 15, wherein at least one of the amino acid sequences of (i), (ii), (iii), or (iv) comprises an amino acid sequence that enhances antigen processing and / or presentation.

17. 17. The composition or pharmaceutical formulation of any one of claims 1 to 16, wherein each of the amino acid sequences of (i), (ii), (iii), or (iv) comprises an amino acid sequence that enhances antigen processing and / or presentation.

18. 18. The composition or pharmaceutical formulation of claim 16 or 17, wherein the antigen processing and / or presentation enhancing amino acid sequence comprises an amino acid sequence corresponding to the transmembrane and cytoplasmic domains of an MHC molecule, preferably an MHC class I molecule.

19. (i) the RNA encoding the antigen processing and / or presentation enhancing amino acid sequence 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 (ii) the antigen processing and / or presentation enhancing amino acid sequence comprises the amino acid sequence of SEQ ID NO: 19 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: 19; A composition or pharmaceutical formulation according to any one of claims 16 to 18.

20. 20. The composition or pharmaceutical formulation of any one of claims 1 to 19, wherein at least one RNA comprises a 3'UTR comprising the nucleotide sequence of SEQ ID NO:21 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:

21.

21. 21. The composition or pharmaceutical formulation of any one of claims 1 to 20, wherein each RNA comprises a 3'UTR comprising the nucleotide sequence of SEQ ID NO:21 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:

21.

22. The composition or pharmaceutical preparation of any one of claims 1 to 21, wherein at least one RNA comprises a polyA sequence.

23. The composition or pharmaceutical preparation of any one of claims 1 to 22, wherein each RNA comprises a polyA sequence.

24. 24. The composition or pharmaceutical preparation of claim 22 or 23, wherein the polyA sequence comprises at least 100 nucleotides.

25. The composition or pharmaceutical preparation according to any one of claims 22 to 24, wherein said polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:

22.

26. 26. The composition or pharmaceutical formulation of any one of claims 1 to 25, wherein the RNA is formulated as a liquid, a solid, or a combination thereof.

27. 27. The composition or pharmaceutical preparation of any one of claims 1 to 26, wherein the RNA is formulated for injection.

28. 28. The composition or pharmaceutical preparation of any one of claims 1 to 27, wherein the RNA is formulated for intravenous administration.

29. 29. A composition or pharmaceutical formulation according to any one of claims 1 to 28, wherein said RNA is or is to be formulated as lipoplex particles.

30. 30. The composition or pharmaceutical formulation of any one of claims 1 to 29, wherein said RNA lipoplex particles are obtained by mixing said RNA with liposomes.

31. 31. The composition or pharmaceutical formulation of claim 29 or 30, wherein at least one RNA encoding the amino acid sequence of (i), (ii), and / or (iii) is or is to be co-formulated as a lipoplex particle with the RNA encoding the amino acid sequence that disrupts immune tolerance.

32. 32. The composition or pharmaceutical formulation of any one of claims 29 to 31, wherein each RNA encoding an amino acid sequence under (i), (ii), and / or (iii) is or is to be co-formulated as a lipoplex particle with said RNA encoding an amino acid sequence that disrupts immune tolerance.

33. The composition or pharmaceutical formulation of any one of claims 1 to 32, which is a pharmaceutical composition.

34. 34. The composition or pharmaceutical formulation of claim 33, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

35. The composition or pharmaceutical preparation of any one of claims 1 to 32, wherein the pharmaceutical preparation is a kit.

36. 36. The composition or pharmaceutical formulation of claim 35, wherein the RNA and optionally the liposomes are in separate vials.

37. 37. The composition or pharmaceutical preparation of claim 35 or 36, further comprising instructions for using the RNA and optionally the liposomes to treat or prevent ovarian cancer.

38. A composition or pharmaceutical formulation according to any one of claims 1 to 37 for pharmaceutical use.

39. 39. The composition or pharmaceutical formulation of claim 38, wherein the pharmaceutical use comprises the therapeutic or prophylactic treatment of a disease or disorder.

40. 40. The composition or pharmaceutical preparation of claim 39, wherein said therapeutic or prophylactic treatment of a disease or disorder comprises treating or preventing ovarian cancer.

41. A composition or pharmaceutical formulation according to any one of claims 1 to 40, for administration to a human.

42. 42. The composition or pharmaceutical formulation of any one of claims 39 to 41, wherein said therapeutic or prophylactic treatment of a disease or disorder further comprises administering an additional therapy.

43. 43. The composition or pharmaceutical preparation of claim 42, wherein the additional therapy comprises one or more selected from the group consisting of: (i) surgery to remove, excise, or debulk a tumor; (ii) radiation therapy; and (iii) chemotherapy.

44. 44. The composition or pharmaceutical preparation of claim 42 or 43, wherein the additional therapy comprises administering an additional therapeutic agent.

45. 45. The composition or pharmaceutical formulation of claim 44, wherein the additional therapeutic agent comprises an anti-cancer therapeutic agent.

46. 46. The composition or pharmaceutical formulation of claim 44 or 45, wherein the additional therapeutic agent is a checkpoint modulator.

47. 47. The composition or pharmaceutical preparation of claim 46, wherein the checkpoint modulator is an anti-PD1 antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD1 antibody and an anti-CTLA-4 antibody.

48. 1. A method of treating ovarian cancer in a subject, comprising administering to said subject at least one RNA, wherein said at least one RNA has the following amino acid sequence: (i) Claudin 6 (CLDN6), an immunogenic variant thereof, or an amino acid sequence comprising an immunogenic fragment of CLDN6 or an immunogenic variant thereof; (ii) an amino acid sequence comprising p53, an immunogenic variant thereof, or an immunogenic fragment of p53 or an immunogenic variant thereof; and (iii) an amino acid sequence comprising a melanoma preferentially expressed antigen (PRAME), an immunogenic variant thereof, or an immunogenic fragment of PRAME or its immunogenic variant. A method for encoding.

49. 49. The method of claim 48, wherein each of the amino acid sequences of (i), (ii), or (iii) is encoded by a separate RNA.

50. (i) the RNA encoding the amino acid sequence of (i) comprises the nucleotide sequence of SEQ ID NO: 2 or 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: 2 or 3; and / or (ii) the amino acid sequence of (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; 50. The method of claim 48 or 49.

51. (i) the RNA encoding the amino acid sequence of (ii) comprises the nucleotide sequence of SEQ ID NO: 6 or 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: 6 or 7; and / or (ii) the amino acid sequence of (ii) comprises the amino acid sequence of SEQ ID NO: 4 or 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: 4 or 5; 51. The method of any one of claims 48 to 50.

52. (i) the RNA encoding the amino acid sequence of (iii) comprises the nucleotide sequence of SEQ ID NO: 10 or 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: 10 or 11; and / or (ii) the amino acid sequence of (iii) comprises the amino acid sequence of SEQ ID NO: 8 or 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: 8 or 9; 52. The method of any one of claims 48 to 51.

53. At least one RNA (iv) an amino acid sequence that disrupts immune tolerance The method of any one of claims 48 to 52, wherein the method is co-administered with RNA encoding the

54. Each RNA is (iv) an amino acid sequence that disrupts immune tolerance The method of any one of claims 48 to 53, wherein the method is co-administered with RNA encoding the

55. 55. The method of claim 53 or 54, wherein the immune tolerance-breaking amino acid sequence comprises a helper epitope, preferably a helper epitope derived from tetanus toxoid.

56. (i) the RNA encoding the immune tolerance-disrupting amino acid sequence comprises the nucleotide sequence of SEQ ID NO: 14 or 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: 14 or 15; and / or (ii) the immune tolerance-disrupting amino acid sequence comprises the amino acid sequence of SEQ ID NO: 12 or 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: 12 or 13; 56. The method of any one of claims 53 to 55.

57. 57. The method of any one of claims 48 to 56, wherein at least one of the amino acid sequences of (i), (ii), (iii) or (iv) is encoded by a coding sequence that has been codon-optimized and / or has an increased G / C content compared to a wild-type coding sequence, and wherein the codon-optimization and / or increased G / C content preferably does not change the sequence of the encoded amino acid sequence.

58. 58. The method of any one of claims 48 to 57, wherein each of the amino acid sequences of (i), (ii), (iii) or (iv) is encoded by a coding sequence that has been codon-optimized and / or has an increased G / C content compared to a wild-type coding sequence, and wherein the codon-optimization and / or increased G / C content preferably does not change the sequence of the encoded amino acid sequence.

59. At least one RNA has a 5' cap ２ ７，２'－Ｏ Gpp ｓ 59. The method of any one of claims 48 to 58, comprising p(5')G.

60. Each RNA is 5' capped ２ ７，２'－Ｏ Gpp ｓ 60. The method of any one of claims 48 to 59, comprising p(5')G.

61. 61. The method of any one of claims 48 to 60, wherein at least one RNA comprises a 5'UTR comprising 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.

62. 62. The method of any one of claims 48 to 61, wherein each RNA comprises a 5'UTR comprising 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.

63. 63. The method of any one of claims 48 to 62, wherein at least one amino acid sequence of (i), (ii), (iii), or (iv) comprises an amino acid sequence that enhances antigen processing and / or presentation.

64. 64. The method of any one of claims 48 to 63, wherein each of the amino acid sequences of (i), (ii), (iii), or (iv) comprises an amino acid sequence that enhances antigen processing and / or presentation.

65. 65. The method of claim 63 or 64, wherein the amino acid sequence that enhances antigen processing and / or presentation comprises an amino acid sequence corresponding to the transmembrane and cytoplasmic domains of an MHC molecule, preferably an MHC class I molecule.

66. (i) the RNA encoding the antigen processing and / or presentation enhancing amino acid sequence 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 (ii) the antigen processing and / or presentation enhancing amino acid sequence comprises the amino acid sequence of SEQ ID NO: 19 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: 19; 66. The method of any one of claims 63 to 65.

67. 67. The method of any one of claims 48 to 66, wherein at least one RNA comprises a 3'UTR comprising the nucleotide sequence of SEQ ID NO:21 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:

21.

68. 68. The method of any one of claims 48 to 67, wherein each RNA comprises a 3'UTR comprising the nucleotide sequence of SEQ ID NO:21 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:

21.

69. 69. The method of any one of claims 48 to 68, wherein at least one RNA comprises a polyA sequence.

70. 70. The method of any one of claims 48 to 69, wherein each RNA comprises a polyA sequence.

71. 71. The method of claim 69 or 70, wherein the polyA sequence comprises at least 100 nucleotides.

72. 72. The method of any one of claims 69 to 71, wherein the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:

22.

73. 73. The method of any one of claims 48 to 72, wherein the RNA is administered by injection.

74. 74. The method of any one of claims 48 to 73, wherein the RNA is administered by intravenous administration.

75. 75. The method of any one of claims 48 to 74, wherein the RNA is formulated as lipoplex particles.

76. 76. The method of any one of claims 48 to 75, wherein the RNA lipoplex particles are obtained by mixing the RNA with liposomes.

77. 77. The method of claim 75 or 76, wherein at least one RNA encoding the amino acid sequence of (i), (ii), and / or (iii) is co-formulated as a lipoplex particle with the RNA encoding the amino acid sequence that disrupts immune tolerance.

78. 78. The method of any one of claims 75 to 77, wherein each RNA encoding the amino acid sequence of (i), (ii) and / or (iii) is co-formulated as a lipoplex particle with the RNA encoding the amino acid sequence that disrupts immune tolerance.

79. The method of any one of claims 48 to 78, wherein the subject is a human.

80. 80. The method of any one of claims 48 to 79, further comprising administering a further therapy.

81. 81. The method of claim 80, wherein the additional therapy comprises one or more selected from the group consisting of: (i) surgery to remove, excise, or debulk the tumor; (ii) radiation therapy; and (iii) chemotherapy.

82. 82. The method of claim 80 or 81, wherein the additional therapy comprises administering an additional therapeutic agent.

83. 83. The method of claim 82, wherein the additional therapeutic agent comprises an anti-cancer therapeutic agent.

84. 84. The method of claim 82 or 83, wherein the additional therapeutic agent is a checkpoint modulator.

85. 85. The method of claim 84, wherein the checkpoint modulator is an anti-PD1 antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD1 antibody and an anti-CTLA-4 antibody.

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