Therapeutic RNA for prostate cancer

Abstract

Compositions, uses and methods for treating prostate cancer are disclosed herein. In one aspect, compositions or pharmaceutical preparations comprising at least one RNA are disclosed herein, wherein the at least one RNA encodes the following amino acid sequences: (i) an amino acid sequence comprising kallikrein-2 (KLK2), an immunogenic variant thereof, or an immunogenic fragment of KLK2 or an immunogenic variant thereof; (ii) an amino acid sequence comprising prostate-specific antigen (PSA), an immunogenic variant thereof, or an immunogenic fragment of PSA or an immunogenic variant thereof; (iii) an amino acid sequence comprising prostatic acid phosphatase (PAP), an immunogenic variant thereof, or an immunogenic fragment of PAP or an immunogenic variant thereof; (iv) an amino acid sequence comprising homeobox B13 (HOXB13), an immunogenic variant thereof, or an immunogenic fragment of HOXB13 or an immunogenic variant thereof; and (v) an amino acid sequence comprising NK3 homeobox 1 (NKX3-1), an immunogenic variant thereof, or an immunogenic fragment of NKX3-1 or an immunogenic variant thereof.

Description

[0001] The present disclosure relates to the field of therapeutic RNA for the treatment of prostate cancer. Prostate cancer is a serious disease that affects thousands of middle-aged or elderly men each year. About 60% of cases occur in men older than 65 years old. The American Cancer Society (ACS) estimates that in 2019, 174,650 American men will be newly diagnosed with this disease. According to the Urology Care Foundation, prostate cancer is the second leading cause of cancer death in American men.

[0002] Disclosed herein are compositions, uses and methods for treating prostate cancer. Administering therapeutic RNA to patients with prostate cancer disclosed herein can reduce tumor size, prolong the time of progressive disease and / or prevent metastasis and / or recurrence of tumors and ultimately prolong survival time. SUMMARY OF THE INVENTION

[0004] In one aspect, provided herein is a composition or pharmaceutical formulation comprising at least one RNA, wherein the at least one RNA encodes the following amino acid sequence:

[0005] (i) comprising an amino acid sequence of Kallikrein-2 (KLK2), an immunogenic variant thereof, or an immunogenic fragment of KLK2 or an immunogenic variant thereof;

[0006] (ii) comprising an amino acid sequence of a prostate specific antigen (PSA), an immunogenic variant thereof, or an immunogenic fragment of PSA or an immunogenic variant thereof;

[0007] (iii) an amino acid sequence comprising prostatic acid phosphatase (PAP), an immunogenic variant thereof, or an immunogenic fragment of PAP or an immunogenic variant thereof;

[0008] (iv) an amino acid sequence comprising homeobox B13 (HOXB13), an immunogenic variant thereof, or an immunogenic fragment of HOXB13 or an immunogenic variant thereof; and

[0009] (v) comprising the amino acid sequence of NK3 Homeobox 1 (NKX3-1), an immunogenic variant thereof, or an immunogenic fragment of NKX3-1 or an immunogenic variant thereof.

[0010] In one embodiment, each of the amino acid sequences in (i), (ii), (iii), (iv) or (v) is encoded by a separate RNA.

[0011] In one embodiment,

[0012] (i) the RNA encoding the amino acid sequence described in (i) comprises the nucleotide sequence of SEQ ID NO: 3 or 4, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the nucleotide sequence of SEQ ID NO: 3 or 4; and / or

[0013] (ii) The amino acid sequence in (i) comprises the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 1 or 2.

[0014] In one embodiment,

[0015] (i) the RNA encoding the amino acid sequence described in (ii) comprises the nucleotide sequence of SEQ ID NO: 7 or 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the nucleotide sequence of SEQ ID NO: 7 or 8; and / or

[0016] (ii) The amino acid sequence in (ii) comprises the amino acid sequence of SEQ ID NO: 5 or 6, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 5 or 6.

[0017] In one embodiment,

[0018] (i) the RNA encoding the amino acid sequence described in (iii) comprises the nucleotide sequence of SEQ ID NO: 11 or 12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the nucleotide sequence of SEQ ID NO: 11 or 12; and / or

[0019] The amino acid sequence in (ii) or (iii) comprises the amino acid sequence of SEQ ID NO: 9 or 10, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 9 or 10.

[0020] In one embodiment,

[0021] (i) the RNA encoding the amino acid sequence described in (iv) comprises the nucleotide sequence of SEQ ID NO: 15 or 16, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the nucleotide sequence of SEQ ID NO: 15 or 16; and / or

[0022] The amino acid sequence in (ii) or (iv) comprises the amino acid sequence of SEQ ID NO: 13 or 14, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 13 or 14.

[0023] In one embodiment,

[0024] (i) the RNA encoding the amino acid sequence described in (v) comprises the nucleotide sequence of SEQ ID NO: 19 or 20, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the nucleotide sequence of SEQ ID NO: 19 or 20; and / or

[0025] (ii) (v) The amino acid sequence described in said method comprises the amino acid sequence of SEQ ID NO: 17 or 18, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 17 or 18.

[0026] In one embodiment, at least one of the amino acid sequences described in (i), (ii), (iii), (iv) or (v) is encoded by a coding sequence that has an increased G / C content compared to the wild-type coding sequence and / or is codon-optimized, wherein codon optimization and / or increased G / C content preferably do not change the sequence of the encoded amino acid sequence. In one embodiment, each of the amino acid sequences described in (i), (ii), (iii), (iv) or (v) is encoded by a coding sequence that has an increased G / C content compared to the wild-type coding sequence and / or is codon-optimized, wherein codon optimization and / or increased G / C content preferably do not change the sequence of the encoded amino acid sequence.

[0027] In one embodiment, at least one RNA is a modified RNA, particularly a stable mRNA. In one embodiment, at least one RNA comprises a modified nucleoside replacing at least one uridine. In one embodiment, at least one RNA comprises a modified nucleoside replacing each uridine. In one embodiment, each RNA comprises a modified nucleoside replacing at least one uridine. In one embodiment, each RNA comprises a modified nucleoside replacing each uridine. In one embodiment, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U).

[0028] In one embodiment, at least one RNA comprises a 5'-cap m2 7，2’-O Gpp s p(5')G. In one embodiment, each RNA comprises a 5'-cap m2 7，2’-O Gpp s p(5')G.

[0029] In one embodiment, at least one RNA comprises a 5'-UTR comprising the nucleotide sequence of SEQ ID NO: 21, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 21. In one embodiment, each RNA comprises a 5'-UTR comprising the nucleotide sequence of SEQ ID NO: 21, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 21.

[0030] In one embodiment, at least one of (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that enhances antigen processing and / or presentation. In one embodiment, each of (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that enhances antigen processing and / or presentation. In one embodiment, 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.

[0031] In one embodiment,

[0032] (i) the RNA encoding the amino acid sequence that enhances antigen processing and / or presentation comprises the nucleotide sequence of SEQ ID NO: 25, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the nucleotide sequence of SEQ ID NO: 25; and / or

[0033] (ii) the amino acid sequence that enhances antigen processing and / or presentation comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 24.

[0034] In one embodiment, the amino acid sequence that enhances antigen processing and / or presentation further comprises an amino acid sequence encoding a secretion signal peptide.

[0035] In one embodiment,

[0036] (i) the RNA encoding the secretory signal peptide comprises the nucleotide sequence of SEQ ID NO: 23, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the nucleotide sequence of SEQ ID NO: 23; and / or

[0037] (ii) the secretory signal peptide comprises the amino acid sequence of SEQ ID NO:22, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO:22.

[0038] In one embodiment, at least one of (i), (ii), (iii), (iv) or (v) amino acid sequences comprises an amino acid sequence that destroys immune tolerance. In one embodiment, each of (i), (ii), (iii), (iv) or (v) amino acid sequences comprises an amino acid sequence that destroys immune tolerance. In one embodiment, the amino acid sequence that destroys immune tolerance comprises a helper epitope, preferably a tetanus toxoid-derived helper epitope.

[0039] In one embodiment,

[0040] (i) the RNA encoding the amino acid sequence that destroys immune tolerance comprises the nucleotide sequence of SEQ ID NO: 27, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the nucleotide sequence of SEQ ID NO: 27; and / or

[0041] (ii) the amino acid sequence that destroys immune tolerance comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 26.

[0042] In one embodiment, at least one RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 28, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 28. In one embodiment, each RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 28, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 28.

[0043] In one embodiment, at least one RNA comprises a poly-A sequence. In one embodiment, each RNA comprises a poly-A sequence. In one embodiment, the poly-A sequence comprises at least 100 nucleotides. In one embodiment, the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 29.

[0044] In one embodiment, the RNA is formulated as a liquid, as a solid, or a combination thereof. In one embodiment, the RNA is formulated for injection. In one embodiment, the RNA is formulated for intravenous administration. In one embodiment, the RNA is formulated or to be formulated into lipoplex particles. In one embodiment, the RNA lipoplex particles can be obtained by mixing the RNA with liposomes.

[0045] In one embodiment, the composition or pharmaceutical preparation is a pharmaceutical composition. In one embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0046] In one embodiment, the pharmaceutical preparation is a kit. In one embodiment, the RNA and optionally the liposomes are in separate vials.

[0047] In one embodiment, the composition or pharmaceutical formulation further comprises instructions for use of the RNA and optionally liposomes for treating or preventing prostate cancer.

[0048] In one aspect, provided herein are compositions or pharmaceutical preparations for pharmaceutical use as described herein. In one embodiment, pharmaceutical uses include therapeutic or prophylactic treatment of a disease or condition. In one embodiment, therapeutic or prophylactic treatment of a disease or condition includes treating or preventing prostate cancer. In one embodiment, the compositions or pharmaceutical preparations described herein are used for administration to a human.

[0049] In one embodiment, the therapeutic or preventive treatment of a disease or condition also includes administering additional treatment. In one embodiment, additional treatment includes one or more selected from the following: (i) surgery to cut, remove or debulk a tumor, (ii) radiotherapy, and (iii) chemotherapy. In one embodiment, additional treatment includes administering additional therapeutic agents. In one embodiment, additional therapeutic agents include anti-cancer therapeutic agents. In one embodiment, additional therapeutic agents are checkpoint regulators. In one embodiment, checkpoint regulators are anti-PD1 antibodies, anti-CTLA-4 antibodies, or a combination of anti-PD1 antibodies and anti-CTLA-4 antibodies.

[0050] In one aspect, provided herein are methods of treating prostate cancer in a subject, comprising administering to the subject at least one RNA, wherein the at least one RNA encodes the following amino acid sequence:

[0051] (i) comprising an amino acid sequence of kallikrein-2 (KLK2), an immunogenic variant thereof, or an immunogenic fragment of KLK2 or an immunogenic variant thereof;

[0052] (ii) an amino acid sequence comprising prostate-specific antigen (PSA), an immunogenic variant thereof, or an immunogenic fragment of PSA or an immunogenic variant thereof;

[0053] (iii) an amino acid sequence comprising prostatic acid phosphatase (PAP), an immunogenic variant thereof, or an immunogenic fragment of PAP or an immunogenic variant thereof;

[0054] (iv) comprising an amino acid sequence of homeobox B13 (HOXB13), an immunogenic variant thereof, or an immunogenic fragment of HOXB13 or an immunogenic variant thereof; and

[0055] (v) comprising the amino acid sequence of NK3 homeobox 1 (NKX3-1), an immunogenic variant thereof, or an immunogenic fragment of NKX3-1 or an immunogenic variant thereof.

[0056] In one embodiment, each of the amino acid sequences in (i), (ii), (iii), (iv) or (v) is encoded by a separate RNA.

[0057] In one embodiment,

[0058] (i) the RNA encoding the amino acid sequence described in (i) comprises the nucleotide sequence of SEQ ID NO: 3 or 4, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the nucleotide sequence of SEQ ID NO: 3 or 4; and / or

[0059] (ii) The amino acid sequence in (i) comprises the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 1 or 2.

[0060] In one embodiment,

[0061] (i) the RNA encoding the amino acid sequence described in (ii) comprises the nucleotide sequence of SEQ ID NO: 7 or 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the nucleotide sequence of SEQ ID NO: 7 or 8; and / or

[0062] (ii) The amino acid sequence in (ii) comprises the amino acid sequence of SEQ ID NO: 5 or 6, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 5 or 6.

[0063] In one embodiment,

[0064] (i) the RNA encoding the amino acid sequence in (iii) comprises the nucleotide sequence of SEQ ID NO: 11 or 12, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the nucleotide sequence of SEQ ID NO: 11 or 12; and / or (ii) the amino acid sequence in (iii) comprises the amino acid sequence of SEQ ID NO: 9 or 10, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 9 or 10.

[0065] In one embodiment,

[0066] (i) the RNA encoding the amino acid sequence described in (iv) comprises the nucleotide sequence of SEQ ID NO: 15 or 16, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the nucleotide sequence of SEQ ID NO: 15 or 16; and / or

[0067] The amino acid sequence in (ii) or (iv) comprises the amino acid sequence of SEQ ID NO: 13 or 14, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 13 or 14.

[0068] In one embodiment,

[0069] (i) the RNA encoding the amino acid sequence described in (v) comprises the nucleotide sequence of SEQ ID NO: 19 or 20, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the nucleotide sequence of SEQ ID NO: 19 or 20; and / or

[0070] (ii) (v) The amino acid sequence described in said method comprises the amino acid sequence of SEQ ID NO: 17 or 18, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 17 or 18.

[0071] In one embodiment, at least one of the amino acid sequences described in (i), (ii), (iii), (iv) or (v) is encoded by a coding sequence that has an increased G / C content compared to the wild-type coding sequence and / or is codon-optimized, wherein codon optimization and / or increased G / C content preferably do not change the sequence of the encoded amino acid sequence. In one embodiment, each of the amino acid sequences described in (i), (ii), (iii), (iv) or (v) is encoded by a coding sequence that has an increased G / C content compared to the wild-type coding sequence and / or is codon-optimized, wherein codon optimization and / or increased G / C content preferably do not change the sequence of the encoded amino acid sequence.

[0072] In one embodiment, at least one RNA is a modified RNA, particularly a stable mRNA. In one embodiment, at least one RNA comprises a modified nucleoside replacing at least one uridine. In one embodiment, at least one RNA comprises a modified nucleoside replacing each uridine. In one embodiment, each RNA comprises a modified nucleoside replacing at least one uridine. In one embodiment, each RNA comprises a modified nucleoside replacing each uridine. In one embodiment, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U).

[0073] In one embodiment, at least one RNA comprises a 5'-cap m2 7，2’-O Gpp s p(5')G. In one embodiment, each RNA comprises a 5'-cap m2 7，2’-O Gpp s p(5')G.

[0074] In one embodiment, at least one RNA comprises a 5'-UTR comprising the nucleotide sequence of SEQ ID NO: 21, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 21. In one embodiment, each RNA comprises a 5'-UTR comprising the nucleotide sequence of SEQ ID NO: 21, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 21.

[0075] In one embodiment, at least one of (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that enhances antigen processing and / or presentation. In one embodiment, each of (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that enhances antigen processing and / or presentation. In one embodiment, 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.

[0076] In one embodiment,

[0077] (i) the RNA encoding the amino acid sequence that enhances antigen processing and / or presentation comprises the nucleotide sequence of SEQ ID NO: 25, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the nucleotide sequence of SEQ ID NO: 25; and / or

[0078] (ii) the amino acid sequence that enhances antigen processing and / or presentation comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 24.

[0079] In one embodiment, the amino acid sequence that enhances antigen processing and / or presentation further comprises an amino acid sequence encoding a secretion signal peptide.

[0080] In one embodiment,

[0081] (i) the RNA encoding the secretory signal peptide comprises the nucleotide sequence of SEQ ID NO: 23, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the nucleotide sequence of SEQ ID NO: 23; and / or

[0082] (ii) the secretory signal peptide comprises the amino acid sequence of SEQ ID NO:22, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO:22.

[0083] In one embodiment, at least one of the amino acid sequences of (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that disrupts immune tolerance. In one embodiment, each of the amino acid sequences of (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that disrupts immune tolerance. In one embodiment, the amino acid sequence that disrupts immune tolerance comprises an auxiliary epitope, preferably an auxiliary epitope derived from tetanus toxoid.

[0084] In one embodiment,

[0085] (i) the RNA encoding the amino acid sequence that destroys immune tolerance comprises the nucleotide sequence of SEQ ID NO: 27, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the nucleotide sequence of SEQ ID NO: 27; and / or

[0086] (ii) the amino acid sequence that destroys immune tolerance comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 26.

[0087] In one embodiment, at least one RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 28, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 28. In one embodiment, each RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 28, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 28.

[0088] In one embodiment, at least one RNA comprises a poly-A sequence. In one embodiment, each RNA comprises a poly-A sequence. In one embodiment, the poly-A sequence comprises at least 100 nucleotides. In one embodiment, the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 29.

[0089] In one embodiment, the RNA is administered by injection. In one embodiment, the RNA is administered by intravenous administration.

[0090] In one embodiment, the RNA is formulated into lipid complex particles. In one embodiment, the RNA lipid complex particles can be obtained by mixing RNA with liposomes.

[0091] In one embodiment, the subject is a human.

[0092] In one embodiment, the method described herein further comprises administering additional treatment. In one embodiment, additional treatment comprises one or more selected from the following: (i) surgery to cut, remove or debulk the tumor, (ii) radiotherapy, and (iii) chemotherapy. In one embodiment, additional treatment comprises administering additional therapeutic agents. In one embodiment, additional therapeutic agents comprise anti-cancer therapeutic agents. In one embodiment, additional therapeutic agents are checkpoint regulators. In one embodiment, checkpoint regulators are anti-PD1 antibodies, anti-CTLA-4 antibodies, or a combination of anti-PD1 antibodies and anti-CTLA-4 antibodies.

[0093] In one aspect, provided herein is an RNA as described herein, e.g., for use in a method described herein:

[0094] (i) encoding an RNA comprising an amino acid sequence of kallikrein-2 (KLK2), an immunogenic variant thereof, or an immunogenic fragment of KLK2 or an immunogenic variant thereof;

[0095] (ii) RNA encoding an amino acid sequence comprising prostate-specific antigen (PSA), an immunogenic variant thereof, or an immunogenic fragment of PSA or an immunogenic variant thereof;

[0096] (iii) RNA encoding an amino acid sequence comprising prostatic acid phosphatase (PAP), an immunogenic variant thereof, or an immunogenic fragment of PAP or an immunogenic variant thereof;

[0097] (iv) encoding an RNA comprising an amino acid sequence of homeobox B13 (HOXB13), an immunogenic variant thereof, or an immunogenic fragment of HOXB13 or an immunogenic variant thereof; and / or

[0098] (v) encoding an RNA comprising an amino acid sequence of NK3 homeobox 1 (NKX3-1), an immunogenic variant thereof, or an immunogenic fragment of NKX3-1 or an immunogenic variant thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 : General structure of RNAs RBL038.1, RBL039.1, RBL040.1, RBL041.1 and RBL045.1.

[0101] Schematic illustration of the general structure of all RNA vaccines with 5'-cap, 5'- and 3'-untranslated regions (UTR), coding sequence with N- and C-terminal fusion tags (sec and P2P16 / MITD, respectively) and poly(A) tail. Please note that the individual elements are not drawn exactly to scale compared to their respective sequence lengths.

[0102] Figure 2 :5'-capping structure β-S-ARCA (D1) (m2 7，2`-O GppSpG).

[0103] Shown in red are β-S-ARCA (D1) and the basic cap analog m 7 Differences between GpppG: building blocks 7 The -OCH3 group at the C2' position of G and the substitution of sulfur for the non-bridging oxygen at the β-phosphate. Due to the presence of a stereogenic P center (marked with an asterisk), the phosphorothioate cap analog β-S-ARCA exists in two diastereomers. These have been named D1 and D2 based on their elution order in reverse phase HPLC.

[0104] Figure 3 : Vector map of plasmid pST4-hAg-Kozak-KLK2-GS-P2P16-GS-MITD-FI-A30L70 used for RBL038.1 production.

[0105] Inserts with sequence elements as marked are shown in different colors. Eam1104I indicates the recognition site for the restriction endonuclease used for linearization. The Kanamycin resistance gene is shown in black.

[0106] Figure 4 : Vector map of plasmid pST4-hAg-Kozak-KLK3-GS-P2P16-GS-MITD-FI-A30L70 used for RBL039.1 production.

[0107] Inserts with sequence elements as marked are shown in different colors. Eam1104I indicates the recognition site for the restriction endonuclease used for linearization. The kanamycin resistance gene is shown in black.

[0108] Figure 5 : Vector map of plasmid pST4-hAg-Kozak-ACPP-GS-P2P16-GS-MITD-FI-A30L70 used for RBLA0.1 production.

[0109] Inserts with sequence elements as marked are shown in different colors. Eam1104I indicates the recognition site for the restriction endonuclease used for linearization. The kanamycin resistance gene is shown in black.

[0110] Figure 6 : Vector map of plasmid pST4-hAg-Kozak-sec-GS-HOXB13-GS-P2P16-GS-MITD-FI-A30L70 used for RBL041.1 production.

[0111] Inserts with sequence elements as marked are shown in different colors. Eam1104I indicates the recognition site for the restriction endonuclease used for linearization. The kanamycin resistance gene is shown in black.

[0112] Figure 7 : Vector map of plasmid pST4-hAg-Kozak-sec-GS-NKX3-1-GS-P2P16-GS-MITD-FI-A30L70 used for RBL045.1 production.

[0113] Inserts with sequence elements as marked are shown in different colors. Eam1104I indicates the recognition site for the restriction endonuclease used for linearization. The kanamycin resistance gene is shown in black.

[0114] Figure 8 : Chemical structures of selected cationic lipids and co-lipids tested during formulation development.

[0115] Fig. 9 : Organ selectivity of RNA-lipid complexes with different charge ratios.

[0116] The positively charged luc-RNA lipoplexes showed high luciferase expression in the lung, whereas the negatively charged RNA lipoplexes showed high and selective luciferase expression in the spleen.

[0117] Fig.10 : The biological activity of RNA lipid complexes depends on the particle size and the size of the liposomes used for formulation.

[0118] 20 μg of luc-RNA was condensed with small (198 nm) and large (381 nm) liposomes for reconstitution of RNA lipid complexes (RNA lipoplexes, RNA (LIP)) and injected into BALB / c mice (n=5). Luciferase expression in spleen was analyzed 6 hours after luc-RNA (LIP) administration (mean ± SD).

[0119] Fig.11: Particle size of RNA lipid complexes reconstituted according to the clinical formulation protocol.

[0120] Particle size of RNA-lipid complexes reconstituted by different experimenters, in different laboratories and with different RNA constructs was analyzed by PCS measurement. For experiments No. 3 and 10, two independent preparations were performed.

[0121] Fig.12 : Size and polydispersity index of RNA-lipid complexes with different charge ratios.

[0122] The particle size (z-average) and polydispersity index of RNA-lipid complexes with different charge ratios (DOTMA:RNA) were measured 10 minutes, 2 hours and 24 hours after preparation.

[0123] Fig.13 : Size and biological activity of RNA-lipid complexes with different charge ratios.

[0124] (A) Particle size (z-average) and polydispersity index of RNA-lipid complexes with different charge ratios (DOTMA:RNA) were measured directly after preparation (10 min). (B) Luciferase expression in spleen was analyzed 6 h after luc-RNA (LIP) (20 μg RNA) administration in BALB / c mice (n=4 to 5).

[0125] Fig.14 : Localization of bioluminescent signal after IV administration of luciferase RNA (LIP).

[0126] Bioluminescent imaging of in vivo (A) and ex vivo (B) transplanted spleen, liver and lung 6 hours after intravenous injection of luc-RNA (LIP) (20 μg RNA) into BALB / c mice (n=3). One representative mouse is shown.

[0127] Fig.15 : RNA (LIP) is selectively internalized by spleen APCs.

[0128] BALB / c mice (n=3) were injected intravenously with Cy5-RNA (40 μg (HED: 9.48 mg)) formulated with rhodamine-labeled liposomes. Uptake of Cy5-labeled RNA (lower row) or rhodamine-labeled liposomes (upper row) by cell populations in the spleen was assessed by flow cytometry 1 hour after lipoplex injection. Representative dot plots are shown.

[0129] Fig.16 : Breakdown of tolerance and antigen-specific cytotoxicity in vivo following immunization with AH5-RNA (LIP).

[0130] BALB / c mice (n=5) were immunized intravenously with AH5-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 gp70-MHC tetramer staining (grey). The line represents the average value of tetramer frequency (A). BALB / c mice (n=5) were immunized intravenously with AH5-RNA (LIP) (40 μg RNA) on days 0, 3, and 8 or were left untreated. On day 12, an in vivo cytotoxicity assay was performed by administering a mixture of CFSEhigh (loaded with AH-5 peptide) and CFSElow (loaded with Inf-HA peptide) splenocytes from naive BALB / c mice. AH5-specific lysis of a representative mouse is shown. All mice immunized with AH5-RNA (LIP) showed more than 90% antigen-specific lysis activity (B).

[0131] Fig.17 : Transient increase in IFN-α after RNA (LIP) vaccination.

[0132] (A) C57BL / 6 mice (n=3) were injected with HA-RNA (LIP) (40 μg RNA), liposomes alone or PBS as a control. Serum concentrations of IFN-α and TNF-α were assessed by ELISA 6 and 24 hours after treatment (mean ± SD). (B) Unexposed or splenectomized C57BL / 6 mice (n=2) were injected intravenously with HA-RNA (LIP) (40 μg RNA). Serum concentrations of IFN-α were assessed by ELISA 6 hours after treatment (mean ± SD).

[0133] Fig.18 : Vaccination with W_pro1 antigen RNA leads to antigen-specific T cell responses.

[0134] Splenocytes from intravenously vaccinated A2 / DR1 mice (n=4 to 5 / group) were restimulated for 20 hours with BMDC electroporated with the corresponding mRNA as indicated. BMDC electroporated with irrelevant mRNA were used as controls (open symbols, gray bars). Effector function was measured using IFN-γ ELISPOT assays. Symbols represent the mean of triplicate wells from individual animals. Bars represent the median of all animals in each group.

[0135] Fig.19 : Mean levels of IFN-α (black bars) and IL-6 (grey bars) in animals of the high-dose group.

[0136] Error bars show standard deviation. IL-6 induction was stronger after the 1st dose (day 1) than after the 5th dose (day 22).

[0137] Fig. 20 : Induction of antigen-specific T cells in the spleen by KLK2-, KLK3-, ACPP-, NKX3-1-, and HOXB13-encoding RNA.

[0138] IFN-γ ELISPOT analysis of T cell effectors from spleens of mice immunized with lipoplex-formulated RNA encoding KLK2 (aa 1-261), KLK3 (aa 1-261), ACPP (aa 1-418), HOXB13 (aa 1-284), or NKX3-1 (aa 1-234). Splenocytes obtained five days after the final immunization were restimulated with a peptide pool spanning the corresponding human proteins, the P2 / P16 / P17 peptides, or with an irrelevant control peptide, CMV pp65 (495-504). Points represent individual animals; horizontal bars represent mean ± SD of three animals.

[0139] Sequence Description

[0140] The following table provides a listing of certain sequences referenced herein.

[0141]

[0142]

[0143]

[0144]

[0145] DETAILED DESCRIPTION OF THE INVENTION

[0147] Although the present disclosure is described in detail below, it should be understood that the present disclosure is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing some specific embodiments and are not intended to limit the scope of the present disclosure, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art are generally understood.

[0148] Preferably, the terms used herein are defined as in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B. Nagel and H. Editor, Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0149] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA techniques as explained in the literature of 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).

[0150] Hereinafter, the elements of the present disclosure will be described. These elements are listed together with some specific embodiments, however, it should be understood that they can be combined in any way and in any number to produce other embodiments. The examples and embodiments of various different descriptions should not be understood as limiting the present disclosure to only some embodiments clearly described. This specification should be understood as disclosing and covering the embodiments that will be clearly described in combination with any number of disclosed elements. In addition, unless the context otherwise indicates, any arrangement and combination of all described elements should be considered to be disclosed by this specification.

[0151] The term "about" means approximately or close to, and in the context of a numerical value or range listed herein, means in one embodiment ±20%, ±10%, ±5%, or ±3% of the stated or claimed numerical value or range.

[0152] Unless otherwise noted herein or obviously contradictory to the context, nouns and similar references without quantifiers used in the context of describing the present disclosure (especially in the context of the claims) should be interpreted as covering one / kind and / or more / kind. The recording of the range of values ​​herein is only intended to be used as a shorthand for individually referring to each individual value falling within the range. Unless otherwise noted herein, each individual value is incorporated into this specification as if it is individually recorded herein. Unless otherwise noted herein or obviously contradictory to the context in other cases, all methods described herein can be carried out in any suitable order. The use of any and all examples or exemplary language (such as "for example") provided herein is only intended to better illustrate the present disclosure, without limiting the scope of the claims. The language in this specification should not be interpreted as indicating any unclaimed element necessary for practicing the present disclosure.

[0153] Unless explicitly stated otherwise, the term "includes / comprising" is used in the context of this document to indicate that in addition to the members of the list introduced by "includes / comprising", further members may optionally be present. However, it is contemplated that as a specific embodiment of the present disclosure, the term "includes / comprising" encompasses the possibility that the further members are not present, i.e., for this purpose, the embodiment "includes / comprising" should be understood to have the meaning of "consisting of..."

[0154] Several documents are cited throughout the text of this specification. Each document cited herein, whether above or below (including all patents, patent applications, scientific publications, manufacturer specifications, instructions for use, etc.), is hereby incorporated by reference in its entirety. Nothing herein should be construed as admitting that the present disclosure is not entitled to predate such disclosure.

[0155] definition

[0156] Definitions applicable to all aspects of the present disclosure are provided below. Unless otherwise indicated, the following terms have the following meanings. Any undefined term has its art-recognized meaning.

[0157] As used herein, terms such as "reduce" or "inhibit" mean the ability to cause an overall decrease in levels, such as about 5% or greater, about 10% or greater, about 20% or greater, about 50% or greater, or about 75% or greater. The term "inhibit" or similar phrases include complete or substantially complete inhibition, i.e., a decrease to zero or substantially to zero.

[0158] In one embodiment, terms such as "increase" or "enhance" refer 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%.

[0159] As used herein, "physiological pH" refers to a pH of about 7.5.

[0160] The term "ionic strength" refers to the mathematical relationship between the number of different types of ionic species in a particular solution and their respective charges. Therefore, the ionic strength I is mathematically represented by the following formula:

[0161]

[0162] where c is the molar concentration of a particular ionic species and z is the absolute value of its charge. The sum ∑ is taken over all the different kinds of ions (i) in the solution.

[0163] According to the present disclosure, in one embodiment, the term "ionic strength" refers to the presence of monovalent ions. Regarding divalent ions, particularly the presence of divalent cations, in one embodiment, their concentration or effective concentration (presence of free ions) is sufficiently low to prevent RNA degradation due to the presence of chelating agents. In one embodiment, the concentration or effective concentration of divalent ions is below the catalytic level for hydrolyzing the 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 substantially absent.

[0164] The term "freezing" involves the solidification of a liquid, usually with the removal of heat.

[0165] The term "lyophilization" or variations thereof refers to the freeze-drying of a substance by freezing the substance and then reducing the surrounding pressure to allow the freezing medium in the substance to sublime directly from the solid phase to the gas phase.

[0166] The term "spray drying" refers to spray drying of a substance by mixing a (heated) gas with a fluid which is atomized (sprayed) in a vessel (spray dryer), wherein the solvent from the formed droplets evaporates, resulting in a dry powder.

[0167] The term "cryoprotectant" relates to substances added to the formulation to protect the active ingredient during the freezing phase.

[0168] The term "lyoprotectant" relates to substances added to the formulation to protect the active ingredient during the drying phase.

[0169] The term "reconstitution" involves adding a solvent (eg, water) to a dry product to return it to a liquid state, eg, its original liquid state.

[0170] The term "recombinant" in the context of the present disclosure means "prepared by genetic engineering." In one embodiment, a "recombinant object" in the context of the present disclosure is not naturally occurring.

[0171] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including a virus) and can be isolated from a source in nature and has not been intentionally modified by man in a laboratory is naturally occurring. The term "found in nature" means "existing in nature" and includes known objects as well as 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.

[0172] In the context of the present disclosure, the term "particle" relates to a structured entity formed by a molecule or a molecular complex. In one embodiment, the term "particle" relates to a micrometer or nanometer sized structure, such as a micrometer or nanometer sized dense structure.

[0173] In the context of the present disclosure, the term "RNA lipid complex particle" relates to a particle comprising a lipid (particularly a cationic lipid) and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA lead to complexation and spontaneous formation of RNA lipid complex particles. Positively charged liposomes can typically be synthesized using a cationic lipid (e.g., DOTMA) and an additional lipid (e.g., DOPE). In one embodiment, the RNA lipid complex particle is a nanoparticle.

[0174] 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.

[0175] The term "mean diameter" refers to the average hydrodynamic diameter of the particles, as measured by dynamic light scattering (DLS) and data analysis using a so-called cumulant algorithm, which provides a so-called Z having a length dimension. 平均值 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 the particles is related to the Z 平均值 The value of is used synonymously.

[0176] The term "polydispersity index" is used herein as a measure of the size distribution of an ensemble of particles (eg nanoparticles).The polydispersity index is calculated by so-called cumulant analysis based on dynamic light scattering measurements.

[0177] The term "ethanol injection technique" refers to a process in which an ethanol solution containing lipids is rapidly injected into an aqueous solution through a needle. This action disperses the lipids throughout the solution and promotes lipid structure formation, such as lipid vesicle formation, such as liposome formation. In general, the RNA lipid complex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. In one embodiment, using the ethanol injection technique, such a colloidal liposome dispersion is formed as follows: an ethanol solution containing lipids (e.g., a cationic lipid (such as DOTMA) and additional lipids) is injected into an aqueous solution under stirring. In one embodiment, the RNA lipid complex particles described herein can be obtained without an extrusion step.

[0178] The term "extrusion" and its variants refer to the production of particles with a fixed cross-sectional profile. In particular, it refers to the miniaturization of particles, thereby forcing the particles to pass through a filter with defined pores.

[0179] The prostate is a small gland found in the lower abdomen of men. It is located below the bladder and around the urethra. The prostate is regulated by the hormone testosterone and produces seminal fluid, also called semen. Semen is the substance containing sperm that exits the urethra during ejaculation.

[0180] "Prostate cancer" as used herein is cancer in the prostate. When an abnormal, malignant growth of cells, which is called a tumor, forms in the prostate, this is called prostate cancer. Most prostate cancers grow slowly; however, some grow relatively quickly. Cancer cells may spread from the prostate to other areas of the body, particularly bones and lymph nodes. It may not cause symptoms initially. In subsequent stages, it may cause dysuria, blood in the urine, or pain in the pelvis, back, or when urinating. About 99% of cases occur in men over 50 years old. Many cases are managed by active surveillance or watchful waiting. Other treatments may include a combination of surgery, radiotherapy, hormone therapy, or chemotherapy. When it occurs only in the prostate, it may be curable. In patients where the disease has spread to the bones, painkillers, bisphosphonates, and targeted therapies, etc. may be useful. The results depend on the age and other health problems of the person, as well as the aggressiveness and extensiveness of the cancer. Globally, it is the second most common type of cancer and the fifth leading cause of cancer-related deaths in men.

[0181] As used herein, the term "co-administered" and variations thereof, etc., refers to the concurrent, simultaneous, or substantially simultaneous administration of two or more agents, as part of a single formulation or as multiple formulations administered by the same or different routes. "Substantially simultaneously," as used herein, means within a period of about 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or 6 hours of each other.

[0182] The present disclosure describes nucleic acid sequences and amino acid sequences that have a certain degree of identity to a given nucleic acid sequence or amino acid sequence, respectively (reference sequence).

[0183] The "sequence identity" between two nucleic acid sequences refers to the percentage of identical nucleotides between the sequences. The "sequence identity" between two amino acid sequences refers to the percentage of identical amino acids between the sequences.

[0184] The terms "same (%)", "identity (%)" or similar terms are particularly intended to refer to the percentage of identical nucleotides or amino acids in an optimal alignment between the sequences to be compared. The percentage is purely statistical, and the differences between the two sequences may (but are not necessarily) be randomly distributed over the entire length of the sequences to be compared. The comparison of two sequences is usually performed by comparing the sequences after optimal alignment over a segment or "comparison window" to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or with the aid of computer programs that use such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in 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 algorithm available on the 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 for the BLASTN algorithm on the NCBI website include: (i) the expected threshold is set to 10; (ii) the word length is set to 28; (iii) the maximum match within the query range is set to 0; (iv) the match / mismatch score is set to 1, -2; (v) the gap cost is set to linear; and (vi) the filter for low complexity regions is being used.In some embodiments, the algorithm parameters for the BLASTP algorithm on the NCBI website include: (i) the expected threshold is set to 10; (ii) the word length is set to 3; (iii) the maximum match within the query range is set to 0; (iv) the matrix is ​​set to BLOSUM62; (v) the gap cost is set to existence 11: extension: 1; and (vi) the condition composition scoring matrix is ​​adjusted.

[0185] The percent identity is calculated by determining the number of identical positions in the sequences being compared, dividing that number by the number of positions being compared (eg, the number of positions in the reference sequence), and multiplying the result by 100.

[0186] 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 full length of the reference sequence. For example, in some embodiments, 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 nucleotides of the contiguous nucleotides. In some embodiments, the degree of identity is given for the full length of the reference sequence.

[0187] A nucleic acid sequence or amino acid sequence having a particular degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, may have at least one functional property of the given sequence, for example, and in some cases, is functionally equivalent to the given sequence. An important property includes immunogenic properties, particularly when administered to a subject. In some embodiments, a nucleic acid sequence or amino acid sequence having a particular degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.

[0188] RNA

[0189] In the present disclosure, the term "RNA" refers to a nucleic acid molecule comprising ribonucleotide residues. In some preferred embodiments, RNA comprises all or most of the ribonucleotide residues. "Ribonucleotide" as used herein refers to a nucleotide having a hydroxyl group at the 2'-position of a β-D-ribofuranosyl group. RNA encompasses but is not limited to double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA), substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that is different from naturally occurring RNA by adding, deleting, replacing and / or changing one or more nucleotides. Such a change may refer to adding a non-nucleotide substance to an internal RNA nucleotide or to an RNA end. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered RNAs are considered to be analogs of naturally occurring RNA.

[0190] In certain embodiments of the present disclosure, RNA is a messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As recognized in the art, mRNA generally comprises a 5' untranslated region (5'-untranslated region, 5'-UTR), a peptide coding region, and a 3' untranslated region (3'-untranslated region, 3'-UTR). In some embodiments, RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, mRNA is produced by in vitro transcription using a DNA template, wherein DNA refers to a nucleic acid comprising deoxyribonucleotides.

[0191] In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0192] In one embodiment, the RNA may have modified nucleosides.In some embodiments, the RNA comprises a modified nucleoside replacing at least one (eg, each) uridine.

[0193] The term "uracil" as used herein describes one of the nucleobases that can be present in RNA nucleic acids. The structure of uracil is:

[0194]

[0195] The term "uridine" as used herein describes one of the nucleosides that can be present in RNA. The structure of uridine is:

[0196]

[0197] UTP (uridine 5'-triphosphate) has the following structure:

[0198]

[0199] Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure:

[0200]

[0201] "Pseudouridine" is an example of a modified nucleoside that is an isomer of uridine in which uracil is linked to the pentose ring by a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond.

[0202] Another exemplary modified nucleoside is N1-methyl-pseudouridine (m1Ψ), which has the following structure:

[0203]

[0204] N1-methyl-pseudo-UTP has the following structure:

[0205]

[0206] Another exemplary modified nucleoside is 5-methyl-uridine (m5U), which has the following structure:

[0207]

[0208] In some embodiments, one or more uridines in the RNA described herein are replaced by modified nucleosides. In some embodiments, the modified nucleosides are modified uridines.

[0209] In some embodiments, the modified uridine replacing uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U).

[0210] In some embodiments, the modified nucleosides replacing one or more uridines in the RNA may be any one or more of the following: 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-hydroxyacetic acid (cmo 5 U), uridine 5-hydroxyacetate (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 s 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurine methyl-uridine (τm 5 U), 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uridine (τm5s2U), 1-taurine methyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 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-thio-uridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-arabino-uridine, 2'-F-uridine, 2'-OH-arabino-uridine, 5-(2-methoxycarbonylvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0211] In some embodiments, at least one RNA comprises a modified nucleoside replacing at least one uridine. In some embodiments, at least one RNA comprises a modified nucleoside replacing each uridine. In some embodiments, each RNA comprises a modified nucleoside replacing at least one uridine. In some embodiments, each RNA comprises a modified nucleoside replacing each uridine.

[0212] In some embodiments, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (ψ). In some embodiments, the modified nucleosides comprise N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleosides comprise 5-methyl-uridine (m5U). In some embodiments, at least one RNA may comprise more than one type of modified nucleosides, and the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (ψ) and N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleosides comprise pseudouridine (ψ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides include pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0213] In some embodiments, the RNA according to the present disclosure comprises 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 by a 5'-cap analog. The term "5'-cap" refers to a structure present at the 5' end of an mRNA molecule, and is typically composed of a guanosine nucleotide linked to the mRNA via a 5'- to 5'-triphosphate linkage. In one embodiment, the guanosine is methylated at position 7. Providing an RNA with a 5'-cap or a 5'-cap analog can be achieved by in vitro transcription, wherein the 5'-cap is co-transcriptionally expressed into the RNA chain, or a capping enzyme can be used to connect to the RNA after transcription.

[0214] In some embodiments, the RNA building block cap is m2 7，3’-O Gppp(m1 2’-O )ApG (sometimes also called m2 7，3’ O G(5')ppp(5')m 2’-O ApG), which has the following structure:

[0215]

[0216] Below is an exemplary cap 1 (Cap1) RNA, which comprises RNA and m2 7，3`O G(5')PPP(5')m 2’-O ApG:

[0217]

[0218] Below is another exemplary Cap 1 RNA (uncapped analog):

[0219]

[0220] In some embodiments, in one embodiment, a cap analog anti-reverse cap (ARCA cap (m2 7’3`O G(5')ppp(5')G)) uses the "Cap0" structure to modify RNA:

[0221]

[0222] The following is a table containing RNA and m2 7，3`O Exemplary cap RNA of G(5')ppp(5')G:

[0223]

[0224] In some embodiments, a cap analog β-S-ARCA (m2 7，2`O G(5')ppSp(5')G) to generate the "cap 0" structure:

[0225]

[0226] The following are the 7，2`O Exemplary cap RNAs of G(5')ppSp(5')G) and RNA:

[0227]

[0228] A particularly preferred cap comprises a 5'-cap m2 7，2`O G(5')ppSp(5')G. In some embodiments, at least one RNA described herein comprises a 5'-cap m2 7，2`O G(5')ppSp(5')G. In some embodiments, each RNA described herein comprises a 5' cap m2 7，2`O G(5')ppSp(5')G.

[0229] In some embodiments, the 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 to a corresponding region in an RNA molecule (e.g., an mRNA molecule). An untranslated region (UTR) may be present at the 5' (upstream) (5'-UTR) and / or the 3' (downstream) (3'-UTR) of an open reading frame. The 5'-UTR, if present, is located at the 5' end, upstream of the start codon of the protein coding region. The 5'-UTR is located downstream of the 5'-cap (if present), for example, directly adjacent to the 5' cap. The 3'-UTR, if present, is located at the 3' end, downstream of the stop codon of the protein coding region, but the term "3'-UTR" preferably does not include a poly-A sequence. Therefore, the 3'-UTR is located upstream of the poly-A sequence (if present), for example, directly adjacent to the poly-A sequence.

[0230] A particularly preferred 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 21. A particularly preferred 3'-UTR comprises the nucleotide sequence of SEQ ID NO:28.

[0231] In some embodiments, at least one RNA comprises a 5'-UTR comprising the nucleotide sequence of SEQ ID NO: 21, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 21. In some embodiments, each RNA comprises a 5'-UTR comprising the nucleotide sequence of SEQ ID NO: 21, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 21.

[0232] In some embodiments, at least one RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 28, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 28. In some embodiments, each RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 28, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 28.

[0233] The term "poly-A tail" or "poly-A sequence" as used herein refers to an uninterrupted or discontinuous sequence of adenylic acid residues that are typically located at the 3' end of an RNA molecule. The poly-A tail or poly-A sequence is known to those skilled in the art and may be after the 3'-UTR in the RNA described herein. An uninterrupted poly-A tail is characterized by continuous adenylic acid residues. In fact, an uninterrupted poly-A tail is typical. The RNA disclosed herein may have a poly-A tail that is attached to the free 3' end of the RNA by a template-independent RNA polymerase after transcription or a poly-A tail that is encoded by a DNA and transcribed by a template-dependent RNA polymerase.

[0234] It has been shown that a poly-A tail of approximately 120 A nucleotides has a beneficial effect on RNA levels in transfected eukaryotic cells and on the levels of protein translated from open reading frames present upstream (5') of the poly-A tail (Holtkamp et al., 2006, Blood, Vol. 108, pp. 4009-4017).

[0235] The poly-A tail may have 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, and in particular about 120 A nucleotides. In this context, "consisting essentially of" means that most of the nucleotides in the poly-A tail, usually 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% are A nucleotides based on the number of nucleotides in the poly-A tail, but the remaining nucleotides are allowed to be nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid). In the present context, "consisting of" means all nucleotides in the poly-A tail, ie 100% by number of nucleotides in the poly-A tail are A nucleotides. The term "A nucleotide" or "A" refers to adenylic acid.

[0236] In some embodiments, the poly-A tail is attached during RNA transcription, such as during the preparation of in vitro transcribed RNA, based on a DNA template containing repeated dT nucleotides (deoxythymidylic acid) in a strand complementary to the coding strand. The DNA sequence encoding the poly-A tail (coding strand) is referred to as a poly (A) box.

[0237] In some embodiments, the poly (A) box present in the DNA coding chain is essentially composed of dA nucleotides, but is interrupted by a random sequence of four nucleotides (dA, dC, dG and dT). The length of such a random sequence can be 5 to 50, 10 to 30 or 10 to 20 nucleotides. Such a box is disclosed in WO 2016 / 005324 A1, which is incorporated by reference herein. Any poly (A) box disclosed in WO2016 / 005324 A1 can be used in the present invention. Contains the following: essentially composed of dA nucleotides but interrupted by a random sequence of four nucleotides (dA, dC, dG, dT) with equal distribution and a length of, for example, 5 to 50 nucleotides. The poly (A) box shows that the constant proliferation of plasmid DNA in Escherichia coli (E. coli) at the DNA level is still associated with the beneficial properties of supporting RNA stability and translation efficiency at the RNA level. Thus, in some embodiments, the poly-A tail contained in the RNA molecule described herein consists essentially of A nucleotides, but is interrupted by a random sequence of four nucleotides (A, C, G, U). Such a random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

[0238] In some embodiments, no nucleotides other than A nucleotides flank the poly-A tail at its 3' end, ie, the poly-A tail is not masked or followed by nucleotides other than A at its 3' end.

[0239] In some embodiments, the poly-A tail comprises the sequence of SEQ ID NO:29.

[0240] 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 shown in SEQ ID NO: 29. 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.

[0241] In the context of the present disclosure, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into a peptide or protein.

[0242] With respect to RNA, the term "expression" or "translation" refers to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of an amino acid sequence to produce a peptide or protein.

[0243] In one embodiment, after administering the RNA described herein, for example, formulated as RNA lipid complex particles, 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 a target cell. In one embodiment, the RNA is translated by the target cell to produce a peptide or protein encoded by it. In one embodiment, the target cell is a splenocyte. 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 lipid complex particles described herein can be used to deliver RNA to such target cells. Therefore, the present disclosure also relates to a method for delivering RNA to a target cell in a subject, the method comprising administering to the subject the RNA lipid complex particles described herein. In one embodiment, the RNA is delivered to the cytosol of a target cell. In one embodiment, the RNA is translated by the target cell to produce a peptide or protein encoded by the RNA.

[0244] According to the present disclosure, the term "RNA encoding" means that, if present in a suitable environment, such as within a cell of a target tissue, the RNA can direct the assembly of amino acids during the translation process to produce a peptide or protein encoded therein. In one embodiment, the RNA is capable of interacting with the cell translation machinery, thereby allowing translation of the peptide or protein. The cell can produce the encoded peptide or protein intracellularly (e.g., in the cytoplasm and / or in the nucleus), can secrete the encoded peptide or protein, or can cause it to be produced on the surface.

[0245] According to the present disclosure, the term "peptide" includes oligopeptides and polypeptides, and refers to a substance comprising about two 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 connected to each other by peptide bonds. The term "protein" refers to large peptides, particularly peptides having at least about 151 amino acids, but the terms "peptide" and "protein" are often used as synonyms herein.

[0246] The term "antigen" refers to a substance comprising an epitope to which an immune response can be generated. In particular, the term "antigen" includes proteins and peptides. In one embodiment, the antigen is presented by cells of the immune system (e.g., antigen presenting cells such as dendritic cells or macrophages). In one embodiment, an antigen or its processed product, such as a T cell epitope, binds to an antibody through a T or B cell receptor or through an immunoglobulin molecule such as an antibody. Therefore, an antigen or its processed product can react specifically 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.

[0247] 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 that contains an epitope that will stimulate the host's immune system to produce a cellular antigen-specific immune response and / or a humoral antibody response to the disease. Thus, a disease associated antigen or an epitope thereof may be used for therapeutic purposes. A disease associated antigen may be associated with a cancer, typically a tumor.

[0248] The term "tumor antigen" refers to a component of a cancer cell, which may be derived from the cytoplasm, cell surface, and cell nucleus. In particular, it refers to those antigens produced intracellularly or as surface antigens on tumor cells.

[0249] The term "epitope" refers to a portion or fragment of a molecule (e.g., 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 comprise a continuous or discontinuous portion of the antigen and can be about 5 to about 100 amino acids in length. In one embodiment, the length of the epitope is about 10 to about 25 amino acids. The term "epitope" includes T cell epitopes.

[0250] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by a T cell when present in the context of an MHC molecule. The term "major histocompatibility complex" and the abbreviation "MHC" include MHC class I and MHC class II molecules, and relate to a gene complex present in all vertebrates. MHC proteins or molecules are important for signal transduction between lymphocytes and antigen presenting cells or diseased cells in an immune response, wherein MHC proteins or molecules bind peptide epitopes and present them to be recognized by T cell receptors on T cells. Proteins encoded by MHC are expressed on the cell surface and display both autoantigens (peptide fragments from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms) to T cells. In the case of class I MHC / peptide complexes, binding peptides are generally about 8 to about 10 amino acids long, although longer or shorter peptides may be effective. In the case of class II MHC / peptide complexes, binding peptides are generally about 10 to about 25 amino acids long, and particularly about 13 to about 18 amino acids long, although longer and shorter peptides may be effective.

[0251] In certain embodiments of the present disclosure, the RNA encodes at least one epitope. In certain embodiments, the epitope is derived from a tumor antigen as described herein.

[0252] RNA administered

[0253] In some embodiments, the compositions described herein comprise RNA encoding Kallikrein-2 (KLK2) protein, RNA encoding prostate-specific antigen (PSA) protein, RNA encoding prostatic acid phosphatase (PAP) protein, RNA encoding homeobox B13 (HOXB13) protein, and RNA encoding NK3 homeobox 1 (NKX3-1) protein. Similarly, the methods described herein comprise administering RNA encoding Kallikrein-2 (KLK2) protein, RNA encoding prostate-specific antigen (PSA) protein, RNA encoding prostatic acid phosphatase (PAP) protein, RNA encoding homeobox B13 (HOXB13) protein, and RNA encoding NK3 homeobox 1 (NKX3-1) protein.

[0254] The Kallikrein-2 (KLK2) protein comprises an amino acid sequence comprising KLK2, an immunogenic variant thereof, or an immunogenic fragment of KLK2 or an immunogenic variant thereof, and may have an amino acid sequence comprising: an amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to an amino acid sequence of SEQ ID NO: 1 or 2. The RNA encoding the KLK2 protein (i) may comprise the nucleotide sequence of SEQ ID NO: 3 or 4, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 3 or 4; and / or (ii) may encode an amino acid sequence comprising: an amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 2.

[0255] The prostate-specific antigen (PSA) protein comprises an amino acid sequence comprising PSA, an immunogenic variant thereof, or an immunogenic fragment of PSA or an immunogenic variant thereof, and may have an amino acid sequence comprising: an amino acid sequence of SEQ ID NO: 5 or 6, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 5 or 6. The RNA encoding the PSA protein (i) may comprise the nucleotide sequence of SEQ ID NO: 7 or 8, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the nucleotide sequence of SEQ ID NO: 7 or 8; and / or (ii) may encode an amino acid sequence comprising: an amino acid sequence of SEQ ID NO: 5 or 6, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 5 or 6.

[0256] The prostatic acid phosphatase (PAP) protein comprises an amino acid sequence containing PAP, an immunogenic variant thereof, or an immunogenic fragment of PAP or an immunogenic variant thereof, and may have an amino acid sequence comprising: an amino acid sequence of SEQ ID NO: 9 or 10, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 9 or 10. The RNA encoding the PAP protein (i) may comprise the nucleotide sequence of SEQ ID NO: 11 or 12, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the nucleotide sequence of SEQ ID NO: 11 or 12; and / or (ii) may encode an amino acid sequence comprising: an amino acid sequence of SEQ ID NO: 9 or 10, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 9 or 10.

[0257] The homeobox B13 (HOXB13) protein comprises an amino acid sequence comprising HOXB13, an immunogenic variant thereof, or an immunogenic fragment of HOXB13 or an immunogenic variant thereof, and may have an amino acid sequence comprising: an amino acid sequence of SEQ ID NO: 13 or 14, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to an amino acid sequence of SEQ ID NO: 13 or 14. The RNA encoding the HOXB13 protein (i) may comprise the nucleotide sequence of SEQ ID NO: 15 or 16, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the nucleotide sequence of SEQ ID NO: 15 or 16; and / or (ii) may encode an amino acid sequence comprising: an amino acid sequence of SEQ ID NO: 13 or 14, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of SEQ ID NO: 13 or 14.

[0258] The NK3 homeobox 1 (NKX3-1) protein comprises an amino acid sequence comprising NKX3-1, an immunogenic variant thereof, or an immunogenic fragment of NKX3-1 or an immunogenic variant thereof, and may have an amino acid sequence comprising: an amino acid sequence of SEQ ID NO: 17 or 18, or an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to an amino acid sequence of SEQ ID NO: 17 or 18. The RNA encoding the NKX3-1 protein (i) may comprise the nucleotide sequence of SEQ ID NO: 19 or 20, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 19 or 20; and / or (ii) may encode an amino acid sequence comprising: an amino acid sequence of SEQ ID NO: 17 or 18, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 17 or 18.

[0259] "Variant" herein means an amino acid sequence that differs from a parent amino acid sequence due to at least one amino acid modification. The parent amino acid sequence may be a naturally occurring or wild-type (WT) amino acid sequence, or may be a modified form of a 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, for example, 1 to about 20 amino acid modifications compared to the parent, and preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0260] "Wild type" or "WT" or "native" herein means an amino acid sequence that occurs 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.

[0261] For the purposes of the present disclosure, "variants" of an amino acid sequence (peptide, protein or polypeptide) include 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, conformations, isoforms, allelic variants, species variants and species homologs, particularly those occurring in nature.

[0262] Amino acid insertion variants include the insertion of a single or two or more amino acids in a specific amino acid sequence. In the case of an amino acid sequence variant with insertion, one or more amino acid residues are inserted into a specific site in the amino acid sequence, although random insertion and suitable screening of the resulting product are also possible. Amino acid addition variants include one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids of amino and / or carboxyl terminal fusions. Amino acid deletion variants are characterized in that one or more amino acids are removed from the sequence, for example, 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids are removed. The deletion can be in any position of the protein. Amino acid deletion variants comprising a deletion at the N-terminal and / or C-terminal end of the protein are also referred to as N-terminal and / or C-terminal truncated variants. Amino acid substitution variants are characterized in that at least one residue in the sequence is removed, and another residue is inserted in its position. It is preferred to consider modifications in positions in non-conserved amino acid sequences between homologous proteins or peptides and / or to consider replacing amino acids with other amino acids with similar properties. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., replacements of similar charged or uncharged amino acids. Conservative amino acid changes involve replacements of one of the amino acid families related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid); basic amino acids (lysine, arginine, histidine); non-polar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes collectively classified as aromatic amino acids. In one embodiment, conservative amino acid replacements include replacements within the following groups:

[0263] Glycine, alanine;

[0264] valine, isoleucine, leucine;

[0265] Aspartic acid, glutamic acid;

[0266] Asparagine, glutamine;

[0267] Serine, threonine;

[0268] lysine, arginine; and

[0269] Phenylalanine, tyrosine.

[0270] 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 will be 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 for 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 total 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 for 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 contiguous amino acids. In some preferred embodiments, the degree of similarity or identity is given over the entire length of the reference amino acid sequence.

[0271] "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 identical amino acids between the sequences.

[0272] An amino acid sequence (peptide, protein or polypeptide) "derived from" a specified amino acid sequence (peptide, protein or polypeptide) refers to the source of the first amino acid sequence. Preferably, the amino acid sequence derived from a specific amino acid sequence has an amino acid sequence that is identical, substantially identical or homologous to the specific sequence or a fragment thereof. The amino acid sequence derived from a specific amino acid sequence may be a variant of the specific sequence or a fragment thereof.

[0273] When the peptide and protein antigens described herein (KLK2 protein, PSA protein, PAP protein, HOXB13 protein and NKX3-1 protein) are provided to a subject by administering RNA encoding the antigen (ie, vaccine antigen), it is preferred that stimulation, sensitization and / or expansion of T cells is caused in the subject. The stimulated, sensitized and / or expanded T cells are preferably directed against target antigens, in particular target antigens expressed by diseased cells, tissues and / or organs, i.e., disease-associated antigens. Therefore, 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 a disease-associated antigen. In the context of the present disclosure, the term "fragment of an antigen" or "variant of an antigen" means a substance that causes stimulation, sensitization and / or expansion of T cells, which are targeted to disease-associated antigens, particularly when expressed on the surface of diseased cells, tissues and / or organs. Therefore, the vaccine antigen administered according to the present disclosure may correspond to or may include a disease-associated antigen, may correspond to or may include a fragment of a disease-associated antigen, or may correspond to or may include an antigen homologous to a disease-associated antigen or its fragment. If the vaccine antigen administered according to the present disclosure includes a fragment of a disease-associated antigen or an amino acid sequence homologous to a fragment of a disease-associated antigen, the fragment or amino acid sequence may include an epitope of a disease-associated antigen or a sequence homologous to an epitope of a disease-associated antigen, wherein T cells bind to the epitope. Therefore, according to the present disclosure, an antigen may include an immunogenic fragment of a disease-associated antigen or an amino acid sequence homologous to an immunogenic fragment of a disease-associated antigen. According to the present disclosure, "immunogenic fragments of antigens" preferably relate to fragments of antigens that can stimulate, sensitize and / or amplify T cells. Preferably, vaccine antigens (similar to disease-associated antigens) provide relevant epitopes to be bound by T cells. Also preferably, vaccine antigens (similar to disease-associated antigens) are expressed on the surface of cells such as antigen presenting cells to provide relevant epitopes to be bound by T cells. Vaccine antigens according to the present invention may be recombinant antigens.

[0274] The term "immunologically equivalent" means immunologically equivalent molecules, such as immunologically equivalent amino acid sequences, for example, exhibit the same or substantially the same immune characteristics and / or play the same or substantially the same immunological effect with respect to the type of immunological action. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effect or characteristics of an antigen or antigen variant. For example, if an amino acid sequence induces an immune response with a specificity for reacting with the reference amino acid sequence when exposed to a T cell bound to the reference amino acid sequence or a cell expressing the reference amino acid sequence, in particular to the stimulation, sensitization and / or amplification of T cells, the amino acid sequence is immunologically equivalent to the reference amino acid sequence. Therefore, a molecule immunologically equivalent to an antigen exhibits the same or substantially the same characteristics and / or plays the same or substantially the same effect as the antigen targeted by the T cell in terms of stimulation, sensitization and / or amplification of T cells.

[0275] As used herein, "activation" or "stimulation" refers to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cell" refers in particular to a T cell that is undergoing cell division.

[0276] The term "priming" refers to the process in which a T cell first comes into contact with its specific antigen and results in differentiation into an effector T cell.

[0277] The term "clonal expansion" or "amplification" refers to a process in which a specific entity multiplies. In the context of the present disclosure, the term is preferably used in the context of an immune response in which lymphocytes are stimulated by an antigen, proliferate, and amplify specific lymphocytes that recognize the antigen. Preferably, clonal expansion results in differentiation of lymphocytes.

[0278] Lipid complex particles

[0279] In certain embodiments of the present disclosure, the RNA described herein may be present in RNA lipid complex particles. After parenteral administration, particularly after intravenous administration, the RNA lipid complex particles described herein and compositions comprising RNA lipid complex particles can be used to deliver RNA to target tissues. Liposomes can be used to prepare RNA lipid complex particles, which can be obtained by injecting a solution of lipids in ethanol into water or a suitable aqueous phase. In one embodiment, the aqueous phase has an acidic pH. In one embodiment, the aqueous phase comprises acetic acid in an amount of, for example, about 5 mM. In one embodiment, liposomes and RNA lipid complex particles comprise 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-trimethylammonium propane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In one embodiment, the 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, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the liposomes and RNA lipid complex particles comprise 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). The liposomes can be used to prepare RNA lipid complex particles by mixing the liposomes with RNA.

[0280] Spleen-targeted RNA lipoplex particles are described in WO 2013 / 143683, which is incorporated herein by reference. It has been found that RNA lipoplex particles with a net negative charge can be used to preferentially target spleen tissue or spleen cells, such as antigen presenting cells, particularly dendritic cells. Therefore, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occurs in the spleen. Therefore, 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, RNA accumulation and / or RNA expression does not or substantially does not occur in the lungs 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. Therefore, 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.

[0281] RNA lipid complex particle diameter

[0282] In one embodiment, the average diameter of the RNA lipid complex particles described herein is about 200nm to about 1000nm, about 200nm to about 800nm, about 250 to about 700nm, about 400 to about 600nm, about 300nm to about 500nm, or about 350nm to about 400nm. In one embodiment, the average diameter of the RNA lipid complex particles is about 250nm to about 700nm. In another embodiment, the average diameter of the RNA lipid complex particles is about 300nm to about 500nm. In an exemplary embodiment, the average diameter of the RNA lipid complex particles is about 400nm.

[0283] 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. For example, the RNA lipoplex particles may exhibit a polydispersity index of about 0.1 to about 0.3.

[0284] Lipids

[0285] In one embodiment, the lipid solution, liposome and RNA lipid complex particles described herein include cationic lipids. "Cationic lipid" used herein refers to a lipid with a net positive charge. Cationic lipids bind negatively charged RNA by electrostatic interactions with lipid matrices. In general, cationic lipids have lipophilic moieties, such as sterols, acyl groups or diacyl chains, and the head groups of lipids usually carry a positive charge. Some examples of cationic lipids include but are not limited to 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dimethylbis(dioctadecyl)ammonium (DDAB), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-diacyloxy-3-dimethylammonium propane, 1,2-dialkoxy-3-dimethylammonium propane, dioctadecyl dimethyl ammonium chloride (DODAC), 2,3-di(tetradecyloxy)propyl-(2-hydroxyethyl)-dimethylammonium (2,3- di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium, DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-trifluoroacetic acid propylammonium (DOSPA). Preferred are DOTMA, DOTAP, DODAC and DOSPA. In some specific embodiments, the cationic lipid is DOTMA and / or DOTAP.

[0286] Additional lipids may be incorporated to adjust the overall positive-negative charge ratio and the physical stability of the RNA lipid complex particles. In certain embodiments, the additional lipids are neutral lipids. "Neutral lipids" as used herein refer to lipids with a net charge of zero. Some examples of neutral lipids include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glyceryl-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glyceryl-3-phosphocholine (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, and cerebrosides. In some specific embodiments, additional lipids are DOPE, cholesterol, and / or DOPC.

[0287] In certain embodiments, the RNA lipid complex particles include both cationic lipids and additional lipids. 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 the at least one cationic lipid compared to the amount of the at least one additional lipid may affect important RNA lipid complex particle characteristics, such as charge, particle size, stability, tissue selectivity, and biological activity of the RNA. Therefore, in some embodiments, the molar ratio of the at least one cationic lipid to the 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 some specific embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. 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.

[0288] Charge ratio

[0289] The charge of the RNA lipid complex particle of the present disclosure is the sum of the charge present in the at least one cationic lipid and the charge present in the RNA. The charge ratio is the ratio of the positive charge present in the at least one cationic lipid to the negative charge present in the RNA. The charge ratio of the positive charge present in the at least one cationic lipid to the negative charge present in the RNA is calculated by the following equation: Charge ratio = [(cationic lipid concentration (mol)) * (total number of positive charges in cationic lipid)] / [(RNA concentration (mol)) * (total number of negative charges in RNA)]. The concentration of RNA and the amount of the at least one cationic lipid can be determined by a person skilled in the art using conventional methods.

[0290] In one embodiment, the charge ratio of positive charge to negative charge in the RNA lipid complex particle at physiological pH is about 1.6: 2 to about 1: 2 or about 1.6: 2 to about 1.1: 2. In some specific embodiments, the charge ratio of positive charge to negative charge in the RNA lipid complex particle at physiological pH is about 1.6: 2.0, about 1.5: 2.0, about 1.4: 2.0, about 13: 2.0, about 1.2: 2.0, about 1.1: 2.0, or about 1: 2.0.

[0291] It has been found that RNA lipoplex particles with such a charge ratio can be used to preferentially target spleen tissue or spleen cells, such as antigen presenting cells, particularly dendritic cells. Thus, in one embodiment, 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, RNA accumulation and / or RNA expression does not or substantially does not occur in the lungs 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.

[0292] A. Salt and ionic strength

[0293] According to the present disclosure, the compositions described herein may include salts, such as sodium chloride. Without wishing to be bound by theory, sodium chloride is used as an ionic osmolality agent for pre-treating RNA before mixing with at least one cationic lipid. In the present disclosure, certain embodiments contemplate alternative organic or inorganic salts of sodium chloride. Alternative salts include, but are not limited to, potassium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium acetate, potassium bicarbonate, potassium sulfate, potassium acetate, disodium phosphate, sodium dihydrogen phosphate, sodium acetate, sodium bicarbonate, sodium sulfate, sodium acetate, lithium chloride, magnesium chloride, magnesium phosphate, calcium chloride, and ethylenediaminetetraacetic acid (EDTA) sodium salt.

[0294] Generally, the composition comprising the RNA lipid complex particles described herein comprises sodium chloride at a concentration preferably 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 lipid complex particles comprises an ionic strength corresponding to such a sodium chloride concentration.

[0295] B. Stabilizer

[0296] The compositions described herein may comprise a stabilizer to avoid substantial loss of product quality, and in particular to avoid substantial loss of RNA activity, during freezing, lyophilization, spray drying or storage, such as storage of a frozen, lyophilized or spray dried composition.

[0297] In one embodiment, the stabilizer is a carbohydrate.The term "carbohydrate" as used herein refers to and encompasses monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides.

[0298] In some embodiments of the present disclosure, the stabilizer is mannose, glucose, sucrose, or trehalose.

[0299] According to the present disclosure, the RNA lipid complex particle compositions described herein have a stabilizer concentration suitable for the stability of the composition, particularly the stability of the RNA lipid complex particles and the stability of the RNA.

[0300] C. pH and buffers

[0301] According to the present disclosure, the RNA lipid complex particle composition described herein has a pH suitable for the stability of the RNA lipid complex particle, and in particular suitable for the stability of the RNA. In one embodiment, the pH of the RNA lipid complex particle composition described herein is about 5.5 to about 7.5.

[0302] According to the present disclosure, compositions comprising a buffer are provided. Without wishing to be bound by theory, the use of a buffer maintains the pH of the composition during manufacture, storage, and use of the composition. In certain embodiments of the present disclosure, the buffer can be sodium bicarbonate, sodium dihydrogen phosphate, disodium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen 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)

[0063] In some embodiments, the buffer may be 1,4-dihydroxy-2-(hydroxymethyl)-1-piperazine-2-yl]- ...

[0303] In one embodiment, the buffer is HEPES.

[0304] In one embodiment, the concentration of the buffer is from about 2.5 mM to about 15 mM.

[0305] D. Chelating agents

[0306] Certain embodiments of the present disclosure contemplate the use of chelating agents. Chelating agents refer to chemical compounds that can form at least two coordinated covalent bonds with metal ions to produce stable water-soluble complexes. Without wishing to be bound by theory, chelating agents reduce the concentration of free divalent ions, which in the present disclosure may additionally induce accelerated RNA degradation. Some examples of suitable chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), EDTA salts, desferrioxamine B, deferoxamine, dithiocarb sodium, penicillamine, calcium valerate, sodium valerate, succimer, 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 an EDTA salt. In an exemplary embodiment, the chelating agent is disodium EDTA dihydrate.

[0307] In some embodiments, the concentration of EDTA is about 0.05 mM to about 5 mM.

[0308] E. Physical State of the Compositions of the Disclosure

[0309] In some embodiments, the composition of the present disclosure is a liquid or a solid. Some non-limiting examples of solids include frozen forms or lyophilized forms. In a preferred embodiment, the composition is a liquid.

[0310] Pharmaceutical compositions of the present disclosure

[0311] The RNA described herein, for example formulated as RNA lipid complex particles, can be used as a pharmaceutical composition or medicament for therapeutic or prophylactic treatment or for the preparation of such a pharmaceutical composition or medicament.

[0312] The compositions of the present disclosure may be administered in the form of any suitable pharmaceutical composition.

[0313] The term "pharmaceutical composition" relates to a formulation comprising a therapeutically effective agent, preferably together with a pharmaceutically acceptable carrier, diluent and / or excipient. The pharmaceutical composition can be used to treat, prevent or alleviate the severity of a disease or condition by administering the pharmaceutical composition to a subject. Pharmaceutical compositions are also referred to in the art as pharmaceutical formulations. In the context of the present disclosure, a pharmaceutical composition comprises the RNA described herein, for example, formulated as RNA lipid complex particles.

[0314] The pharmaceutical compositions of the present disclosure preferably comprise one or more adjuvants or can be administered with one or more adjuvants. The term "adjuvant" relates to a compound that prolongs, enhances or accelerates an immune response. Adjuvants include heterogeneous groups of compounds such as oil emulsions (e.g., Freund's adjuvant), mineral compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immunostimulatory complexes. Some examples of adjuvants include, but are not limited to: LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, 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, INFa, INF-γ, GM-CSF, LT-a. Other known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils, such as ISA 51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys.

[0315] The pharmaceutical compositions according to the present disclosure are generally administered in a "pharmaceutically effective amount" and in a "pharmaceutically acceptable formulation."

[0316] The term "pharmaceutically acceptable" refers to the non-toxicity of a substance that does not interact with the effects of the active ingredients of the pharmaceutical composition.

[0317] The term "pharmaceutically effective amount" refers to an amount that achieves a desired response or desired effect alone or in combination with another dose. In the case of treating a specific disease, the desired response preferably involves inhibition of the disease process. This includes slowing down the progression of the disease, and in particular interrupting or reversing the progression of the disease. The desired response in disease treatment can also be a delay in the onset of the disease or the condition or prevention of the onset. The effective amount of the composition described herein will depend on: the condition to be treated, the severity of the disease, the individual parameters of the patient, 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. Therefore, the dosage of the composition described herein may depend on a variety of such parameters. In the case of an insufficient response in the patient at the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used.

[0318] In some embodiments, the effective amount comprises an amount sufficient to cause the tumor / lesion to shrink. In some embodiments, the effective amount is an amount sufficient to reduce the tumor growth rate (e.g., inhibit tumor growth). In some embodiments, the effective amount is an amount sufficient to delay the development of the tumor. In some embodiments, the effective amount is an amount sufficient to prevent or delay tumor recurrence. In some embodiments, the effective amount is an amount sufficient to increase the immune response of the subject to the tumor, so as to reduce, delay, improve and / or prevent tumor growth and / or size and / or metastasis. The effective amount can be administered in one or more administrations. In some embodiments, the administration of an effective amount (e.g., a composition comprising mRNA) can: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow down and prevent cancer cells from infiltrating into peripheral organs to a certain extent; (iv) inhibit (e.g., slow down and / or block or prevent to a certain extent) metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) alleviate one or more symptoms associated with cancer to a certain extent.

[0319] The pharmaceutical compositions of the present disclosure may contain salts, buffers, preservatives, and optionally other therapeutic agents.In one embodiment, the pharmaceutical compositions of the present disclosure contain one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0320] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0321] The term "excipient" as used herein refers to a substance that may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Some examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0322] The term "diluent" refers to a diluting agent and / or a thinning agent. In addition, the term "diluent" includes any one or more of a fluid, a liquid or solid suspension and / or a mixed medium. Some examples of suitable diluents include ethanol, glycerol and water.

[0323] The term "carrier" refers to a component that can be natural, synthetic, organic, inorganic, in which the active ingredients are combined to promote, enhance or achieve the use of the pharmaceutical composition. The carrier used herein can be one or more compatible solid or liquid fillers, diluents or encapsulating materials suitable for application to the object. 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 naphthalene and particularly biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure comprises isotonic saline.

[0324] Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro ed. 1985).

[0325] The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0326] Administration routes of the pharmaceutical compositions of the present disclosure

[0327] In one embodiment, the pharmaceutical composition described herein can be administered intravenously, intraarterially, subcutaneously, intradermally, intranodally or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for topical or systemic administration. Systemic administration may include enteral administration involving absorption by the gastrointestinal tract, or parenteral administration. "Parenteral administration" used herein refers to administration in any manner other than by 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 administered intravenously.

[0328] Uses of the pharmaceutical compositions of the present disclosure

[0329] The RNA described herein, for example formulated as RNA lipid complex particles, can be used in the therapeutic or prophylactic treatment of a disease, wherein provision of an amino acid sequence encoded by the RNA to a subject results in a therapeutic or prophylactic effect.

[0330] The term "disease" refers to an abnormal condition that affects an individual's body. Disease is usually interpreted as a medical condition associated with specific symptoms and signs. Disease can be caused by factors that are initially from an external source, such as an infectious disease, or disease can be caused by internal dysfunction, such as an autoimmune disease. In people, "disease" is usually more widely used to refer to any condition that causes the following: pain, dysfunction, distress, social problems, or death of the afflicted individual, or problems similar to those of the contact individual. Broadly speaking, disease sometimes includes injury, disability, illness, syndrome, infection, isolated symptoms, abnormal behavior, and atypical changes in structure and function, and in other contexts and for other purposes, these can be considered as distinguishable categories. Disease usually affects individuals not only physically but also emotionally, because contracting multiple diseases and living with multiple diseases can change people's views on life and people's personality.

[0331] In the context of the present invention, the term "treatment" and its variants or "therapeutic intervention" refers to the management and care of a subject for the purpose of combating a condition, such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition suffered by a subject, such as the administration of a therapeutically effective compound to alleviate symptoms or complications, delay the progression of a disease, disorder or condition, relieve or alleviate symptoms and complications, and / or cure or eliminate a disease, disorder or condition as well as prevention of a condition, wherein prevention is understood as the management and care of an individual for the purpose of combating a disease, disorder or condition, and includes the administration of an active compound to prevent the onset of symptoms or complications.

[0332] The term "therapeutic treatment" refers to any treatment that improves health and / or prolongs (increases) the life span of an individual. The treatment may eliminate the disease in an individual, prevent or slow the progression of the disease in an individual, inhibit or slow the progression of the disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual currently suffering from or previously suffering from the disease.

[0333] The term "prophylactic treatment" or "preventive therapy" relates to any treatment aimed at preventing the occurrence of a disease in an individual. The terms "prophylactic treatment" or "preventive treatment" are used interchangeably herein.

[0334] The terms "individual" and "subject" are used interchangeably herein. They refer to humans or other mammals (e.g., mice, rats, rabbits, dogs, cats, cattle, pigs, sheep, horses, or primates) that may be afflicted with a disease or condition (e.g., cancer) or are susceptible to a disease or condition (e.g., cancer) but may or may not have a disease or condition. In many embodiments, the individual is a human. Unless otherwise indicated, the terms "individual" and "subject" do not indicate a specific age, and therefore encompass adults, the elderly, children, and newborns. In some embodiments of the present disclosure, an "individual" or "subject" is a "patient".

[0335] The term "patient" refers to an individual or subject being treated, particularly an individual or subject who is ill.

[0336] 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 treat cancer diseases involving cells expressing one or more tumor antigens. In one embodiment, the cancer is prostate cancer. In one embodiment, the tumor antigen is KLK2, PSA, PAP, HOXB13 and / or NKX3-1.

[0337] A pharmaceutical composition comprising RNA can be administered to a subject to induce an immune response in the subject to one or more antigens or one or more epitopes encoded by the RNA, which can be therapeutic or partially or completely protective. Those skilled in the art will know that one of the principles of immunotherapy and vaccination is based on the fact that an immunoprotective response to a disease is produced by immunizing a subject with an antigen or epitope, which is immunologically related to the disease to be treated. Therefore, the pharmaceutical composition described herein can be applied to induce or enhance an immune response. Therefore, the pharmaceutical composition described herein can be used in the preventive and / or therapeutic treatment of a disease (particularly prostate cancer) involving an antigen or epitope.

[0338] As used herein, "immune response" refers to the body's integrated response to an antigen or a cell expressing an antigen, and refers to a cellular immune response and / or a humoral immune response. A cellular immune response includes, but is not limited to, a cellular response to a cell expressing an antigen and characterized by presenting the antigen with a class I or class II MHC molecule. Cellular responses are associated with T lymphocytes, which can be classified as helper T cells (also known as CD4+ T cells), which act by regulating the immune response or as killer cells (also known as cytotoxic T cells, CD8 + T cells or CTLs) play a central role in inducing apoptosis in infected cells or cancer cells. In one embodiment, administration of a pharmaceutical composition of the present disclosure involves stimulating anti-tumor CD8 +T cell response. In a specific embodiment, the tumor antigen is presented together with a class I MHC molecule.

[0339] The present disclosure contemplates an immune response that may be protective, preventative, prophylactic and / or therapeutic. As used herein, "inducing an immune response" may indicate that there was no immune response to a particular antigen prior to induction, or it may indicate that there was a basal level of immune response to a particular antigen prior to induction that was enhanced after induction. Thus, "inducing an immune response" includes "enhancing an immune response."

[0340] 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.

[0341] The terms "immunization" or "vaccination" describe the process of administering an antigen to an individual with the purpose of inducing an immune response, for example for therapeutic or prophylactic reasons.

[0342] In one embodiment, the present disclosure contemplates an embodiment in which RNA lipid complex particles as described herein are administered that target spleen tissue. The RNA encodes a peptide or protein comprising, for example, an antigen or epitope as described herein. The RNA is taken up by antigen presenting cells (e.g., dendritic cells) in the spleen to express the peptide or protein. After optional processing and presentation by the antigen presenting cells, an immune response to the antigen or epitope can be generated, resulting in preventive and / or therapeutic treatment of diseases involving the antigen or epitope. In one embodiment, the immune response induced by the RNA lipid complex particles described herein includes presentation of antigens or fragments thereof, such as epitopes, by antigen presenting cells, such as dendritic cells and / or macrophages, and activation of cytotoxic T cells caused by the presentation. For example, a peptide or its processed product or protein encoded by the RNA can be presented by a major histocompatibility complex (MHC) protein expressed on an antigen presenting cell. The MHC peptide complex can then be recognized by immune cells (e.g., T cells or B cells), resulting in their activation.

[0343] Thus, in one embodiment, after administration, the RNA in the RNA lipid complex particles described herein is delivered to the spleen and / or expressed in the spleen. In one embodiment, the RNA lipid complex particles are delivered to the spleen for activation of splenic antigen presenting cells. Thus, in one embodiment, after administration of the RNA lipid complex particles, RNA delivery and / or RNA expression occurs in antigen presenting cells. Antigen presenting cells may be professional antigen presenting cells or non-professional antigen presenting cells. Professional antigen presenting cells may be dendritic cells and / or macrophages, and even more preferably splenic dendritic cells and / or splenic macrophages.

[0344] Therefore, the present disclosure relates to the RNA lipid complex particles described herein or pharmaceutical compositions comprising the RNA lipid complex particles for use in inducing or enhancing an immune response, preferably an immune response against prostate cancer.

[0345] In one embodiment, systemic administration of the RNA lipid complex particles described herein or a pharmaceutical composition comprising the RNA lipid complex particles results in targeting and / or accumulation of the RNA lipid complex particles or RNA in the spleen but not in the lungs and / or liver. In one embodiment, the RNA lipid complex particles release RNA in the spleen and / or enter cells in the spleen. In one embodiment, systemic administration of the RNA lipid complex particles described herein or a pharmaceutical composition comprising the RNA lipid complex particles delivers RNA to antigen presenting cells in the spleen. In a specific embodiment, the antigen presenting cells in the spleen are dendritic cells or macrophages.

[0346] The term "macrophage" refers to a subgroup of phagocytes produced by differentiation of monocytes. Macrophages activated by inflammation, immune cytokines or microbial products non-specifically engulf and kill foreign pathogens by hydrolysis and oxidative attack in macrophages, resulting in degradation of pathogens. Peptides from degraded proteins are displayed on the macrophage surface, where they can be recognized by T cells, and they can directly interact with antibodies on the B cell surface, thereby causing T cells and B cells to activate and further stimulate an immune response. Macrophages belong to the category of antigen presenting cells. In one embodiment, macrophages are spleen macrophages.

[0347] The term "dendritic cell" (DC) refers to another subtype of phagocytes, which belongs to the category of antigen presenting cells. In one embodiment, dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells are initially converted into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation potential. Immature dendritic cells constantly sample pathogens in the surrounding environment, such as viruses and bacteria. Once they are in contact with presentable antigens, they will be activated to become mature dendritic cells, and begin to migrate to the spleen or migrate to lymph nodes. Immature dendritic cells engulf pathogens and degrade their proteins into small pieces (piece), and after maturation, these fragments are presented at their cell surfaces using MHC molecules. At the same time, they have raised cell surface receptors that act as co-receptors in T cell activation, such as CD80, CD86 and CD40, which greatly enhance their ability to activate T cells. They also raised CCR7, which is a chemotactic receptor that induces dendritic cells to reach the spleen through the bloodstream, or to reach lymph nodes through the lymphatic system. Here they act as antigen presenting cells and activate helper T cells and killer T cells and B cells by presenting their antigens together with non-antigen specific co-stimulatory signals. Therefore, dendritic cells can actively induce T cell or B cell related immune responses. In one embodiment, the dendritic cells are spleen dendritic cells.

[0348] The term "antigen presenting cell" (APC) is a cell of a variety of cells that is capable of displaying, capturing and / or presenting at least one antigen or antigenic fragment on its cell surface (or at its surface). Antigen presenting cells can be distinguished into professional antigen presenting cells and non-professional antigen presenting cells.

[0349] The term "professional antigen presenting cells" refers to antigen presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules required for interaction with naive T cells. If T cells interact with the MHC class II molecule complex on the antigen presenting cell membrane, the antigen presenting cells produce co-stimulatory molecules that induce T cell activation. Professional antigen presenting cells include dendritic cells and macrophages.

[0350] The term "non-professional antigen presenting cells" refers to antigen presenting cells that do not constitutively express MHC class II molecules, but constitutively express MHC class II molecules after being stimulated by certain cytokines such as interferon-γ. Some exemplary non-professional antigen presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic β cells or vascular endothelial cells.

[0351] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of the antigen (e.g., degradation of a protein into peptides), and to the association (e.g., by binding) of one or more of these fragments with an MHC molecule for presentation to specific T cells by cells such as antigen presenting cells.

[0352] The term "antigen-related disease" or "epitope-related disease" refers to any disease involving an antigen or epitope, e.g., a disease characterized by the presence of an antigen or epitope. The antigen- or epitope-related disease may be a cancer disease or simply cancer. As described above, the antigen may be a disease-associated antigen, e.g., a tumor-associated antigen, and the epitope may be derived from such an antigen.

[0353] The term "cancer disease" or "cancer" refers to or describes a physiological condition in an individual that is generally characterized by unregulated cell growth. Some examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularly, some examples of such cancers include bone cancer, blood cancer lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, cancer of the sex and reproductive organs, Hodgkin's Disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) neoplasms, neuroectodermal cancer, spinal axis tumors, gliomas, meningiomas, and pituitary adenomas. One specific form of cancer that may be treated by the compositions and methods described herein is prostate cancer.The term "cancer" according to the present disclosure also includes cancer metastasis.

[0354] Due to the synergistic effects produced, combination strategies in cancer treatment can be desirable, which can be much stronger than the effects of a single treatment method. In one embodiment, the pharmaceutical composition is administered together with an immunotherapeutic agent. "Immunotherapeutic agent" as used herein refers to any agent that can participate in activating a specific immune response and / or immune effector function. 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 a variety of mechanisms, including inducing apoptosis, blocking components of signal transduction pathways, or inhibiting the proliferation of tumor cells. In certain embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies can induce cell death through antibody-dependent cell-mediated cytotoxicity (ADCC), or bind to complement proteins, resulting in direct cytotoxicity, known as complement-dependent cytotoxicity (CDC). Some non-limiting examples of anti-cancer antibodies and potential antibody targets (in parentheses) that can be used in combination with the present disclosure include: Abavolumab (CA-125), Abciximab (CD41), Adalimumab (EpCAM), Afutuzumab (CD20), Alacizumab pegol (VEGFR2), Atumotuzumab Pentetate (CEA), Amatuximab (MORAb-009), Maanatumomab (TAG-72), Apolizumab (HLA-DR), Acitumomab (CEA), Atezolizumab (PD-L1), Baviximab (phosphatidylserine), Betumumab (CD22), Belimumab (BAFF), Bevacizumab (VEGF-A), Mobivac (Bivatuzumab mertansine)(CD44v6), blinatumomab(CD 19), brentuximab(CD30TNFRSF8), mocantuzumab(mucin CanAg), recantuzumab(MUC1), calosomal pendetide(prostate cancer cells), carlumab(CNT0888), catumaxomab(EpCAM, CD3), cetuximab(EGFR), pocitaluzumab(Citatuzumabbogatox (EpCAM), citumumab (IGF-1 receptor), claudiximab (claudin), clivatuzumabtetraxetan (MUC1), citumumab (TRAIL-R2), daclizumab (CD40), dalotuzumab (insulin-like growth factor I receptor), denosumab (RANKL), demuximab (B-lymphoma cells), drozitumab (DR5), emeticholic acid (GD3 ganglioside), edrecolomab (EpCAM), elotuzumab (SLAMF7), enatuzumab (PDL192), ensituximab (NPC-1C), epratuzumab (CD22), ertuinomab (HER2 / neu, CD3), Irelizumab (integrin ανβ3), Farletuzumab (folate receptor 1), FBTA05 (CD20), Ficlatuzumab (SCH900105), Figitumumab (IGF-1 receptor), Flanvotumab (glycoprotein 75), Fresolimumab (TGF-β), Galiximab (CD80), Ganetumab (IGF-I), Gemtuzumab Ozomicin (CD33), Gevokizumab (IL-Iβ), Girentuximab (carbonic anhydrase 9 (CA-IX)), Glembatumumab-vedotin ( vedotin)(GPNMB), Ibritumomab Tiuxetan (CD20), Icrucumab (VEGFR-1), Igovolumab (CA-125), Indatuximab ravtansine (SDC1), Intetumumab (CD51), Intuzumab Ointuzumab (CD22), Ipilimumab (CD 152), Iratumumab (CD30), Labetuximab (CEA), Lexatumumab (TRAIL-R2), Rivimab (HBsAg), Lintuzumab (CD33), Lorvotuzumabmertansine)(CD56), rucamumab(CD40), ruximab(CD23), mapamumab(TRAIL-R1), matuzumab(EGFR), mepolizumab(IL-5), milatuzumab(CD74), mitumomab(GD3 ganglioside), mogamulizumab(CCR4), moxetumomab( pasudotox)(CD22), tadalafil (C242 antigen), tadalafil (5T4), natumomab (RON), necitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4), ofatumumab (CD20), olaratumab (PDGF-Ra), onartuzumab (human scatter factor receptor kinase), oportuzumab ( monatox)(EpCAM), Ogovolumab(CA-125), Oxelumab(OX-40), Panitumumab(EGFR), Patritumab(HER3), Pemtumomab(MUC1), Pertuzumab(HER2 / neu), Pintumomab(Adenocarcinoma Antigen), Protumumab(Vimentin), Racotumomab(N-glycolylneuraminic acid), Radretumab( Fibronectin extra domain-B), Rabies virus glycoprotein, Ramucirumab (VEGFR2), Rilotumumab (HGF), Rituximab (CD20), Robatumumab (IGF-1 receptor), Samalizumab (CD200), Ciroizumab (FAP), Siltuximab (IL-6), Tabalumab (BAFF), Talizumab (alpha-fetoprotein), Pertamizumab (CD 19), Tenatumomab (tenascin C), Teprotumumab (CD221), cimetuzumab (CTLA-4), tegazumab (TRAIL-R2), TNX-650 (IL-13), tositumomab (CD20), trastuzumab (HER2 / neu), TRBS07 (GD2), tesitumomab (CTLA-4), tucotuzumabcelmoleukin)(EpCAM), Ublituximab(MS4A1), Urelumab(4-1 BB), Volociximab(integrin α5β1), Vortumomab(tumor antigen CTAA 16.88), Zalutumumab(EGFR), and Zalutumumab(CD4).

[0355] 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. Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PD-L1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PD-L2" include B7-DC, Btdc, and CD273. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 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, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a specific embodiment, 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 the binding of PD-L2 to its binding partner. In a specific embodiment, the PD-L2 binding partner is PD-1. The PD-1 binding antagonist can be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an 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). Some 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.

[0356] In one embodiment, the PD-1 binding antagonist is an immunoadhesin comprising an 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), which is a fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0357] 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).

[0358] 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.

[0359] The citation of documents and studies cited herein is not intended to admit that any of the foregoing is relevant prior art. All statements regarding the contents of these documents are based on the information available to the applicant and do not constitute any admission that the contents of these documents are correct.

[0360] The following description is presented to enable one of ordinary skill in the art to make and use multiple embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the embodiments described herein will be apparent to one of ordinary skill in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of multiple embodiments. Therefore, multiple embodiments are not intended to be limited to the embodiments described and shown herein, but are consistent with the scope of the claims. Example

[0361] Example 1: Intravenous vaccine for the treatment of prostate cancer

[0362] Described herein is a second generation vaccine for intravenous (IV) administration. It consists of RNA targeting five antigens expressed in prostate cancer, which are respectively complexed with liposomes into serum-stable RNA lipid complexes (RNA (LIP)). RNA (LIP) delivers the encoded vaccine antigens to antigen presenting cells (APCs) in lymphoid organs, which leads to effective induction of antigen-specific immune responses.

[0363] The vaccine for IV injection consists of five different RNA drug products targeting five prostate cancer-related antigens. This RNA cancer vaccine for prostate cancer consists of RBL038.1, RBL039.1, RBL040.1, RBL041.1 and RBL045.1. Each RNA cancer vaccine is composed of an RNA drug substance encoding the following respectively and is selected based on its selective expression in prostate cancer: antigens kallikrein-2 (KLK2), kallikrein-3 (KLK3, also known as prostate-specific antigen (PSA)), acid phosphatase, prostate (Acid phosphatase, prostate, ACPP, also known as prostate acid phosphatase (PAP)), homeobox B13 (HOXB13) and NK3 homeobox 1 (NKX3-1).

[0364] The RNA is reconstituted into RNA lipid complexes (RNA(LIP)) and then administered.

[0365] RNA drug products for reconstitution can be provided in vials containing 1.1 mL of the corresponding RNA drug product at a concentration of 0.25 mg / mL. Sterile isotonic NaCl solution (40 mL, 0.9%) can be delivered as the primary diluent and liposomes (4.0 mL, at a concentration of 1.4 mg / mL) as the excipient for reconstitution.

[0366] Specialized materials for reconstitution such as syringes and cannulas as well as additional isotonic saline solution to allow further dilution of the RNA (LIP) are available as clinical standards.

[0367] Drug Substance:

[0368] RBL038.1, β-S-ARCA(D1)-hAg-Kozak-KLK2-GS-P2P16-GS-MITD-FI-A30L70

[0369] Encoded antigen: Human kallikrein-2 (corresponding gene ID (HG19): uc002ptu.3, uc002ptv.3, uc002ptt.3, uc010ycl.2, uc010ycm.2, uc010yck.2, uc010eog.3)

[0370] RBL039.1, β-S-ARCA(D1)-hAg-Kozak-KLK3-GS-P2p16-GS-MITD-FI-A30L70

[0371] Encoded antigen: Human prostate-specific antigen, also known as human kallikrein-3 (corresponding gene IDs (HG19: uc002pts.1, uc002ptr.1, uc021uyl.1, uc010eof.1)

[0372] RBL040.1, β-S-ARCA(D1)-hAg-Kozak-ACPP-GS-P2P16-GS-MITD-FI-A30L70

[0373] Encoded antigen: Human prostatic acid phosphatase, also known as human acid phosphatase, prostate (corresponding gene ID (HG19): uc003eop.4)

[0374] RBL041.1, β-S-ARCA(D1)-hAg-Kozak-sec-GS-HOXB13-GS-P2P16-GS-MITD-FI-A30L70

[0375] Encoded antigen: Human homeobox B13 (corresponding gene ID (HG19): uc002ioa.3r)

[0376] RBL045.1, β-S-ARCA(D1)-hAg-Kozak-sec-GS-NKX31-GS-P2P16-GS-MITD-FI-A30L70

[0377] Encoded antigen: NK3 homeobox 1 (corresponding gene ID (HG19): uc011kzx.2).

[0378] The active ingredient in each drug substance is a single-stranded 5'-capped mRNA, which is translated into the corresponding protein after entering the antigen presenting cell (APC).

[0379] Figure 1 The general structure of antigen encoding RNA is shown, which is determined by the respective nucleotide sequence of the linearized plasmid DNA used as an in vitro RNA transcription template. In addition to the wild type or codon-optimized sequence encoding the target protein, each RNA also contains common structural elements (5'-cap, 5'-UTR, 3'-UTR, poly (A) tail; see below) optimized for mediating maximum RNA stability and translation efficiency. In addition, sec (secretion signal peptide) and MITD (MHC class I transport domain) are fused to the antigen coding region and translated into N-terminal or C-terminal tags respectively. Both fusion tags are shown to improve antigen processing and presentation on both MHC class I and MHC class II complexes. For some antigens given below, the sec fusion tag is not necessary and is therefore omitted. β-S-ARCA (D1) ( Figure 2) is used as a specific capping structure at the 5' end of the RNA drug substance.

[0380] like Figure 1 The general sequence elements of the mRNA depicted are given below.

[0381] KLK2, PSA (KLK3), PAP (ACPP), HOXB13, and NKX3-1: codon-optimized sequences encoding the respective target proteins.

[0382] hAg-Kozak: 5'-UTR sequence of human α-globin mRNA with an optimized 'Kozak sequence' to increase translation efficiency.

[0383] sec / MITD: A fusion protein tag derived from a sequence encoding the human MHC class I complex (HLA-B51, haplotype A2, B27 / B51, Cw2 / Cw3) that has been shown to enhance antigen processing and presentation. sec corresponds to a 78 bp fragment encoding a secretion signal peptide that directs 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 trafficking domain. Note that KLK2, PSA (KLK3), and PAP (ACPP) each have their own secretion signal peptide. Therefore, no sec fusion tag was added to these antigens.

[0384] GS / Linker: A sequence encoding a short linker peptide consisting mainly of the amino acids glycine (G) and serine (S) as commonly used in fusion proteins.

[0385] P2P16: A sequence encoding a tetanus toxoid-derived helper epitope to break immune tolerance.

[0386] FI element: The 3'-UTR is a combination of two sequence elements derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial-encoded 12S ribosomal RNA (called I). These were identified through an in vitro selection process for sequences that confer RNA stability and increase total protein expression.

[0387] A30L70: A poly(A) tail measuring 110 nucleotides in length consisting of a stretch of 30 adenosine residues followed by a 10-nucleotide linker sequence and an additional 70 adenosine residues designed to enhance RNA stability and translation efficiency in dendritic cells.

[0388] The complete nucleotide sequences of the five RNA drug substances RBL038.1, RBL039.1, RBL040.1, RBL041.1 and RBL045.1 are given below:

[0389] Nucleotide sequence of RBL038.1.

[0390] The nucleotide sequence is shown with individual sequence elements (as indicated by bold letters). In addition, the sequence of the translated protein is shown below the coding nucleotide sequence in italic letters (* = stop codon).

[0391]

[0392]

[0393]

[0394] Nucleotide sequence of RBL039.1.

[0395] The nucleotide sequence is shown with individual sequence elements (as indicated by bold letters). In addition, the sequence of the translated protein is shown below the coding nucleotide sequence in italic letters (* = stop codon).

[0396]

[0397]

[0398]

[0399] Nucleotide sequence of RBL040.1.

[0400] The nucleotide sequence is shown with individual sequence elements (as indicated by bold letters). In addition, the sequence of the translated protein is shown below the coding nucleotide sequence in italic letters (* = stop codon).

[0401]

[0402]

[0403]

[0404]

[0405] Nucleotide sequence of RBL041.1.

[0406] The nucleotide sequence is shown with individual sequence elements (as indicated by bold letters). In addition, the sequence of the translated protein is shown below the coding nucleotide sequence in italic letters (* = stop codon).

[0407]

[0408]

[0409]

[0410]

[0411] Nucleotide sequence of RBL045.1.

[0412] The nucleotide sequence is shown with individual sequence elements (as indicated by bold letters). In addition, the sequence of the translated protein is shown below the coding nucleotide sequence in italic letters (* = stop codon).

[0413]

[0414]

[0415]

[0416] Used to generate RBL038.1(pST4-hAg-Kozak-KLK2-GS-P2P16-GS-MITD-FI-A30L70), RBL039.1(pST4-hAg-Koza k-KLK3-GS-P2P16-GS-MITD-FI-A30L70), RBL040.1(pST4-hAg-Kozak-ACPP-GS-P2P16-GS-MITD-FI- Separate plasmid DNAs of RBL041.1 (pST4-hAg-Kozak-sec-GS-HOXB13-GS-P2P16-GS-MITD-FI-A30L70), RBL041.1 (pST4-hAg-Kozak-sec-GS-HOXB13-GS-P2P16-GS-MITD-FI-A30L70), and RBL045.1 (pST4-hAg-Kozak-sec-GS-NKX3-1-GS-P2P16-GS-MITD-FI-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 also contains a promoter for T7 RNA polymerase, a recognition sequence for a class II endonuclease for linearization, a kanamycin resistance gene, and an origin of replication (ori).

[0417] Plasmid DNA pST4-hAg-Kozak-sec-GS-SIINFEKL-GS-Ova-GS-P2P16-GS-MITD-FI-A30L70 was used as the starting point for the DNA template for the generation of RBL038.1, RBL039.1, RBL040.1, RBL041.1 and RBL045.1.

[0418] The vector map is Figures 3 to 7 Shown in.

[0419] The circular plasmid DNA was linearized with a suitable restriction enzyme to obtain the starting material for RNA transcription. Here, the enzyme Eam1104I (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) was selected because linearization with such a class II restriction endonuclease allows transcription of RNA encoding a "no" poly(A) tail, i.e., without additional nucleotides at the 3' end. It can be shown that this provides higher protein expression.

[0420] RNA (LIP) products can be prepared in a two-step procedure including: (i) diluting the RNA concentrate with a NaCl solution, and (ii) forming an RNA lipid complex by adding liposomes. For RNA (LIP) preparation, liposomes can be added to the diluted RNA. For lipids, synthetic cationic lipids DOTMA and naturally occurring phospholipids DOPE can be used. The IV injection product is a formulation with pharmaceutical and physiological characteristics that allows RNA to be selectively targeted to APCs primarily present in the spleen. RNA lipid complexes are formed by first condensing RNA in a suitable ionic environment and then incubating with positively charged liposomes.

[0421] For RNA condensation, a variety of monovalent and divalent ions, peptides, and buffers were applied at different concentrations. Monovalent ions, such as sodium and ammonium, were tested at concentrations up to 1.5 M. Divalent ions, especially Ca, were tested at concentrations up to 50 mM. 2+ Mg 2+ 、Zn 2+ and Fe 2+ In addition, various commercially available buffer solutions were tested.

[0422] Liposomes containing cationic lipids and different co-lipids were extensively tested for RNA (LIP) formation. Liposomes differing in charge, phase, size, lamellarity, and surface functionalization were investigated. Liposomes that were available at GMP grade and previously tested in clinical trials or used in market-approved products were considered. Figure 8 ).

[0423] Using the above liposome components, RNA lipid complexes were assembled with different cationic lipids: RNA and different charge ratios, where the charge ratio was calculated by the number of positive charges from the lipids and the negative charges from the RNA nucleotides (i.e., from the RNA phosphate groups). More specifically, the calculation of the charge ratio was performed as follows:

[0424] Assume that RNA is composed of nucleotides with an average molar mass of 330Da, each of which carries a negatively charged phosphate group. Therefore, a 1mg / mL RNA solution accounts for about 3mM of negative charge. On the other hand, a positive charge per monovalent cationic lipid is considered. For example, the molar mass of the cationic lipid DOTMA is 670Da, and the DOTMA concentration is that the liposome of 2mg / mL provides a positive charge concentration of 3mM. Therefore, in this case, the (+:-) charge ratio is considered to be 1: 1. The concentration of the uncharged co-lipids present in most cases does not contribute to this calculation.

[0425] The chemical and physicochemical properties of the liposomes and the RNA lipid complexes formed on this basis (i.e. with respect to chemical composition, particle size, zeta potential) 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. In addition, electron microscopy, small angle X-ray scattering (SAXS), calorimetry, field flow fractionation, analytical ultracentrifugation and spectroscopic techniques were applied during formulation development. Through this procedure, optimized formulations for further drug development were identified.

[0426] Suitable liposome formulations were tested in vitro and in vivo. In order to optimize the targeting of APCs present primarily in the spleen, the expression of luciferase as a reporter gene was observed in vivo. It could be shown that at a suitable charge ratio (negative or positive charge excess), colloidally stable nanoparticle lipoplex formulations with discrete particle sizes could be formed. Furthermore, it was shown in vivo that negatively charged luciferase-RNA lipoplex formulations showed 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 adjusted as desired, such as Fig. 9 , where organ selectivity of RNA lipoplexes from the same liposomes at different mixing ratios of cationic lipids to RNA is shown. A large number of lipid compositions can verify the observation that negatively charged lipoplexes target spleen APCs. Liposomes composed of the cationic lipid DOTMA and the auxiliary phospholipid DOPE were identified as the most suitable for forming suitable RNA lipoplexes for intended spleen APC targeting in terms of particle characteristics. Optimized spleen targeting selectivity and efficacy were observed when the negative charge was slightly in excess, consisting of excess RNA. RNA lipoplexes with slightly more positive charge and showing comparable efficacy are not suitable for development of drug products because they are too unstable as colloids and there is a high risk of aggregation and precipitation under these conditions.

[0427] Furthermore, it can be shown that for a given RNA, the biological activity of the formulation increases with the particle size of the RNA lipoplex. More specifically, it can be shown that RNA lipoplexes formed from larger liposomes (e.g., about 400 nm) are inherently larger and exhibit greater biological activity than RNA lipoplexes prepared with smaller liposomes (e.g., about 200 nm). Fig.10 ). Therefore, it is preferred to use liposomes larger than 200 nm for RNA (LIP) formation.

[0428] Based on the above findings, a robust and reproducible protocol for RNA(LIP) preparation was developed. By using defined components and a defined preparation protocol, RNA lipid complexes self-assemble to form with expected physicochemical characteristics and biological activity. As an example, Fig.11 Particle sizes from multiple independently prepared RNA lipoplexes are given in. The limited scatter in the RNA lipoplex particle sizes obtained indicates the robustness of the reconstitution procedure.

[0429] To determine the limit and robustness of the RNA(LIP) preparation, the particle size was measured at different charge ratios (mixing ratio between cationic lipid and nucleotide) ranging from 1.0:2.0 to 1.9:2.0. Fig.12 In Figure 2, the results from size measurements of RNA lipoplexes after mixing liposomes with RNA in different ratios are shown. Particle size was measured at different time points after RNA (LIP) preparation. For ratios of 1.0:2.0 to 1.6:2.0, comparable particle sizes were obtained that were stable over time. For ratios of 1.7:2.0 and higher, the particle size of the RNA lipoplexes increased both initially and over time. This finding was most pronounced after 24 hours.

[0430] Based on these data, charge ratios of 1.0:2.0 to 1.6:2.0 are considered suitable to obtain acceptable particle characteristics for RNA lipid complex products. At higher ratios (1.7:2.0 and above), particle size increased, leading to potential deviations in product quality. No changes in particle characteristics were observed for lower charge ratios, however, lower ratios were not considered due to potential lower activity in this range (data not shown). The experiments were repeated for 1.1:2.0 to 1.6:2.0, and except for size measurements ( Fig.13 In addition to A), the biological activity ( Fig.13B). In agreement with previous experiments, the particle size was almost constant. The same applies to biological activity (luciferase expression). In summary, all RNA-lipid complexes tested at charge ratios delivered RNA to APCs without significant changes in physicochemical properties or biological performance. Therefore, it is believed that 1.1:2.0 to 1.6:2.0 produce RNA-lipid complexes of equivalent quality.

[0431] Example 2: Non-clinical data

[0432] This example reviews the nonclinical studies conducted to elucidate the mode of action, pharmacodynamics, antitumor activity, pharmacokinetics, and potential toxicity of the RNA(LIP) vaccine. The key findings are summarized in Table 1.

[0433] The first part of this section provides a brief overview of the scientific basis and preparatory work used to develop the vaccine platform itself (Section 1) and a brief overview of the target characteristics of the W_pro1 target.

[0434] The following sections describe the primary pharmacodynamic studies of RNA(LIP), namely the induction of antigen-specific T cells in vivo, and the antitumor activity of RNA(LIP) vaccination (Section 2).

[0435] Secondary pharmacodynamic studies show results testing RNA(LIP)-mediated induction of proinflammatory cytokines (Section 3). Pharmacodynamic non-GLP studies were performed in cynomolgus monkeys to complement the cytokine kinetic data, as well as the hematological changes observed in mice. In vitro studies analyzing cytokine secretion by human and cynomolgus monkey blood cells after incubation with RNA(LIP) preparations are also summarized in this section.

[0436] Section 4 summarizes the safety pharmacology studies targeting the respiratory and nervous systems.

[0437] A brief overview of in vivo biodistribution, pharmacokinetics and metabolism is given in Section 5.

[0438] GLP-compliant repeated-dose toxicity studies that incorporated immunotoxicity studies were conducted and are presented and discussed in Section 6.

[0439] Table 1: Summary of the main pharmacological and toxicological characteristics of RNA (LIP) vaccines.

[0440]

[0441]

[0442]

[0443] Chapter 1: Scientific Basis

[0444] Sequence features enhance RNA translation and intracellular stability

[0445] The RNA vaccine platform has been developed and systematically optimized for more than 20 years to safely and effectively induce antigen-specific CD8+ and CD4+ T cell responses against the encoded antigens.

[0446] As described above, the active ingredient (drug substance) is a single-stranded, capped messenger RNA (mRNA) that is translated into a protein antigen after entering an antigen presenting cell. The mRNA vaccine format is pharmacologically optimized (Table 2) by (i) a modified cap analog for stabilizing translationally active RNA, (ii) an optimized 5'- and 3'-UTR for improving stability and RNA translation, (iii) a signal peptide and MITD sequence for improving MHC class I and class II antigen processing, (iv) a tetanus toxoid-derived helper epitope that breaks immune tolerance by providing nonspecific CD4+ T cell help, and (v) an extended free-terminal poly (A) tail that further enhances RNA stability and translation efficiency.

[0447] Table 2: RNA structural elements for optimization in immunopharmacology.

[0448]

[0449]

[0450] Targeting of lymphoid-resident antigen-presenting cells by antigen-encoding RNA

[0451] To deliver RNA systemically to dendritic cells, each individual RNA drug product of W_pro1 is formulated to form an RNA lipid complex (RNA(LIP)) that allows IV administration. The RNA(LIP) formulation is engineered to protect the RNA from plasma ribonuclease degradation and is optimized to selectively deliver the formulated RNA DP to dendritic cells that reside primarily in the spleen ( Fig.14 ) and other antigen-presenting cells (APCs) in lymphoid organs, where selective RNA uptake by dendritic cells and macrophages has been shown ( Fig.15 ).

[0452] Once the RNA lipid complex is taken up by APCs in the spleen, the mRNA is expressed, processed and presented by MHC molecules. This leads to a highly efficient induction of antigen-specific CD8+ and CD4+ T cell responses and T cell memory, which is further supported by the immunostimulatory environment in the spleen induced by TLR signaling mediated by RNA (LIP).

[0453] Induction of antigen-specific T cell responses

[0454] To explore the efficacy of RNA(LIP) immunization and investigate the ability of RNA(LIP) to break tolerance to endogenously expressed antigens, BALB / c mice were immunized with RNA(LIP) encoding the AH5 epitope of the murine leukemia virus (muLV)-derived gp70 protein. Repeated immunization with AH5-RNA(LIP) resulted in a significant expansion of gp70-specific CD8+ T cells ( Fig.16 A). CD8+ T cells induced by vaccination are able to lyse targets in vivo in an antigen-specific manner ( Fig.16 B), which means that functional antigen-specific CD8+ T cells with lytic capacity can be induced by RNA(LIP) immunization.

[0455] Induction of cell activation

[0456] mRNA is a ligand for human Toll-like receptors (TLRs) and is therefore able to induce immunomodulatory effects. After cellular uptake of in vitro transcribed (IVT)-RNA, recognition of TLRs occurs in the endosomal compartments where these receptors are primarily located. This triggers a cascade of signaling events that ultimately lead to activation and maturation of DCs, as shown by maturation of splenic DCs after intravenous administration of RNA (LIP) vaccines to mice. Other consequences of these immunomodulatory effects are the subsequent activation of splenic T, B, NK cells and macrophages and the reversible induction of proinflammatory cytokines.

[0457] Most importantly, injection of model RNA encoding influenza hemagglutinin HA (LIP) showed strong induction of IFN-α in mice ( Fig.17 A), which in splenectomized mice was shown to originate from the spleen ( Fig.17 B). Notably, RNA alone (LIP) showed induction of IFN-α, whereas liposomes alone did not result in induction of IFN-α in mice.

[0458] The transient cell activation and cytokines observed in mice treated with RNA (LIP) vaccines are consistent with findings that RNA vaccines can bind to and trigger TLRs. Others have also shown that RNA formulated as particles and RNA formulated in aqueous solution can activate TLRs. TLR activation has been shown to induce lymphopenia, leading to type I interferon-dependent recycling events of leukocytes. Consistent with this, activation of dendritic cells and other spleen cell populations was severely hampered in TLR7- / - or IFNAR- / - mice. Therefore, studies in IFNAR- / - mice administered RNA (LIP) do show that the transient hematological changes observed after IV administration are primarily mediated by IFN-α downstream effects.

[0459] P2P16 tetanus-derived helper epitope in W_pro1

[0460] Each W_pro1RNA DP contains the so-called P2P16 amino acid sequence of tetanus toxoid (TT) from Clostridium tetani. These sequences support overcoming the self-tolerance mechanism to effectively induce immune responses to self-antigens by providing tumor non-specific T cell help during sensitization.

[0461] The tetanus toxoid heavy chain contains epitopes that can promiscuously bind to MHC class II alleles and induce CD4+ memory T cells in nearly all individuals vaccinated with tetanus. In addition, it is known that the combination of TT helper epitopes with tumor-associated antigens can enhance CD4+ memory T cells by providing CD4+ memory T cells during sensitization compared to administration of tumor-associated antigens alone. + To reduce the stimulation of CD8 + T cell risk, two peptide sequences known to contain promiscuous binding helper epitopes were selected to ensure binding to as many MHC class II alleles as possible. Based on the data from the in vitro studies cited above, the well-known epitopes P2 (QYIKANSKFIGITEL; TT positions 830 to 844) and P16 (MTNSVDDALINSTKIYSYFPSVISKVNQGAQG; TT positions 578 to 609) were selected.

[0462] Pharmacology

[0463] The mode of action of RNA(LIP) vaccination relies on (i) the recruitment of antigen-specific T lymphocytes following peptide presentation of RNA-encoded antigens by professional APCs and (ii) TLR-mediated immunomodulatory effects that lead to cell activation and the induction of proinflammatory cytokines (e.g., type I interferons), thereby enhancing the vaccination effect.

[0464] In Section 2, we report the activation and expansion of target antigen-specific T cells after immunization with cancer antigen-encoding RNA, as well as the antitumor effects of antigen RNA (LIP) vaccination.

[0465] Extensive in vitro and in vivo studies were performed to investigate potential secondary effects of RNA(LIP) vaccine administration, such as pro-inflammatory cytokine induction and hematological changes resulting from the expected immunomodulatory effects of RNA(LIP).

[0466] In Section 3, a group of studies evaluating the extent of cellular activation of human peripheral blood cells (PBMCs) and blood cells in heparinized whole blood are discussed. In addition, the extent of vaccination-induced cytokine induction and hematological changes in cynomolgus monkeys treated with doses higher than the highest expected clinical dose in humans is shown. Finally, a side-by-side comparison of in vitro cytokine induction in blood samples from human donors and cynomolgus monkeys is performed, and the data generated in these studies are used to support the definition of a safe starting dose for an ongoing clinical trial in malignant melanoma (Lipo-MERIT) and other trials investigating RNA (LIP) immunotherapy

[0467] An overview of nonclinical studies evaluating the secondary pharmacodynamics of RNA(LIP) using human blood cells, mice and cynomolgus monkeys as test systems is given in Section 3.

[0468] We anticipate that the observed secondary pharmacodynamic effects observed for liposomally formulated RNA are not sequence dependent and therefore this study is equally applicable to other RNA drug products in use.

[0469]

[0470] Chapter 2: Main Pharmacodynamics

[0471] Several in vitro and in vivo experiments were performed to demonstrate the immunogenicity of RNA (LIP) vaccination with many different mRNAs encoding antigens specific for melanoma, breast cancer, HPV+ head and neck cancer, ovarian cancer, and other cancer types. In vivo studies were performed in mice with R&D and GMP grade materials.

[0472] Induction of antigen-specific T cell responses using W_pro1 mRNA

[0473] To obtain more information about the in vivo induction of antigen-specific T cells by prostate-specific antigens KLK2, PSA (KLK3), PAP (ACPP), HOXB13, and NKX3-1, use R&D-quality RNA and GMP-quality liposome-prepared RNA (LIP) products.

[0474] The RNA (LIP) product was injected intravenously into transgenic mice manipulated to express the human leukocyte antigens HLA-A*0201 and -DRB1*01. Using these mice, the sensitization and expansion of T cells specific for HLA-restricted epitopes can be examined in vivo. A2 / DR1 mice were vaccinated four to five times by injection of 30 μg of each antigen RNA complexed with liposomes, followed by isolation of splenocytes (5 days after the last vaccination). After restimulation with bone marrow-derived dendritic cells (BMDC) electroporated with the corresponding mRNA or an irrelevant control mRNA, the sensitization efficiency of the test items was evaluated by IFN-γ ELISPOT assay.

[0475] For all antigens, four vaccinations with RNA(LIP) preparations were sufficient to sensitize specific T cell responses in treated animals ( Fig.18 ).

[0476] These results indicate that RNA(LIP) vaccine can effectively induce T cell responses against W_pro1 antigen in vivo.

[0477] The mRNA used in the study was manufactured under R&D conditions. Additional immunogenicity studies in A2 / DR1 mice with RBL038.1, RBL039.1, RBL040.1, RBL041.1 and RBL045.1 manufactured under GMP conditions will be performed. The immunogenicity is expected to be comparable to the results from earlier studies.

[0478] In vivo antitumor activity of antigen-specific T cells induced by model antigen RNA

[0479] In relation to the known challenges in identifying murine tumor models, no additional studies were performed targeting the W_pro1 antigens, as there are no murine homologs of these antigens. Instead, we developed appropriate tumor models targeting the ovalbumin-derived SIINFEKL epitope; human papillomavirus-derived E6 / E7 antigens and gp70 as models of foreign and mouse autoantigens, respectively, for vaccination.

[0480] A summary of the in vivo antitumor effects induced by RNA(LIP) is given in Table 3.

[0481] Table 3: Summary of the in vivo anti-tumor effects of RNA (LIP).

[0482]

[0483] Section 3: Secondary Pharmacodynamics

[0484] To investigate potential secondary effects of administering RNA lipid complex vaccines, such as the induction of inflammatory cytokines and hematological changes induced by the expected immunomodulatory effects, we have conducted extensive in vitro and in vivo studies using human blood cells and cynomolgus monkeys as test systems. To our knowledge, secondary pharmacodynamic effects triggered by TLRs and activation of innate immunity by therapeutic mRNA are not sequence-dependent, and the studies presented in this chapter were conducted with an equal mixture of ATM-quality liposome-formulated RBL001.1, RBL002.2, RBL003.1, and RBL004.1. These RNAs encode melanosome antigens and have been used in clinical trials. The studies have not yet been repeated with W_pro1 RNA DP encoding TAAs.

[0485] As described below, the extent of cytokine induction, hematological changes, complement activation, and clinical chemistry induced by RNA(LIP) vaccination was studied in cynomolgus monkeys treated with doses corresponding to the expected doses in humans. In addition, the extent of cytokine release in response to RNA lipid complex treatment in human and cynomolgus monkey peripheral blood cells (PBMCs) and blood cells in heparinized whole blood was studied in non-GLP and GLP studies.

[0486] In addition, 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.

[0487]

[0488] In vitro activation of PBMC and whole blood from healthy human donors and cynomolgus monkeys

[0489] In addition to its characteristic of encoding protein antigens, RNA also has immunomodulatory effects, which derive from its ability to induce cell activation processes through TLR triggering. On the one hand, the immunomodulatory ability of RNA vaccines enhances the induction of antigen-specific T cell responses, and this should be considered the main pharmacodynamic effect. On the other hand, too strong or nonspecific immune cell activation may lead to undesirable secondary effects and should have been addressed in preclinical studies.

[0490] To investigate the extent of cellular activation of human blood cells, heparinized whole blood and PBMC (isolated from heparinized whole blood) from four healthy donors were incubated in vitro with an aliquot of ATM-quality liposome-formulated RNAs (RBL001.1, RBL002.2, RBL003.1 and RBL004.1) encoding melanosomal tumor-associated antigens. Since RNA activation of TLRs is not sequence-dependent, this study was not repeated with W_pro1 RNA.

[0491] According to the clinical formulation protocol, RNA (LIP) of each of the four RNA drug products was prepared separately. In this first study (Study 1, STR-30207-013), a concentration range of 0.014 μg RNA / mL to 3.333 μg RNA / mL, equivalent to a human dose of 0.07 mg to 16.65 mg total RNA, was selected (Table 5). As the primary endpoint, cell activation was 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 (PBMC) or plasma (whole blood) after 6 hours and 24 hours, respectively.

[0492] Table 5: Description of doses for in vitro studies based on expected clinical dose cohorts.

[0493] The following values ​​represent total RNA (μg) / mL whole blood or culture medium, respectively.

[0494]

[0495] [1] Assume an average total blood volume of 5 L.

[0496] After PBMCs were incubated with RNA (LIP) mixtures, there was detectable dose-dependent activation for all eight tested analytes, but with high variation in concentration levels. Cytokine responses were dominated by five of the eight selected markers, i.e., IP-10, IFN-γ, TNF-α, IL-1β, and IL-6 (see Table 4 for summary). IFN-α, IL-2, and IL-12 showed only slight induction at the highest tested dose level.

[0497] In contrast, after incubation with RNA (LIP), no IFN-γ, TNF-α, IL-1β, IL-2 and IL-12 secretion was detected in the whole blood test system. Here, it was observed that the dose-dependent secretion of IP-10 and IL-6 increased. For IFN-α, only a low-level baseline secretion was observed, which was comparable to the diluent control and would not be further increased by incubation with RNA (LIP) (see Table 4 for summary).

[0498] In summary, the findings in PBMCs showed clear differences compared to whole blood, indicating a higher sensitivity of the test system using PBMCs. Although elevated cytokine levels were detected in PBMCs for all eight tested analytes, cytokine detection was limited to IFN-α, IP-10, and IL-6 when whole blood samples were used as the test system.

[0499] Table 4: Summary of results for PBMC and whole blood in all donors (Study STR-30207-013).

[0500]

[0501]

[0502]

[0503] To further investigate cytokine release from human cells in response to RNA (LIP) in vitro and to compare and categorize in vivo data from mouse immunotoxicity studies (see below) and cynomolgus monkey studies (see below), an additional GLP-compliant in vitro study (LPT No. 31031) was performed at an external CRO. The primary objectives of this study were to determine (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. Study LPT No. 31031 was designed as follows: In vitro induction of proinflammatory cytokines in healthy human donors and cynomolgus monkeys was tested in two test systems, namely PBMC and whole blood. The same dose range and dose steps as for RNA (LIP) in Study No. STR-30207-013 were tested. The test item was also a mixture of ATM-quality, individually prepared, liposomally formulated RBL001.1, RBL002.2, RBL003.1 and RBL004.1 RNAs. As mentioned above, the data generated with these IVT-RNAs also account for the DP of RNA encoding TAAs, since TLR activation is RNA sequence-independent. In total, samples from four individuals of each species were analyzed. As primary endpoint, cell activation was determined after 6, 24, and 48 hours by secretion of proinflammatory cytokines into the cell culture medium (PBMC) or plasma (whole blood), respectively.

[0504] The observed cytokine responses are summarized in Table 6 for the whole blood test system and in Table 7 for the PBMC test system, respectively.

[0505] Table 6: Summary of cytokine responses in the whole blood test system.

[0506]

[0507]

[0508] Table 7: Summary of cytokine responses in the PBMC test system (LPT No. 31031).

[0509]

[0510]

[0511] Table 8 shows data generated in study LPT No.31031, in which whole blood from four cynomolgus monkeys and four healthy donors was analyzed after 6 hours and 24 hours of incubation with six different doses of RNA (LIP). The analysis focused on the proinflammatory cytokines TNF-α, IL-6 and IFN-γ, as they were primarily upregulated in human PBMCs in study No.STR-30207-013. As shown, the in vitro cytokine responses in the two species were highly comparable. For IL-6, after 24 hours of incubation, a 122-fold induction in cynomolgus monkeys and a 108-fold induction in healthy donors were observed, respectively. At the highest dose level, only low levels of TNF-α were detectable in both species. After 24 hours of incubation, very low IFN-γ induction was observed only at the highest dose level in cynomolgus monkeys.

[0512] Most importantly, strong study-related cytokine induction of these three proinflammatory cytokines was observed only at dose levels ≥ 5,500 μg, which is higher than the highest expected dose level of 100 μg in patients and > 100-fold higher than the planned dose for the initial vaccination cycle (= 50 μg RNA). Of note, the results from healthy donors confirm the findings from the in vitro study STR-30207-013, and the pattern of cynomolgus monkey cytokine responses observed in the whole blood study system is similar to the findings from the in vivo study LPT No. 29928, in which only IL-6 was detectable in cynomolgus monkeys treated with RNA (LIP) (see below).

[0513] Table 9 shows the data generated in the study LPT No.31031, in which PBMCs from four cynomolgus monkeys and four healthy donors were analyzed after incubation with six different doses of RNA (LIP) for 6 hours and 24 hours. In human and cynomolgus monkey PBMCs, the induction of IL-6 and TNF-α is comparable for the following: (i) absolute amount of induced cytokines (difference between species is less than factor 2), (ii) kinetics (early induction of IL-6 and TNF-α after 6 hours), and (iii) RNA (LIP) dosage levels that cause cytokine induction. After RNA (LIP) stimulation for 24 hours, IFN-γ was detected in PBMCs from two species treated with only an intermediate dose of RNA (LIP), although the degree in people was higher. In short, for IL-6 and TNF-α, the cytokine profiles induced by RNA (LIP) in PBMCs are comparable between species. The results obtained in this study indicate that cynomolgus monkeys are a relevant species for assessing RNA (LIP)-mediated cytokine induction and that human PBMCs constitute a more sensitive system for capturing IFN-γ induction.

[0514] Table 8: In vitro induction of proinflammatory cytokines IL-6, TNF-α and IFN-γ in cynomolgus monkeys and healthy human donors in the whole blood test system.

[0515] This table shows data generated in study LPT No.31031: cytokine levels (pg / mL) of IL-6 (upper), TNF-α (middle), and IFN-γ (lower) detected after incubation of whole blood with different doses of RNA (LIP). The red code indicates the height of the cytokine level, where darker red indicates higher cytokine levels. The first column indicates the total dose level applied in the clinical setting. The second column indicates the amount of RNA used in the in vitro test system assuming a 5L blood volume. * = Data not collected.

[0516]

[0517]

[0518] [1] Assume an average total blood volume of 5 L.

[0519] Table 9: In vitro induction of proinflammatory cytokines IL-6, TNF-α and IFN-γ in cynomolgus monkeys and healthy human donors in the PBMC test system.

[0520] This table shows the data generated in study LPT No.31031: cytokine levels (pg / mL) of IL-6 (upper), TNF-α (middle), and IFN-γ (lower) detected after incubation of PBMC with different doses of RNA (LIP). The red code indicates the height of the cytokine level, where darker red indicates higher cytokine levels. The first column indicates the total dose level applied in the clinical setting. The second column indicates the amount of RNA used in the in vitro test system assuming a 5L blood volume. * = Data not collected.

[0521]

[0522]

[0523] [1] Assume an average total blood volume of 5 L.

[0524] When comparing the results found in the whole blood and PBMC test systems, it is apparent that the pro-inflammatory cytokines in the PBMC test system were generally more extensive, reached higher absolute values, and initiated at lower dose levels compared to the whole blood test system.

[0525] In this most sensitive in vitro system tested, a dramatic increase in cytokine levels as measured after 24 hours was observed over a 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 the in vitro system, the expected starting dose of 25 μg is 25-fold lower than the dose level that marks the onset of strong in vitro cytokine induction.

[0526] In addition to the GLP study LPT No.31031, a non-GLP in vitro study (report RB 14 001 B) using a similar experimental setup was also performed, with samples from three individuals of each species tested, where similar observations were made to determine the results from the GLP study (data not shown). Combining all studies, the findings emphasized (i) the similarity (comparability) of the two species of cynomolgus monkeys and humans for stimulation of cells after incubation with the test item. In addition, these observations indicate that (ii) the whole blood test system more closely reflects the in vivo situation than PBMC. In the whole blood test system, cytokine induction was generally less significant and was only observed in the highest dose group, and the main induction of IL-6 was similar to the findings of the cynomolgus monkey in vivo study (see below).

[0527] We acknowledge the more significant findings in PBMCs, which were considered artifactual, but also more sensitive in an in vitro test system, and therefore integrated the results from this more sensitive test system in the strategy to define a safe initial starting dose.

[0528] In vivo testing of secondary pharmacology in cynomolgus monkeys

[0529] In order to more accurately understand the dynamics of RNA (LIP) and the correlation of the secondary effects of RNA (LIP) with cytokine expression, a non-GLP study was performed in male cynomolgus monkeys (see Table 10 for treatment regimens and dosages, and see Table 11 for detailed study design and amounts of all formulation components). Animals in groups 1 to 5 (2 males per dose) were treated with four melanosome RNA (LIP) vaccines RBL001.1, RBL002.2, RBL003.1 and RBL004.1 (ATM quality), and then control solutions were given with slow bolus injections (about 10 seconds), with an interval of 30 minutes between each injection (i.e., the last injection was given after 1.5 hours). The results of this study should also apply to W_pro1 injections, because the secondary effects are not sequence-dependent.

[0530] Doses up to 42-fold higher than the highest clinical dose were tested within the study. Additionally, animals in dose group 6 received a single dose of 4 x 3.6 μg RNA on day 22 following a single dose of 4 x 88.6 μg RNA on day 1.

[0531] Table 10: Study regimen and doses related to expected doses in patients (LPT No. 29928).

[0532] Treatments: Animals 1 to 10 (Groups 1 to 5) were treated 5 times with four subsequent injections of NaCl (saline) (Group 1), liposomes at the same dose as high dose animals (Group 2), and RNA (LIP) 1 to 4 (ATM quality, Groups 3 to 5). Animals in Group 6 received a single treatment with 4 x 88.6 μg (total 354 μg RNA) on test day 1, followed by a single treatment with 4 x 3.6 μg (total 14.4 μg RNA) on test day 22. Dosages: Doses are shown as total RNA amount (mg / kg body weight) and as total RNA dose (μg / individual, estimated patient weight 70 kg).

[0533]

[0534] Table 11: Design of pharmacodynamic study in cynomolgus monkeys (LPT No. 29928).

[0535]

[0536]

[0537] [1] NaCl was considered the most appropriate control group. Compared to the liposome-formulated RNA forming RNA of defined size and charge (LIP), the pure liposomes applied in Group 2 had significant differences in physical characteristics (e.g., charge and structure), leading to different pharmacological properties and changes in in vivo biodistribution.

[0538] Clinical observation

[0539] Overall, treatment was very well tolerated. For local and systemic tolerance observations (including behavior, appearance, feces, mortality, body weight, and food and water intake), no abnormal signs of intolerance were noted in any animal.

[0540] Cytokine analysis

[0541] Cytokine release into plasma was studied with two kinetics for IFN-α, IFN-γ, TNF-α, IL-1β, IL-2, IL-6, IL-10, IL-12p70 and IP-10, before dosing (predose), after completion of treatment (i.e., after completion of the injection cycle of all 4 RNA (LIP) products), and 0.5, 2, 5, 9, 24 and 48 hours after the 1st and after the 5th injection.

[0542] At the tested dose, only IL-6 showed a dose-dependent and test-item-related induction. Cmax levels were reached 30 minutes after the completion of treatment and returned to pre-dose levels after 24 hours ( Fig.19 ). Animal 11 (Group 6) was an outlier, showing a very strong response, and its IL-6 peak level was 1,071 pg / mL, which was about 5x higher than in other animals in the same dose group. Notably, after the 5th treatment, IL-6 induction was much lower, indicating an adaptive effect of IL-6 in monkeys.

[0543] Very low levels of IFN-α induction were observed only in animals of the high-dose group 6, reaching a maximum level after 5 hours, which returned to pre-dose levels after 24 hours ( Fig.19 ). In contrast to observations in cultured human cells and in mice, IP-10 induction was not observed in monkeys. The reason why IP-10 was not observed in this study remains open, as IP-10 induction was observed in monkeys following TLR activation with agonists as reported by others.

[0544] The other cytokines tested (IFN-γ, TNF-α, IL-1β, IL-2, IL-10, and IL-12p70) were not altered. Liposomes alone had no effect on cytokine release.

[0545] hematology

[0546] Standard hematology parameters were tested after the 1st and after the 5th injection, before dosing, 5, 9, 24 and 48 hours after completion of treatment (including an additional 2 hours after the 5th dose). In addition, hematology was tested daily from test day 4 to day 12, and 1 week and 3 weeks after the last dose.

[0547] It was found that the transient decrease of lymphocytes and the transient increase of neutrophils were the relevant findings of the tested items, in a dose-dependent manner. In high-dose animals, lymphocytes declined very quickly 5 hours after completing the treatment, until 5 times (the lowest amount in the 6th group of animals was about 1,000 lymphocytes / μL). This effect is short-lived and recovered in about 48 hours. It is worth noting that the lymphocyte depletion degree was also observed to be lower in the liposome group animals, but was not observed in the NaCl control group (Table 12). The adaptation effect of IL-6 induction was not observed.

[0548] As a result of treatment, an increase in neutrophils was also observed in the NaCl control group, however, when compared to controls, significant differences were observed in groups 3 to 6. The maximum effect was observed 10 hours after treatment and was 44%, 34%, 89% and 91% in groups 3, 4, 5 and 6, respectively, relative to controls.

[0549] Treatment-related transient effects were observed (also in the NaCl group) for eosinophils, leukocytes and reticulocytes (probably due to continuous blood sampling).

[0550] Table 12: Results of absolute lymphocyte counts [1,000 / μL] in cynomolgus monkeys (mean values ​​of n=2).

[0551]

[0552]

[0553] Complement activation

[0554] C3a was measured prior to dosing, 0.5, 2, 5, 9, 24, and 48 hours after completion of treatments 1 and 5, and 1 and 3 weeks after the last dose. No subject-related changes were observed, and all values ​​were considered to be within the normal range of biological variability.

[0555] Clinical Chemistry

[0556] Standard parameters were tested prior to dosing, 24 hours after each dose, and for an additional 4 days after the 3rd and 4th doses, and 1 and 3 weeks after the last dose.

[0557] No test article-related effects were assessed on biochemical parameters for animals in the liposome-treated groups and for animals treated with the test article, compared to control animals and / or background data available at the CRO conducting the study. In part, due to the small number of animals used per group, the data showed some scatter.

[0558] No test item-related changes were noted for serum levels of bile acids, bilirubin, cholesterol, creatinine, glucose, phosphate, total protein, triglycerides, urea, calcium, chloride, potassium, and sodium, nor for serum proteins (albumin, globulin, and albumin / globulin ratio).

[0559] 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), gamma-glutamyl transferase (γ-GT), and glutamate dehydrogenase (GLDH) were considered to be within the limits of normal biological variability.

[0560] On test day 23, higher values ​​were noted for the enzyme activities of LDH, α-amylase, and CK for animal No. 11 treated with 4 x 3.5 μg RNA / animal on test day 22. However, these changes were considered to be stress related (due to the monkeys being restrained in the infusion chair) and not related to the test subject.

[0561] Although assessed as unrelated to the tested subject, minor changes in CK were evaluated in more detail. Differential analysis of the CK isozymes CK-BB, CK-MB, and CK-MM revealed that the increased CK activity noted for individual animals in groups 4, 5, or 6 compared to control animals tested on days 9, 16, or 23 was primarily due to an increase in the CK-MM fraction. In general, no increases were noted for CK-BB and CK-MB, thus confirming that the increase in total CK levels was stress related.

[0562] Cardiovascular examination

[0563] ECG and blood pressure measurements did not show any effects on the cardiovascular system.

[0564] Screening for sequence homology between RNA DP encoding TAA and the human proteome

[0565] All mRNA sequences used in the W_pro1 method are fused in-frame with as many as two flank glycine / serine (GS)-rich linker sequences. These fused stitching points can produce new antigen fusion proteins or peptides, which may potentially trigger undesirable autoimmune responses if they are homologous to human proteins. Therefore, a BLASTp-based homology search is performed against the database of established human proteins to determine whether the stitching points associated with the linker sequence and the antigen have sequence homology with known human proteins.

[0566] The fusion protein sequence to be analyzed was disassembled into smaller peptide sequences by using a sliding window with a length of 9 to 15 and a step size of one amino acid residue. All resulting peptides were compared with the reference database using the BLASTp command of the blast software package (e-value cutoff of 10, no gaps allowed).

[0567] For 100% homologous peptide subsequences, no significant alignment to the human protein sequence could be found.

[0568] Chapter 4: Safety Pharmacology

[0569] ICH guideline S7A describes a series of core studies including functional assessments of the respiratory system, central nervous system (CNS), and cardiovascular system that should be performed prior to human exposure to any medicinal product. Therefore, the safety pharmacology of RNA(LIP) was tested as an integrated part of the six GLP toxicology studies described below.

[0570] In pivotal repeated-dose toxicity studies, potential effects on CNS and respiratory system function were evaluated and no relevant effects were shown in any of the animals tested.

[0571] For the cardiovascular system, a risk analysis of the potential effects of RNA (LIP) vaccines was performed. The RNA distributed throughout the body degrades in the circulation, and the RNA formulated as RNA (LIP) is removed from the blood within a few minutes, and is mainly distributed to the spleen and liver, as shown in the biodistribution study (see below). The data obtained do not show that RNA (LIP) can accumulate in the cardiovascular system. It is expected that the potential systemic side effects of RNA (LIP) vaccination are related to the short-term increase of IFN-α, and it is expected that this will not cause cardiovascular side effects, as recorded in thousands of patients who have received IFN-α. Therefore, the GLP cardiovascular safety pharmacology study that meets ICHS7A / B is not carried out. However, the supportive ECG and blood pressure data from non-GLP pharmacology studies in cynomolgus monkeys treated with RNA (LIP) are available, and the assessment of cardiovascular function after the treatment with RNA (LIP) vaccine is solved.

[0572] In conclusion, no test item or treatment-related changes in the respiratory, nervous, and cardiovascular systems were observed in mice (respiratory system and CNS function) and cynomolgus monkeys (cardiovascular function) at any dose group tested.

[0573] Respiratory safety

[0574] Respiratory safety was included in repeated dose toxicity studies in mice using GLP-compliant RNA DP encoding TAAs (LPT Nos. 28864 and 30283, see below for study design). For example, in a study using RNA (LIP) (LPT No. 30283), plethysmography was tested in four animals / sex / group treated with control buffer, low dose and high dose (5 and 50 μg of RNA formulated with 9 μg and 90 μg of liposomes, respectively). A positive control of animals treated with 30 mg carbamoyl-β-methylcholine chloride (bethanecol) / kg bw was also included. Plethysmography was performed one day after the 4th to 7th dose. Tests included evaluation of respiratory rate, tidal volume, minute ventilation, inspiratory time, expiratory time, peak expiratory volume and inspiratory flow, expiratory time and airway resistance index. None of the lung parameters tested showed any test-item-related changes in the treated animals compared to the control group. Only animals in the positive control group showed the expected changes.

[0575] CNS safety

[0576] CNS safety was included in repeated dose toxicity studies in mice using GLP-compliant RNA DP encoding TAAs (LPT Nos. 28864 and 30283, see below for study design). For example, in a study using RNA (LIP) (LPT No. 30283), observational screening was tested in five animals / sex approximately 24 hours after the 5th dose with control buffer, low dose, and high dose (5 μg and 50 μg of RNA formulated with 9 μg and 90 μg of liposomes, respectively). The following tests were included in the observational screening: righting reflex, temperature, salivation, startle response, respiration, mouth breathing, urination, seizures, piloerection, diarrhea, pupil size, pupil response, tearing, impaired gait, stereotypy, toe pinch, tail pinch, wire maneuver, hind-leg splay, positional passivity, tremor, positive geotropism, limb rotation, and auditory function. In addition, functional tests to evaluate grip strength and voluntary movements were included.

[0577] Neurological screening did not reveal any test item-related effects in mice attributable to neurotoxicity. These findings were confirmed by the results of a GLP-compliant repeated-dose toxicity study, LPT No. 28864, conducted using a different RNA for the Lipo-MERIT study.

[0578] Cardiovascular safety

[0579] Because RNA is degraded within seconds in the circulation and there is no indication that RNA(LIP) would accumulate in the cardiovascular system, cardiovascular safety studies according to ICH S7 were not performed.

[0580] However, supporting data from a non-GLP pharmacology study in cynomolgus monkeys with RNA targeting a melanoma-associated antigen (LIP) used in the Lipo-MERIT study are available. 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 at three time points after the 4th dose, before dosing, 5 hours after completion of dosing, and 24 hours after dosing.

[0581] In cynomolgus monkeys, treatment with RNA (LIP) was very well tolerated (no clinical observation 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 tested items. In addition, serum levels of CK-MB and troponin-I were measured to exclude the possibility of necrosis of myocardial tissue. All measured parameters were negative, supporting the fact that RNA (LIP) had no toxic effects on the cardiovascular system at the dose levels tested in the study.

[0582] Discussion and Conclusion

[0583] The mode of action and main pharmacodynamics of RNA(LIP) have been extensively studied in mice and in human in vitro test systems. Preclinical studies have shown that RNA(LIP) vaccines target spleen and lymphoid tissues after IV administration. RNA(LIP) vaccines trigger a dual effect, namely the induction of antigen-specific T cell responses and the cell activation process and immune modulation after TLR triggering.

[0584] The data generated established that all antigenic RNA guide constructs applied in vivo induced antigen-specific T cell responses, including tetanus toxoid helper epitopes.

[0585] The functional properties of RNA (LIP) preparations are (i) RNA protection in serum and (ii) effective in vivo targeting of APCs that can present antigenic peptides and become activated after TLR7 triggering. The immunomodulatory activity of RNA leads to dose-dependent cytokine induction in human samples, mice and cynomolgus monkeys, which all show different degrees of induction of IFN-α, IP-10 and IL-6, depending on the test species or the test system used. Cytokine induction mediated by RNA (LIP) in PBMC is expected because there is good evidence from self RNA research and literature. In addition to these expectations, the moderate induction of IFN-α and the induction of chemokine IP-10 (CXCL10) are more likely to reflect the onset of the expected pharmacological effect rather than undesirable immunotoxicological events.

[0586] Data generated in mice indicate that splenocytes are the major source of TLR7-dependent IFN-α secretion, as IFN-α secretion is reduced in TLR7- / - mice. We believe that the observed transient and fully reversible cytokine responses serve as the expected pharmacodynamic effects that contribute to the effective induction of vaccine-induced anti-tumor T cell responses. The favorable immune properties are combined with the good tolerability of RNA (LIP) vaccines in mice and cynomolgus monkeys.

[0587] We also investigated the secondary effects of treatment with RNA(LIP) vaccines in several in vitro and in vivo studies using human, cynomolgus monkey, and mouse test systems. Of particular interest are the immunomodulatory effects of RNA(LIP) vaccines, as these are stronger than the effects we observed with unformulated RNA vaccines administered into lymph nodes, which resulted in only local cell activation and cytokine induction.

[0588] Experiments using whole blood samples and PBMC from human and cynomolgus monkey donors were performed to exclude nonspecific or uncontrolled cell activation of human immune cells by RNA (LIP) vaccines, but still showed a modest induction of cytokines as expected. In these experiments, human cells and cynomolgus monkeys were treated with doses covering and above the highest expected clinical dose group.

[0589] Although it is found that there are differences between donors, different in vitro test systems (PBMC vs. whole blood through cultivation) or species in terms of cytokine levels, the cytokine patterns observed in all studies and the transient nature of cytokine responses are similar, with only a few exceptions, such as, in cynomolgus monkeys, IP-10 induction is not observed. Human PBMC demonstrates IP-10 induction, and the low response of IL-6, and when checking in whole blood, the level is even lower IFN-α. Cynomolgus monkeys demonstrate very low IFN-α responses, and do not demonstrate any IP-10 induction and more significant IL-6 responses under the tested dose level. Mouse demonstrates the strong response of IFN-α, IP-10 and IL-6, but with about 10 times of high dosage (based on the dosage of every kg bw) tested in monkey. On the one hand, the difference of cytokine expression between mouse and monkey can be explained by testing different dosages. On the other hand, mouse has the different activities of TLR7 / 8, which can also be the reasonable explanation of different cytokine expression patterns.

[0590] The cytokine response pattern observed in cynomolgus monkeys was better reflected by the whole blood test system than by the PBMC test system, where a more extensive and higher cytokine response at lower dose levels was observed. Nevertheless, the findings in the more sensitive PBMCs were integrated into the strategy to define a safe starting dose for patients. A side-by-side comparison of cytokine secretion in human and cynomolgus monkey whole blood revealed that the two species were highly comparable for the induction of proinflammatory cytokines after RNA(LIP) treatment, indicating that cynomolgus monkeys are an appropriate animal model for predicting secondary pharmacodynamic effects that may occur after RNA(LIP) vaccination in patients.

[0591] In addition to cellular activation processes and cytokine induction following exposure to RNA(LIP), BioNTech also evaluated hematological changes in mouse and cynomolgus monkey studies. Here, transient lymphopenia was observed equally in mice and monkeys at all dose levels. Overall, monkeys treated with RNA(LIP) showed similar responses in cytokine profiles and hematological parameters to those observed in monkeys treated with other TLR agonists. This is consistent with the hypothesis that the primary activation process of cytokine expression by RNA(LIP) occurs through TLR stimulation. In wild-type, TLR7 - / - and IFNAR - / - Extensive pharmacodynamic studies in mice have shown that the hematological findings are secondary to the cytokines induced by RNA(LIP). RNA(LIP) as well as unformulated naked RNA have been shown to be able to activate TLRs. TLR activation has been shown to induce lymphopenia and B cell accumulation in the spleen. Supportingly, histopathological data generated in toxicology testing showed that transient lymphoid hyperplasia was found in the spleen, but not in any other organ or tissue. This is consistent with the lymphopenia observed in the blood and emphasizes the intended targeting of RNA(LIP), and the subsequent intended attraction of effector cells to lymphoid organs.

[0592] Safety pharmacology studies performed demonstrated the safety profile of RNA(LIP). Neurological screening did not show any test item related effects on mice in any of the tests performed. None of the lung parameters tested showed any changes in mice treated with RNA(LIP). There were no indications for cardiovascular effects in cynomolgus monkeys. Overall, RNA(LIP) showed a very good overall safety profile in terms of safety pharmacology parameters.

[0593] Chapter 5: Pharmacokinetics

[0594] Although pharmacokinetic studies are not typically performed during cancer vaccine development, we have performed in vivo studies to determine the biodistribution of intravenously injected RNA lipid complexes and the presence or persistence of residual plasmids due to impurities in the drug product.

[0595] In vitro transcribed RNA is composed of ribonucleotides and therefore has the same structure as 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 ribonucleases cause RNA to decompose rapidly.

[0596] As shown below, the distribution / disposition and potential accumulation of RNA in spleen, liver and lungs was investigated in pharmacokinetic studies. In addition, potential plasmid DNA impurities were quantified in gonads from mice treated with RNA (LIP).

[0597] The biodistribution and persistence of the synthetic cationic lipid DOTMA were investigated in the first in vivo study. Synthetic DOPE is indistinguishable from the body's own natural phospholipid DOPE and should follow natural metabolic pathways, and therefore biodistribution and accumulation were not further investigated.

[0598]

[0599] Biodistribution

[0600] RNA

[0601] During a GLP repeated-dose toxicity study (LPT No. 28864) for the clinical trial Lipo-MERIT, the biodistribution of RNA (LIP) was studied in detail in mice by sampling organs. Under non-GLP conditions, a quantitative real-time reverse transcription PCR (RT-qPCR) method developed at IMGM Laboratories GmbH, Martinsried, Germany, was applied to analyze the sum of all IVT-RNAs of organs (Study ID: RS297). Overall, RNA was cleared very quickly from the blood with an estimated half-life of about 5 minutes. After 48 hours and after 7 days, only marginal levels of RNA were detected in the blood and organs, indicating that it is rapidly degraded and does not persist.

[0602] Residual plasmid impurities

[0603] The biodistribution of residual plasmid impurities from RNA (LIP) vaccination was investigated using samples from a GLP repeated dose toxicity study (LPT No. 28864). A method for analyzing residual plasmid impurities in organ samples was developed in accordance with GLP at BioNTech IMFS GmbH, Idar-Oberstein, Germany. All samples tested were below or slightly above the lower limit of detection (LLOD), indicating that plasmid DNA did not accumulate or persist in the gonads (Study ID: 36X130313).

[0604] DOTMA

[0605] The biodistribution of two synthetic lipids used in RNA(LIP) formulations can provide insight into the body distribution of lipoplex carrier particles over time. The synthetic cationic lipid DOTMA was chosen for biodistribution studies because it is not a naturally occurring molecule and can therefore be easily detected in the context of biological matrices.

[0606] In an exploratory study of the biodistribution of DOTMA, lipids were extracted from blood and seven selected organs collected after IV injection of RNA(LIP) into mice. Here, an aliquot mixture of liposomally formulated IVT-RNA of ATM quality was used. This preliminary study included five mice, one of which remained untreated, two received a single injection of 60 μg RNA, and two received two injections of 60 μg RNA each at 20 day intervals. All mice were sacrificed 24 hours after the time point of the last injection. Quantification of DOTMA was performed by LC / MS measurement. The purpose of the experiment was to test the general feasibility of the extraction and quantification scheme and to obtain the first hint of the biodistribution of DOTMA after RNA(LIP) vaccination.

[0607] DOTMA could be clearly determined from all organs investigated and significant differences between findings in different organs could be observed. In accordance with the proposed mode of action, the highest DOTMA findings were in the spleen.

[0608] On the basis of these first results, a study of single administration of RNA (LIP) was performed (report_BN_14_004). The DOTMA concentration in selected organs was evaluated over a period of up to 28 days (day 0, day 1, day 4, day 7, day 14, day 21 and day 28). In this experiment, 200 μL RNA (LIP) containing 20 μg RNA and 26 μg DOTMA was administered (in the first study, 60 μg was administered per injection). The DOTMA concentration in the administered product was 195 μM. Three mice / time point were studied.

[0609] It is evident that DOTMA is mainly present in the spleen and liver, with indications of slightly different accumulation kinetics. In all other organs / tissues studied (lungs, heart, kidneys, lymph nodes, fat pads, bone marrow, brain), the findings were lower than the aforementioned by a factor of 10 to 50. Based on the data in the liver and spleen, the pharmacokinetics of DOTMA can be estimated: maximum concentrations are detected several days after administration. Within 20 days, DOTMA concentrations decrease to about 50% of the maximum value. These findings support the hypothesis that DOTMA is cleared from organs within an acceptable time scale and fail to make out an indication for the risk of permanent accumulation in any organ.

[0610] In a subsequent study, DOTMA concentrations in selected organs were assessed before (control group), during and after eight weekly RNA (LIP) injections, each containing 20 μg RNA (RBL005.2) and 26 μg DOTMA (reporter_RB_15_004_V02). Organs were sampled from mice one hour after the first RNA (LIP) administration and then every other week after the previous application. After the completion of eight application cycles, mice were sacrificed after an additional 3, 6, 9, 12 and 15 weeks to study DOTMA clearance in the organs. Repeated administration and organ sampling of the RNA (LIP) test items were performed in-house, while extraction and quantification of DOTMA from the provided organ samples were performed by Charles River Laboratories Edinburgh Ltd. (Study No. 322915). The results are completely consistent with our previous studies: Again, the highest DOTMA concentrations were observed in the spleen as the main target organ, followed by the liver. In all other organs, no more than about 5% of the concentration present in the spleen samples was found (data not shown). DOTMA concentrations increased with increasing numbers of RNA(LIP) injections and then continued to decline during the recovery period after the last administration. 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 in the recovery period after the 8th dose.

[0611] In summary, after IV RNA (LIP) administration, DOTMA was delivered to the spleen (and other organs) rapidly (in less than one hour) as an indicator of lipid carriers. In addition to the spleen, DOTMA accumulated mainly in the liver, but RNA translation was not observed in the liver. In absolute numbers, the amount of DOTMA present in these two organs was close to the total cumulative amount of DOTMA injected, while the amount of DOTMA in all other organ samples was almost negligible.

[0612] Based on the evaluation of animals from the recovery group, the accumulated DOTMA was cleared from the organs after repeated RNA (LIP) administration with kinetics that could reasonably be represented by a first-order decay, with an approximate half-life of about 6 to 7 weeks. Such clearance kinetics are also consistent with the findings from repeated administrations in which transient accumulation was observed.

[0613] Taken together, these findings support the hypothesis that DOTMA is cleared from organs within an acceptable time frame and that the potential risk of permanent lipid accumulation in plasma, liver, spleen, lung, heart, brain, kidney, uterus, lymph nodes, and bone marrow is fairly low.

[0614] Discussion and Conclusion

[0615] IVT-RNA, composed of ribonucleotides, has the same structure as RNA produced by human cells, with only the 5'-cap as a different structure. Therefore, IVT-RNA undergoes the same degradation process as natural mRNA. Especially in the extracellular space and serum, abundant ribonucleases lead to rapid degradation of RNA.

[0616] The results of RNA (LIP) biodistribution studies show that soon after RNA (LIP) injection, high levels of RNA in the blood. RNA is quickly cleared from the blood and found in the spleen and liver subsequently, although at much lower levels, and only marginal amounts can be found in the lungs. Since the RNA distributed to the liver may cause transient immune activation by TLR triggering, after the first injection and throughout the study, liver enzymes will be closely monitored in patients. After 48 hours and 7 days, only residual amounts of RNA are found in blood and organs, indicating that RNA does not accumulate or persist in any organ. Comparison of Cmax levels after the 1st and 8th injections does not show any accumulation effect either.

[0617] In gonads, no plasmid DNA was detected or samples were slightly above LLOD, indicating that there is only a small risk of integration of plasmid remnants (e.g., the kanamycin resistance gene) into the genome of germline cells.

[0618] The biodistribution of DOTMA has been shown to be primarily present in the spleen and liver, establishing the spleen as the primary target organ for RNA(LIP) vaccination, and significantly low exposure in plasma and other tissues after single and eight repeated RNA(LIP) administrations. DOTMA is cleared from plasma, spleen, and liver with a comparable terminal half-life of approximately 6 to 7 weeks. A more systematic analysis of the biodistribution and accumulation of DOTMA will be performed prior to conducting advanced clinical testing.

[0619] Chapter 6: Toxicology

[0620] The toxicology program for the RNA vaccine platform includes several pharmacology studies testing RNA (LIP) vaccination in different dose ranges, as well as repeated dose toxicity studies including local tolerance and safety pharmacology parameters and immunotoxicity studies. The studies were performed under GLP conditions at an external CRO (LPT, Hamburg, Germany) using RNA and liposome batches comparable to clinical trial materials in terms of manufacturing process and analytical quality control.

[0621] The GLP compliance study included a 6-week repeat dose toxicity study in which eight different RNADPs encoding breast cancer antigens (LPT No. 30283) were administered IV to C57BL / 6 mice.

[0622] In addition, a complementary GLP-compliant 6-week repeated dose toxicity study (LPT No. 28864) was conducted using an RNA vaccine platform targeting multiple melanoma-specific antigens. Although different RNA sequences were tested, the toxicity data were also related to the application of RNA DP encoding TAAs and can add important information because the same type of liposomes was used for RNA (LIP) preparation. Due to the fact that possible side effects are related to the intrinsic molecular properties of liposome-formulated RNA that are independent of RNA sequence and length, the toxicity profiles of the formulated RNA in these two studies should be the same or at least comparable.

[0623] In addition, an additional 4-week repeated-dose toxicity study was conducted to evaluate the similarity of the liposomes used in the 6-week repeated-dose toxicity study to a pH-adapted liposomal formulation whose buffer conditions were slightly adjusted for long-term stability reasons (LPT No. 30586).

[0624]

[0625] Selection of relevant species

[0626] We consider mice as a relevant species for testing potential direct toxic effects of RNA (LIP) vaccines for the following main reasons:

[0627] The mouse as a model system provides all relevant features of innate and adaptive immunity associated with characterizing the direct toxic effects of RNA DP encoding TAAs. Mice exhibit all expected primary and secondary pharmacological effects, from induction of CD4+ / CD8+ T cell responses to immunomodulatory effects that enhance immune responses and lead to subsequent TLR triggering, cell activation, and cytokine secretion.

[0628] The mouse system contains a wealth of available tools and techniques for studying biological effects that far exceed the number of experimental possibilities in other species (e.g., availability of transgenic mouse models, MHC tetramers, antibodies, etc.). This enables a more in-depth analysis of all unexpected events.

[0629] The on-target effects of vaccines cannot be adequately studied in animal species. Therefore, the use of other animal species will not provide additional information, and the use of higher mammals should therefore not be considered.

[0630] Single-dose toxicology

[0631] Dose range finding studies are usually performed to adjust the dose for pivotal toxicity studies and to obtain preliminary information on target organs and signs of toxicity. We conducted several pharmacology studies to test RNA(LIP) over different dose ranges using protocols similar to the intended clinical protocols. During these studies, administration of RNA(LIP) was found to induce favorable pharmacodynamic effects and was well tolerated.

[0632] In addition, previous toxicity studies have shown that, at high doses, the tolerance of naked RNA administered by IV in mice is also very good. Liposomes containing DOTMA or DOPE as synthetic lipid components have been tested in many clinical studies, and several approved liposome drug products have shown very good tolerance. Some liposome preparations have been applied to reduce drug-specific toxicity, such as the nephrotoxicity or hepatotoxicity of high-dose nucleic acids, or the toxicity of small molecules (e.g., doxorubicin or clofazimine).

[0633] The following data is based on data generated from a series of internal studies and literature research:

[0634] A tolerable dose in mice that would provide an adequate safety margin for a first dose used in humans can be extrapolated from the pharmacology studies performed.

[0635] A single dose administration will not be sufficient to induce a significant immune response. The maximum immune response is observed after at least three administrations.

[0636] RNA vaccines and lipid complex formulations were generally well tolerated.

[0637] Based on these conclusions, we decided not to conduct a single-dose toxicity study but to proceed directly to a repeated-dose toxicity study.

[0638] Repeated dose toxicology

[0639] The safety and toxicology of RNA(LIP) products from the RNA vaccine platform were analyzed in several GLP-compliant repeated-dose toxicity studies involving IV-administered RNA(LIP) products. Table 13 provides an overview of the GLP repeated-dose toxicity studies that support Phase 1 and Phase 2 clinical testing of RNA(LIP) vaccines.

[0640] Table 13: Design of GLP repeated dose toxicity study.

[0641]

[0642]

[0643] ATM preparations

[0644] The composition, formulation and strength of AMT are planned to be as close as possible to the intended drug product for use in humans.

[0645] Minor changes to the RNA(LIP) preparation process must be made for the following reasons:

[0646] Achieving high doses increases the likelihood of capturing potential dose-dependent toxicological effects and thereby meeting the criteria for toxicity testing as outlined in the ICH S6 or M3(R2) guidelines.

[0647] To prevent administration of a volume higher than the maximum feasible in mice, a volume of 250 μL in a slow bolus injection is recommended. Higher injection volumes are not ethically recommended and carry the risk of losing mice during injection.

[0648] The differences from the clinical protocol are:

[0649] Patients will receive the different RNA(LIP) products in a serial manner. This is not possible in mice due to volume limitations. RNA(LIP) for mice is prepared separately, then mixed, and all four RNA lipid complexes are injected simultaneously in a total volume of 250 μL.

[0650] For the formulation of RNA(LIP) for treating patients, 150 mM NaCl will be used. In order to achieve higher doses in toxicity studies, a more concentrated NaCl solution must be used for RNA(LIP) formation.

[0651] For the preparation of RNA(LIP) for patient treatment, a concentration of 0.25 mg / mL of RNA drug product will be used. In order to achieve high doses in toxicity studies, a higher concentrated RNA (i.e., 1 mg / mL) must be used to prepare the RNA(LIP) product in Study LPT No. 28864.

[0652] In this experiment, half concentrated acetic acid stabilized liposomes (L4) relative to the same amount of acetic acid stabilized liposomes (L2) used previously will be used. No further bridging studies are planned as the same characteristics as L2 liposomes were given.

[0653] Our position is that the above-mentioned changes to the formulation regimen of ATM had little or no impact on the outcome or conduct of the study.

[0654] Study Design

[0655] See Table 13 for study design.According to ICH S6 and S8, the data from standard toxicity studies were evaluated for signs of immunotoxic potential.Following studies were conducted according to 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 and clinical chemistry and cytokines (IL-1 β, IL-2, IL-6, IL-10, IL-12, TNF-α, INF-α, INF-γ and IP-10).

[0656] Safety pharmacology studies were included to test the respiratory and central nervous systems as described in Section 4.

[0657] result

[0658] Toxicological evaluation in a 6-week repeated dose toxicity study using a vaccine platform revealed only slight effects (Table 14) that were primarily attributable to the expected pharmacological mode of action of RNA (LIP). The expected immunomodulatory effects of RNA (LIP) are TLR activation and cytokine release. Induction of IFN-α in mice results in secondary effects, such as leukopenia, thrombocytopenia, and improvement of liver parameters (e.g., ALAT), effects typically described for patients treated with IFN-α.

[0659] Consistent with this, the changes related to the tested items in the treated animals were mainly transient (Table 14). In addition, transient activation of cytokines IP-10, IFN-α, IL-6 and IFN-γ was observed. All inductions were returned to normal levels (except IP-10 levels, which were still slightly higher than normal levels) after 24 hours.

[0660] In all treatment groups, the hematological findings in mice mainly included lymphopenia and low, reversibly reduced total leukocytes, neutrophils, reticulocytes and thrombocytopenia. These findings were fully reversible. The lymphoid hyperplasia of the spleen observed in histopathology was fully restored and outlined the expected effects and the expected targeting of the test substances and lymphocytes to the spleen.

[0661] The observed slight changes in liver parameters (eg, GLDH, LDH, ALAT, and ASAT) mainly affected the high dose group and were not noted in animals in the recovery group, indicating that the effects fully recovered in at least three weeks or less. No hepatotoxicity was detected by histopathology.

[0662] Since there were no findings in the low dose group in study LPT No. 30283, the NOAEL was met at a dose of 5 μg total RNA per animal (ie, approximately 0.2 mg / kg bw in mice).

[0663] An additional 4-week repeated dose toxicity study was performed to address changes in the liposome buffer composition (LPT No. 30586). The data demonstrated that the new liposomes were fully comparable to the liposomes used in the main study in terms of the parameters measured.

[0664] Table 14: Summary of toxicology findings in repeated dose toxicity studies with RNA (LIP) (LPT Nos. 28864, 30283 and 30586).

[0665] All described findings were statistically significant compared with the control group.

[0666]

[0667]

[0668]

[0669]

[0670]

[0671]

[0672]

[0673] Genotoxicity

[0674] The components of the RNA(LIP) product (lipids and RNA) are not suspected of having genotoxic potential. No impurities or components of the delivery system were tested for genotoxicity. Based on the recommendations given in the ICH Guideline S6(R1) for Preclinical Safety Evaluation of Pharmaceuticals Derived from Biotechnology (June 2011), no genotoxicity studies are planned.

[0675] Carcinogenicity

[0676] RNA itself and lipids used as carriers have no carcinogenic or tumorigenic potential. According to ICH S1A, long-term carcinogenicity studies are not necessary in the absence of reasons for concern from laboratory and toxicology studies and in the absence of an intended long-term use of the drug.

[0677] Reproductive and developmental toxicity

[0678] Repeated dose toxicity studies in mice treated with RNA(LIP) included macroscopic and microscopic evaluation of male and female reproductive tissues. No findings were noted in these studies, therefore, specific fertility and developmental toxicity studies will not be conducted prior to the initiation of Phase 1 studies with the RNA(LIP) vaccine. RNA(LIP) is not expected to produce direct cytotoxic effects on reproductive tissues, as supported by experience from other cancer vaccines, indicating no effects on reproduction and development. Because effects on reproduction cannot be excluded, females of reproductive potential will have to use effective contraception during treatment. No further long-term or reproductive toxicity studies are currently planned.

[0679] Local tolerance

[0680] According to ICH recommendations, the test for local tolerance was evaluated in a GLP repeat-dose toxicity study for IV injection. No signs of local intolerance were observed during the study.

[0681] Other toxicity studies

[0682] Antigenicity

[0683] Since IVT-RNA is rapidly degraded extracellularly within seconds to minutes, no anti-drug antibody (ADA) formation is expected. Therefore, specific antigenicity testing regarding antibody induction is not planned.

[0684] Immunotoxicity

[0685] Since the aim is to activate the immune system by RNA (LIP) products, special attention is paid to immunotoxicological parameters to exclude unexpected activation or inhibition. Immunotoxicological examinations were implemented in both 6-week repeated dose toxicity studies (LPT No. 28864 and 30283). In addition to monitoring cytokine levels in serum, the following relevant parameters were also considered to evaluate immunotoxicity: body weight, body temperature, lymphoid organ weight, macroscopic and histopathology of lymphoid organs, absolute and relative differential blood counts, total serum protein, albumin / immunoglobulin ratio, myeloid / erythroid ratio in bone marrow, coagulation parameters.

[0686] hematology

[0687] In both studies, a decrease in lymphocytes, white blood cell counts (mainly due to lymphocyte reduction) and platelets was observed in all treatment groups on test day 44, approximately 24 hours after the 8th injection. After two weeks, all effects were fully restored. The results of the LPT No. 28864 and 30283 studies are shown in Table 15 and Table 16, respectively.

[0688] Table 15: Hematology data (LPT No. 28864).

[0689] Samples for hematological determination were collected on test day 44 (approximately 24 hours after the 8th injection).

[0690]

[0691]

[0692] *Statistically significant, p≤0.05; **Statistically significant, p≤0.01 (Dunnett's test).

[0693] Table 16: Hematology data (LPT No. 30283).

[0694] Samples for hematological determination were collected on test day 44 (approximately 24 hours after the 8th injection).

[0695]

[0696]

[0697] *Statistically significant, p≤0.05; **Statistically significant, p≤0.01 (Dunnett's test).

[0698] Cytokine determination

[0699] The secretion of the following cytokines was analyzed in repeated dose toxicity studies: IL-1β, IL-2, IL-6, IL-10, IL-12p70, TNF-α, IFN-γ, and IP-10, which are known to be sensitive indicators of immune activation or TLR7 signaling. In the toxicity study, LPT No.28864 mice showed a dose-dependent and test item-related increase in the cytokine IP-10. IP-10 levels increased transiently and reached a maximum 6 hours after the 4th injection. After 24 hours, the levels were still significantly higher than the control levels, but had returned to normal levels. Compared with the control group, after 6 hours, IP-10 showed a maximum induction of 28 times and 16 times (in males and females, respectively). 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 maximum 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. After 24 hours, all effects were completely reversible (except for the TNF-α level in the females of Group 4). The results are summarized in Table 17.

[0700] Table 17: Cytokine levels in plasma (LPT No. 28864).

[0701] Samples for cytokine determination were collected 6 and 24 hours after the 4th injection.

[0702]

[0703] *Statistically significant, p≤0.05; **Statistically significant, p≤0.01 (Dunnett's test).

[0704] For the determination of cytokines in study LPT No.30283, serum samples were collected 6 hours and 24 hours after the 5th injection. It was found that serum levels of IL-2, IL-6, IP-10, IFN-α and IFN-γ increased (Table 18). For IL-6, a significant dose-dependent induction was noted. For IFN-γ, induction was noted after high-dose treatment (statistically significant at p≤0.01), more significantly in male animals. For IFN-α, induction was observed after low doses and after high-dose treatment (statistically significant at p≤0.01 or p≤0.05), more significantly for female animals in Group 5 (RNA Group 2, high dose). The induction of all the above cytokines subsided 24 hours after administration.

[0705] Relatively low but dose-related induction of IL-2 was noted for both male and female animals at 6 and 24 hours following low- or high-dose treatment.

[0706] 6 hours after high dose treatment, significant dose-dependent effects of IP-10 were detected for male and female animals at all dose levels compared to the control group. 24 hours after administration, IP-10 levels were still elevated at all dose levels compared to the control group. This induction of IP-10 reflects the expected pharmacological effects and is not considered to be an undesirable immunotoxicological event.

[0707] Table 18: Cytokine levels in plasma (LPT No. 30283).

[0708] Samples for cytokine determination were collected 6 and 24 hours after the fifth injection.

[0709]

[0710]

[0711] incr.: A significant increase was noted compared to the control group, however, since the control group value was set to "0.0", the increase could not be expressed as a fold.

[0712] *Statistically significant, p≤0.05; **Statistically significant, p≤0.01 (Dunnett's test).

[0713] During the study comparing L1 and L2 liposomes, cytokine determination was also performed (LPT No. 30586). Here, cytokines were analyzed 6 hours and 24 hours after the 4th immunization. No test item-related changes between the groups were noted.

[0714] Discussion and Conclusion

[0715] Treatment with RNA (LIP) was well tolerated in mice, as shown by many antigen-encoding RNAs evaluated in three different repeated dose toxicity studies (LPT Nos. 28864, 30283, and 30586). Overall, treatment with up to 8 IV injections was well tolerated, including in animals in the high-dose group. No premature deaths related to the test items were observed in studies No. 30283 and 30586. One animal died prematurely after administration of the test item in study No. 28864. Since there were no findings in the low-dose group of study No. 30283, the NOAEL was reached at a dose of 5 μg total RNA per animal (i.e., approximately 0.2 mg / kg bw in mice). In addition, vaccination with RNA (LIP) products was also very well tolerated in non-GLP pharmacology studies in 12 cynomolgus monkeys (no clinical observation findings).

[0716] Toxicological evaluation of RNA(LIP) in repeated dose toxicity studies in mice revealed effects attributable to the test item, including transient induction of cytokines, hematological changes, and elevations in liver enzymes. The effects observed were primarily the induction of the cytokines IP-10, IFN-α, IFN-γ, and IL-6 in the in vivo studies reported here and in the in vitro studies described and discussed above.

[0717] Notably, in mice, proinflammatory cytokines such as TNF-α, IFN-γ, or IL-2 were not upregulated in an excessive manner. 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 and other cytokines will be carefully monitored throughout clinical studies and analyzed directly in patients.

[0718] Effects such as lymphopenia and elevation of liver enzymes have also been reported in mice and monkeys following treatment with plasmid lipid complexes and activation via TLRs and are generally observed as secondary effects driven by IFN-α secretion and are often described for patients treated with recombinant IFN-α, which has been marketed for many years for the treatment of a variety of neoplastic and non-neoplastic diseases.

[0719] The changes in liver parameters of the high-dose group observed in mice indicate that the liver may be a toxic target of the higher dose of RNA formulated through liposomes. These changes include an increase in liver weight, an increase in GLDH, LDH, ASAT and ALAT levels in plasma. These changes are considered to be slight and are not observed in the animals of the recovery group, indicating that the effect is fully recovered in at least three weeks. In addition, histopathology does not reveal any hepatotoxicity. In cynomolgus monkeys, the biochemical parameters of the animals of the liposome-treated group and the animals treated with the test items are considered to be within the limits of normal biological variability compared to the control animals. On the 9th, 16th or 23rd day of the test, the CK increase noted for the individual animals of the 4th, 5th or 6th groups, compared to the control animals, is mainly attributable to the CK-MM fraction and is considered to be related to stress.

[0720] The mild elevation of liver parameters in mice may be a response caused by immunomodulatory effects that may be triggered by phagocytosis of RNA (LIP) by liver target cells, such as Kupffer cells. In contrast to the effects observed in the mouse spleen (lymphoid hyperplasia), this did not result in leukocyte recruitment to the liver, indicating that the desired pharmacological effects, such as TLR activation and lymphocyte trafficking, are restricted to lymphoid organs.

[0721] Complement activation by liposomally formulated materials has been reported previously. For RNA (LIP) vaccination, slightly elevated C5a levels were observed in female mice, but this was considered an event of low biological relevance. In addition, mice are not considered a good model for extrapolating complement effects to humans.

[0722] In general, the immune responses seen in all three RNA (LIP) repeated dose toxicity studies (LPT No. 28864, 30283 and 30586) showed a comprehensive picture of increased spleen weight, cytokine / chemokine activation and lymphocyte transport. This reflects the induction of the expected pharmacological events and emphasizes the relevance of mice as a correct test model for toxicity studies. In the bridging study LPT No. 30583 evaluating the toxicity of the pH-adjusted L2 liposome formulation, no significant differences were observed between the two liposome formulations. In this experiment, L4 pH-adjusted liposomes, which were half concentrated as L2 liposomes, were applied. Due to the same characteristics as L2 liposomes, no further bridging studies were planned.

[0723] Example 3: Induction of antigen-specific T cells in the spleen by KLK2-, KLK3-, ACPP-, NKX3-1-, and HOXB13-encoding RNA

[0724] On days 1, 8, 15 and 22, A2 / DR1 mice were immunized with 200 μL of 0.15 mg / mL RNA(Lip) solution corresponding to 30 μg RNA(LIP).

[0725] Five days after the end of the four RNA(Lip) injections, splenocytes were obtained from immunized A2 / DR1 mice and in vivo induction of antigen-specific T cells was determined by ELISPOT analysis. To test immunogenicity, isolated splenocytes were restimulated with peptide pools (15mer, overlapping 11 amino acids) spanning the corresponding human proteins, KLK2, KLK3 (PSA), ACPP (PAP), NKX3-1, or HOXB13. Results Fig. 20 . According to unpaired t-tests, IFN-γ+ spot counts induced by KLK2-, ACPP-, and HOXB13 encoding RNA (Lip) were statistically higher than restimulation with the control peptide CMV pp65 (P = 0.0056; P > 0.0001; P = 0.0095). In all immunized mice, restimulation of splenocytes with the control P2 / P16 / P17 peptides also resulted in high spot counts, indicating successful immunization. On all ELISPOT plates, the negative control medium alone induced only the lowest spot counts, regardless of the animal from which the splenocytes were derived.

Claims

1. A pharmaceutical composition comprising RNA, wherein the RNA encodes the following amino acid sequence: (i) an amino acid sequence of kallikrein-2 (KLK2), wherein the amino acid sequence consists of the amino acid sequence of SEQ ID NO: 2; (ii) an amino acid sequence of prostate-specific antigen (PSA), wherein the amino acid sequence consists of the amino acid sequence of SEQ ID NO: 6; (iii) an amino acid sequence of prostatic acid phosphatase (PAP), wherein the amino acid sequence consists of the amino acid sequence of SEQ ID NO: 10; (iv) an amino acid sequence of homeobox B13 (HOXB13), wherein the amino acid sequence consists of the amino acid sequence of SEQ ID NO: 14; and (v) an amino acid sequence of NK3 homeobox 1 (NKX3-1), wherein the amino acid sequence consists of the amino acid sequence of SEQ ID NO: 18, wherein Each of the amino acid sequences described in (i), (ii), (iii), (iv) or (v) is encoded by a separate RNA; Each RNA comprises a 5'UTR consisting of the nucleotide sequence of SEQ ID NO: 21; Each RNA comprises a 3'UTR consisting of the nucleotide sequence of SEQ ID NO: 28; and Each RNA contains a poly-A sequence.

2. The pharmaceutical composition of claim 1, wherein: The RNA encoding the amino acid sequence described in (i) consists of the nucleotide sequence of SEQ ID NO:

4.

3. The pharmaceutical composition of claim 1, wherein: The RNA encoding the amino acid sequence described in (ii) consists of the nucleotide sequence of SEQ ID NO:

8.

4. The pharmaceutical composition of claim 1, wherein: The RNA encoding the amino acid sequence described in (iii) consists of the nucleotide sequence of SEQ ID NO:

12.

5. The pharmaceutical composition of claim 1, wherein: The RNA encoding the amino acid sequence described in (iv) consists of the nucleotide sequence of SEQ ID NO:

16.

6. The pharmaceutical composition of claim 1, wherein: The RNA encoding the amino acid sequence described in (v) consists of the nucleotide sequence of SEQ ID NO:

20.

7. The pharmaceutical composition of claim 1, wherein at least one RNA comprises a 5' cap m2 7,2’-O Gpp s p(5')G.

8. The pharmaceutical composition of claim 1, wherein each RNA comprises a 5' cap m2 7,2’-O Gpp s p(5')G.

9. The pharmaceutical composition of claim 1, wherein the amino acid sequence in (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that enhances antigen processing and / or presentation encoded by the nucleotide sequence of SEQ ID NO:

25.

10. The pharmaceutical composition of claim 1, wherein the amino acid sequence in (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that destroys immune tolerance encoded by the nucleotide sequence of SEQ ID NO:

27.

11. The pharmaceutical composition of claim 1, wherein the poly-A sequence consists of the nucleotide sequence of SEQ ID NO:

29.

12. The pharmaceutical composition of claim 1, wherein the RNA is formulated as a liquid, formulated as a solid, or a combination thereof.

13. The pharmaceutical composition of claim 1, wherein the RNA is formulated for injection.

14. The pharmaceutical composition of claim 1, wherein the RNA is formulated for intravenous administration.

15. The pharmaceutical composition of claim 1, wherein the RNA is formulated or is to be formulated into lipid complex particles.

16. The pharmaceutical composition of claim 15, wherein the lipid complex particles are obtainable by mixing the RNA with liposomes.

17. The pharmaceutical composition of claim 1, wherein each RNA comprises: -m2 7,2’-O Gpp s p(5')G 5' cap; -5'UTR consisting of the nucleotide sequence of SEQ ID NO:21; - a 3'UTR consisting of the nucleotide sequence of SEQ ID NO: 28; and - a poly-A sequence consisting of the nucleotide sequence of SEQ ID NO:

29.

18. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

19. The pharmaceutical composition of claim 1, which is used for treating or preventing prostate cancer.

20. The pharmaceutical composition of claim 1, for administration to humans.

21. Use of an RNA encoding the following amino acid sequence for the preparation of a medicament for treating prostate cancer in a subject, wherein the medicament is formulated for administration of the RNA to the subject, (i) an amino acid sequence of kallikrein-2 (KLK2), wherein the amino acid sequence consists of the amino acid sequence of SEQ ID NO: 2; (ii) an amino acid sequence of prostate-specific antigen (PSA), wherein the amino acid sequence consists of the amino acid sequence of SEQ ID NO: 6; (iii) an amino acid sequence of prostatic acid phosphatase (PAP), wherein the amino acid sequence consists of the amino acid sequence of SEQ ID NO: 10; (iv) an amino acid sequence of homeobox B13 (HOXB13), wherein the amino acid sequence consists of the amino acid sequence of SEQ ID NO: 14; and (v) an amino acid sequence of NK3 homeobox 1 (NKX3-1), wherein the amino acid sequence consists of the amino acid sequence of SEQ ID NO: 18, Each of the amino acid sequences described in (i), (ii), (iii), (iv) or (v) is encoded by a separate RNA; Each RNA comprises a 5'UTR consisting of the nucleotide sequence of SEQ ID NO: 21; Each RNA comprises a 3'UTR consisting of the nucleotide sequence of SEQ ID NO: 28; and Each RNA contains a poly-A sequence.

22. The use according to claim 21, wherein: The RNA encoding the amino acid sequence described in (i) consists of the nucleotide sequence of SEQ ID NO:

4.

23. The use of claim 21, wherein: The RNA encoding the amino acid sequence described in (ii) consists of the nucleotide sequence of SEQ ID NO:

8.

24. The use of claim 21, wherein: The RNA encoding the amino acid sequence described in (iii) consists of the nucleotide sequence of SEQ ID NO:

12.

25. The use of claim 21, wherein: The RNA encoding the amino acid sequence described in (iv) consists of the nucleotide sequence of SEQ ID NO:

16.

26. The use of claim 21, wherein: The RNA encoding the amino acid sequence described in (v) consists of the nucleotide sequence of SEQ ID NO:

20.

27. The use of claim 21, wherein at least one RNA comprises a 5' cap m2 7,2’-O Gpp s p(5')G.

28. The use of claim 21, wherein each RNA comprises a 5' cap m2 7,2’-O Gpp s p(5')G.

29. The use of claim 21, wherein the amino acid sequence in (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that enhances antigen processing and / or presentation encoded by the nucleotide sequence of SEQ ID NO:

25.

30. The use of claim 21, wherein the amino acid sequence in (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that breaks immune tolerance encoded by the nucleotide sequence of SEQ ID NO:

27.

31. The use of claim 21, wherein the poly-A sequence consists of the nucleotide sequence of SEQ ID NO:

29.

32. The use of claim 21, wherein the RNA is administered by injection.

33. The use of claim 21, wherein the RNA is administered by intravenous administration.

34. The use of claim 21, wherein the RNA is formulated into lipid complex particles.

35. The use according to claim 34, wherein the lipoplex particles are obtainable by mixing the RNA with liposomes.

36. The use of claim 21, wherein each RNA comprises: -m2 7,2’-O Gpp s p(5')G 5' cap; -5'UTR consisting of the nucleotide sequence of SEQ ID NO:21; - a 3'UTR consisting of the nucleotide sequence of SEQ ID NO: 28; and - a poly-A sequence consisting of the nucleotide sequence of SEQ ID NO:

29.

37. The use of claim 21, wherein the subject is a human.

38. The use of claim 21, wherein the method of treating prostate cancer further comprises administering an additional therapy.

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