RU2838161C2 - Therapeutic rna for prostate cancer - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: group of inventions relates to pharmaceutics and medicine. 1 and 2 objects represent a pharmaceutical composition and a kit for treating or preventing prostate cancer, containing RNA coding amino acid sequences containing kallikrein-2 (KLK2), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), Homeobox B13 (HOXB13), NK3 Homeobox 1 (NKX3-1), their immunogenic variants or immunogenic fragments or their immunogenic variants. 3 and 4 objects – use of the pharmaceutical composition or kit as a drug for treating or preventing prostate cancer. 5 object is a method of treating prostate cancer in a subject, comprising administering to the subject at least one RNA coding amino acid sequences containing kallikrein-2 (KLK2), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), Homeobox B13 (HOXB13), NK3 Homeobox 1 (NKX3-1), their immunogenic variants or immunogenic fragments or their immunogenic variants.

EFFECT: stimulation of proliferation/priming of T-cells and induction of antigen-specific T-cell response in a subject against prostate cancer cells expressing one or more specified tumour antigens.

83 cl, 20 dwg, 18 tbl, 3 ex

Description

The invention 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 and older men each year. About 60 percent of cases occur in men over 65 years of age. The American Cancer Society (ACS) estimates that 174,650 American men will be diagnosed with the disease in 2019. According to the Urology Care Foundation, prostate cancer is the second leading cause of cancer death among men in the United States.

Disclosed herein are compositions, uses, and methods for treating prostate cancer. Administration of the therapeutic RNAs described herein to a patient suffering from prostate cancer may reduce tumor size, prolong time to disease progression, and/or protect against tumor metastasis and/or recurrence, and ultimately increase survival time.14

The essence of the invention

In one aspect, the present application provides a composition or medical formulation comprising at least one RNA, wherein the at least one RNA encodes the following amino acid sequences:

(i) the amino acid sequence of kallikrein-2 (KLK2), its immunogenic variant, or an immunogenic fragment of KLK2 or its immunogenic variant;

(ii) the amino acid sequence of prostate-specific antigen (PSA), its immunogenic variant, or an immunogenic fragment of PSA or its immunogenic variant;

(iii) the amino acid sequence of prostatic acid phosphatase (PAP), its immunogenic variant, or an immunogenic fragment of PAP or its immunogenic variant;

(iv) the amino acid sequence of Homeobox B13 (HOXB13), an immunogenic variant thereof, or an immunogenic fragment of HOXB13 or an immunogenic variant thereof; and

(v) the amino acid sequence of NK3 Homeobox 1 (NKX3-1), its immunogenic variant, or an immunogenic fragment of NKX3-1 or its immunogenic variant.

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

In one incarnation

(i) an RNA encoding the amino acid sequence of (i) comprises the nucleotide sequence of SEQ ID NO: 3 or 4 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: 3 or 4; and/or

(ii) the amino acid sequence of (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.

In one incarnation

(i) an RNA encoding the amino acid sequence of (ii) comprises 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) the amino acid sequence of (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.

In one incarnation

(i) an RNA encoding the amino acid sequence of (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 of (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.

In one incarnation

(i) an RNA encoding the amino acid sequence of (iv) comprises 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) the amino acid sequence of paragraph (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.

In one incarnation

(i) an RNA encoding the amino acid sequence of (v) comprises the nucleotide sequence of SEQ ID NO: 19 or 20 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: 19 or 20; and/or

(ii) the amino acid sequence of (v) 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.

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

In one embodiment, the at least one RNA is a modified RNA, in particular a stabilized mRNA. In one embodiment, the at least one RNA comprises a modified nucleoside in place of at least one uridine. In one embodiment, the at least one RNA comprises a modified nucleoside in place of each uridine. In one embodiment, each RNA comprises a modified nucleoside in place of at least one uridine. In one embodiment, each RNA comprises a modified nucleoside in place of each uridine. In one embodiment, the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyluridine (m5U).

In one embodiment, at least one RNA comprises a 5'-cap m ₂ ^7,2'-O Gppsp (5') G. In one embodiment, each RNA comprises a 5'-cap m ₂ ^7,2'-O Gppsp (5') G.

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.

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

In one incarnation

(i) an RNA encoding an 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

(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.

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

In one incarnation

(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

(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.

In one embodiment, at least one amino acid sequence according to (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that breaks immunological tolerance. In one embodiment, each amino acid sequence according to (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that breaks immunological tolerance. In one embodiment, the amino acid sequence that breaks immunological tolerance comprises helper epitopes, preferably helper epitopes derived from tetanus toxoid.

In one incarnation

(i) an RNA encoding an amino acid sequence that breaks immunological 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

(ii) the amino acid sequence that breaks immunological 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 nucleotide sequence of SEQ ID NO: 26.

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.

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.

In one embodiment, the RNA is included in the composition as a liquid, as a solid, or a combination thereof. In one embodiment, the RNA is included in a composition for injection. In one embodiment, the RNA is included in a composition for intravenous administration. In one embodiment, the RNA is or will be included in the composition as lipoplex particles. In one embodiment, the RNA lipoplex particles can be prepared by mixing RNA with liposomes.

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

In one embodiment, the medicinal composition is a kit. In one embodiment, the RNA and, optionally, the liposomes are in separate vials.

In one embodiment, the composition or medicinal formulation further comprises instructions for using the RNA and, optionally, liposomes for the treatment or prevention of prostate cancer.

In one aspect, the present application provides a composition or medical formulation as disclosed herein for pharmaceutical use. In one embodiment, the pharmaceutical use comprises therapeutic or prophylactic treatment of a disease or disorder. In one embodiment, the therapeutic or prophylactic treatment of a disease or disorder comprises treatment or prevention of prostate cancer. In one embodiment, the composition or medical formulation as described herein is for administration to a human.

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

In one aspect, the present application provides a method 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 sequences:

(i) the amino acid sequence of kallikrein-2 (KLK2), its immunogenic variant, or an immunogenic fragment of KLK2 or its immunogenic variant;

(ii) the amino acid sequence of prostate-specific antigen (PSA), its immunogenic variant, or an immunogenic fragment of PSA or its immunogenic variant;

(iii) the amino acid sequence of prostatic acid phosphatase (PAP), its immunogenic variant, or an immunogenic fragment of PAP or its immunogenic variant;

(iv) the amino acid sequence of Homeobox B13 (HOXB13), an immunogenic variant thereof, or an immunogenic fragment of HOXB13 or an immunogenic variant thereof; and

(v) the amino acid sequence of NK3 Homeobox 1 (NKX3-1), its immunogenic variant, or an immunogenic fragment of NKX3-1 or its immunogenic variant.

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

In one incarnation

(i) an RNA encoding the amino acid sequence of (i) comprises the nucleotide sequence of SEQ ID NO: 3 or 4 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: 3 or 4; and/or

(ii) the amino acid sequence of (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.

In one incarnation

(i) an RNA encoding the amino acid sequence of (ii) comprises 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) the amino acid sequence of (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.

In one incarnation

(i) an RNA encoding the amino acid sequence of (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 of (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.

In one incarnation

(i) an RNA encoding the amino acid sequence of (iv) comprises 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) the amino acid sequence of (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.

In one incarnation

(i) an RNA encoding the amino acid sequence of (v) comprises the nucleotide sequence of SEQ ID NO: 19 or 20 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: 19 or 20; and/or

(ii) the amino acid sequence of (v) 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.

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

In one embodiment, the at least one RNA is a modified RNA, in particular a stabilized mRNA. In one embodiment, the at least one RNA comprises a modified nucleoside in place of at least one uridine. In one embodiment, the at least one RNA comprises a modified nucleoside in place of each uridine. In one embodiment, each RNA comprises a modified nucleoside in place of at least one uridine. In one embodiment, each RNA comprises a modified nucleoside in place of each uridine. In one embodiment, the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyluridine (m5U).

In one embodiment, at least one RNA comprises a 5'-cap m ₂ ^7,2'-O Gppsp (5') G. In one embodiment, each RNA comprises a 5'-cap m ₂ ^7,2'-O Gppsp (5') G.

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.

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

In one incarnation

(i) an RNA encoding an 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

(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.

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

In one incarnation

(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

(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.

In one embodiment, at least one amino acid sequence according to (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that breaks immunological tolerance. In one embodiment, each amino acid sequence according to (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that breaks immunological tolerance. In one embodiment, the amino acid sequence that breaks immunological tolerance comprises helper epitopes, preferably helper epitopes derived from tetanus toxoid.

In one incarnation

(i) an RNA encoding an amino acid sequence that breaks immunological 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

(ii) the amino acid sequence that breaks immunological 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 nucleotide sequence of SEQ ID NO: 26.

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.

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.

In one embodiment, the RNA is administered by injection. In one embodiment, the RNA is administered intravenously.

In one embodiment, the RNA is included in the lipoplex particles (represented by lipoplex particles). In one embodiment, the RNA lipoplex particles can be obtained by mixing the RNA with liposomes.

In one incarnation the subject is a human being.

In one embodiment, the method described herein also comprises administering an additional therapy. In one embodiment, the additional therapy comprises one or more agents selected from the group consisting of: (i) surgery to excise, resect, or remove the tumor, (ii) radiation therapy, and (iii) chemotherapy. In one embodiment, the additional therapy comprises administering an additional therapeutic agent (agent). In one embodiment, the additional therapeutic agent comprises an anti-tumor therapeutic agent. In one embodiment, the additional therapeutic agent is a checkpoint modulator. In one embodiment, the checkpoint modulator is an anti-PD1 antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD1 antibody and an anti-CTLA-4 antibody.

In one aspect, the present application provides an RNA as disclosed herein, such as,

(i) RNA encoding the amino acid sequence of kallikrein-2 (KLK2), its immunogenic variant, or an immunogenic fragment of KLK2 or its immunogenic variant;

(ii) RNA encoding the amino acid sequence of prostate-specific antigen (PSA), its immunogenic variant, or an immunogenic fragment of PSA or its immunogenic variant;

(iii) RNA encoding the amino acid sequence of prostatic acid phosphatase (PAP), its immunogenic variant, or an immunogenic fragment of PAP or its immunogenic variant;

(iv) RNA encoding the amino acid sequence of Homeobox B13 (HOXB13), its immunogenic variant, or an immunogenic fragment of HOXB13 or its immunogenic variant; and/or

(v) RNA encoding 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, for use in the method disclosed herein.

Brief description of the drawings

Figure 1. General RNA structure of RBL038.1, RBL039.1, RBL040.1, RBL041.1 and RBL045.1

Schematic representation of the general structures of all RNA vaccines with 5' cap, 5' and 3' untranslated regions (UTRs), coding sequences with N- and C-terminal fusion targets (sec and P2P16/MITD, respectively) and a poly(A) tail. Note that individual elements are not drawn to scale compared to their respective sequence lengths.

Figure 2. 5'-capping structure of beta-S-ARCA (D1) (m ₂ ^7,2'-O GppSpG)

The differences between beta-S-ARCA (D1) and the major cap analogue m7GpppG are shown in red: the -OCH3 group at the C2' position of the ^m7G building block and the substitution of the non-bridging oxygen in the beta-phosphate by sulfur. Due to the presence of the stereogenic P-centre (marked with an asterisk), the phosphorothioate cap analogue beta-S-ARCA exists as two diastereomers. Based on their elution order in reversed-phase HPLC, they have been designated as D1 and D2.

Figure 3. Vector map of the plasmid pST4-hAg-Kozak-KLK2-GS-P2P16-GS-MITD-FI-A30L70 for the production of RBL038.1

The insert with sequence elements labeled is shown in different colors. Eam1104I indicates the restriction endonuclease recognition site used for linearization. The kanamycin resistance gene is shown in black.

Figure 4. Vector map of the plasmid pST4-hAg-Kozak-KLK3-GS-P2P16-GS-MITD-FI-A30L70 for the production of RBL039.1

The insert with sequence elements labeled is shown in different colors. Eam1104I indicates the restriction endonuclease recognition site used for linearization. The kanamycin resistance gene is shown in black.

Figure 5. Vector map of the plasmid pST4-hAg-Kozak-ACPP-GS-P2P16-GS-MITD-FI-A30L70 for the production of RBL40.1

The insert with sequence elements labeled is shown in different colors. Eam1104I indicates the restriction endonuclease recognition site used for linearization. The kanamycin resistance gene is shown in black.

Figure 6. Vector map of the plasmid pST4-hAg-Kozak-sec-GS-HOXB13-GS-P2P16-GS-MITD-FI-A30L70 for the production of RBL041.1

The insert with sequence elements labeled is shown in different colors. Eam1104I indicates the restriction endonuclease recognition site used for linearization. The kanamycin resistance gene is shown in black.

Figure 7. Vector map of the plasmid pST4-hAg-Kozak-sec-GS-NKX3-1-GS-P2P16-GS-MITD-FI-A30L70 for the production of RBL045.1

The insert with sequence elements labeled is shown in different colors. Eam1104I indicates the restriction endonuclease recognition site used for linearization. The kanamycin resistance gene is shown in black.

Figure 8. Chemical structures of selected cationic lipids and lipid copolymers tested during formulation development

Figure 9. Organ selectivity of RNA lipoplexes with different charge ratios

Positively charged luc-RNA lipoplexes show high luciferase expression in the lungs, while negatively charged RNA lipoplexes show high selectivity for luciferase expression in the spleen.

Figure 10. Biological activity of RNA lipoplexes depends on the particle size and the size of the liposomes used to prepare the formulation

20 μg luc-RNA was condensed with small (198 nm) and large (381 nm) liposomes to reconstitute RNA lipoplexes (RNA(LIP)) and administered to BALB/c mice (n = 5). Luciferase expression in the spleen was analyzed 6 h after luc-RNA(LIP) administration (mean ± standard deviation).

Figure 11. Particle sizes of RNA lipoplexes reconstituted according to the protocol for clinical formulations

The particle sizes of RNA lipoplexes reconstituted by different experimenters, in different laboratories, and with different RNA constructs were analyzed using PCS measurements. Two independent formulations were prepared for experiments #3 and #10.

Figure 12. Size and polydispersity index for RNA lipoplexes with different charge ratios

Particle size (z-mean) and polydispersity index were measured for RNA lipoplexes with different charge ratios (DOTMA:RNA) at 10 min, 2 h and 24 h after preparation.

Figure 13. Size and biological activity of RNA lipoplexes with different charge ratios

(A) Particle size (z-mean) and polydispersity index were measured for RNA lipoplexes with different charge ratios (DOTMA:RNA) immediately after preparation (10 min). (B) Luciferase expression in the spleen was analyzed 6 h after luc-RNA (LIP) administration (20 μg RNA) in BALB/c mice (n = 4–5).

Figure 14. Localization of bioluminescence signal after intravenous administration of luciferase RNA (LIP)

Bioluminescence imaging 6 hours after intravenous injection of luc RNA (LIP) (20 μg RNA) into BALB/c mice (n = 3) in vivo (A) and into explanted spleen, liver, and lungs ex vivo (B). One representative mouse is shown.

Figure 15. RNA (LIP) is selectively internalized by splenic APC

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 (bottom row) or rhodamine-labeled liposomes (top row) by cell populations in the spleen was assessed by flow cytometry 1 h after lipoplex injection. Representative dot plots are shown.

Figure 16. Breaking of tolerance and antigen-specific cytotoxicity in vivo following immunization with AH5-RNA (LIP)

BALB/c mice (n = 5) were immunized intravenously with AH5-RNA(LIP) (40 μg RNA) on days 0, 3, 8, and 15 (green). Antigen-specific CD8+ T cell frequencies were monitored in blood by gp70-MHC tetramer staining (gray). The line represents the mean tetramer frequency (A). BALB/c mice (n = 5) were immunized intravenously with AH5-RNA(LIP) (40 μg RNA) on days 0, 3, 8, or left untreated. On day 12, in vivo cytotoxicity assay was performed by injecting a mixture of CFSEhigh (loaded with AH-5 peptide) and CFSElow (loaded with Inf-HA peptide) spleen cells from naïve BALB/c mice. AH5-specific lysis was shown for one representative mouse. All mice immunized with AH5-RNA(LIP) showed more than 90% antigen-specific lytic activity (B).

Figure 17. Transient increase in IFN-α after RNA vaccination (LIP)

(A) C57BL/6 mice (n = 3) were treated with HA-RNA (LIP) (40 μg RNA), liposomes alone, or PBS as a control. Serum IFN-α and TNF-α concentrations were assessed by ELISA at 6 and 24 h post-treatment (mean ± SD). (B) Native or splenectomized C57BL/6 mice (n = 2) were intravenously treated with HA-RNA (LIP) (40 μg RNA). Serum IFN-α concentrations were assessed by ELISA at 6 h post-treatment (mean ± SD).

Figure 18. Vaccination with W_pro1 antigen RNA results in antigen-specific T-cell responses

Splenocytes from intravenously vaccinated A2/DR1 mice (n = 4–5 per group) were restimulated for 20 h with BMDCs electroporated with the relevant mRNAs as indicated. BMDCs electroporated with irrelevant mRNA were used as controls (open symbols, gray bars). Effector function was measured by IFN-γ ELISPOT assay. Symbols indicate the mean values of triplicate wells for individual animals. Bars indicate the median value of all animals per group.

Figure 19. Mean levels of IFN-α (black bars) and IL-6 (gray bars) in animals from the high dose group

Error bars show standard deviations. IL-6 induction was significantly greater after the 1st dose (day 1) than after the 5th dose (day 22).

Figure 20. Induction of antigen-specific T cells in the spleen by KLK2-, KLK3-, ACPP-, NKX3-1- and HOXB13-encoding RNA

IFN-γ ELISPOT analysis of effector T cells from the spleens of mice immunized with lipoplex 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 last immunization were restimulated with either a peptide pool spanning the relevant human protein, the P2/P16/P17 peptides, or an irrelevant control peptide, CMV pp65 (495-504). Dots represent individual animals; horizontal bars indicate the mean ± SD of three animals.

Description of sequences

The following table provides a list of specific sequences referred to in this description.

Detailed description of the invention

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

It is preferred that the terms used in this application be defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G.W. Leuenberger, B. Nagel, and H. Kölbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology as explained in the literature in the art (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

The elements of the present invention will now be described. These elements are listed with specific embodiments, but it should be understood that they can be combined in any way and in any number to create additional embodiments. The various examples and embodiments described should not be construed as limiting the present invention to only the expressly described embodiments. It should be understood that this description discloses and covers embodiments that combine the expressly described embodiments with any number of the disclosed elements. Moreover, any permutations and combinations of all of the described elements should be considered disclosed in this description unless the context indicates otherwise.

The term “about” means approximately or nearly, and in the context of a numerical value or range set forth herein, in one embodiment means ±20%, ±10%, ±5%, or ±3% of the stated or claimed numerical value or range.

The terms "a" and "an" and "the" and similar references when used in the context of the description of the invention (especially in the context of the claims) are to be construed as embracing both the singular and the plural unless otherwise indicated herein or the context clearly contradicts them. The recitation of ranges in this specification is intended merely to serve as a means of abbreviating the individual designation of each distinct value falling within the range. Unless otherwise indicated herein, each distinct value is included in the specification as if it were individually recited herein. All methods described in this application may be performed in any suitable order unless otherwise indicated herein or the context clearly dictates otherwise. The use of any and all examples or introductory words before an example (e.g., "such as") provided in this application is intended merely to better illustrate the invention and does not impose a limitation on the scope of the claims. No statement in the description should be construed as indicating any non-claimed element essential to the practical implementation of the invention.

Unless specifically stated otherwise, the term "comprising" is used in the context of the present description to indicate that additional elements may optionally be present in addition to the list elements introduced by the word "comprising". However, as a specific embodiment of the present invention, the term "comprising" is intended to cover the possibility of the absence of additional elements, i.e., for the purposes of this embodiment, "comprising" should be understood to mean "consisting of".

Several documents are cited throughout this specification. Each of the documents cited in this specification (including all patents, patent applications, scientific publications, manufacturer's descriptions, instructions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. Nothing in this specification should be construed as an admission that the present invention cannot claim an earlier date of disclosure.

Definitions

The following definitions will be provided that apply to all aspects of the present invention. The following terms have the meanings given below unless otherwise indicated. Any undefined terms have their art-recognized meaning.

Terms such as "reduce" or "inhibit" as used herein mean the ability to cause an overall decrease in the level, preferably by 5% or more, 10% or more, 20% or more, more preferably by 50% or more, and most preferably by 75% or more. The term "inhibits" or similar expressions include complete or substantially complete inhibition, i.e., a decrease to zero or substantially to zero.

Terms such as “increasing” or “improving” in one embodiment 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%.

As used herein, the term “physiological pH” refers to a pH of approximately 7.5.

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. Thus, ionic strength I is mathematically represented by the formula

in which c is the molar concentration of a particular ionic substance and z is the absolute value of its charge. The sum Σ is taken over all the different types of ions (i) in the solution.

According to the invention, the term "ionic strength" in one embodiment refers to the presence of monovalent ions. With regard to the presence of divalent ions, in particular divalent cations, their concentration or effective concentration (free ion presence) due to the presence of chelating agents in one embodiment is low enough to prevent RNA degradation. In one embodiment, the concentration or effective concentration of divalent ions is below the catalytic level of hydrolysis of phosphodiester bonds between RNA nucleotides. In one embodiment, the concentration of free divalent ions is 20 μM or less. In one embodiment, free divalent ions are absent or substantially absent.

The term "freezing" refers to the solidification of a liquid, usually with the removal of heat.

The term "lyophilize" or "lyophilization" refers to the freeze-drying of a substance by freezing it and then reducing the ambient pressure to allow the frozen medium in the substance to sublimate directly from the solid phase to the gas phase.

The term "spray drying" refers to the spray drying of a substance by mixing a (heated) gas with a liquid which is atomized (sprayed) into a vessel (spray dryer) where the solvent in the resulting droplets evaporates, resulting in a dry powder.

The term "cryoprotectant" refers to a substance that is added to a formulation to protect the active ingredients during the freezing stages.

The term lyoprotectant refers to a substance that is added to a formulation to protect the active ingredients during the drying stages.

The term "reducing agent" refers to the addition of a solvent, such as water, to a dried product to return it to a liquid state, such as its original liquid state.

The term "recombinant" in the context of the present invention means "obtained by genetic engineering". In one embodiment, a "recombinant object" in the context of the present invention does not occur in nature.

The term "natural" as used in the present description denotes the fact that an object can be found in the natural environment. For example, a peptide or nucleic acid that is present in an organism (including viruses) can be isolated from a natural (natural) source and that has not been specially modified by man in a laboratory is natural. The term "found in nature" means "present in nature" and includes known objects, as well as objects that have not yet been discovered and/or isolated from the natural environment, but which may be discovered and/or isolated in the future from a natural source.

In the context of the present invention, the term "particle" refers to a structured object formed by molecules or complexes of molecules. In one embodiment, the term "particle" refers to a micro- or nano-sized structure, such as a compact micro- or nano-sized structure.

In the context of the present description, the term "RNA lipoplex particle" refers to a particle that comprises a lipid, in particular a cationic lipid, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA lead to complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes can typically be synthesized using a cationic lipid, such as DOTMA, and additional lipids, such as DOPE. In one embodiment, the RNA lipoplex particle is a nanoparticle.

As used herein, the term "nanoparticle" refers to a particle comprising RNA and at least one cationic lipid and having an average diameter suitable for intravenous administration.

The term "mean diameter" refers to the average hydrodynamic diameter of particles measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-mean with the length dimension, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, "mean diameter", "diameter" or "size" in relation to particles are used as synonyms for the stated Z-mean value.

The term "polydispersity index" is used in this specification as a measure of the size distribution of a set of particles, such as nanoparticles. The polydispersity index is calculated from dynamic light scattering measurements using a so-called cumulant analysis.

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 the formation of a lipid structure, such as the formation of lipid vesicles, such as the formation of liposomes. Typically, the RNA lipoplex particles described herein can be prepared by adding RNA to a dispersion of colloidal liposomes. Using the ethanol injection technique, such a dispersion of colloidal liposomes is, in one embodiment, formed as follows: an ethanol solution containing lipids, such as cationic lipids, such as DOTMA, and additional lipids, is injected into an aqueous solution with stirring. In one embodiment, the RNA lipoplex particles described herein can be prepared without an extrusion step.

The term "extrude" or "extrusion" refers to the creation of particles having a fixed cross-sectional profile. Specifically, it refers to the reduction in particle size when the particle is forced through filters with defined pores.

The prostate gland is a small gland that is located in the lower abdomen of a man. It is located under the bladder and surrounds the urethra. The prostate gland is regulated by the hormone testosterone and produces seminal fluid, also known as semen. Seminal fluid is the substance containing sperm that comes out of the urethra during ejaculation.

As used herein, "prostate cancer" means a malignant tumor in the prostate gland. When an abnormal, malignant growth of cells called a tumor forms in the prostate gland, it is called prostate cancer. Most prostate cancers grow slowly; however, some grow relatively quickly. The malignant tumor cells can spread from the prostate gland to other parts of the body, especially the bones and lymph nodes. At first, this may not cause any symptoms. In later stages, it may cause difficulty urinating, blood in the urine, or pain in the pelvis, back, or when urinating. About 99% of cases occur in men over 50 years of age. In many cases, active surveillance, or watchful waiting, is used. Other treatments may include a combination of surgery, radiation therapy, hormonal therapy, or chemotherapy. When the cancer is confined to the prostate gland, it may be curable. For those whose disease has spread to the bones, painkillers, bisphosphonates, and targeted therapy, among other treatments, may be helpful. Results depend on a person’s age and other health problems, as well as how aggressive and extensive the cancer is. Globally, prostate cancer is the second most common type of cancer and the fifth leading cause of cancer-related death in men.

As used herein, the term "co-administered" or "co-administration" or similar term refers to the simultaneous, concurrent, or substantially simultaneous administration of two or more agents as part of a single preparation, or as multiple preparations that are administered by the same or different routes.

The term “substantially at the same time,” as used herein, means a period within approximately 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or 6 hours of each other.

The invention describes nucleic acid sequences and amino acid sequences having a certain degree of identity with a given nucleic acid sequence or amino acid sequence, respectively (reference sequence).

The "sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences. The "sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.

The terms "% identical", "% identity" or similar terms are intended to refer specifically to the percentage of nucleotides or amino acids that are identical in an optimal alignment between the sequences being compared. The percentage reported is purely statistical and the differences between the two sequences may, but need not, be randomly distributed over the entire length of the sequences being compared. Comparison of two sequences is typically performed after optimal alignment against a segment or "comparison window" in order to identify local regions of the corresponding sequences. Optimal alignment for comparison may be performed manually or by using the local homology algorithm Smith and Waterman, 1981, Ads App. Math. 2, 482, by the local homology algorithm Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by the similarity search algorithm Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or using computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Drive, Madison, WI). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm available on the U.S. National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch &BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) an expectation threshold of 10; (ii) a word size of 28; (iii) a maximum number of matches in a query range of 0; (iv) the match/mismatch scores are 1, -2; (v) the gap cost is set to Linear; and (vi) a filter is used for low complexity regions. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) an expectation threshold of 10; (ii) a word size set to 3; (iii) the maximum number of hits in a query range set to 0; (iv) the matrix set to BLOSUM62; (v) the gap cost set to Existence: 11 extension: 1; and (vi) an adjustment to the conditional compositional scoring matrix.

Percent identity is obtained by determining the number of identical positions that the sequences being compared correspond to, dividing this number by the number of positions being compared (e.g. the number of positions in a reference sequence), and multiplying this result by 100.

In some embodiments, the degree of identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides in some embodiments in contiguous nucleotides. In some embodiments, the degree of identity is given for the entire length of the reference sequence.

Nucleic acid sequences or amino acid sequences having a certain degree of identity with a given nucleic acid sequence or amino acid sequence, respectively, can have, for example, at least one functional property of said given sequence, and in some cases are functionally equivalent to said given sequence. One important property includes an immunogenic property, in particular when administered to a subject. In some embodiments, a nucleic acid sequence or amino acid sequence having a specific degree of identity with a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.

RNA

As used herein, the term "RNA" refers to a nucleic acid molecule that includes ribonucleotide residues. In preferred embodiments, the RNA comprises all or most ribonucleotide residues. As used herein, the term "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially purified RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide material to the nucleotides of the internal RNA or to the end(s) of the RNA. It is also contemplated in the present description that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the purposes of the present invention, these altered RNAs are considered analogs of natural RNA.

In some embodiments of the present invention, the RNA is messenger RNA (mRNA), which refers to an RNA transcript that encodes a peptide or protein. As established in the art, mRNA typically comprises a 5'-untranslated region (5'-UTR), a peptide coding region, and a 3'-untranslated region (3'-UTR). In some embodiments, the RNA is produced by transcription or chemical synthesis in vitro. In one embodiment, mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid containing deoxyribonucleotides.

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

In one embodiment, the RNA may comprise modified nucleosides. In some embodiments, the RNA comprises a modified nucleoside in place of at least one (e.g., each) uridine.

As used herein, the term "uracil" describes one of the nitrogenous bases that can occur in the nucleic acid RNA. The structure of uracil is:

As used herein, the term "uridine" describes one of the nucleosides that can occur in RNA. The structure of uridine is:

.

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

.

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

.

"Pseudouridine" is one example of a modified nucleoside that is an isomer of uridine where uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

Another example of a modified nucleoside is N1-methyl-pseudouridine (m1Ψ), which has the structure:

.

N1-methyl-pseudo-UTP has the following structure:

.

Another example of a modified nucleoside is 5-methyluridine (m5U), which has the structure:

.

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

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

In some embodiments, the modified nucleoside that replaces one or more uridines in the RNA may be one or more of 3-methyluridine (m3U), 5-methoxyuridine (mo5U), 5-azauridine, 6-aza-uridine, 2-thio-5-azauridine, 2-thiouridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine (ho5U), 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromuridine), uridine 5-hydroxyacetic acid (cmo5U), methyl ester of uridine 5-hydroxyacetic acid (mcmo5U), 5-carboxymethyl uridine (cm5U), 1-carboxymethyl pseudouridine, 5-carboxyhydroxymethyluridine (chm5U), 5-carboxyhydroxymethyluridine methyl ester (mchm5U), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxycarbonyl-2-methoxycarbonyl (mcm5s2U), 5-aminomethyl-2-thiouridine (nm5s2U), 5-methylaminomethyluridine (mnm5U), 1-ethyl pseudouridine, 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-methylaminomethyl-2-selenouridine (mnm5se2U), 5-carbamoylmethyluridine (ncm5U), 5-carboxymethylaminomethyluridine (cmnm5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 5-propynyl uridine, 1-propynyl pseudouridine, 5-taurinomethyl uridine (τm5U), 1-taurinomethyl pseudouridine, 5-taurinomethyl pseudouridine, 5-taurinomethyl pseudouridine, 5-taurinomethyl pseudouridine, 5-taurinomethyl pseudouridine, 5-taurinomethyl pseudouridine (τm5s2U), 1-taurinomethyl-4-thio pseudouridine), 5-methyl-2-thiouridine (m5s2U), 1-methyl-4-thio pseudouridine (m1s4ψ), 4-thio-1-methyl pseudouridine, 3-methyl pseudouridine (m3ψ), 2-thio-1-methyl pseudouridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deazapseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m5D), 2-thiodihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxy-pseudouridine, 4-methoxy-2-thiopseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl) pseudouridine (acp3 ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U), α-thiouridine, 2′-O-methyluridine (Um), 5,2′-O-dimethyluridine (m5Um), 2′-O-methylpseudouridine (ψm), 2-thio-2′-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 3,2'-O-dimethyluridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 1-thiouridine, deoxythymidine, 2'-F-arauridine, 2'-F-uridine, 2'-OH-arauridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine or any other modified uridine known in the art.

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

In some embodiments, the modified nucleoside is independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises 5-methyluridine (m5U). In some embodiments, at least one RNA can comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (ψ) and N1-methylpseudouridine (m1ψ). In some embodiments, the modified nucleosides comprise pseudouridine (ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleosides comprise N1-methyl-pseudouridine (m1ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U).

In some embodiments, the RNA of the present invention comprises a 5' cap. In one embodiment, the RNA of the present disclosure does not comprise unprotected 5' triphosphates. In one embodiment, the RNA may be modified with a 5' cap analogue. The term "5' cap" refers to a structure found at the 5' end of an mRNA molecule and typically consists of a guanosine nucleotide attached to the mRNA via a 5'-5' triphosphate bond. In one embodiment, this guanosine is methylated at the 7-position. Provision of RNA with a 5' cap or a 5' cap analogue may be achieved by in vitro transcription, in which the 5' cap is co-transcriptionally expressed on the RNA strand, or may be attached to the RNA post-transcriptionally using capping enzymes.

In some embodiments, the "capping" structural element for the RNA is m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG (also sometimes referred to as

m ₂ ^7,3`O G(5')ppp(5')m ^2'-O ApG), which has the following structure:

.

Below is an approximate structure of cap1 RNA, which includes RNA and m ₂ ^7,3`O G(5')ppp(5')m ^2'-O ApG:

.

Below is another example of cap1 RNA:

.

In some embodiments, the RNA is modified with "Cap0" structures using, in one embodiment, a cap analog, an anti-reverse cap (ARCA Cap (m ₂ ^7,3`O G(5')ppp(5')G)) with the structure:

.

Below is an example of Cap0 RNA containing RNA and _m2 ^7,3`O G(5')ppp(5')G:

.

In some embodiments, the "Cap0" structures are generated using the cap analog Beta-S-ARCA ( _m27,2`O G(5')ppSp(5')G ⁾ with the structure:

.

Below is an example of RNA-Cap0 containing Beta-S-ARCA ( _m2 ^7,2`O G(5')ppSp(5')G) and RNA:

.

A particularly preferred Cap comprises a 5'-cap of _m2 ^{7,2`O</sup> G(5')ppSp(5')G. In some embodiments, at least one RNA described herein comprises a 5'-cap of <sub>m2</sub> <sup>7,2`O} G(5')ppSp(5')G. In some embodiments, each RNA described herein comprises a 5'cap of _m2 ^7,2`O G(5')ppSp(5')G.

In some embodiments, the RNA of the present invention 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 the corresponding region in an RNA molecule, such as an mRNA molecule. The untranslated region (UTR) may be located upstream of the open reading frame (5' UTR) and/or downstream of the open reading frame (3' UTR). The 5' UTR, when present, is located 5' upstream of the start codon of the protein coding region. The 5' UTR is downstream of the 5' cap structure (if present), such as immediately adjacent to the 5' cap structure. The 3' UTR, when present, is located at the 3' end, downstream of the termination codon of the protein coding region, but the term "3' UTR" preferably does not include the poly-A sequence. Thus, the 3' UTR is upstream of the poly(A) sequence (if present), e.g., immediately adjacent to the poly(A) sequence.

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.

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.

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.

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

A poly-A tail of approximately 120 nucleotides has been shown to have beneficial effects on RNA levels in transfected eukaryotic cells, as well as on the levels of protein translated from the open reading frame located upstream (5') of the poly-A tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

The poly-A tail can be 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 the context of the invention, the term "consists essentially of" means that the majority of the nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides in the poly-A tail are A nucleotides, but it is possible for the remaining nucleotides to be A nucleotides, other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). The term "consists of" means that all nucleotides in the poly-A tail, i.e. 100% of the nucleotides in the poly-A tail, are A nucleotides. The term "nucleotide A" or "A" refers to adenylate.

In some embodiments, the poly-A tail is added during transcription of RNA, such as during the production of in vitro transcribed RNA from a DNA template containing repeating dT (deoxythymidylate) nucleotides in the strand complementary to the coding strand. The DNA sequence encoding the poly-A tail (coding strand) is called a poly (A) cassette.

In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of four nucleotides (dA, dC, dG and dT). Such a random sequence may have a length of 5 to 50, 10 to 30 or 10 to 20 nucleotides. Such a cassette is disclosed in WO 2016/005324 A1, incorporated herein by reference. Any poly(A) cassette described in WO 2016/005324 A1 may be used in the present invention. A poly(A) cassette, which essentially consists of dA nucleotides but is interrupted by a random sequence having an equal distribution of four nucleotides (dA, dC, dG, dT) and having a length of, for example, from 5 to 50 nucleotides, exhibits, at the DNA level, constant replication of plasmid DNA in E. coli, and is associated, at the RNA level, with useful properties with respect to maintaining RNA stability and translation efficiency. Therefore, in some embodiments, the poly-A tail contained in the RNA molecule disclosed in the present application essentially consists of A nucleotides but is interrupted by a random sequence of four nucleotides (A, C, G, U). Such a random sequence can have a length of from 5 to 50, from 10 to 30, or from 10 to 20 nucleotides.

In some embodiments, no nucleotides other than A nucleotides flank the poly-A tail at its 3' end, i.e., the poly-A tail is not masked and does not contain a nucleotide other than A at its 3' end. In some embodiments, the poly-A tail comprises the sequence of SEQ ID NO: 29.

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 be 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 include the poly-A tail of 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.

In the context of the present invention, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA. The RNA can subsequently be translated into a peptide or protein.

In the case of RNA, the term "expression" or "translation" refers to the process that occurs in the cell's ribosomes by which a strand of mRNA directs the assembly of a sequence of amino acids to form a peptide or protein.

In one embodiment, after administration of the RNA described herein, for example prepared as RNA lipoplex 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 the target cell. In one embodiment, the RNA is translated by the target cell to form the peptide or protein that it encodes. In one embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an antigen-presenting cell, such as a professional antigen-presenting cell in the spleen. In one embodiment, the target cell is a dendritic cell or a macrophage. The RNA lipoplex particles described herein can be used to deliver RNA to such a target cell. Accordingly, the present invention also relates to a method of delivering RNA to a target cell of a subject, comprising administering to the subject the RNA lipoplex particles disclosed herein. In one embodiment, the RNA is delivered to the cytosol of the target cell. In one embodiment, the RNA is translated by the target cell to form a peptide or protein encoded by the RNA.

According to the invention, the term "encodes RNA" means that the RNA, if present in an appropriate environment, such as in the cells of the target tissue, can direct the assembly of amino acids to form the peptide or protein that it encodes during the translation process. In one embodiment, the RNA is capable of interacting with the cellular translation machinery to ensure 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 display it on the cell surface.

As used herein, the term "peptide" includes oligo- and polypeptides and refers to substances that include 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 linked to each other by peptide bonds. The term "protein" refers to larger peptides, particularly peptides having at least about 151 amino acids, but the terms "peptide" and "protein" are generally used interchangeably herein.

The term "antigen" refers to an agent comprising an epitope against which an immune response can be generated. The term "antigen" includes, in particular, proteins and peptides. In one embodiment, the antigen is presented by cells of the immune system, such as antigen-presenting cells, for example, dendritic cells or macrophages. The antigen or a product of its processing, such as a T-cell epitope, in one embodiment binds to a T- or B-cell receptor or to an immunoglobulin molecule, such as an antibody. Accordingly, the antigen or a product of its processing can specifically react with antibodies or T-lymphocytes (T-cells). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, and the epitope is derived from such an antigen.

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 epitopes that will stimulate the host immune system to mount a cellular antigen-specific immune response and/or an antibody humoral response against the disease. Therefore, a disease-associated antigen or its epitope can be used for therapeutic purposes. Disease-associated antigens may be associated with an oncological disease, usually tumors.

The term "tumor antigen" refers to a component of malignant tumor cells that can originate from the cytoplasm, cell surface, and cell nucleus. In particular, it refers to those antigens that are produced intracellularly or as surface antigens on tumor cells.

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

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

In some embodiments of the present invention, the RNA encodes at least one epitope. In some embodiments, the epitope is derived from a tumor antigen as disclosed herein.

RNAs used

In some embodiments, the compositions disclosed herein comprise RNA encoding kallikrein-2 protein (KLK2), RNA encoding prostate-specific antigen (PSA) protein, RNA encoding prostate-specific acid phosphatase (PAP) protein, RNA encoding Homeobox B13 protein (HOXB13), and RNA encoding NK3 Homeobox 1 protein (NKX3-1). Similarly, the methods described herein comprise administering RNA encoding kallikrein-2 protein (KLK2), RNA encoding prostate-specific antigen (PSA) protein, RNA encoding prostate-specific acid phosphatase (PAP) protein, RNA encoding Homeobox B13 protein (HOXB13), and RNA encoding NK3 Homeobox 1 protein (NKX3-1).

The kallikrein-2 (KLK2) protein comprises the amino acid sequence of KLK2, an immunogenic variant thereof, or an immunogenic fragment of KLK2 or an immunogenic variant fragment, and may have an amino acid sequence comprising 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. The RNA encoding the KLK2 protein (i) may comprise the nucleotide sequence of SEQ ID NO: 3 or 4, 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: 3 or 4; and/or (ii) may encode an amino acid sequence comprising 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.

The prostate-specific antigen (PSA) protein comprises the amino acid sequence of PSA, an immunogenic variant thereof, or an immunogenic fragment of PSA or an immunogenic variant fragment, and may have an amino acid sequence comprising 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. 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 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.

The prostatic acid phosphatase (PAP) protein comprises the amino acid sequence of PAP, an immunogenic variant thereof, or an immunogenic fragment of PAP or an immunogenic variant fragment, and may have an amino acid sequence comprising 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. 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 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.

The Homeobox B13 (HOXB13) protein comprises the amino acid sequence of HOXB13, an immunogenic variant thereof, or an immunogenic fragment of HOXB13 or an immunogenic variant fragment, and may have an amino acid sequence comprising 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. 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 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.

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

By "variant" as used herein is meant an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid modification. The parent amino acid sequence may be a natural or wild type (WT) amino acid sequence, or may be a modified version 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, such as from 1 to about 20 amino acid modifications, and preferably from 1 to about 10 or from 1 to about 5 amino acid modifications compared to the parent.

The terms "wild type", "WT" or "native" as used herein refer to the amino acid sequence that occurs in nature, including allelic variants. A wild type peptide or protein has an amino acid sequence that has not been intentionally modified.

For the purposes of the present invention, "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-translational modified variants, conformations, isoforms, allelic variants, species variants and species homologues, in particular those that occur in nature.

Amino acid insertional variants include insertions of one or more amino acids into a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in the amino acid sequence, although random insertion is also possible with appropriate screening of the resulting product. Amino acid addition variants include fusions at the amino and/or carboxy terminus of one or more amino acids, for example, 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. Deletions can be at any position in the protein. Amino acid deletion variants that contain a deletion at the N-terminus and/or C-terminus of a protein are also called N-terminus and/or C-terminus truncation variants. Amino acid substitution variants are characterized in that at least one residue in the sequence is deleted and another residue is inserted in its place. Preference is given to modifications at those positions of the amino acid sequence that are not conserved between homologous proteins or peptides and/or to substitutions of amino acids with others having similar properties. Preferably, amino acid substitutions in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid substitution involves substitution of one amino acid from a family of amino acids in which the amino acids have similar side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions in the following groups:

glycine, alanine;

valine, isoleucine, leucine;

aspartic acid, glutamic acid;

asparagine, glutamine;

serine, threonine;

lysine, arginine; And

phenylalanine, tyrosine.

Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence which is a variant of said 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 which 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 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 preferred embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence.

"Sequence similarity" indicates the percentage of amino acids that are either identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical in the sequences.

An amino acid sequence (peptide, protein or polypeptide) "derived from" a particular amino acid sequence (peptide, protein or polypeptide) indicates the origin of the first amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, substantially identical or homologous to this particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of this particular sequence or a fragment thereof.

The peptide and protein antigen described in the present application (KLK2 protein, PSA protein, PAP protein, HOXB13 protein and NKX3-1 protein), delivered to a subject by administering RNA encoding the antigen, i.e. a vaccine antigen, preferably results in stimulation, priming and/or expansion of T cells in the subject. Said stimulated, primed and/or expanded T cells are preferably directed against a target antigen, in particular a target antigen expressed by diseased cells, tissues and/or organs, i.e. a disease-associated antigen. Thus, the vaccine antigen may comprise a disease-associated antigen or a fragment or variant thereof. In one embodiment, such a fragment or variant is immunologically equivalent to a disease-associated antigen. In the context of the present invention, the term "antigen fragment" or "antigen variant" means an agent that leads to stimulation, priming and/or expansion of T cells that are directed to a T cell target that is a disease-associated antigen, in particular when it is expressed on the surface of affected cells, tissues and/or organs. Thus, the vaccine antigen administered according to the present invention may correspond to or may comprise an antigen associated with a disease, may correspond to or may comprise a fragment of an antigen associated with a disease, or may correspond to or may comprise an antigen that is homologous to an antigen associated with a disease or a fragment thereof. If the vaccine antigen administered according to the invention comprises a fragment of a disease-associated antigen or has an amino acid sequence that is homologous to a fragment of a disease-associated antigen, said fragment or amino acid sequence may comprise an epitope of the disease-associated antigen or a sequence homologous to an epitope of the disease-associated antigen, wherein T cells bind to said epitope. Thus, according to the invention, the administered antigen may comprise an immunogenic fragment of an antigen associated with a disease or have an amino acid sequence homologous to an immunogenic fragment of an antigen associated with a disease. An "immunogenic fragment of an antigen" according to the present description preferably refers to a fragment of an antigen that is capable of stimulating, priming and/or causing expansion of T cells. Preferably, the vaccine antigen (similar to an antigen associated with a disease) comprises a corresponding epitope for binding by T cells. It is also preferred that the vaccine antigen (similar to the disease-associated antigen) is expressed on the surface of a cell, such as an antigen-presenting cell, to provide an appropriate epitope for binding by T cells. The vaccine antigen according to the invention may be a recombinant antigen.

The term "immunologically equivalent" means that an immunologically equivalent molecule, such as an immunologically equivalent amino acid sequence, exhibits the same or substantially the same immunological properties and/or exerts the same or substantially the same immunological effects, for example with respect to the type of immunological effect. In the context of the present invention, the term "immunologically equivalent" is preferably used in relation to the immunological effects or properties of antigens or antigen variants. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if said amino acid sequence, when exposed to T cells binding to the reference amino acid sequence or to cells expressing the reference amino acid sequence, elicits an immune response having the same specificity as the reference amino acid sequence, in particular with respect to stimulation, priming and/or expansion of T cells. Thus, a molecule that is immunologically equivalent to an antigen exhibits the same or substantially the same properties and/or exerts the same or substantially the same effects in stimulating, priming and/or expanding T cells as the antigen to which the T cells are targeted.

"Activation" or "stimulation" in the context of the present invention refers to a state of a T cell that has been sufficiently stimulated to cause detectable cellular proliferation. Activation may also be associated with induced cytokine production and detectable effector functions. The term "activated T cells" refers, among other things, to T cells that undergo cell division.

The term "priming" refers to the process by which a T cell first contacts its specific antigen and differentiates into effector T cells.

The term "clonal expansion" or "multiplication" refers to a process by which a specific entity is multiplied. In the context of the present invention, the term is preferably used in relation to an immunological response in which lymphocytes are stimulated by an antigen, proliferate, and a specific lymphocyte recognizing said antigen is amplified. Preferably, clonal expansion results in differentiation of lymphocytes.

Lipoplex particles

In some embodiments of the present invention, the RNA described herein may be in RNA lipoplex particles. The RNA lipoplex particles and compositions comprising these particles disclosed herein are useful for delivering RNA to a target tissue following parenteral administration, in particular following intravenous administration. The RNA lipoplex particles may be prepared using liposomes, which may be prepared 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, for example in an amount of about 5 mM. In one embodiment, the liposomes and RNA lipoplex 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, at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the liposomes and RNA lipoplex 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 produce RNA lipoplex particles by mixing the liposomes with RNA.

Lipoplex RNA particles targeting the spleen are described in WO 2013/143683, incorporated herein by reference. It has been found that lipoplex RNA particles having a net negative charge can be used to preferentially target spleen tissue or spleen cells, such as antigen-presenting cells, in particular dendritic cells. Accordingly, after administration of the lipoplex RNA particles, RNA accumulation and/or RNA expression occurs in the spleen. Thus, the lipoplex RNA particles of the present invention can be used to express RNA in the spleen. In one embodiment, after administration of the lipoplex RNA particles, there is no or virtually no RNA accumulation and/or RNA expression in the lungs and/or liver. In one embodiment, after administration of the lipoplex RNA particles, there is RNA accumulation and/or RNA expression in antigen-presenting cells, such as professional antigen-presenting cells in the spleen. Thus, the RNA lipoplex particles of the present invention can be used to express RNA in said antigen-presenting cells. In one embodiment, the antigen-presenting cells are dendritic cells and/or macrophages.

Diameter of RNA lipoplex particles

The RNA lipoplex particles described herein have an average diameter that, in one embodiment, is in the range of from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In one embodiment, the RNA lipoplex particles have an average diameter in the range of from about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter in the range of from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

In one embodiment, the RNA lipoplex particles described herein have 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 can exhibit a polydispersity index in the range of about 0.1 to about 0.3.

Lipid

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

An additional lipid may be included to control the overall positive to negative charge ratio and physical stability of the RNA lipoplex particles. In some embodiments, the additional lipid is a neutral lipid. As used herein, the term "neutral lipid" refers to a lipid having zero net charge. Examples of neutral lipids include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingoemielin, cephalin, cholesterol, and cerebroside. In particular embodiments, the additional lipid is DOPE, cholesterol, and/or DOPC.

In some embodiments, the RNA lipoplex particles include both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE. Without wishing to be bound by theory, the inventors note that the amount of at least one cationic lipid compared to the amount of at least one additional lipid can affect important characteristics of the RNA lipoplex particles, such as charge, particle size, stability, tissue selectivity, and bioactivity of the RNA. Accordingly, in some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is from about 10:0 to about 1:9, from about 4:1 to about 1:2, or from about 3:1 to about 1:1. In particular embodiments, the molar ratio can be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of at least one cationic lipid to at least one additional lipid is about 2:1.

Charge ratio

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

In one embodiment, at physiological pH, the ratio of positive charges to negative charges in the RNA lipoplex particles is from about 1.6:2 to about 1:2, or from about 1.6:2 to about 1.1:2. In particular embodiments, the ratio of positive charges to negative charges in the RNA lipoplex particles at physiological pH is about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0.

It has been found that RNA lipoplex particles having the above charge ratio can be used to preferentially target spleen tissue or spleen cells, such as antigen-presenting cells, in particular dendritic cells. Accordingly, 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 invention can be used to express RNA in the spleen. In one embodiment, after administration of the RNA lipoplex particles, there is no or virtually no RNA accumulation and/or RNA expression 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 invention can be used to express RNA in such antigen-presenting cells. In one embodiment, the antigen-presenting cells are dendritic cells and/or macrophages.

A. Salt and ionic strength

According to the present invention, the compositions disclosed herein may comprise salts, such as sodium chloride. Without wishing to be bound by theory, the inventors note that sodium chloride acts as an ionic osmolality agent to precondition the RNA prior to mixing with at least one cationic lipid. In some embodiments, organic or inorganic salts alternative to sodium chloride are contemplated herein. Alternative salts include, but are not limited to, potassium chloride, dipotassium phosphate, monopotassium phosphate, potassium acetate, potassium bicarbonate, potassium sulfate, potassium acetate, disodium phosphate, monosodium phosphate, sodium acetate, sodium bicarbonate, sodium sulfate, sodium acetate, lithium chloride, magnesium chloride, magnesium phosphate, calcium chloride, and sodium salts of ethylenediaminetetraacetic acid (EDTA).

Typically, the compositions comprising the RNA lipoplex particles described herein comprise sodium chloride at a concentration that is preferably in the range of 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 compositions comprising the RNA lipoplex particles have an ionic strength corresponding to such concentrations of sodium chloride.

B. Stabilizer

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

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

In embodiments of the present invention, the stabilizer is mannose, glucose, sucrose or trehalose.

According to the present invention, the RNA lipoplex particle compositions disclosed herein have a stabilizer concentration suitable for the stability of the composition, in particular for the stability of the RNA lipoplex particles and the stability of the RNA.

C. pH value and buffer

According to the present invention, the RNA lipoplex particle compositions disclosed herein have a pH suitable for stabilizing the RNA lipoplex particles and, in particular, for stabilizing RNA. In one embodiment, the RNA lipoplex particle compositions described herein have a pH of about 5.5 to about 7.5.

According to the present invention, the claimed compositions include a buffer. Without being limited by theory, the inventors note that the use of a buffer maintains the pH of the composition during its production, storage and use. In some embodiments of the present invention, the buffer may be sodium bicarbonate, monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate, [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), 2-(bis(2-hydroxyethyl)amino)acetic acid (bicine), 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris), N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine), 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonic acid (TAPSO), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino] ethanesulfonic acid (TES), 1,4-piperazine diethanesulfonic acid (PIPES), dimethylarsinic acid, 2-morpholin-4-ylethanesulfonic acid (MES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), or phosphate buffered saline (PBS). Other suitable buffers may include acetic acid and its salts, citric acid and its salts, boric acid and its salts, and phosphoric acid and its salts.

In one embodiment, the buffer is HEPES.

In one embodiment, the buffer has a concentration of from about 2.5 mM to about 15 mM.

D. Chelating agent

Some embodiments of the present invention involve the use of a chelating agent. Chelating agents refer to chemical compounds that are capable of forming at least two coordinating covalent bonds with a metal ion, thereby forming a stable water-soluble complex. Without wishing to be bound by theory, the inventors believe that chelating agents reduce the concentration of free divalent ions that could otherwise cause accelerated degradation of RNA. Examples of suitable chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), an EDTA salt, desferrioxamine B, deferoxamine, sodium dithiocarb, penicillamine, calcium pentetate, sodium pentetate, 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 a salt thereof. In some embodiments, the chelating agent is EDTA or an EDTA salt. In an exemplary embodiment, the chelating agent is disodium EDTA dihydrate.

In some embodiments, EDTA has a concentration of from about 0.05 mM to about 5 mM.

E. Physical state of the proposed compositions

The composition according to the present invention is a liquid or a solid. Non-limiting examples of a solid include a frozen form or a lyophilized form. In a preferred embodiment, the composition is a liquid.

Proposed pharmaceutical compositions

The RNA described in the present application, for example in the form of RNA lipoplex particles, is used for the preparation of pharmaceutical compositions or medicinal products for therapeutic or prophylactic treatment.

The compositions of the present invention can be administered in any pharmaceutically acceptable form.

The term "pharmaceutical composition" refers to a composition comprising a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and/or excipients. Said pharmaceutical composition is useful for treating, preventing or reducing the severity of a disease or disorder by administering said pharmaceutical composition to a subject. A pharmaceutical composition is also known in the art as a pharmaceutical preparation. In the context of the present invention, the pharmaceutical composition comprises RNA as described in the present application, for example, prepared as RNA lipoplex particles.

The pharmaceutical compositions of the present invention preferably comprise one or more adjuvants or can be administered with one or more adjuvants. The term "adjuvant" refers to a compound that prolongs, enhances or accelerates an immune response. Adjuvants include a heterogeneous group of compounds such as oil emulsions (e.g., Freund's adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin) or immunostimulatory complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors and cytokines such as monokines, lymphokines, interleukins, chemokines. The chemokines may 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 an oil such as Montanide® ISA51. Other suitable adjuvants for use in the present invention include lipopeptides such as Pam3Cys.

The pharmaceutical compositions of the present invention are typically administered in a “pharmaceutically effective amount” and in a “pharmaceutically acceptable formulation.”

The term "pharmaceutically acceptable" refers to the non-toxicity of the material and does not interact with the active component of the pharmaceutical composition.

The term "pharmaceutically effective amount" refers to an amount that provides the desired response or desired effect, alone or with additional doses. In the case of treating a specific disease, the desired response preferably relates to inhibition of the course of the disease. This includes slowing the progression of the disease and, in particular, stopping or reversing the progression of the disease. The desired response in the treatment of a disease may also be delaying the onset or preventing the onset of said disease or condition. An effective amount of the compositions described herein will depend on the condition being treated, the severity of the disease, individual patient parameters including age, physiological condition, size and weight, duration of treatment, type of concomitant therapy (if any), the particular route of administration and similar factors. Accordingly, the administered doses of the compositions described herein may depend on a variety of such parameters. In the event that the patient's response is insufficient at the initial dose, higher doses (or effectively higher doses achieved by another, more precisely localized route of administration) may be used.

In some embodiments, an effective amount comprises an amount sufficient to reduce the size of the tumor/lesion. In some embodiments, an effective amount is an amount sufficient to reduce the rate of tumor growth (e.g., to suppress tumor growth). In some embodiments, an effective amount is an amount sufficient to delay tumor development. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. In some embodiments, an effective amount is an amount sufficient to enhance the subject's immune response to the tumor such that tumor growth and/or size and/or metastasis are reduced, delayed, reduced, and/or prevented. An effective amount can be administered by one or more administrations. In some embodiments, administration of an effective amount (e.g., a composition comprising mRNA) can: (i) reduce the number of malignant tumor cells; (ii) reduce the size of the tumor; (iii) suppress, slow, somewhat slow, and stop the infiltration of malignant tumor cells into peripheral organs; (iv) inhibit (e.g., to some extent, slow down and/or block or prevent) metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor onset and/or recurrence; and/or (vii) alleviate to some extent one or more symptoms associated with cancer.

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

Suitable preservatives for use in the pharmaceutical compositions of the present invention include, but are not limited to, benzalkonium chloride, chlorobutanol, paraben and thimerosal.

As used herein, the term "excipient" refers to a substance that may be present in a pharmaceutical composition of the present invention but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surface-active agents, preservatives, stabilizers, emulsifiers, buffers, flavors, or colorants.

The term "diluent" refers to a diluting and/or liquefying agent. In addition, the term "diluent" includes any one or more liquids, liquid or solid suspensions and/or mixed media. Examples of suitable diluents include ethanol, glycerol and water.

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

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

Pharmaceutical carriers, excipients or diluents may be selected taking into account the intended route of administration and standard pharmaceutical practice.

Methods of administration of the proposed pharmaceutical compositions

In one embodiment, the pharmaceutical compositions described herein can be administered intravenously, intraarterially, subcutaneously, intradermally, intranodally, or intramuscularly. In some embodiments, the pharmaceutical composition is formulated for local or systemic administration. Systemic administration can include enteral administration, which includes absorption through the gastrointestinal tract, or parenteral administration. As used herein, the term "parenteral administration" refers to administration by any route other than the gastrointestinal tract, such as by intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for systemic administration. In another preferred embodiment, systemic administration is by intravenous administration.

Application of the proposed pharmaceutical compositions

The RNA described in this application, for example in the form of RNA lipoplex particles, can be used in the therapeutic or prophylactic treatment of diseases in which providing a subject with amino acid sequences encoded by the RNA results in a therapeutic or prophylactic effect.

The term disease refers to an abnormal condition that affects an individual. Disease is often understood as a medical condition associated with specific symptoms and signs. Disease may be caused by factors from an external source, such as an infectious disease, or it may be caused by internal dysfunctions, such as an autoimmune disease. When applied to humans, the term disease is often used in a broader sense to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the person with the disease, or similar problems for those who come into contact with the person. In this broader sense, disease sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behavior, and atypical variations in structure and function, while in other contexts and for other purposes they may be considered separate categories. Diseases usually affect individuals not only physically but also emotionally, as acquiring a disease and living with multiple diseases can change one's outlook on life and personality.

In the context of the present invention, the terms "treatment", "treat" or "therapeutic intervention" refer to the observation and care of a subject in order to combat a condition such as a disease or disorder. The terms include the full range of treatment of a condition from which a subject suffers, where treatment, for example, means the administration of a therapeutically effective compound to alleviate symptoms or complications, to slow the progression of a disease, disorder or condition and/or to cure or eliminate a disease, disorder or condition, as well as to prevent a condition; while prevention should be understood as the observation and care of an individual in order to combat a disease, condition or disorder and includes the administration of active compounds to prevent the occurrence of symptoms or complications.

The term "therapeutic treatment" refers to any treatment that improves the health status and/or prolongs (increases) the life expectancy of an individual. Said treatment may eliminate a disease in an individual, halt or slow the progression of a disease in an individual, suppress or slow the progression of a disease in an individual, reduce the frequency or severity of symptoms in an individual, and/or reduce relapses in an individual who currently has or has previously had a disease.

The terms "prophylactic treatment" or "preventive treatment" refer to any treatment that is intended to prevent the occurrence of a disease in an individual. The terms "prophylactic treatment" or "preventive treatment" are used interchangeably herein.

The terms "individual" and "subject" are used interchangeably herein. They refer to a human or other mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that may be affected by or susceptible to a disease or disorder (e.g., cancer), but may or may not have the disease or disorder. In many embodiments, the individual is a human. Unless otherwise specified, the terms "individual" and "subject" do not denote a particular age and thus include adults, the elderly, children, and neonates. In embodiments of the present invention, "individual" or "subject" is a "patient."

The term "patient" means the individual or subject to be treated, in particular a sick individual or subject.

In one embodiment of the present invention, the objective is to provide an immune response against malignant tumor cells expressing one or more tumor antigens and to treat a cancer disease involving cells expressing one or more tumor antigens. In one embodiment, the cancer disease is prostate cancer. In one embodiment, the tumor antigens are KLK2, PSA, PAP, HOXB13 and/or NKX3-1.

A pharmaceutical composition comprising RNA can be administered to a subject to induce an immune response in the subject against one or more antigens or one or more epitopes encoded by the RNA, which may be therapeutic or partially or completely protective. A person skilled in the art knows that one of the principles of immunotherapy and vaccination is based on the fact that an immunoprotective response to a disease is induced by immunizing the subject with an antigen or epitope that is immunologically relevant to the disease to be treated. Accordingly, the pharmaceutical compositions described in the present application are useful for inducing or enhancing an immune response. Thus, the pharmaceutical compositions described herein are useful for the prophylactic and/or therapeutic treatment of a disease associated with an antigen or epitope, in particular prostate cancer.

As used herein, the term "immune response" refers to an integrated response of the body to an antigen or a cell expressing the 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 directed against cells expressing the antigen and is characterized by the presentation of the antigen using MHC class I or class II molecules. A cellular response refers to T lymphocytes, which can be classified as T helper cells (also called CD4+ T lymphocytes), which play a central role in regulating the immune response, or killer cells (also called cytotoxic T cells, CD8+ T cells, or CTLs), which induce apoptosis in infected cells or malignant tumor cells. In one embodiment, administration of the pharmaceutical composition of the present invention comprises stimulating an antitumor CD8+ T-cell response against malignant tumor cells expressing one or more tumor antigens. In a specific embodiment, the tumor antigens are presented by MHC class I molecules.

The present invention contemplates an immune response that may be protective, preventive, prophylactic and/or therapeutic. As used herein, the term "induces [or inducing] an immune response" may indicate the absence of an immune response against a particular antigen prior to induction, or may indicate that prior to induction, there was a baseline immune response against a particular antigen that was enhanced following induction. Therefore, the term "induces [or inducing] an immune response" encompasses the concept of "enhances [or enhances] an immune response."

The term "immunotherapy" refers to the treatment of a disease or condition by inducing or enhancing an immune response. The term "immunotherapy" includes immunization with an antigen or vaccination with an antigen.

The terms immunization or vaccination describe the process of introducing an antigen into an individual to induce an immune response, such as for therapeutic or prophylactic purposes.

In one embodiment, the present invention provides for the administration of RNA lipoplex particles targeting spleen tissue. As disclosed herein, the RNA encodes a peptide or protein comprising an antigen or epitope. The RNA is taken up by antigen-presenting cells of the spleen, such as dendritic cells, to express the peptide or protein. After optional processing and presentation by the antigen-presenting cells, an immune response against the antigen or epitope may occur, resulting in prophylactic and/or therapeutic treatment of a disease involving the antigen or epitope. In one embodiment, the immune response induced by the RNA lipoplex particles described herein comprises presentation of an antigen or a fragment thereof, such as an epitope, by antigen-presenting cells, such as dendritic cells and/or macrophages, and activation of cytotoxic T cells following the presentation. For example, RNA-encoded peptides or proteins, or their processed products, can be presented by major histocompatibility complex (MHC) molecules expressed on antigen-presenting cells. The MHC peptide complex can then be recognized by immune cells such as T cells or B cells, leading to their activation.

Thus, in one embodiment, the RNA included in the lipoplex particles described in the present application is delivered to the spleen and/or expressed in the spleen after administration. In one embodiment, the RNA lipoplex particles are delivered to the spleen to activate splenic antigen-presenting cells. Thus, in one embodiment, after administration of the RNA lipoplex particles, the RNA is delivered and/or the RNA is expressed in antigen-presenting cells. The antigen-presenting cells can be professional antigen-presenting cells or non-professional antigen-presenting cells. The professional antigen-presenting cells can be dendritic cells and/or macrophages, even more preferably splenic dendritic cells and/or splenic macrophages.

Accordingly, the present invention relates to RNA lipoplex particles or a pharmaceutical composition comprising RNA lipoplex particles as disclosed herein for inducing or enhancing an immune response, preferably an immune response against prostate cancer.

In one embodiment, systemic administration of RNA lipoplex particles or a pharmaceutical composition comprising RNA lipoplex particles as disclosed herein results in targeting and/or accumulation of RNA lipoplex particles in the spleen rather than in the lungs and/or liver. In one embodiment, the RNA lipoplex particles release RNA in the spleen and/or enter spleen cells. In one embodiment, systemic administration of RNA lipoplex particles or a pharmaceutical composition comprising such particles as disclosed herein 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.

The term "macrophage" refers to a subset of phagocytic cells that are formed by the differentiation of monocytes. Macrophages, which are activated by inflammation, non-specifically engulf foreign pathogens, immune cytokines, or microbial products and destroy the pathogens internally through hydrolytic and oxidative attack, resulting in their degradation. Peptides from the degraded proteins are displayed on the surface of macrophage cells, where they can be recognized by T cells, and directly interact with antibodies on the surface of B cells, resulting in the activation of T and B cells and further stimulation of the immune response. Macrophages belong to the class of antigen-presenting cells. In one embodiment, macrophages are splenic macrophages.

The term "dendritic cell" (DC) refers to another subtype of phagocytic cells belonging to the class of antigen-presenting cells. In one embodiment, dendritic cells are derived from hematopoietic progenitor cells in the bone marrow. These progenitor cells initially transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T-cell activation potential. Immature dendritic cells constantly scan the environment for pathogens such as viruses and bacteria. Once they come into contact with the presented antigen, they are activated into mature dendritic cells and begin to migrate to the spleen or lymph node. Immature dendritic cells phagocytose pathogens and cleave their proteins into small pieces, and after maturation, present these fragments on their cell surface using MHC molecules. At the same time, they activate cell surface receptors that act as co-receptors for T cell activation, such as CD80, CD86, and CD40, greatly enhancing their ability to activate T cells. They also activate CCR7, a chemotactic receptor that induces dendritic cells to travel through the bloodstream to the spleen or through the lymphatic system to a lymph node. Here, they act as antigen-presenting cells and activate T helper and killer T cells, as well as B cells, by presenting them with antigens along with non-antigen-specific costimulatory signals. In this way, dendritic cells can actively induce a T cell- or B cell-associated immune response. In one embodiment, the dendritic cells are splenic dendritic cells.

The term "antigen-presenting cell" (APC) means a cell of a plurality of cells capable of displaying, acquiring and/or presenting at least one antigen or antigen fragment on its cell surface. Antigen-presenting cells are divided into professional antigen-presenting cells and non-professional antigen-presenting cells.

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. When a T cell interacts with the MHC class II molecule complex on the membrane of an antigen-presenting cell, the antigen-presenting cell produces a costimulatory molecule that causes activation of the T cell. Professional antigen-presenting cells include dendritic cells and macrophages.

The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules but do so when stimulated by certain cytokines such as interferon-gamma. Examples of non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.

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

The term "antigen-mediated disease" or "epitope-mediated disease" refers to any disease that is associated with an antigen or epitope, such as a disease that is characterized by the presence of an antigen or epitope. An antigen-mediated or epitope-mediated disease may be a cancer or simply a malignancy. As mentioned above, the antigen may be a disease-associated antigen, such as a tumor-associated antigen, and the epitope may be derived from such an antigen.

The terms "oncological disease" ("cancer") or "malignancy" refer to or describe a physiological condition of an individual that is typically characterized by unregulated cell growth. Examples of oncological diseases include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specifically, examples of such types of cancer include malignant bone tumors, hematological malignancies, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, cancer of the genitourinary system, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine 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, glioma, meningioma, and pituitary adenoma. One particular form of cancer that can be treated using the compositions and methods disclosed herein is prostate cancer. The term "cancer" as used herein also includes metastases of malignant tumors.

Combination strategies for treating cancer may be desirable because of the resulting synergistic effect, which may be significantly greater than the effect of a monotherapeutic approach. In one embodiment, the pharmaceutical composition is administered with an immunotherapeutic agent. As used herein, the term "immunotherapeutic agent" refers to any agent that can participate in the activation of a specific immune response and/or immune effector function(s). The present invention contemplates the use of an antibody as an immunotherapeutic agent. Without wishing to be bound by theory, the inventors believe that antibodies are capable of achieving a therapeutic effect against malignant tumor cells through a variety of mechanisms, including inducing apoptosis, blocking components of signal transduction pathways, or inhibiting tumor cell proliferation. In some embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies can induce cell death through antibody-dependent cell-mediated cytotoxicity (ADCC) or bind complement proteins, resulting in direct cellular toxicity known as complement-dependent cytotoxicity (CDC). Non-limiting examples of anti-tumor antibodies and potential target antibodies (in parentheses) that can be used in combination with the present invention include: abagovomab (CA-125), abciximab (CD41), adecatumumab (EpCAM), afutuzumab (CD20), alacizumab pegol (VEGFR2), altumomab pentetate (CEA), amatuximab (MORAb-009), anatumomab mafenatox (TAG-72), apolizumab (HLA-DR), arcitumomab (CEA), atezolizumab (PD-L1), bavituximab (phosphatidylserine), bectumomab (CD22), belimumab (BAFF), bevacizumab (VEGF-A), bivatuzumab mertansine (CD44 v6), blinatumomab (CD 19), brentuximab vedotin (CD30 TNFRSF8), cantuzumab mertansine (CanAg mucin), capromab pendetide (prostate carcinoma cells), carlumab (CNT0888), catumaxomab (EpCAM, CD3), cetuximab (EGFR), citatuzumab bogatox (EpCAM), cixutumumab (IGF-1 receptor), claudeximab (claudin), clivatuzumab tetraxetane (MUC1), conatumumab (TRAIL-R2), dacetuzumab (CD40), dalotuzumab (insulin-like growth factor I receptor), denosumab (RANKL), detumomab (B-lymphoma cell), drositumab (DR5), ecromeximab (GD3 ganglioside), edrecolomab (EpCAM), elotuzumab (SLAMF7), enavatuzumab (PDL192), ensituximab (NPC-1C), epratuzumab (CD22), ertumaxomab (HER2/neu, CD3), etaracizumab (integrin ανβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), ficlatuzumab (SCH 900105), figitumumab (IGF-1 receptor), flanvotumab (glycoprotein 75), frezolimumab (TGF-β), galiximab (CD80), ganitumab (IGF-I), gemtuzumab ozogamicin (CD33), gevokizumab (IL-Ιβ), girentuximab (carbonic anhydrase 9 (CA-IX)), glembatumumab vedotin (GPNMB), ibritumomab tiuxetan (CD20), icrucumab (VEGFR-1), igovoma (CA-125), indatuximab ravtansine (SDC1), intetumumab (CD51), inotuzumab ozogamicin (CD22), ipilimumab (CD 152), iratumumab (CD30), labetuzumab (CEA), lexatumumab (TRAIL-R2), libivirumab (hepatitis B surface antigen), lintuzumab (CD33), lorvotuzumab mertansine (CD56), lucatumumab (CD40), lumiliximab (CD23), mapatumumab (TRAIL-R1), matuzumab (EGFR), mepolizumab (IL-5), milatuzumab (CD74), mitumomab (ganglioside GD3), mogamulizumab (CCR4), moxetumomab pasudotox (CD22), nacolomab tafenatox (C242 antigen), naptumomab estafenatox (5T4), namatumab (RON), necitumab (EGFR), nivolumab (IgG4), ofatumumab (CD20), olaratumab (PDGF-Ra), onartuzumab (human hepatocyte growth factor receptor kinase), oportuzumab monatox (EpCAM), oregovomab (CA-125), oxelumab (OX-40), panitumumab (EGFR), patritumab (HER3), pemtumoma (MUC1), pertuzuma (HER2/neu), pintumomab (adenocarcinoma antigen), pritumumab (vimentin), racotumomab (N-glycolylneuraminic acid), radretumab (fibronectin extra domain-B), rafivirumab (rabies virus glycoprotein), ramucirumab (VEGFR2), rilotumumab (HGF), rituximab (CD20), robatumumab (IGF-1 receptor), samalizumab (CD200), mibrotuzumab (FAP), miltuximab (IL-6), tabalumab (BAFF), tacatizumumb tetraxetane (alpha-fetoprotein), taplitumomab paptox (CD 19), tenatumomab (tenascin C), teprotumumab (CD221), ticilimumab (CTLA-4), tigatuzumab (TRAIL -R2), TNX-650 (IL-13), tositumomab (CD20), trastuzumab (HER2/neu), TRBS07 (GD2), tremelimumab (CTLA-4), tucotuzumab celmoleukin (EpCAM), ublituximab (MS4A1), urelumab (4-1 BB), volociximab (integrin α5β1), votumumab (tumor antigen CTAA 16.88), zalutumumab (EGFR), and zanolimumab (CD4).

In one embodiment, the immunotherapeutic agent is a PD-1 axis binding antagonist. A PD-1 axis binding antagonist includes, but is not limited to, a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist. 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 partners. In a particular aspect, the PD-1 ligand binding partners are PD-L1 and/or PD-L2. In another embodiment, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a specific embodiment, the PD-L1 binding partners are 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 partners. 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). 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.

In one embodiment, the PD-1 binding antagonist is an immunoadhesin that comprises 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, is PD-L2-Fc), which is a fusion-soluble receptor described in WO2010/027827 and WO201 1/066342.

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).

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 a typical embodiment, the anti-PD-L1 antibody is atezolizumab.

Reference to documents and studies in this description does not constitute an admission that any of the above relates to prior art. All statements regarding the contents of these documents are based on information available to the applicants and do not constitute an admission that the contents of these documents are correct.

The following information is provided to enable one skilled in the art to make and use various embodiments of the invention. The description of specific devices, methods, and applications is provided as examples only. Various modifications to the examples disclosed herein will be apparent to those skilled in the art, and the generic principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments of the claimed invention. Thus, the various embodiments are not intended to limit the invention to the examples disclosed and shown, but should fall within the scope of the appended claims.

Examples

Example 1: Intravenous vaccine for the treatment of prostate cancer

This application describes a second-generation vaccine for intravenous (i.v.) administration. It consists of RNAs targeting five antigens expressed in prostate cancer that are individually complexed with liposomes to form serum-stable lipoplex RNAs (LIPs). The LIPs deliver encoded vaccine antigens to antigen-presenting cells (APCs) in lymphoid organs, resulting in efficient induction of antigen-specific immune responses.

The intravenous vaccine consists of five different RNA-based drug formulations targeting five prostate cancer-associated antigens. These formulations are RBL038.1, RBL039.1, RBL040.1, RBL041.1 and RBL045.1. Each RNA cancer vaccine consists of a single RNA drug substance that encodes the antigens kallikrein-2 (KLK2), kallikrein-3 (KLK3, also known as prostate-specific antigen (PSA)), prostatic acid phosphatase (ACPP, also known as prostatic acid phosphatase (PAP)), Homeobox B13 (HOXB13) and NK3 Homeobox 1 (NKX3-1), respectively, which were selected based on their selective expression in prostate cancer.

Before administration, the RNAs will be converted (reduced) into lipoplex RNAs (LIPs).

RNA-based dosage forms for reconstitution may be supplied in vials containing 1.1 ml of the corresponding finished RNA-based dosage form at a concentration of 0.25 mg/ml. Sterile isotonic NaCl solution (40 ml, 0.9%) as the main diluent and liposomes (4.0 ml at a concentration of 1.4 mg/ml) may be supplied as an excipient for reconstitution.

Specialty materials such as syringes and cannulas for reconstitution, as well as additional isotonic saline for further dilution of RNA (LIP) can be supplied as clinical standard goods.

Medicinal substance (drug substance):

RBL038.1, beta-S-ARCA(D1)-hAg-Kozak-KLK2-GS-P2P16-GS-MITD-FI-A30L70

Encoded antigen: human kallikrein-2 (corresponding gene identifier (HG19): uc002ptu.3, uc002ptv.3, uc002ptt.3, uc010ycl.2, uc010ycm.2, uc010yck.2, uc010eog.3)

RBL039.1, beta-S-ARCA (D1)-hAg-Kozak-KLK3-GS-P2P16-GS-MITD-FI-A30L70

Encoded antigen: human prostate-specific antigen, also known as human kallikrein-3 (corresponding gene identifier (HG19: uc002pts.1, uc002ptr.1, uc021uyi.1, uc010eof.1)

RBL040.1, beta-S-ARCA (D1)-hAg-Kozak-ACPP-GS-P2P16-GS-MITD-FI-A30L70

Encoded antigen: human prostatic acid phosphatase, also known as human prostatic acid phosphatase (corresponding gene identifier (HG19): uc003eop.4)

RBL041.1, beta-S-ARCA(D1)-hAg-Kozak-sec-GS-HOXB13-GS-P2P16-GS-MITD-FI-A30L70

Encoded antigen: human HOMEOBOX B13 (corresponding gene identifier (HG19): uc002ioa.3r)

RBL045.1, beta-S-ARCA (D1) -hAg-Kozak-sec-GS-NKX31-GS-P2P16-GS-MITD-FI-A30L70

Encoded antigen: NK3 homeobox 1 (corresponding gene identifier (HG19): uc011kzx.2)

The active principle in each drug substance is a single-stranded mRNA with a 5'-cap, which is translated into the corresponding protein upon entry into antigen-presenting cells (APCs).

Figure 1 shows a schematic representation of the general structure of antigen-coding RNAs, which is defined by the corresponding nucleotide sequence of the linearized plasmid DNA used as a template for in vitro RNA transcription. In addition to the wild-type or codon-optimized sequences encoding the target protein, each RNA contains common structural elements optimized to maximize RNA stability and translation efficiency (5' cap, 5' UTR, 3' UTR, poly(A) tail; see below). In addition, sec (secretory signal peptide) and MITD (MHC class I transport domain) are fused to the antigen-coding regions and translated as an N- or C-terminal tag, respectively. Both fusion tags have been shown to improve antigen processing and presentation in MHC class I and MHC class II complexes. For some antigens, as noted below, the sec fusion tag is not required and is therefore omitted. Beta-S-ARCA (D1) (Figure 2) is used as a specific capping structure at the 5' end of RNA-based drugs.

The general elements of the mRNA sequence, as shown in Figure 1, are given below.

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

hAg-Kozak: The 5' UTR sequence of human alpha globin mRNA with an optimized "Kozak sequence" for increased translation efficiency.

sec/MITD: protein fusion tags derived from the sequence encoding the human MHC class I complex (HLA-B51, haplotype A2, B27/B51, Cw2/Cw3) that have been shown to enhance antigen processing and presentation. Sec corresponds to a 78 bp fragment encoding the secretory signal peptide that directs translocation of the nascent polypeptide chain to the endoplasmic reticulum. MITD corresponds to the transmembrane and cytoplasmic domain of the MHC class I molecule, also called the MHC class I trafficking domain. Note that KLK2, PSA (KLK3), and PAP (ACPP) each have their own secretory signal peptide. Accordingly, a second fusion tag was not added to these antigens.

GS/Linker: Sequences encoding short linker peptides, predominantly composed of the amino acids glycine (G) and serine (S), which are commonly used for fusion proteins.

P2P16: sequence encoding helper epitopes derived from tetanus toxoid for breaking immunological tolerance.

The FI:3'-UTR element is a combination of two sequence elements derived from the amino-terminal enhancer of cleavage (AES) mRNA (called F) and encoded by the mitochondrial 12S ribosomal RNA (called I). They were identified by an ex vivo selection process for sequences that confer RNA stability and increase total protein expression.

A30L70: a 110-nucleotide poly(A) tail consisting of a stretch of 30 adenosine residues followed by a 10-nucleotide linker sequence and another 70 adenosine residues designed to enhance RNA stability and translation efficiency in dendritic cells.

The complete nucleotide sequences of the drug substances based on five RNAs, RBL038.1, RBL039.1, RBL040.1, RBL041.1 and RBL045.1, are given below:

Nucleotide sequence of RBL038.1.

The nucleotide sequence is shown with individual sequence elements indicated in bold. In addition, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (* = stop codon).

Nucleotide sequence of RBL039.1.

The nucleotide sequence is shown with individual sequence elements indicated in bold. In addition, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (* = stop codon).

Nucleotide sequence of RBL040.1.

The nucleotide sequence is shown with individual sequence elements indicated in bold. In addition, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (* = stop codon).

Nucleotide sequence of RBL041.1.

The nucleotide sequence is shown with individual sequence elements indicated in bold. In addition, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (* = stop codon).

Nucleotide sequence of RBL045.1.

The nucleotide sequence is shown with individual sequence elements indicated in bold. In addition, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (* = stop codon).

Individual plasmid DNAs for obtaining RBL038.1 (pST4-hAg-Kozak-KLK2-GS-P2P16-GS-MITD-FI-A30L70), RBL039.1 (pST4-hAg-Kozak-KLK3-GS-P2P16-GS -MITD-FI-A30L70), RBL040.1 (pST4-hAg-Kozak-ACPP-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 constructed using a combination of gene synthesis and recombinant DNA technology. In addition to the sequence encoding the transcribed regions, the plasmid DNAs contain a promoter for T7 RNA polymerase, a recognition sequence for a class II endonuclease used for linearization, a kanamycin resistance gene, and an origin of replication (ori).

Plasmid DNA pST4-hAg-Kozak-sec-GS-SIINFEKL-GS-Ova-GS-P2P16-GS-MITD-FI-A30L70 served as the starting point for the construction of DNA templates for RBL038.1, RBL039.1, RBL040.1, RBL041.1, and RBL045.1.

Vector maps are shown in figures 3 through 7.

The circular plasmid DNA is 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 chosen because linearization with this class IIs restriction enzyme ensures transcription of RNAs encoding a “free” oligo(A)-tail, i.e., the 3’-end. This can be demonstrated to result in higher protein expression.

The RNA product (LIP) can be prepared by a two-step procedure involving (i) dilution of the RNA concentrate with NaCl solution and (ii) formation of RNA lipoplex by adding liposomes. To prepare the RNA (LIP), liposomes can be added to the diluted RNA. The lipids that can be used are the synthetic cationic lipid DOTMA and the naturally occurring phospholipid DOPE. The product for intravenous injection is a formulation with pharmaceutical and physiological characteristics that allow selective targeting of RNA to APCs, which are mainly found in the spleen. RNA lipoplexes are formed by first condensing the RNA with a suitable ionic environment and then incubating with positively charged liposomes.

Various monovalent and divalent ions, peptides and buffers at different concentrations were used for RNA condensation. Monovalent ions such as sodium and ammonium were tested at concentrations up to 1.5 M. Divalent ions, in particular Ca2+, Mg2+, Zn2+ and Fe2+, were tested at concentrations up to 50 mM. In addition, various commercially available buffer solutions were tested.

To form RNA-(LIP) liposomes containing cationic lipid and different colipids were extensively tested. Liposomes that differ in charge, phase state, size, lamellarity and surface functionalization were examined. Only lipid components available in GMP grade, that have been previously tested in clinical trials or that are used for approved products on the market were considered (Figure 8).

Using the liposomal components described above, RNA lipoplexes were assembled with different cationic lipid:RNA ratios and different charge ratios, where the charge ratio was calculated from the number of positive charges of the lipids and the negative charges of the RNA nucleotides, i.e., the phosphate groups of the RNA. In particular, the charge ratio calculation was performed as follows.

It was assumed that RNA consists of nucleotides with an average molar mass of 330 Da, each carrying a phosphate group with one negative charge. Therefore, there are approximately 3 mM negative charges per RNA solution with a concentration of 1 mg/mL. On the other hand, one positive charge per monovalent cationic lipid was taken into account. For example, the cationic lipid DOTMA has a molar mass of 670 Da, liposomes with a concentration of DOTMA of 2 mg/mL were assigned to a positive charge concentration of 3 mM. Therefore, in this case, the charge ratio (+:-) was taken to be 1:1. The concentration of uncharged colipids, which were present in most cases, does not affect this calculation.

The chemical and physicochemical properties of the liposomes and RNA lipoplexes based on them (i.e., chemical composition, particle size, zeta potential) were thoroughly investigated. For regular quality control of the product, the chemical composition was determined by HPLC analysis and the particle size was measured by photon correlation spectroscopy (PCS). Zeta potential was also measured by PCS. In addition, electron microscopy, small-angle X-ray scattering (SAXS), calorimetry, field-assisted flow fractionation, analytical ultracentrifugation, and spectroscopic methods were used in the formulation development process. Using this procedure, optimized formulations were identified for further pharmaceutical development.

Suitable liposome formulations were tested in vitro and in vivo. In order to optimize targeting of APCs mainly located in the spleen, the expression of luciferase as a reporter gene was monitored in vivo. It could be shown that colloidally stable nanoparticle lipoplex formulations with discrete particle sizes could be formed at suitable charge ratios (excess negative or positive charge). Furthermore, it was shown in vivo that negatively charged formulations of RNA lipoplexes with luciferase and exhibit high selectivity for the spleen, which serves as a reservoir for professional APCs. By changing the charge ratio, the selectivity of luciferase expression in the spleen could be adjusted as desired, as shown in Figure 9, which displays the selectivity of RNA lipoplexes from the same liposomes with different mixing ratios of cationic lipids and RNA. The observation that negatively charged lipoplexes target spleen APCs can be confirmed for a large number of lipid formulations. Liposomes composed of the cationic lipid DOTMA and the auxiliary phospholipid DOPE were identified as the most suitable in terms of particle characteristics for the formation of suitable lipoplex RNAs for targeting spleen APCs. Optimized selectivity and spleen targeting efficiency were observed with a slight excess of negative charge generated by excess RNA. Lipoplex RNAs that were slightly more positively charged and showed comparable efficiency were not suitable for pharmaceutical product development because they were too colloidally unstable and there was a high risk of aggregation and precipitation under these conditions.

Furthermore, it could be shown that for a given RNA, the biological activity of the formulations increased with increasing particle size of the RNA lipoplexes. More specifically, it could be shown that RNA lipoplexes formed from larger liposomes (e.g., approximately 400 nm) were themselves larger than lipoplexes obtained from smaller liposomes (e.g., approximately 200 nm) and exhibited higher biological activity (Figure 10). Therefore, it is preferable to use liposomes larger than 200 nm for the formation of RNA (LIPs).

Based on the results described above, a robust and reproducible protocol for the preparation of RNA-(LIP) was developed. Using the specified components and a defined preparation protocol, RNA lipoplexes are formed by self-assembly with specified physicochemical characteristics and biological activity. As an example, Figure 11 shows the sizes of RNA LIP particles from different independent compositions. The limited variation in the sizes of the obtained RNA lipoplex particles demonstrates the robustness of the recovery procedure.

To determine the limits and robustness of the RNA (LIP) preparation, particle sizes were measured for different charge ratios from 1.0:2.0 to 1.9:2.0 (mixing ratios between cationic lipid and nucleotides). Figure 12 shows the results of RNA lipoplex size measurements after mixing liposomes with RNA in different ratios. Particle size was measured at different time points after RNA (LIP) preparation. Ratios from 1.0:2.0 to 1.6:2.0 yield particles of comparable size that are stable over time. For ratios of 1.7:2.0 and higher, the particle size of RNA lipoplexes increases both initially and over time. This result is most pronounced after 24 hours.

Based on these data, charge ratios from 1.0:2.0 to 1.6:2.0 were considered suitable to obtain acceptable particle characteristics for the RNA lipoplex products. At higher ratios (1.7:2.0 and above), the particle size increased, which could potentially lead to a decrease in product quality. For lower charge ratios, no change in particle characteristics was observed, however, lower ratios were not considered due to the potentially lower activity in this range (data not shown). The experiment was repeated for the range from 1.1:2.0 to 1.6:2.0 and in addition to the size measurements (Figure 13A), the biological activity was examined (Figure 13B). Consistent with previous experiments, the particle sizes were virtually constant. The same was true for the biological activity (luciferase expression). In summary, RNA lipoplexes of all charge ratios tested delivered RNA to APCs without significant changes in physicochemical properties or biological characteristics. Thus, the range from 1.1:2.0 to 1.6:2.0 is considered to result in RNA lipoplexes of equivalent quality.

Example 2: Non-clinical data

This example reviews the preclinical studies that were conducted to elucidate the mechanism of action, pharmacodynamics, antitumor activity, pharmacokinetics, and potential toxicity of the RNA (LIP) vaccine. The key findings are summarized in Table 1.

The first part of this section provides a brief overview of the scientific background and preparatory work for developing the vaccine platform itself (Section 1), as well as a brief overview of the target characteristics of the W_pro1 targets.

The next section describes 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).

The secondary pharmacodynamics studies present the results of testing RNA-(LIP)-mediated induction of proinflammatory cytokines (Section 3). A non-GLP pharmacodynamics study in cynomolgus monkeys was conducted to clarify the cytokine kinetics data and hematological changes observed in mice. This section also summarizes in vitro studies analyzing cytokine secretion by human and cynomolgus monkey blood cells following incubation with RNA-(LIP) formulations.

The pharmacological safety studies for the respiratory and neurological systems are summarized in Section 4.

Brief reviews of biodistribution, pharmacokinetics and in vivo metabolism are provided in Section 5.

GLP-compliant repeated dose toxicity studies, including immunotoxicity studies, are presented and discussed in Section 6.

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

Category Signs Drug class Complex liposomal mRNAs encoding prostate cancer-specific antigens.
In vitro transcription by T7 polymerase using DNA templates.
Batch evaluation using in vitro translation (activity assay).
For intravenous injection in patients with prostate cancer for neoadjuvant chemotherapy of the primary tumor followed by interval surgery. Leading structure The RNA guide structures targeting prostate cancer antigens are codon optimized and contain stabilizing untranslated sequences and a modified cap analog to enhance stability and translational capability. Moreover, all guide structures consist of full-length mRNA with sequence elements that enhance stability and translational capability and are in most cases flanked by 5' and 3' ends encoding secretory and transmembrane domains that enhance protein processing and presentation. In addition, tetanus toxoid-derived helper epitopes P2 and P16 are fused in frame to the target antigens. Mode of action The lipoplex formulation protects RNA from RNase-mediated degradation, making intravenous administration possible.
Selective uptake of intravenously administered and circulating RNA-(LIP) by professional APCs in the lymphatic compartment, particularly by splenic resident APCs.
Translation of RNA-encoded antigens and processing of antigens into peptide epitopes.
Induction of antigen-specific T lymphocytes by presentation of RNA-encoded epitopes as peptides complexed with the MHC molecule on the surface of professional APCs in the context of costimulatory signals.
Activation and proliferation of tumor-specific T cells.
mRNA signaling through TLR binding and TLR-mediated immunomodulatory effects leading to cellular activation and induction of proinflammatory cytokines (e.g. IFN-α, IFN-γ, IP-10, TNF-α, IL-6 and IL-10) enhances the vaccine effect. Antitumor activity in animal tumor models In vivo destruction of target cells activated by antigen.
Inhibition of tumor growth rate and eradication of large established tumors following SC tumor challenge in fast-growing syngeneic mouse tumor models.
Increased life expectancy of tumor-bearing animals. Relevant animal species Mice are considered a suitable species to measure the biological activity and immunological effects resulting from the administration of vaccines derived from the RNA-(LIP) vaccine platform. Undesirable adverse reactions due to the expected immune activation via TLRs and subsequent induction of proinflammatory cytokines can also be assessed in mice, but need to be complemented by human in vitro data, as TLR expression differs between mice and humans. There is no relevant animal species that can adequately capture potential detrimental effects caused by potentially autoreactive or cross-reactive vaccine-induced T cell responses, given the differences in MHC when using human antigen sequences in mice. Pharmacokinetics Rapid degradation of RNA in the blood (half-life about 5 minutes) and organs within 48 hours. Temporary presence of RNA in the spleen and liver.
Remnants of matrix DNA do not accumulate or persist in the gonads.
Transient accumulation of DOTMA in the spleen and liver after repeated RNA application (LIP). DOTMA is cleared from the organs with an approximate half-life of 6-7 weeks. Pharmacological safety studies In safety pharmacology studies (central nervous system (CNS) and respiratory system) in mice, no adverse effects were observed.
Cardiovascular safety as demonstrated by confirmatory data from a non-GLP pharmacological study in cynomolgus monkeys. Toxicology Intravenous administration of multiplex RNA (LIP) was very well tolerated in mice, as demonstrated for a number of leading mRNA structures evaluated in three different repeated dose toxicity studies (LPT #28864, 30283, and 30586).
Mild and transient lymphopenia is thought to correspond to TLR-induced cytokine induction, the putative pharmacologic effect.

Section 1: Scientific basis

Sequence features that enhance RNA translation and intracellular stability

The RNA vaccine platform has been designed and systematically optimized over two decades to ensure safety and efficient induction of antigen-specific CD8+ and CD4+ T cell responses against encoded antigens.

As described above, the active component (drug substance) is a single-stranded, capped messenger RNA (mRNA), which is translated into a protein antigen upon entry into antigen-presenting cells. The mRNA vaccine format is pharmacologically optimized (Table 2) by (i) a modified cap analogue to stabilize the translationally active RNA, (ii) optimized 5' and 3' UTRs to enhance RNA stability and translation, (iii) a signal peptide and MITD sequence that enhance antigen processing by MHC class I and II molecules, (iv) tetanus toxoid-derived helper epitopes to break immunological tolerance by providing non-specific help to CD4+ T lymphocytes, and (v) an extended poly(A) tail that further enhances RNA stability and translation efficiency.

Table 2. Structural elements of RNA for immunopharmacological optimization

Sign Effect Optimized analogue of the cap Stabilizes and increases the amount of translational active RNA 5'-UTR, 3'-UTR Non-coding sequences that enhance RNA translation stability and efficiency Signal peptide, MITD sequence Enhances antigen processing by MHC class I and class II molecules Helper epitopes of tetanus toxoid Provide assistance to CD4+ T-lymphocytes that are not specific to tumor antigens Extended free end of poly (A) tail Non-coding sequence that increases RNA stability and translation efficiency

Targeting of antigen-coding RNA to lymphoid-resident antigen-presenting cells

For systemic delivery of RNA to dendritic cells, each individual W_pro1 RNA drug product is formulated to form RNA lipoplexes (RNA-LIPs) that can be administered intravenously. The RNA-LIP formulation was designed to protect the RNA from degradation by plasma RNases and optimized for selective delivery of the formulated RNA DPs to antigen-presenting cells (APCs), predominantly located in the spleen (Figure 14) and other lymphatic organs, where selective uptake of the RNA by dendritic cells and macrophages has been shown (Figure 15).

Once RNA lipoplexes are taken up by APCs in the spleen, the mRNA is expressed, processed and presented via MHC molecules. This results in potent 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 RNA-mediated TLR (LIP) signaling.

Induction of antigen-specific T-cell responses

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

Induction of cellular activation processes

The mRNA is a ligand for human Toll-like receptors (TLRs) and is thus capable of inducing immunomodulatory effects. Upon cellular uptake of transcribed (IVT)-RNA in vitro, TLR recognition occurs in endosomal compartments where these receptors are primarily localized. This initiates cascades of signaling events that ultimately lead to DC activation and maturation, as demonstrated by splenic DC maturation following intravenous administration of RNA (LIP) vaccine to mice. Further consequences of these immunomodulatory effects include subsequent activation of T, B, NK cells and splenic macrophages and reversible induction of proinflammatory cytokines.

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

The transient cell activation and cytokines observed in mice treated with RNA(LIP) vaccines are consistent with the finding that RNA vaccines can bind to and prime TLRs. Particulate RNA as well as RNA in aqueous solutions have also been shown to activate TLRs. TLR activation has been shown to induce lymphopenia, leading to interferon-dependent leukocyte recycling. Consistent with this, activation of dendritic cells as well as other splenic cell populations was greatly impaired in TLR7 -/- or IFNAR -/- mice. Accordingly, studies in IFNAR -/- mice treated with RNA(LIP) indeed showed that the transient hematological changes observed following intravenous administration were primarily mediated by the downstream effects of IFN-α.

Tetanus toxoid-derived P2P16 helper epitopes in W_pro1

Each W_pro1 DP RNA preparation contains so-called P2P16 amino acid sequences derived from tetanus toxoid (TT) of Clostridium tetani. These sequences support overcoming self-tolerance mechanisms to efficiently induce immune responses to autoantigens by assisting tumor-non-specific T cells during priming.

The heavy chain of tetanus toxoid contains epitopes that can randomly bind to MHC class II alleles and induce memory CD4+ T cells in nearly all tetanus-vaccinated individuals. In addition, the combination of TT helper epitopes with tumor-associated antigens is known to improve immune stimulation compared to tumor-associated antigen alone by providing CD4+ T cell-mediated help during priming. To reduce the risk of CD8+ T cell stimulation, two peptide sequences known to contain randomly binding helper epitopes were selected to ensure binding to as many MHC class II alleles as possible. Based on the data from the above ex vivo studies, well-known epitopes P2 (QYIKANSKFIGITEL; TT830-844) and P16 (MTNSVDDALINSTKIYSYFPSVISKVNQGAQG; TT578-609) were selected.

Pharmacology

The mechanism of action of the RNA-(LIP) vaccine is based on (i) the recruitment of antigen-specific T lymphocytes following presentation of peptides derived from RNA-encoded antigens by professional APCs, and (ii) TLR-mediated immunomodulatory effects that lead to cellular activation and induction of pro-inflammatory cytokines such as type I interferons, thereby enhancing the effects of vaccination.

Section 2 reports the activation and expansion of targeted antigen-specific T cells upon immunization with RNA encoding cancer antigens and the antitumor effects of LIP RNA vaccination.

Extensive in vitro and in vivo studies have been conducted to investigate potential secondary effects of RNA(LIP) vaccine administration, such as the induction of proinflammatory cytokines and hematological changes, which are caused by the putative immunomodulatory effect of RNA(LIP).

Section 3 discusses a series of studies that evaluate the extent of cellular activation in human peripheral blood cells (PBMC) and blood cells in heparinized whole blood. In addition, the extent of vaccination-induced cytokine induction and hematological changes in cynomolgus monkeys treated with doses exceeding the highest anticipated clinical dose in humans are shown. Finally, side-by-side comparisons of in vitro cytokine induction in blood samples from human donors and cynomolgus monkeys were performed, and data from these studies were used to support the determination of a safe starting dose for the ongoing Lipo-MERIT clinical trial in malignant melanoma and other RNA-based immunotherapy (LIP) trials.

A review of preclinical studies using human, mouse and cynomolgus macaque blood cells as test systems to assess the secondary pharmacodynamics of RNA-(LIP) is provided in Section 3.

It is expected that the secondary pharmacodynamic effects observed for RNA in liposomes are sequence independent and that the presented studies are therefore also applicable to other RNA-based formulations in use.

Summary of Key Findings In vitro and in vivo studies were conducted to investigate the mechanism of action of RNA (LIP) vaccines.
The leading antigen-encoding RNA structures RBL038.1, RBL039.1, RBL040.1, RBL041.1 and RBL045.1 induced antigen-specific T cell responses in vivo in mice expressing human HLA molecules. Moreover, using murine model antigens, the antitumor effects of RNA-(LIP) vaccination were demonstrated in prophylactic and therapeutic immunization studies in in vivo mouse tumor models.
In addition, sequence-independent pharmacological effects of RNA(LIP) vaccination were analyzed. RNA(LIP) vaccines induced transient activation of antigen-presenting cells, leading to subsequent induction of inflammatory cytokines such as IFN-α, IFN-γ, IL-6, and IP-10. Secretion of cytokines including IFN-α was shown to originate from spleen cells and was mediated by TLR7 signaling following RNA(LIP) treatment, which was accompanied by transient and completely reversible hematological changes. Notably, RNA(LIP)-mediated cytokine induction and subsequent hematological changes were strongly reduced in mice lacking the interferon receptor-α/β (IFNAR -/-).

Section 2. Primary Pharmacodynamics

Several in vitro and in vivo experiments were conducted to prove the immunogenicity of RNA-(LIP) vaccination with a number of different mRNAs encoding antigens specific for melanoma, breast cancer, HPV+ head and neck cancer, ovarian cancer and other types of cancer. In vivo studies were conducted using R&D and GMP-grade materials in mice.

Induction of antigen-specific T cell responses by W_pro1 mRNA

To obtain further information on the in vivo induction of antigen-specific T cells by prostate-specific antigens KLK2, PSA (KLK3), PAP (ACPP), HOXB13 and NKX3-1, RNA-based (LIP) products were generated using R&D grade RNA and GMP-like quality liposomes.

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

For all antigens, four RNA preparation (LIP) vaccinations were sufficient to prime specific T cell responses in treated animals (Figure 18).

These results indicate that RNA(LIP) vaccines can effectively induce T cell responses against W_pro1 antigens in vivo.

The mRNAs used in the studies were manufactured under R&D conditions. Additional immunogenicity studies in A2/DR1 mice will be performed with RBL038.1, RBL039.1, RBL040.1, RBL041.1 and RBL045.1 manufactured under GMP conditions. Immunogenicity is expected to be comparable to the results of the earlier studies.

Antitumor activity of antigen-specific T cells in vivo induced by model antigen RNAs

Due to known problems in identifying mouse tumor models, no additional studies were conducted on the W_pro1 antigens, as mouse homologues of these antigens do not exist. Instead, we developed suitable tumor models for the ovalbumin-derived SIINFEKL epitope, as well as the human papillomavirus-derived E6/E7 and gp70 antigens as models for foreign and mouse self-antigen vaccination, respectively.

A summary of the in vivo antitumor effects induced by RNA-(LIP) is shown in Table 3.

Table 3. Summary of antitumor effects of RNA (LIP) in vivo

Study Way Result Prophylactic models B16F10-OVA and CT26
(STR-30207-008) Three cycles of intravenous immunization of mice with SIINFEKL-RNA-(LIP) or gp70-RNA-(LIP) prior to subcutaneous challenge with B16F10-OVA or CT26 tumor, respectively. Untreated mice died within 22-28 days after tumor challenge, all immunized mice were protected during monitoring. Therapeutic models B16F10-OVA and CT26
(STR-30207-010) Subcutaneous infection with B16F10-OVA or CT26 tumor followed by immunization with SIINFEKL-RNA-(LIP) or gp70-RNA-(LIP) for macroscopic tumor sizes. Untreated or liposome-treated mice died within 22-28 days of tumor challenge. Immunized mice showed significant tumor growth delay and tumor shrinkage in some cases. CT26 Therapeutic Model - Metastatic Model IV
(STR-30207-012) Metastatic tumor induction by intravenous injection of CT26-Luc cells followed by three gp70-RNA-(LIP) immunizations starting on day 4. Measurement of tumor growth by in vivo imaging of Luc-expressing cells and metastasis analysis 17 days after tumor induction. Untreated mice developed metastases as measured by increased Luc signals and macroscopic examination. Treated animals showed decreased Luc signals after the start of treatment and no lung metastases at the end of the study. Therapeutic model TC-1 – SC
(STR-30207-018) Subcutaneous challenge of the tumor with TC-1 cells followed by three immunizations with HPV E6/E7 (LIP) 13 days later. Untreated or liposome-treated mice died within 22-28 days of tumor challenge. Immunized mice showed strong antitumor activity with tumors reduced to 1 ^cm3 during treatment and complete cure in 30% of animals.

Section 3: Secondary Pharmacodynamics

To study potential secondary effects of lipoplex-RNA vaccine administration, such as induction of inflammatory cytokines and hematological changes induced by putative immunomodulatory effects, the authors performed extensive in vitro and in vivo studies using human and cynomolgus macaque blood cells as test systems. To the authors' knowledge, secondary pharmacodynamic effects triggered by TLRs and activation of innate immunity by therapeutic mRNA are independent of the sequence of the studies presented in this section, which were performed with an equal part mixture of ATM grade liposomal formulations RBL001.1, RBL002.2, RBL003.1 and RBL004.1. These RNAs encoding melanosome antigens were used in clinical trials. The studies were not repeated with W_pro1 RNA DP encoding TAA.

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

In addition, bioinformatic sequence homology searches of the RNA vaccine with the human proteome were performed to exclude potential cross-reactivity of induced T cells.

Summary of Key Findings Secondary effects were studied in vivo in cynomolgus monkeys by examining cytokine release, hematology, clinical biochemistry and cardiovascular parameters. Cynomolgus monkeys showed a strong transient induction of IL-6 and a very weak induction of IFN-α at the 354 μg RNA dose level, which we consider to be a putative pharmacodynamic effect of lipoplex treatment. This was accompanied by a transient lymphopenia that resolved within 48 hours. There were no signs of cardiovascular toxicity.
In addition, cytokine release (IP-10, IFN-α, IFN-γ, TNF-α, IL-1β, IL-2, IL-6, and IL-12) was analyzed in cultured human peripheral blood cells (PBMCs) and in cultured human whole blood from different donors after treatment with ATM-grade RNA (LIP). In cultured PBMCs, dose-dependent induction was observed for all analytes analyzed. However, at doses representing clinical dose levels and above, induction was observed for five of the eight markers studied, namely IP-10, IFN-γ, TNF-α, IL-1β, and IL6. In cultured whole blood, no changes in cytokine levels were detected for the analytes IFN-γ, TNF-α, IL-1β, IL-2, and IL-12. The chemokine IP-10 (CXCL10) level increased in a dose-dependent manner but was not significantly increased compared to control at dose levels intended for clinical use. Increased IL-6 induction, although at a low level, was detected in one of four donors. IFN-α was not significantly increased at any dose compared to vehicle control.
To obtain further information on the extent of proinflammatory cytokine induction and to test whether the cytokine profile observed in the cynomolgus macaque in vivo could be adequately captured in vitro, additional GLP and non-GLP pharmacodynamic studies were conducted using PBMC and whole blood assay systems. These studies showed a much better reflection of the in vivo observations in the whole blood assay system than in the PBMC-based assay system.
A side-by-side comparison of cytokine secretion in human and cynomolgus monkey samples in a whole blood-based assay and a PBMC-based assay showed that both species were highly comparable in the induction of proinflammatory cytokines when incubated with RNA-(LIP).

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

In addition to its ability to encode protein antigens, RNA has immunomodulatory effects that derive from its ability to induce cellular activation processes via TLR activation. On the one hand, the immunomodulatory capacity of an RNA vaccine enhances the induction of antigen-specific T-cell responses and should be considered as a primary pharmacodynamic effect. On the other hand, too strong or non-specific activation of immune cells may lead to undesirable secondary effects and should already be considered in preclinical studies.

To examine the extent of cellular activation of human blood cells, heparinized whole blood and PBMCs (isolated from heparinized whole blood) from four healthy donors were incubated in vitro with a mixture of equal parts of liposome-embedded RNA encoding melanoma tumor-associated antigens (RBL001.1, RBL002.2, RBL003.1, and RBL004.1) of ATM grade. Since TLR activation by RNA is sequence independent, the study was not repeated with W_pro1 RNA.

RNA-(LIP) for each of the four RNA-based drugs was prepared separately according to the clinical formulation protocol. In this first study (Study 1, STR-30207-013), a concentration range of 0.014 μg RNA/mL to 3.333 μg RNA/mL was chosen, equivalent to human doses of 0.07 mg to 16.65 mg total RNA (Table 4). As a primary endpoint, cellular activation was determined at 6 and 24 h 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), respectively.

Table 4. Dose determination for in vitro studies based on anticipated clinical dose cohorts

Values represent µg total RNA per ml whole blood or medium, respectively.

Research Report No: STR-30207-013 Dose level µg RNA per ml blood volume Total dose [µg RNA] [1] 1 0,014 70 2 0.041 205 3 0.123 615 4 0.371 1850 5 1,111 5550 7 3,333 16650

^[1] The average total blood volume is assumed to be 5 L.

Following incubation of PBMC with the RNA-(LIP) mixture, dose-dependent activation was detectable for all eight analytes tested, albeit with wide variations in concentration levels. The cytokine response was dominated by five of the eight markers selected, namely IP-10, IFN-γ, TNF-α, IL-1β and IL-6 (see Table 4 for summary). IFN-α, IL-2 and IL12 showed only minor induction at the highest dose levels tested.

In contrast, no secretion of IFN-γ, TNF-α, IL-1β, IL-2, and IL-12 was detected in the whole blood assay system after incubation with RNA-(LIP). In this case, increased dose-dependent secretion of IP-10 and IL-6 was observed. For IFN-α, only a low-level basal secretion was observed, which was comparable to the diluent control and was not further increased by incubation with RNA-(LIP) (see Table 5 for summary).

Thus, the PBMC data showed clear differences compared to whole blood, indicating a higher sensitivity of the assay to PBMC. While elevated cytokine levels for all eight tested analytes were detected in PBMC, cytokine detection was limited to IFN-α, IP-10, and IL-6 when whole blood samples were used as the assay.

Table 5. Summary of results for PBMC and whole blood in all donors (study STR-30207-013)

Cytokine/
Sample analyzed Test system PBMC Whole blood IFN-α Increased secretion is detected after 24 hours in all donors.
Dose dependent induction
Values are low and do not increase significantly at doses of 0.014–0.37 μg/ml RNA. Secretion is detected at very low levels in some donor samples 3/4
No clear increase was detected compared to the control at any dilution. IP-10 Increased secretion is detected after 24 hours in all donors.
Dose dependent induction
In 4/4 donors, elevated levels are detected at 0.37 μg/ml RNA.
In 3/4 of donors, elevated levels are detected at 0.12 μg/ml RNA. Increased secretion is detected after 24 hours in all donors.
Dose dependent induction
In 2/4 of donors, elevated levels are found at 0.37 mcg/ml. IL-6 Increased secretion is detected after 6 and 24 hours in all donors.
Dose dependent induction
In 4/4 donors, elevated levels are detected at 0.37 μg/ml RNA.
In 3/4 of donors, elevated levels are detected at 0.12 μg/ml RNA after 24 hours. Increased secretion is detected at very low levels only at the highest dose in 2/4 of donors. IFN-γ Increased secretion is detected after 24 hours in all donors.
Dose dependent induction
In 4/4 donors, elevated levels are detected at 0.37 μg/ml RNA. Increased secretion is not detected TNF-α Increased secretion is detected after 6 and 24 hours in all donors.
Dose dependent induction
In 4/4 donors, elevated levels were detected at 0.37 μg/ml RNA only after 6 hours. Increased secretion is not detected IL-1β Increased secretion is detected after 6 and 24 hours in all donors.
Dose dependent induction
In 4/4 donors, elevated levels are detected at 0.37 μg/ml RNA.
In 2/4 of donors, elevated levels are detected at 0.12 μg/ml RNA after 24 hours. Increased secretion is not detected IL-2 Increased secretion is detected after 6 and 24 hours in all donors.
Dose dependent induction
Values are low and do not increase at doses of 0.014–0.37 μg/ml RNA. Increased secretion is not detected IL-12 Increased secretion is detected after 24 hours in all donors.
Dose dependent induction
In 2/4, elevated levels are detected at 0.37 μg/ml RNA. Increased secretion is not detected

To further investigate the cytokine release by human cells in response to RNA(LIP) in vitro and to compare and categorize the data from the in vivo mouse immunotoxicity studies (see below) and the cynomolgus monkey study (see below), an additional GLP-compliant in vitro study was conducted at an external CRO (LPT #31031). The primary objective of this study was to confirm (i) whether the results in cynomolgus monkeys are comparable to those in humans and (ii) which assay system better reflects the pattern of cytokine response observed in cynomolgus monkeys in vivo. Study LPT #31031 was designed as follows: In vitro induction of pro-inflammatory cytokines in healthy human donors and cynomolgus monkeys was tested in two assay systems, namely PBMC and whole blood. The same dose range and dosing stages of RNA(LIP) were tested as in Study No. STR-30207-013. The test item was again a mixture of separately prepared ATM-grade liposomal RNAs RBL001.1, RBL002.2, RBL003.1 and RBL004.1. As mentioned above, the data obtained with these IVT-RNAs also take into account the DP RNAs encoding TAA, since TLR activation is not RNA sequence dependent. A total of four samples from each species were analyzed. As a primary endpoint, cellular activation was determined at 6, 24 and 48 h by secretion of proinflammatory cytokines into cell culture medium (PBMC) or plasma (whole blood), respectively.

The observed cytokine responses are summarized in Table 6 for the whole blood-based assay system and Table 7 for the PBMC-based assay system, respectively.

Table 6. Summary of cytokine responses in the whole blood assay system

Analysis system: whole blood Cytokine/Analyte Result TNF-α Increased secretion is found in 4/4 monkeys and 4/4 humans.
Maximum absolute cytokine levels at maximum dose:
Monkeys: 1/4: <100 pg/mL; 1/4: 100–500 pg/mL; 2/4: 500–1000 pg/mL
Humans: 1/4: <100 pg/ml; 3/4: 500–1000 pg/ml IFN-γ Increased secretion is found in only 2/4 of monkeys
Maximum absolute cytokine levels at maximum dose:
Monkeys: 2/4 <100 pg/ml IL-6 Increased secretion is found in 4/4 monkeys and 4/4 humans.
Maximum absolute cytokine levels at maximum dose:
Monkeys: 2/4: 100–500 pg/ml; 2/4: 500–1000 pg/ml
Humans: 1/4: 500–1000 pg/ml; 3/4: 1000–5000 pg/ml IP-10 Increased secretion is found in only 4/4 people.
Maximum absolute cytokine levels at maximum dose:
Humans: 4/4: 500–1000 pg/ml. IL-1β Increased secretion is found in 4/4 monkeys and 4/4 humans.
Maximum absolute cytokine levels at maximum dose:
Monkeys: 2/4: <100 pg/mL; 2/4: 100–500 pg/mL
Humans: 2/4: <100 pg/ml; 2/4: 100–500 pg/ml IL-12 Increased secretion is found in only 4/4 people.
Maximum absolute cytokine levels at maximum dose:
Humans: 3/4: <100 pg/ml; 1/4: 100–500 pg/ml IL-2 no increased secretion is found in either monkeys or humans

Table 7. Summary of cytokine responses in the PBMC-based assay system (LPT #31031)

Test system: PBMC Cytokine/Analyte Result TNF-α Increased secretion is found in 4/4 monkeys and 4/4 humans.
Maximum absolute cytokine levels at maximum dose:
Monkeys: 4/4: 1,000–5,000 pg/mL.
Humans: 4/4: 1,000–5,000 pg/ml. IFN-γ Increased secretion is found in 4/4 monkeys and 4/4 humans.
Maximum absolute cytokine levels at maximum dose:
Monkeys: 2/4: 100–500 pg/ml; 2/4: 500–1000 pg/ml
Humans: 2/4: 1,000–5,000 pg/ml; 2/4: 5,000–10,000 pg/ml IL-6 Increased secretion is found in 4/4 monkeys and 4/4 humans.
Maximum absolute cytokine levels at maximum dose:
Monkeys: 1/4: 1,000–5,000 pg/mL; 3/4: 5,000–10,000 pg/mL
Humans: 2/4: 5,000–10,000 pg/ml; 2/4: 10,000–15,000 pg/ml IP-10 Increased secretion is found in only 4/4 people.
Maximum levels at maximum dose:
Humans: 4/4: 100–500 pg/ml. IL-1β Increased secretion is found in 4/4 monkeys and 4/4 humans.
Maximum absolute cytokine levels at maximum dose:
Monkeys: 4/4: 1000–5000 pg/ml.
Humans: 4/4: 1000–5000 pg/ml. IL-12 Increased secretion is found in 4/4 monkeys and 4/4 humans.
Maximum absolute cytokine levels at maximum dose:
Monkeys: 4/4: <100 pg/ml
Humans: 1/4: 100–500 pg/ml; 3/4: 500–1000 pg/ml IL-2 no increased secretion is found in either monkeys or humans

Table 8 shows data obtained in LPT study #31031, in which whole blood from four cynomolgus macaques and four healthy donors was analyzed after 6 and 24 h of incubation with six different doses of RNA-(LIP). The analysis focused on the proinflammatory cytokines TNFα, IL-6, and IFN-γ, as they were preferentially upregulated in human PBMCs in study #STR-30207-013. As shown, the in vitro cytokine responses were highly comparable between the two species. For IL-6, a 122-fold induction was observed in cynomolgus macaques and a 108-fold induction was observed in healthy donors, respectively, after 24 h of incubation. Only low levels of TNF-α could be detected in both species at the highest dose level. Very low IFN-γ induction was observed only at the highest dose level in cynomolgus monkeys after 24 h of incubation.

Most importantly, strong induction of these three test-item-associated proinflammatory cytokines was observed only at dose levels ≥ 5500 μg, which is higher than the highest intended dose level of 100 μg for patients and which is > 100-fold higher than the planned dose for the initial vaccination cycle (= 50 μg RNA). Notably, the results from healthy donors confirmed the in vitro results of the STR-30207-013 study, and the cytokine response pattern of cynomolgus monkeys observed in the whole blood-based assay system resembled the results of the in vivo LPT study #29928, in which only IL-6 could be detected in RNA-(LIP)-treated cynomolgus monkeys (see below).

Table 9 shows data from LPT study #31031, in which PBMCs from four cynomolgus monkeys and four healthy donors were analyzed after 6 and 24 h of incubation with six different doses of RNA(LIP). Induction of IL-6 and TNF-α was comparable in human and cynomolgus PBMCs with respect to (i) the absolute amounts of cytokines induced (<2-fold difference between species), (ii) kinetics (early induction of IL-6 and TNF-α at 6 h), and (iii) the RNA(LIP) dose level that resulted in cytokine induction. IFN-γ was detected in PBMCs from both species treated with intermediate doses of RNA(LIP) only after 24 h of RNA(LIP) stimulation, although to a greater extent in humans. Overall, RNA-(LIP)-induced cytokine profiles in PBMCs were comparable across species for IL-6 and TNF-α. The results obtained in this study suggest that cynomolgus macaques are a suitable species for assessing RNA-(LIP)-mediated cytokine induction and that human PBMCs represent a more sensitive system for capturing IFN-γ induction.

Table 8. In vitro induction of proinflammatory cytokines IL-6, TNF-α and IFN-γ in cynomolgus macaques and healthy human donors in a whole blood-based assay system

The table presents data from LPT study #31031: cytokine levels (pg/mL) of IL-6 (top), TNF-α (middle), and IFN-γ (bottom) detected after incubation of whole blood with different doses of RNA-(LIP). Red color code indicates the height of cytokine levels, and darker red indicates higher cytokine levels. The first column shows the common dose levels used in the clinical setting. The second column shows the amount of RNA used in the in vitro test system, based on a blood volume of 5 L. * = data not collected.

^[1] The average total blood volume is assumed to be 5 L.

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

The table presents data from LPT study #31031: cytokine levels (pg/mL) of IL-6 (top), TNF-α (middle), and IFN-γ (bottom) detected after incubation of PBMC with different doses of RNA-(LIP). Red color code indicates the height of cytokine levels, and darker red indicates higher cytokine levels. The first column indicates the common dose levels used in the clinical setting. The second column indicates the amount of RNA used in the in vitro test system based on a blood volume of 5 L. * = data not collected.

^[1] The average total blood volume is assumed to be 5 L.

When comparing the results obtained in whole blood and the PBMC assay, it became clear that proinflammatory cytokines in the PBMC assay were generally broader, reached higher absolute values, and started at lower dose levels compared to the whole blood assay.

In this most sensitive in vitro test system, dramatic increases in cytokine levels measured at 24 hours were observed over a dose range of 615 μg to 1,850 μg RNA for IL-6, 1,850 to 5,550 μg RNA for IFN-γ, and 5,550 μg RNA to 16,650 μg RNA for TNF-α. Even for IL-6, which was the most sensitive cytokine marker in the in vitro system, the proposed starting dose of 25 μg is 25-fold lower than the dose level that marks the onset of potent cytokine induction in vitro.

In addition to the GLP LPT study #31031, a non-GLP in vitro study (Report_RB_14_001_B) was conducted with a similar experimental setup testing samples from three individuals of each species, in which similar observations were made confirming the results of the GLP study (data not shown). Taken together, the results highlight (i) the comparability of both species, cynomolgus monkeys and humans, with respect to cell stimulation following incubation with the test sample. Furthermore, these observations indicate (ii) that the whole blood assay system more accurately reflects the in vivo situation than PBMC. In the whole blood assay system, cytokine induction was generally less pronounced and observed only in the highest dose groups, and the predominant induction of IL-6 was reminiscent of the results of the in vivo cynomolgus monkey study (see below).

The authors acknowledge the more robust results of the PBMC-based assay, which is considered an artificial but also more sensitive in vitro test system, and therefore integrated the results obtained using this more sensitive test system into the strategy for determining a safe starting dose.

In vivo testing of secondary pharmacology in cynomolgus monkeys

To further understand the kinetics and correlation of RNA-(LIP) secondary effects with cytokine expression, a non-GLP study was conducted in male cynomolgus monkeys (see Table 10 for treatment and dose schedule and Table 11 for details of study design and amounts of all formulation components). Animals (two males per dose) in groups 1 through 5 were treated with the four melanosomal RNA (LIP) vaccines RBL001.1, RBL002.2, RBL003.1 and RBL004.1 (ATM quality) and then given control solutions as slow bolus injections (approximately 10 s) with an interval of 30 min between each injection (i.e. the last injection was given after 1.5 h). The results of this study should also apply to W_pro1 injection since the secondary effects are not sequence dependent.

The study tested doses up to 42 times the maximum clinical dose. In addition, animals in Dose Group 6 received a single dose of 4 x 3.6 μg RNA on day 22 after receiving a single dose of 4 x 88.6 μg RNA on day 1.

Table 10. Study schedule and doses based on expected patient doses (LPT #29928)

group Animal ID Days of use Dose
[mg/kg body weight] Dose
total RNA [µg] 1 1,2 Days 1, 4, 8, 15, 22 NaCl NaCl 2 3.4 Days 1, 4, 8, 15, 22 Liposomes Liposomes 3 5.6 Days 1, 4, 8, 15, 22 0,0086 43 4 7.8 Days 1, 4, 8, 15, 22 0.0256 128 5 9,10 Days 1, 4, 8, 15, 22 0,0708 354 6 11,12 Day 1 0,0708 354 6 11,12 Day 22 0.029 14

Treatment: Animals 1–10 (groups 1–5) received 5 x 4 consecutive injections of NaCl (saline) (group 1), liposomes at the same dose as high-dose animals (group 2), and RNA-(LIP)1–4 (ATM quality, groups 3–5). Animals in group 6 received a single treatment of 4 x 88.6 μg (total 354 μg RNA) on test day 1 followed by a single treatment of 4 x 3.6 μg (total 14.4 μg RNA) on test day 22. Doses: Doses are shown as total RNA in mg/kg body weight and as total RNA dose (μg per person, patients are assessed at 70 kg body weight).

Table 11. Design of the pharmacodynamic study in cynomolgus monkeys (LPT #29928)

Design of a pharmacodynamic study of RBL001.1, RBL002.2, RBL003.1 and RBL004.1 following intravenous administration in cynomolgus monkeys. Test object RBL001.1, RBL002.2, RBL003.1 and RBL004.1 RNA (LIP) Introduction 5 injections on days 1, 4, 8, 15 and 22, except for group 6 Path Bolus intravenously into the cephalic vein of the left or right arm
Bolus administration (approximately 10 sec) of RBL001.1, RBL002.2, RBL003.1 and RBL004.1 with 30-minute intervals between each RNA (LIP) Dose groups 1. NaCl control [1]
2. liposome control
3. small dose
4. average dose
5. high dose
6. single high dose on day 1 followed by a recovery period
additional very low dose on day 22 Doses
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Group 6 Days of use
1, 4, 8, 15, 22
1, 4, 8, 15, 22
1, 4, 8, 15, 22
1, 4, 8, 15, 22
1, 4, 8, 15, 22
1
22 RNA [mcg]
-
-
4 x 10.75
4 x 32
4 x 88.6
4 x 88.6
4 x 3.6 Lipids [mcg]
-
4 x 181
4 x 22
4 x 65
4 x 181
4 x 181
4 x 8 DOTMA [mcg]
-
4 x 116
4 x 14
4 x 42
4 x 116
4 x 116
4 x 5 DOPE [mcg]
-
4 x 65
4 x 8
4 x 23
4 x 65
4 x 65
4 x 3 Group size 2 males per group, 12 animals in total

^[1] NaCl is considered to be the most suitable control group. Unlike RNA in liposomes, which forms PH- (LIP) of a certain size and charge, pure liposomes used in group 2 differ significantly in terms of physical characteristics such as charge and structure, resulting in different pharmacological properties and altered biodistribution in vivo.

Clinical observation

Overall, the treatment was very well tolerated. No abnormal signs of intolerance were observed in any animal with respect to local and systemic tolerance observations (including behavior, appearance, feces, mortality, body weight, and food and water consumption).

Cytokine analysis

Cytokine release into plasma was studied for IFN-α, IFN-γ, TNF-α, IL-1β, IL-2, IL-6, IL-10, IL12p70 and IP-10 in two kinetic studies after the 1st and after the 5th injection, pre-dose, 0.5, 2, 5, 9, 24 and 48 h after completion of treatment (i.e., after completion of the injection cycle of all 4 RNA-(LIP) products).

At the doses tested, only IL-6 showed dose-dependent and test-subject-related induction. Cmax levels were reached 30 minutes after completion of treatment and returned to pre-dose levels by 24 hours (Figure 19). Animal 11 (Group 6) was an exception, showing a very strong response and had peak IL-6 levels of 1071 pg/mL, approximately 5-fold higher than other animals in the same dose group. It should be noted that IL-6 induction was much lower after the 5th treatment, suggesting an adaptation effect to IL-6 in the monkeys.

IFN-α induction was observed only at very low levels in high-dose group 6 animals, reaching peak levels at 5 h and returning to pre-dose levels by 24 h (Figure 19). In contrast to observations in cultured human cells and in vivo in mice, no IP-10 induction was observed in monkeys. It remains unclear why IP10 was not observed in this study, as IP-10 induction has been observed in monkeys following TLR agonist activation as reported by others.

Other cytokines tested (IFN-γ, TNF-α, IL-1β, IL-2, IL-10, and IL-12p70) were not altered. Liposomes themselves did not affect cytokine release.

Hematology

Standard hematology parameters were tested after the 1st and 5th injections, predose, 5, 9, 24, and 48 hours after completion of treatment (2 hours were additionally included after the 5th dose). In addition, hematology was checked daily from days 4 to 12, and 1 and 3 weeks after the last dose.

A transient decrease in lymphocytes and a transient increase in neutrophils were found as test element related results in a dose dependent manner. The lymphocyte count decreased very rapidly 5 h after completion of treatment, up to 5-fold in high dose animals (lowest count approximately 1000 lymphocytes/µl in group 6 animals). The effect was short-lived and recovered after approximately 48 h. It should be noted that lymphocyte depletion was also observed in the liposome group animals to a lesser extent, but not in the NaCl control group (Table 12). The adaptation effect observed for IL-6 induction was absent.

Increase in neutrophils was also observed in the NaCl control group due to treatment, however, significant difference was observed in groups 3 to 6 compared to control. The maximum effects were observed at 10 hours after treatment and were 44%, 34%, 89% and 91% compared to control in groups 3, 4, 5 and 6, respectively.

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

Table 12. Results for absolute lymphocyte count [1000/µl] in cynomolgus macaques (mean values n = 2)

Dosage Test day/hours after RNA4 Group 1: NaCl Group 2: liposome Group 3: 43 µg Group 4: 128 mcg Group 5: 354 mcg Group 6: 354 mcg single Group 6: 14.4 µg 1 Day 1, pre-dose 6,170 9,130 6,190 6,490 7,060 5,700 n.d. day 1, 5 h 4,845 3,295 2,095 2,665 1,490 1,065 n.d. day 1, 9 h 8,605 6,365 4,250 4,200 1,850 1.78 n.d. day 2 5,165 8,950 4,065 4,300 4,190 3.37 n.d. day 3 5,725 9,075 4,745 5,375 5,250 5,425 n.d. 2 day 4 5,775 8,185 5,175 5,265 5,530 5.87 n.d. day 5 5,565 8,850 4,480 4,170 4,600 5,845 n.d. day 6 5,865 8,560 5,590 6,660 7,200 5,495 n.d. day 7 7,090 9,645 6,225 6,780 8,660 6,885 n.d. 3 day 8 5,980 10,555 6,020 6,955 8,775 6,325 n.d. day 9 4,870 7,870 3,465 4,260 4,960 5,430 n.d. day 10 5,495 8,125 4,350 5,885 7,025 5,400 n.d. day 11 6,360 8,325 4,725 6,045 8,555 5,640 n.d. day 12 5,305 7,555 5,290 5,155 7,950 5,815 n.d. 4 day 15 8,180 11,030 9,540 9,170 9,855 n.d. n.d. day 16 4,765 7,320 2,880 3,365 3,490 n.d. n.d. day 19 5,545 8,230 5,215 5,485 8,035 6,205 n.d. 5 day 22, 2 h 4,360 4,385 2,055 2,645 2,120 n.d. 2,955 day 22, 5 h 5,525 5,225 2,955 3,325 2,445 n.d. 3,485 day 22, 9 am 8,560 12,525 5,490 4,650 3,695 n.d. 4,625 day 23 4,645 7,655 3,505 3,720 4,685 n.d. 4,350 day 24 5,780 8,360 4,760 4.50 6,270 n.d. 5,505 day 30 6,120 8,190 5,265 6,370 6,360 n.d. 5,615

Complement activation

C3a was measured pre-dose, 0.5, 2, 5, 9, 24 and 48 hours after completion of the 1st and 5th treatments, 1 and 3 weeks after the last dose. No test subject related variations were observed and all values were considered to be within the normal range of biological variability.

Clinical Chemistry

Standard parameters were tested before administration, 24 hours after each dose, and additionally 4 days after the 3rd and 4th doses, as well as 1 and 3 weeks after the last dose.

Effects on biochemical parameters unrelated to the test item were not assessed for liposome-treated animals and for test item-treated animals compared to control animals and/or baseline data available from the CRO conducting the study. Some scatter in the data is due to the small number of animals in each group.

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

Serum enzyme activities of alanine aminotransferase (ALAT), alkaline phosphatase (AP), aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH), alpha-amylase, creatine kinase (CK, including CK-BB, CK-MB, and CK-MM isoforms), gamma-glutamyl transferase (gamma-GT), and glutamate dehydrogenase (GLDH) were considered to be within the range of normal biological variability.

On day 23 of testing, high values of LDH, alpha-amylase, and CK enzyme activities were noted for animals #11 treated with 4 x 3.5 μg RNA/animal on day 22 of testing. However, these changes are considered to be related to the stress of restraining the monkey in the infusion chair rather than to the test object.

Although assessed as unrelated to the test subjects, small changes for CK were assessed in more detail. Differential analysis of the CK isoenzymes CK-BB, CK-MB, and CK-MM showed that increased CK activity noted in individual animals of groups 4, 5, or 6 compared to control animals on testing days 9, 16, or 23 was mainly due to an increase in the CK-MM fraction. Generally, no increase was noted for CK-BB and CK-MB, confirming that the increase in total CK levels was related to stress.

Cardiovascular system research

ECG and blood pressure measurements showed no effect on the cardiovascular system.

Sequence homology screening between TAA-encoding RNA DPs and the human proteome

All mRNA sequences used in the W_pro1 approach are fused in frame with up to two flanking glycine/serine-rich (GS) linker sequences. The junctions may generate novel antigen fusion proteins or peptides that could potentially induce an unwanted autoimmune response if they are homologous to human proteins. Therefore, a BLASTp-based homology search against a database of known human proteins was used to determine whether the junctions associated with the linker sequences and the antigen shared sequence homology with known human proteins.

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

For peptide subsequences with 100% homology, no significant alignments with human protein sequences were found.

Section 4: Safety pharmacological studies

ICH S7A describes a core group of studies, including respiratory, central nervous system (CNS) and cardiovascular function, that should be performed with any pharmaceutical product prior to human exposure. Therefore, the safety pharmacology of RNA-(LIP) was tested as an integral part of six GLP toxicology studies, which are described below.

Potential effects on CNS and respiratory function were assessed in the pivotal repeated dose toxicity studies and did not reveal any test-related effects in animals.

A risk analysis of potential cardiovascular effects of RNA(LIP) vaccines was performed. Systemically distributed RNA is degraded in the bloodstream and RNA in the form of RNA(LIP) is cleared from the blood within minutes and is distributed primarily to the spleen and liver as shown in biodistribution studies (see below). The data do not suggest that RNA(LIP) will accumulate in the cardiovascular system. Potential systemic adverse effects of RNA(LIP) vaccination are expected to be related to a transient increase in IFN-α, which is not expected to result in cardiovascular adverse events as has been reported in thousands of patients receiving IFN-α. Therefore, a GLP cardiovascular safety pharmacology study compliant with ICH S7A/B was not conducted. However, supporting ECG and blood pressure data are available from a non-GLP pharmacology study in cynomolgus monkeys treated with RNA-(LIP) and relate to the assessment of cardiovascular function following treatment with RNA-(LIP) vaccines.

Thus, no test subject or treatment related changes in the respiratory, neurological and cardiovascular systems were observed in any dose group tested in mice (respiratory system and CNS function) and cynomolgus monkeys (cardiovascular function).

Respiratory safety

Respiratory safety was included in repeated dose toxicity studies in mice using DP RNA encoding GLP compliant TAA (LPT #28864 and 30283, see study design below). For example, plethysmography was tested in a RNA-(LIP) study (LPT #30283) with four animals/sex/group receiving either buffer control, low dose, or high dose (5 and 50 μg RNA in 9 μg and 90 μg liposomes, respectively). A positive control of animals receiving 30 mg carbamyl-β-methylcholine chloride (bethanecol)/kg body weight was also included. Plethysmography was performed every other day after the 4th to 7th dose. The tests included assessment of respiratory rate, tidal volume, minute volume, inspiratory time, expiratory time, peak expiratory and inspiratory flow, expiratory time, and airway resistance index. None of the pulmonary parameters tested showed any changes related to the test object in the treated animals compared to the control group. Only animals from the positive control group showed the expected changes.

CNS safety

CNS safety was included in repeated dose toxicity studies in mice using DP RNAs encoding GLP-compliant TAA (LPT #28864 and 30283, see study design below). For example, in the RNA-(LIP) study (LPT #30283), observational screening was tested in five animals/sex approximately 24 hours after the 5th administration of buffer control, low dose, and high dose (5 μg and 50 μg RNA with 9 μg and 90 μg liposomes, respectively). The observational screening included the following tests: righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, seizures, piloerection, diarrhea, pupil size, pupillary response, lacrimation, gait abnormality, stereotypy, toe squeeze test, tail squeeze test, wire test, hind limb extension test, positional passivity, tremor, positive geotropism, limb rotation, and auditory function. In addition, functional tests were included to assess grip strength and motor activity.

Neurological screening did not reveal any effects of the test elements in mice that could be attributed to neurological toxicity. These findings were confirmed by the results of the GLP-compliant LPT repeat dose toxicity study #28864 conducted for the Lipo-MERIT study using various RNAs.

Cardiovascular safety

A cardiovascular safety study according to ICH S7 was not conducted because RNA is degraded in the bloodstream within seconds and there is no indication that RNA (LIP) will accumulate in the cardiovascular system.

However, there is supporting data from a non-GLP pharmacology study in cynomolgus monkeys with RNA-(LIP) targeting melanoma-associated antigens used in the Lipo-MERIT study. In this study, twelve cynomolgus monkeys were treated in six groups (see study flow chart in Table 11), and ECG and blood pressure measurements were performed after the 4th dose at three time points pre-dose, 5 h after completion of dosing, and 24 h post-dose.

Treatment with RNA-(LIP) was very well tolerated by cynomolgus monkeys (no clinical follow-up data available). None of the measured parameters (blood pressure, heart rate, QTc values, QT intervals, P-segment, PQ, QRS) showed any effects related to the test subjects. In addition, serum levels of CK-MB and troponin-I were measured to exclude the possibility of necrotic damage to the cardiac muscle tissue. All measured parameters were negative, indicating the absence of cardiovascular toxicity from RNA-(LIP) at the dose levels tested in the study.

Discussion and Conclusions

Extensive studies on the mechanism of action and primary pharmacodynamics of RNA-(LIP) have been conducted in mice and in human in vitro test systems. Preclinical studies show that RNA-(LIP) vaccines target the spleen and lymphoid tissue after intravenous administration. RNA-(LIP) vaccine induces dual action, namely, induction of antigen-specific T-cell responses and cellular activation and immunomodulation processes following TLR triggering.

The data obtained confirm that all leading antigen RNA structures used in vivo induce antigen-specific T-cell responses, including tetanus toxoid helper epitopes.

The functional properties of the RNA-(LIP) formulation are (i) protection of RNA in serum and (ii) efficient in vivo targeting of APCs that are capable of presenting antigen peptides and are also activated following TLR7 priming. The immunomodulatory activity of RNA resulted in a dose-dependent induction of cytokines in human, mouse and cynomolgus monkey samples, all of which showed induction of IFN-α, IP-10 and IL-6 to varying degrees depending on the species tested or the assay system used. RNA(LIP)-mediated cytokine induction in PBMC was expected, as there is good evidence from our own RNA studies and the literature. Apart from these expectations, the moderate induction of IFN-α and the induction of the chemokine IP10 (CXCL10) rather reflect the onset of the intended pharmacological effect than an unwanted immunotoxicological event.

Our mouse data indicate that splenocytes are the major source of TLR7-dependent IFN-α secretion, as it is reduced in TLR7 -/- mice. We interpret the observed transient and completely reversible cytokine responses as a putative pharmacodynamic effect contributing to the efficient induction of vaccine-induced antitumor T cell responses. The favorable immunological properties were combined with good tolerability of RNA(LIP) vaccines in mice and cynomolgus monkeys.

We also studied the secondary effects of RNA(LIP) vaccine treatment in several in vitro and in vivo studies using human, cynomolgus monkey and mouse assay systems. Particular attention was paid to the immunomodulatory effects of RNA(LIP) vaccines, as they were stronger than what we observed for unformulated RNA vaccines administered into the lymph nodes, which resulted only in local cellular activation and cytokine induction.

Experiments were performed using whole blood and PBMC samples from human and cynomolgus monkey donors, excluding non-specific or uncontrolled cellular activation of human immune cells by RNA (LIP) vaccines, but demonstrating modest cytokine induction as expected. In these experiments, human and cynomolgus monkey cells were treated at doses spanning the highest clinical dose cohorts and above.

Although differences were found between donors, different in vitro assays (cultured PBMCs versus whole blood) or species in cytokine levels, the observed cytokine patterns and the temporal patterns of cytokine responses were similar across studies with minor exceptions, such as no IP-10 induction in cynomolgus monkeys. Human PBMCs showed IP-10 induction and low IL-6 and IFN-α responses, which were even lower when tested in whole blood. Cynomolgus monkeys showed a very low IFN-α response, no IP-10 induction and a more pronounced IL-6 response at the dose levels tested. Mice showed strong responses to IFN-α, IP-10 and IL-6, but at doses approximately 10-fold higher than those tested in monkeys (based on doses per kg body weight). On one hand, the differences in cytokine expression between mice and monkeys could be explained by the different doses tested. On the other hand, mice have different TLR7/8 activity, which may also be a plausible explanation for the different cytokine expression patterns.

The cytokine response patterns observed in the cynomolgus macaque in vivo were better reflected by the whole blood-based assay system compared to the PBMC-based assay system, which showed a broader, higher cytokine response at lower dose levels. However, the results in the more sensitive PBMC were integrated into the strategy for determining a safe starting dose for patients. A side-by-side comparison of cytokine secretion in human and cynomolgus macaque whole blood showed that both species were highly comparable in inducing proinflammatory cytokines upon RNA-(LIP) treatment, suggesting that the cynomolgus macaque is an adequate animal model to predict secondary pharmacodynamic effects that may occur following RNA-(LIP) vaccination of patients.

In addition to the cellular activation and cytokine induction processes following RNA(LIP) exposure, BioNTech assessed hematological changes in mice and cynomolgus monkeys. Here, transient lymphopenia was observed similarly in mice and monkeys at all dose levels. Overall, RNA(LIP)-treated monkeys showed a similar response in cytokine profile and hematological parameters as monkeys treated with another TLR agonist. This is consistent with the idea that the primary processes of cytokine expression activation by RNA(LIP) occur through TLR stimulation. Extensive pharmacodynamic studies in wild-type, TLR7 -/- and IFNAR/ mice suggest that the hematological findings are secondary to the cytokine effects induced by RNA(LIP). Both RNA(LIP) and naked RNA have been shown to activate TLRs. TLR activation has been shown to induce lymphopenia and B-cell accumulation in the spleen. In support, histopathology data obtained during toxicology testing showed that transient lymphoid hyperplasia was detected in the spleen but not in any other organ or tissue. This is consistent with the observed lymphopenia in the blood and highlights the putative targeting of RNA-(LIP) and thus also the putative recruitment of effector cells to the lymphoid organ.

The safety pharmacology studies performed suggest a safe profile for RNA-(LIP). Neurological screening did not reveal any effects of the test elements on mice in any of the tests performed. None of the pulmonary parameters tested showed any changes in mice treated with RNA-(LIP). There were no signs of cardiovascular effects in cynomolgus monkeys. Overall, RNA-(LIP) demonstrates a very good overall safety profile with respect to the safety pharmacology parameters.

Section 5: Pharmacokinetics

Although pharmacokinetic studies are not typically performed during cancer vaccine development, the authors conducted in vivo studies to determine the biodistribution of intravenously administered RNA lipoplexes and the presence or retention of residual plasmids due to impurities in the finished dosage form.

RNA transcribed in vitro consists of ribonucleotides and is therefore identical in structure to RNA synthesized by human cells, except for the structure of the 5' cap. Consequently, RNA is subject to the same degradation processes as natural mRNA. Excessive amounts of RNases, especially in the extracellular space and serum, lead to rapid degradation of RNA.

As described below, the distribution/location and potential accumulation of RNA in the spleen, liver and lungs were studied in pharmacokinetic studies. In addition, potential plasmid DNA contaminants were quantified in the gonads of mice treated with RNA-(LIP).

The biodistribution and stability of the synthetic cationic lipid DOTMA were investigated in the first in vivo studies. Synthetic DOPE cannot be distinguished from the native natural phospholipid DOPE, it must undergo natural metabolic pathways, and therefore biodistribution and accumulation were not further investigated.

Summary of Key Findings The biodistribution of RNA-(LIP), residual plasmid DNA contaminants and the synthetic lipid DOTMA were analyzed in in vivo studies in mice.
RNA: For in vivo biodistribution analysis of RNA-(LIP), IVT-RNA levels in samples from the GLP-compliant repeat dose toxicity study were analyzed using a RT-qPCR-based method at a third party. Organ samples for RNA analysis included blood, spleen, liver, and lung. A semi-quantitative assay was developed to determine total RNA and organ samples were analyzed under non-GLP conditions. The method was based on RT-qPCR. RNA was rapidly cleared from blood with an estimated half-life of approximately 5 min. It was subsequently detected in liver, spleen, and lung at levels much lower than in blood.
Residual plasmid impurities: A quantitative analytical method for the detection of residual plasmid impurities was developed and gonad samples were analyzed in accordance with GLP at BioNTech IMFS GmbH. The method is based on a quantitative PCR method for the detection of the kanamycin resistance gene in the plasmid.
Residual plasmid contaminants were not detected or only slightly exceeded the lower detection limit. The signals were similar after the 1st or 8th injection, indicating that both RNA and residual DNA contaminants do not accumulate and persist in the organs studied.
DOTMA: To analyze the in vivo biodistribution of RNA-(LIP), DOTMA was extracted from blood and seven selected organs that were collected after intravenous injection of RNA-(LIP) into mice, and DOTMA content was quantified by LC/MS analysis under non-GLP conditions. DOTMA was rapidly (less than an hour) delivered to the spleen (and other organs) after intravenous administration of RNA-(LIP). The highest levels of DOTMA were in the spleen and liver, while the amount of DOTMA in all other organ samples was quite negligible. Accumulated DOTMA after repeated RNA-(LIP) administration is cleared from the organs with kinetics that can be reasonably represented by first-order decay with an approximate half-life of about 6-7 weeks.

Biodistribution

RNA

The biodistribution of (LIP) RNA was studied in detail in mice by organ sampling during a GLP-repeated dose toxicity study (LPT #28864) conducted for the Lipo-MERIT clinical trial. Organs were analyzed for total IVT RNA using a real-time quantitative reverse transcription PCR (RT-qPCR) method developed at IMGM Laboratories GmbH, Martinsried, Germany under non-GLP conditions (study ID: RS297). The RNA was thus cleared very rapidly from the blood with an estimated half-life of approximately 5 min. After 48 hours and after seven days, the RNA was detectable only at marginal levels in the blood and organs, suggesting that it is rapidly degraded and not retained.

Residual plasmid impurities

The biodistribution of residual plasmid impurities from RNA-(LIP) vaccination was investigated using samples from a GLP repeated dose toxicity study (LPT #28864). A GLP-compliant assay for residual plasmid impurities in organ samples was developed at BioNTech IMFS GmbH, Idar-Oberstein, Germany. All samples tested were either below or slightly above the lower limit of detection (LLOD), suggesting that plasmid DNA does not accumulate or persist in the gonads (Study ID: 36X130313).

DOTMA

The biodistribution of two synthetic lipids used in RNA formulations (LIPs) can provide insight into the physical distribution of the lipoplex carrier particle over time. The synthetic cationic lipid DOTMA was chosen for biodistribution studies because it is a non-naturally occurring molecule and can therefore be easily detected in the background of a biological matrix.

In a preliminary study of DOTMA biodistribution, lipid was extracted from blood and seven selected organs that were collected after intravenous injection of RNA-(LIP) into mice. In this case, a mixture of equal parts of IVT-RNA prepared using ATM grade liposomes was used. This initial study included five mice, of which one was left untreated, two received a single injection of 60 μg RNA, and two received two injections of 60 μg RNA each, 20 days apart. All mice were sacrificed 24 hours after the last injection. DOTMA was quantified using LC/MS measurements. The aim of the experiments was to test the general feasibility of the extraction and quantification protocols and to provide initial insights into the biodistribution of DOTMA following RNA-(LIP) vaccination.

DOTMA could be clearly detected in all organs examined, and clear differences between the results in different organs could be observed. In accordance with the proposed mechanism of action, the highest DOTMA values were found in the spleen.

Based on these initial results, a single-injection RNA (LIP) study was conducted (Report_BN_14_004). DOTMA concentrations in selected organs were assessed over a period of up to 28 days (d0, d1, d4, d7, d14, d21, and d28). In this experiment, 200 μl of RNA (LIP) containing 20 μg RNA and 26 μg DOTMA were injected (60 μg per injection was administered in the first study). The concentration of DOTMA in the injected product was 195 μM. Three mice were tested at each time point.

Apparently, DOTMA was predominantly found in the spleen and liver with slightly different accumulation kinetics. In all other organs/tissues examined (lungs, heart, kidneys, lymph nodes, adipose tissue, bone marrow, brain) the results were 10-50 times lower than in these. Based on the liver and spleen data, the pharmacokinetics of DOTMA could be estimated: the maximum concentration was detected a few days after administration. Within 20 days, the DOTMA concentration decreased to approximately 50% of the maximum values. These data support the assumption that DOTMA is eliminated from the organs in an acceptable time frame and no indication can be made of the risk of chronic accumulation in any organ.

In a subsequent study, DOTMA concentrations in selected organs were assessed before (control), during, and after eight weeks of LIP injections, each containing 20 μg RNA (RBL005.2) and 26 μg DOTMA (Report_RB_15_004_V02). Organ samples were collected from mice one hour after the first LIP injection and then every two weeks thereafter. Following completion of the eight LIP cycles, mice were sacrificed at 3, 6, 9, 12, and 15 weeks to assess DOTMA clearance in organs. Repeat LIP injections and organ sampling were performed in-house, while DOTMA extraction and quantification from provided organ samples were performed by Charles River Laboratories Edinburgh Ltd. (Study #322915). The results are in good agreement with our previous studies: again, the highest DOTMA concentrations were observed in the spleen as the main target organ, followed by the liver. No more than 5% of the concentration present in the spleen samples was detected in all other organs (data not shown). DOTMA concentrations increased with the number of RNA injections (LIP) and then decreased continuously during the recovery phase after the last application. The terminal half-lives of DOTMA in plasma (7.07 weeks), spleen (6.76 weeks) and liver (6.57 weeks) were comparable to those obtained in animals during the recovery period after the 8th dose.

Thus, DOTMA as a lipid carrier indicator is rapidly (less than an hour) delivered to the spleen (and other organs) after intravenous RNA injection (LIP). In addition to the spleen, DOTMA predominantly accumulates in the liver, where, however, RNA translation is not observed. In absolute numbers, the amount of DOTMA detected in these two organs was close to the total cumulative amount of DOTMA that was completely injected, whereas the amounts of DOTMA in all other organ samples were rather insignificant.

Based on assessment of animals in the recovery phase groups, accumulated DOTMA following repeated RNA-(LIP) administration is cleared from organs with kinetics that can be reasonably represented by a first-order decay mechanism with an approximate half-life of 6-7 weeks. This clearance kinetics is also consistent with the results of repeated administrations in which transient accumulation was observed.

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

Discussion and Conclusions

IVT-RNA, composed of ribonucleotides, has a structure identical to RNA produced by human cells, with only a 5' cap as a special structure. Thus, IVT-RNAs are subject to the same degradation processes as natural mRNA. Excessive amounts of RNases, especially in the extracellular space and serum, lead to rapid degradation of RNA.

Results from biodistribution studies of RNA-(LIP) show high levels of RNA in the blood shortly after RNA-(LIP) injection. RNA is rapidly cleared from the blood and is subsequently detected, albeit at much lower levels, in the spleen and liver, while only trace amounts can be detected in the lungs. Since RNA distributed in the liver may result in transient immune activation via TLR activation, liver enzymes will be closely monitored in patients after the first injection and throughout the study. Only trace amounts of RNA were detected in the blood and organs after 48 hours and seven days, suggesting that RNA does not accumulate or persist in any organ. Also, comparison of Cmax levels after the 1st and 8th injections did not show any accumulation effects.

In the gonads, plasmid DNA was either not detected or samples were slightly above LLOD, suggesting that there is only a low risk of integration of plasmid remnants, such as the kanamycin resistance gene, into the germline cell genome.

DOTMA biodistribution was shown to be primarily found in the spleen and liver, confirming the spleen as the primary target organ for LIP vaccination, with significantly less exposure in plasma and other tissues following single and eight repeated LIP administrations. DOTMA was cleared from plasma, spleen, and liver with comparable terminal half-lives of approximately 6-7 weeks. A more systematic analysis of DOTMA biodistribution and accumulation will be conducted prior to larger clinical trials.

Section 6: Toxicology

The toxicology program for the RNA vaccine platform included several pharmacology studies to test RNA (LIP) vaccination across dose ranges and repeat dose toxicity studies, including local tolerance and safety pharmacology parameters, as well as immunotoxicity studies. The studies were conducted under GLP conditions in an external CRO (LPT, Hamburg, Germany) using RNA and liposome batches comparable to clinical trial materials within manufacturing processes and analytical quality control.

GLP-compliant studies included a 6-week repeated dose toxicity study with intravenous administration of eight different RNA DPs encoding breast cancer antigens in C57BL/6 mice (LPT #30283).

In addition, an additional 6-week GLP-compliant repeat dose toxicity study was conducted using an RNA vaccine platform targeting a range of melanoma-specific antigens (LPT #28864). Although different RNA sequences were tested, the toxicity data are also relevant for the use of DP RNA encoding TAA and may add important information since the same type of liposome was used to prepare the RNA (LIP). The toxicity profile of the incorporated RNAs in both studies should be the same or at least comparable due to the fact that potential adverse effects are related to the intrinsic molecular properties of the RNA incorporated into the liposomes, which are independent of the sequence and length of the RNA.

In addition, an additional 4-week repeat-dose toxicity study was conducted to evaluate the comparability of the liposomes used in the 6-week repeat-dose toxicity study with a pH-adapted liposomal formulation whose buffer conditions were slightly adjusted for long-term stability reasons (LPT #30586).

Summary of Key Findings In LPT studies #28864 and 30283 using melanosome antigen and breast cancer antigen RNA, no toxicological effects were observed that could be attributed to RNA-(LIP) vaccination.
No signs of local or systemic intolerance reactions were observed in vaccinated animals. The test objects did not affect body weight, food intake, drinking water consumption, functional observation tests, grip strength of the forelimbs and hindlimbs, and spontaneous motor skills.
Mild and transient lymphopenia was observed in animals of all dose groups, which is considered to be consistent with the expected pharmacological effect of RNA-(LIP) vaccination due to TLR activation and cytokine induction. Most importantly, these effects were completely restored after two weeks.
The chemokine IP-10 (CXCL10) and IFN-α were found to be transiently induced in a dose-dependent manner, likely due to the onset of the putative pharmacological effect.
Induction of IP-10 and IFN-α was maximal at 6 hours after the 5th injection and had almost or completely returned to normal levels by 24 hours.
Transient significant inductions of IL-6 and IFN-γ were detected only in male animals of the high dose groups, whereas only moderate inductions were observed for IL-2, regardless of sex and dose level.
Test element-related increases in ALAT, ASAT, GLDH and LDH were noted in the high-dose group, but these effects were transient and completely disappeared after three weeks. Histopathological examinations revealed no evidence of liver toxicity.
Spleen weight increases were observed in animals of all dose groups. In animals of the medium and high dose groups, these effects had not fully recovered after three weeks. Urinary status and bone marrow were unaffected by any of the dose levels tested. No test-related changes were noted at necropsy or histopathology. Minimal to mild lymphoid hyperplasia of the white pulp of the spleen due to the pharmacologic mechanism of action was observed in animals vaccinated with the high dose. These effects had completely resolved after three weeks.
It is important to note that since no effects were observed in the low dose group of LPT study #30283 (5 µg total RNA), a NOAEL of 5 µg total RNA per animal (i.e., approximately 0.2 mg/kg body weight in mice) was achieved for RNA (LIP) vaccines.
In conclusion, intravenous injection of vaccine antigens with multiple liposomes was very well tolerated in mice. When comparing the toxicity profiles of the liposomes used in the main studies and the slightly pH-adapted liposomal formulation, no toxicologically significant differences were observed between the two liposomal preparations (LPT #30586).

Selection of appropriate species

The authors consider mice to be a suitable species for testing potentially toxic direct effects of RNA (LIP) vaccines for the following main reasons:

The mouse model system provides all the relevant characteristics of innate and adaptive immunity relevant to characterizing the direct toxic effects of RNA-DP encoding TAA. Mice exhibit all the expected primary and secondary pharmacological effects from the induction of CD4+/CD8+ T cell responses to immunomodulatory effects that enhance the immunological response and lead to subsequent TLR triggering, cellular activation and cytokine secretion.

The mouse system includes a wealth of available tools and methods for investigating biological effects that far exceed the experimental capabilities in other species (e.g., availability of transgenic mouse models, MHC tetramers, antibodies, etc.). This allows for a more in-depth analysis of unexpected events.

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

Single Dose Toxicology

Dose-ranging studies are typically performed to justify doses for the main toxicity study and to provide initial information on target organs and toxicity signatures. The authors conducted several pharmacology studies testing RNA-(LIP) across a range of doses with schedules similar to the intended clinical regimen. These studies found that RNA-(LIP) administration produced favorable pharmacodynamic effects and was well tolerated.

In addition, previous toxicity studies have shown that intravenously administered naked RNA is very well tolerated in mice also at high doses. Liposomes containing DOTMA or DOPE as synthetic lipid components have been tested in numerous clinical studies, and several approved liposomal formulations have shown very good tolerability. Some liposomal formulations have been used to reduce specific drug toxicities, such as nephrotoxicity or hepatotoxicity of nucleic acids at high doses or toxicity of small molecules such as doxorubicin or clofazimine.

Based on data obtained from a number of internal studies and literature sources, the following conclusion was made:

Tolerable doses for mice that would provide a sufficient safety margin for the first dose in humans can be derived from pharmacological studies.

A single dose will not be sufficient to induce a significant immune response. The maximum immune response was observed after at least three applications.

RNA vaccines and lipoplex formulations are generally well tolerated.

Based on these findings, we decided not to conduct single-dose toxicity studies but instead conducted a repeat-dose toxicity study directly.

Multiple Dose Toxicology

The RNA(LIP) product of the RNA vaccine platform has been analyzed for safety and toxicity in several GLP-compliant multiple-dose toxicity studies using intravenous administration of RNA(LIP) products. Table 13 provides an overview of the GLP-compliant multiple-dose toxicity studies that support the Phase 1 and 2 clinical trials using RNA(LIP) vaccines.

Table 13. Design of GLP repeated dose toxicity studies

Study Study design 6-week IV repeat dose RNA (LIP) toxicity study in C57BL/6 mice
(LPT No. 28864) A total of eight intravenous injections were given into the tail vein on days 1, 4, 8, 11, 15, 22, 29, and 43, followed by a 3-week recovery period.
Vaccines: RBL001.1, RBL002.2, RBL003.1 and RBL004.1 RNA (LIP)
4 groups (14 animals/sex/group):
1. control: carrier
2. Low dose: 4×3.75μg (total RNA: 15μg; total DOTMA 17.4μg, total DOPE: 9.3μg)
3. Average dose: 4×7.5μg (total RNA: 30μg; total DOTMA 34.8μg; total DOPE: 18.6μg)
4. High dose: 4×15μg (total RNA: 60μg; total DOTMA: 69.6μg; total DOPE: 37.2μg) 6-week RNA toxicity study (LIP), coformulated with RBLTet.1, administered intravenously to C57BL/6 mice
(LPT No. 30283) A total of eight intravenous injections into the tail vein were given on days 1, 4, 8, 11, 15, 22, 29, and 43, followed by a recovery period of 3 weeks.
Vaccine Pool 1:
RBL008.1, RBL005.2, RBL006.2, RBL007.1, together with RBLTet.1 (auxiliary epitope P2P16 of tetanus toxoid).
Vaccine Pool 2:
RBL008.1, RBL009.1, RBL010.1, RBL011.1, together with RBLTet.1 (auxiliary epitope P2P16 of tetanus toxoid).
5 groups (14 animals/sex/group):
1. control: carrier
2. Low dose: Vaccine pool 1 (0.6 μg RBLTet.1 + 4 × 1.1 μg antigen RNA; Total DOTMA: 5.8 μg; Total DOPE: 3.1 μg)
3. High dose: Vaccine pool 1 (6 μg RBLTet.1 + 4 × 11 μg antigen RNA; total DOTMA: 58.0 μg; total DOPE: 31.0 μg)
4. Low dose: Vaccine pool 2 (0.6 μg RBLTet.1 + 4 × 1.1 μg antigen RNA; Total DOTMA: 5.8 μg; Total DOPE: 3.1 μg)
5. High dose: Vaccine pool 2 (6 μg RBLTet.1 + 4 × 11 μg antigen RNA; Total DOTMA: 58.0 μg; Total DOPE: 31.0 μg) A 4-week repeat dose toxicity study of two liposomal formulations of RBL008.1 administered intravenously in C57BL/6 mice
(LPT No. 30586) A total of five intravenous injections into the tail vein on days 1, 4, 8, 11 and 25, followed by a recovery period of 2 weeks.
Vaccine: RBL008.1, L1 liposomes, L2 liposomes
2 groups (9 animals/sex/group):
1:20 µg RBL008.1 + 40 µg L1 liposomes/animal
2: 20 μg RBL008.1 + 40 μg L2 liposomes/animal

ATM Composition

The composition, formulation and specifications of the AMT were planned as close as possible to the intended finished dosage form for human use.

Minor changes in the process of obtaining RNA-(LIP) had to be made for the following reasons:

To obtain a high dose that increases the likelihood of detecting potential dose-related toxicological effects and thus meets the toxicity testing criteria set out in ICH S6 or M3 (R2) guidelines.

To prevent injection above the maximum volume allowed in mice, which is 250 µl by slow bolus injection. Higher injection volumes are not recommended from an ethical point of view and carry the risk of losing mice during injection.

Differences from the clinical protocol:

Patients will receive the different RNA-(LIP) products sequentially. In mice, this was not possible due to volume limitations. The RNA-(LIP)s for mice were prepared individually, then mixed, and all four RNA lipoplexes were administered simultaneously in a total volume of 250 µl.

To create RNA-(LIP) for treatment of patients, 150 mM NaCl will be used. To obtain higher doses in toxicity studies, it was necessary to use NaCl solutions with higher concentrations to form RNA-(LIP).

To prepare RNA (LIP) for treatment of patients, finished RNA dosage forms with a concentration of 0.25 mg/mL will be used. In order to obtain a high dose in the toxicity study, it was necessary to use more concentrated RNA, i.e. 1 mg/mL, to obtain RNA (LIP) products in the LPT study #28864.

This trial will use acetic acid stabilised liposomes (L4) which are concentrated to half the previously used equivalently produced acetic acid stabilised liposomes (L2). No further bridging studies are planned as the same characteristics are given as for L2 liposomes.

Our position is that the mentioned changes in the ATM protocol have little or no impact on the results or conduct of the studies.

Study design

For study design, see Table 13. According to ICH S6 and S8, data from routine toxicity studies were assessed for evidence of immunotoxic potential. The following studies were performed in accordance with FDA, ICH, and CHMP guidance documents: mortality, histopathology (especially spleen), gross pathology and organ weight, clinical observations, ophthalmology, local tolerability, injection site reactions, body weight, food intake, routine hematology parameters, clinical chemistry, and cytokines (IL-1β, IL-2, IL-6, IL-10, IL-12, TNF-α, INF-α, INF-γ, and IP-10).

Safety pharmacology studies were included to test the respiratory and central nervous system as described in Section 4.

Results

Toxicological evaluation in 6-week repeated-dose toxicity studies using the vaccine platform revealed only minor effects that can be primarily attributed to the expected pharmacological mode of action of RNA-(LIP) (Table 14). The desired immunomodulatory effects of RNA-(LIP) are TLR activation and cytokine release. Induction of IFN-α in mice results in secondary effects such as leukopenia, decreased platelets, and increased liver parameters such as ALAT, effects that are commonly described in patients treated with IFN-α.

In line with this, the changes associated with the test objects in the treated animals were mostly transient (Table 14). In addition, transient activation of the cytokines IP-10, IFN-α, IL-6 and IFN-γ was observed. All inductions returned to normal levels after 24 h (except for IP-10 levels, which were still slightly above normal levels).

Hematological findings in mice included primarily lymphocytopenia and low reversible decreases in total leukocytes, neutrophils, reticulocytes, and thrombocytopenia in all treatment groups. These findings were completely reversible. Lymphoid hyperplasia observed for the spleen on histopathology was completely restored, indicating the desired effect and putative targeting of the test substance and lymphocytes to the spleen.

The observed moderate changes in liver parameters such as GLDH, LDH, ALAT and ASAT affected mainly the high dose groups and were not seen in animals from the recovery group, suggesting full recovery of effects within at least three weeks or less. Histopathological examination did not reveal any toxic effects on the liver.

Because no effects were observed in the low dose group in LPT study #30283, the NOAEL was observed at a dose of 5 µg total RNA per animal (i.e., approximately 0.2 mg/kg body weight in mice).

A further 4-week repeat dose toxicity study was conducted to investigate the change in the liposomal buffer composition (LPT #30586). The data obtained confirm that the new type of liposomes is absolutely comparable in the measured parameters to the one used in the main study.

Table 14. Summary of toxicological results from repeated dose toxicity studies using RNA-(LIP) (LPT Nos. 28864, 30283, and 30586)

All described results were statistically significant compared to the control group.

Category Study No. Conclusions Mortality LPT #28864 One animal in the high dose group died prematurely immediately after the 5th injection. The animal had an enlarged spleen and moderate extramedullary hematopoiesis. The death was considered related to the administration of the test object. LPT #30283 No premature deaths occurred during the study. LPT #30586 One animal from the L2 liposome group died prematurely 4 days after the 4th injection. No premortem symptoms were observed. The animal showed enlarged ovaries and autolysis of all organs. It was considered that death occurred spontaneously. Body weight, food consumption LPT #28864
LPT #30283
LPT #30586 There was no effect on food intake, body weight or weight gain in any study. Local tolerance LPT #28864
LPT #30283
LPT #30586 No evidence of local impairment of tolerance was observed in any of the studies. Clinical signs LPT #28864 A slight decrease in mobility was observed in nine animals of the high dose group after the 5th injection, beginning 5 min after injection and continuing for approximately 15 min. Thereafter, the animals showed normal behavior. This was again observed in one animal after the 6th and 7th injections. Mild ataxia was simultaneously observed in one female.
However, although they were only seen in the high-dose group, these findings are considered to be spontaneous due to a single event, likely due to handling of the animals during the administration procedure. This assumption is supported by the fact that no biologically significant changes were measured in the motor activity assessment as part of the neurological screening. LPT #30283 No signs of systemic intolerance were observed in any animal. LPT #30586 No signs of systemic intolerance were observed in any animal. Neurological screening LPT #28864 It was started 24 hours after the 5th dose. No effects related to the test element were noted in any animal. LPT #30283 It was started 24 hours after the 5th dose. No effects related to the test element were noted in any animal. LPT #30586 Not done. Plethysmography LPT #28864 It was started 24 hours after the 8th dose. No effects related to the test objects were noted. LPT #30283 It was started 24 hours after the 4th dose. No effects related to the test objects were noted. LPT #30586 Not done. Hematology and coagulation LPT #28864
LPT #30283 A decrease in leukocytes, lymphocytes and platelets and an increase in neutrophils and large unstained cells were observed in all dose groups. The effects were completely restored and are considered consistent with the pharmacological effect of RNA (LIP) due to TLR activation and IFNα induction (see also section 2.2.2.3). LPT #30586 No changes in test-related hematological parameters were observed between the two groups. Clinical biochemistry LPT #28864 Some changes compared to control animals were found in liver enzymes as shown below. Histopathological examinations showed no evidence of liver toxicity (clinical chemistry samples were taken shortly before necropsy).
Cholesterol levels were increased in all dose groups. Alanine aminotransferase (ALAT), lactate dehydrogenase (LDH) and glutamate dehydrogenase (GLDH) levels were significantly increased mainly in the high dose group (60 μg/animal). LPT #30283 Cholesterol levels were increased in all dose groups. ALAT, aspartate aminotransferase (ASAT), LDH and GLDH levels were significantly increased in the high dose groups (50 μg/animal), more pronounced in male animals. LPT #30586 No test-related changes were observed in clinical biochemistry parameters between the two groups. Urine analysis LPT #28864
LPT #30283
LPT #30586 Urine parameters were not affected by any of the studies. Ophthalmology and auditory system LPT #28864
LPT #30283
LPT #30586 None of the studies had any effect. Cytokines LPT #28864 Observations in dose groups 2, 3 and 4:
IP-10: dose-dependent induction peaking at 6 h after the 4th administration (up to 29-fold induction). Levels were close to normal levels at 24 h (4-5 times higher than controls).
IL-6: dose-dependent induction peaking at 6 h after the 4th administration (up to 8-fold induction). Levels were normal at 24 h.
IL-10: induction peaking at 6 h after the 4th administration (induction up to 3.5-fold) in females only. Levels were normal at 24 h.
TNF-α: low induction peaking at 6 h after the 4th administration (up to 2.5-fold induction). Levels were close to normal levels at 24 h (2-fold in females in the high dose group).
Observations in dose group 4 (high dose) only:
IFN-γ: Induction in males (518%) and females (88%) 6 hours after the 4th dose returned to normal levels by 24 hours. LPT #30283 Observations in all dose groups:
IP-10: dose-dependent induction peaking at 6 h after the 5th administration (up to 41-fold induction). Levels were close to normal levels at 24 h (4-5 times higher than controls).
Observations in high dose groups (groups 3 and 5):
IL-6: induction peaking at 6 h after the 5th administration (up to 20-fold induction). Levels were normal at 24 h.
IFN-α: induction peaking at 6 h after the 5th administration (induction up to 55-fold). Levels were normal at 24 h.
IFN-γ: Induction in group 3 (men 176 times) and group 5 (men 49 times) 6 hours after the 5th dose, returned to normal levels after 24 hours. LPT #30586 No test-related changes were observed between the two groups when measuring cytokines. Addition LPT #28864 Slight increase in C5a 24 hours after the 6th dose in dose group 3 (13/70% in males/females) and dose group 4 (42/90%). LPT #30283 Slight increase (up to 71%) of C5a 24 h after the 5th dose in all dose groups, only in females. Levels normalized after 48 h. LPT #30586 Not defined. Macroscopic pathological studies LPT #28864
LPT #30283
LPT #30586 No macroscopic systemic changes related to the study subjects were noted for any dose group during the treatment or recovery period in any study. Organ masses LPT #28864 A slight increase in liver weight (19%) was observed in males of all dose groups and in females of group 3 (13%). The effect was completely restored after 3 weeks. Histopathological examination revealed no evidence of liver toxicity. This finding has no biological significance since liver weight was within the normal range of biological variation.
Increases in spleen weight (61–107%) were observed in animals of all dose groups. The effects were not fully restored after 3 weeks in animals of the medium and high dose groups. LPT #30283 Increases in spleen weight were dose-dependent and were observed in animals of all dose groups (33–53% in the low dose group and 72–85% in the high dose group). The effects were not fully restored after 3 weeks (19 to 22% in the low dose group and 40 to 74% in the high dose group). LPT #30586 No changes in organ weight were observed between the two groups. Bone marrow LPT #28864
LPT #30283
LPT #30586 The myeloid:erythroid cell ratio was not affected by any of the studies. Histopathology LPT #28864 The examination was limited to controls and groups 3 and 4.
Spleen: lymphoid hyperplasia of the white pulp in animals of groups 3 and 4.
Thymus: pronounced atrophy in female animals only in animals of the 3rd and 4th groups.
All effects were fully restored after 3 weeks.
Lymphoid hyperplasia was not observed in any other organ. The observed effects in lymphoid organs are related to the pharmacological mechanism of action and are not considered an adverse reaction. LPT #30283 The examination was limited to controls and groups 3 and 5.
Spleen: lymphoid hyperplasia of the white pulp in animals of the high-dose groups. All effects were completely restored after 3 weeks.
Lymphoid hyperplasia was not observed in any other organ. The observed effects in lymphoid organs are due to the pharmacological mechanism of action and are not considered an adverse reaction. LPT #30586 Histopathological examination was limited to the primary study animals treated with 20 μg RBL008.1/animal + 40 μg L2 liposomes/animal (Group 2). No morphological lesions were observed that were considered to be related to the administration of RBL008.1 in combination with L2 liposomes. All observations were considered to be within the normal range of background changes that can be observed in untreated mice of this age and strain.

Genotoxicity

The components of RNA (LIP) products (lipids and RNA) are expected to have genotoxic potential. No impurities or components of the delivery system require genotoxicity testing. In accordance with the recommendations of the ICH Guideline on Nonclinical Safety Evaluation of Biotechnological Pharmaceuticals S6 (R1) (June 2011), genotoxicity studies are not planned.

Carcinogenicity

The RNA itself and the lipids used as carriers do not have carcinogenic or tumorigenic effects. According to ICH S1A, long-term carcinogenicity studies are not required in cases where there is no cause for concern based on laboratory and toxicological studies and if long-term use of the medicinal product is not expected.

Reproductive and developmental toxicity

Macroscopic and microscopic examinations of male and female reproductive tissues were included in repeated dose toxicity studies in mice treated with RNA(LIP). No effects were observed in these studies, so no specific fertility or developmental toxicity studies will be conducted prior to initiation of phase 1 studies with RNA(LIP) vaccines. No direct cytotoxic effects on reproductive tissues are expected from RNA(LIP), consistent with experience with other cancer vaccines showing no effects on reproduction or development. Since effects on fertility cannot be excluded, women of childbearing potential will be advised to use effective contraception during treatment. No further studies on long-term or reproductive toxicity are planned at this time.

Local tolerance

In accordance with ICH recommendation, local tolerability testing was assessed in a GLP repeated dose toxicity study for intravenous injection. No signs of local intolerance were observed during the studies.

Other toxicity studies

Antigenicity

Due to the rapid extracellular degradation of IVT-RNA within seconds to minutes, formation of anti-drug antibodies (ADA) is not expected. Therefore, no specific antigenicity tests for antibody induction are planned.

Immunotoxicity

Since the intent is to activate the immune system with RNA products (LIPs), special attention was paid to immunotoxicological parameters to exclude unintended activation or suppression. Immunotoxicology testing was performed in both 6-week repeated-dose toxicity studies (LPT #28864 and 30283). In addition to monitoring serum cytokine levels, the following relevant parameters were considered for the assessment of immunotoxicity: body weight, body temperature, lymph organ weights, lymph organ gross and histopathology, absolute and relative differential blood count, total serum protein, albumin/immunoglobulin ratio, bone marrow myeloid/erythroid ratio, coagulation parameters.

Hematology

Decreases in lymphocytes, white blood cells (primarily due to decreased lymphocytes), and platelets were observed in all treatment groups on day 44 of testing, approximately 24 hours after the 8th injection in both studies. All effects were completely reversed after two weeks. The results are shown in Table 15 and Table 16 for LPT studies #28864 and 30283, respectively.

Table 15. Hematological data (LPT No. 28864)

Samples for hematology determination were taken on day 44 of testing (approximately 24 hours after the 8th injection).

Changes in hematological parameters compared to the control group (mean values) at the end of the treatment period (44th day of testing) [%] Parameter Group 2
15 mcg/animal Group 3 30 mcg/animal Group 4 60 mcg/animal males females males females males females White blood cells (WBC) –72 ** –60 ** –78 ** –61 ** –68 ** –69 ** Neutrophil granulocytes (neutral) -rel. +100 +50 +163 +58 +74 +36 -abs. -46 * -30 -44* -29 -47* -51 Lymphocytes (Lym) -rel. -16 -10 -19 No -10 No -abs. –77 ** –64 ** –82 ** –65 ** –71 ** –71 ** Monocytes (mono) -rel. +89 -21 +68 No -24 -29 -abs. –44 ** -64 -59 ** -43 –72 ** -79 * Eosinophilic granulocytes (Eos) -rel. +41 +68 +90 +45 -34 -23 -abs. -59 ** -43 -59 ** -38 –78 ** -73 * Large unstained cells (LUC) -rel. +385 +315 +257 +239 +259 +275 -abs. +59 +28 -25 No +13 No Basophilic granulocytes (Baso) -rel. +255 +144 +445 +50 +273 +80 Platelets (PCT) –35 ** –38 ** –32 ** –39 ** –46 ** –39 **

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

Table 16. Hematological data (LPT No. 30283)

Samples for hematology determination were taken on day 44 of testing (approximately 24 hours after the 8th injection).

Changes in hematological parameters compared to the control group (mean values) at the end of the treatment period (44th day of testing) [%] Parameter Group 2:
5 µg/animal (RNA set 1)
+ 9 mcg/animal (liposomes) Group 3:
50 µg/animal (RNA set 1)
+ 90 mcg/animal (liposomes) Group 4:
5 µg/animal (RNA kit 2)
+ 9 mcg/animal (liposomes) Group 5:
50 µg/animal (RNA kit 2)
+ 90 mcg/animal (liposomes) males females males females males females males females White blood cells (WBC) -15 -54 -65 -39 –51 ** -48 –71 ** -57 ** Platelets (PCT) -18 * No –48 ** –43 ** –16 ** No –45 ** –46 ** Reticulocytes (Ret) No –30 ** –27 ** –34 ** –17 ** –25 ** –27 ** –38 ** Neutrophilic
granulocytes (neutral) -rel. +125 +24 +199 +32 +81 +22 +234 +72 Lymphocytes
(Lim) -rel. -11 -5 -21 -8 -11 -4 -20 -9 -abs. -21 -56 * -73 -43 -56 * -49 * –89 ** –61 * Monocytes (mono) -rel. No No -12 -74 No No -41 -47 -abs. No No -74 * -93 * No No –76 ** -71 Large unstained cells (LUC) -rel. +86 +29 +516 +640 +194 +195 +320 +295 -abs. No No +80 * +336 * +30 +50 No +71 * Basophilic granulocytes
(Baso) -rel. No No +540 +290 No No +300 +90 Platelets (PCT) -18 * No –48 ** –43 ** –16 ** No –45 ** –46 **

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

Determination of cytokines

The excretion of the following cytokines was analyzed in repeated dose toxicity studies: IL-1β, IL2, 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 of LPT #28864 in mice, a dose-dependent and test-subject-related increase in the cytokine IP-10 was found. IP-10 levels increased transiently and were maximal at 6 hours after the 4th injection. At 24 hours, levels were still significantly elevated compared to control levels, but had almost returned to normal levels. Compared to the control group, IP-10 showed a maximal induction of 28- and 16-fold (in males and females, respectively) at 6 hours. Significant increases were also observed for TNF-α (females only, groups 2, 3, and 4), IL-10 (females only, groups 3 and 4), IL-6 (males, group 4), and IFN-γ (male, group 4). The maximum induction was 3-fold for TNF-α, 4-fold for IL-10, 7- and 8-fold for IL-6, and 6- and 2-fold for IFN-γ. All effects were completely reversible after 24 h (except for TNF-α levels in group 4 females). The results are summarized in Table 17.

Table 17. Plasma cytokine levels (LPT #28864)

Samples for cytokine determination were collected 6 and 24 h after the 4th injection.

Test item-related changes in cytokine levels (mean values), expressed as x-fold increase over control group level, if applicable Cytokine Time Group 2
15 mcg/animal Group 3 30 mcg/animal Group 4 60 mcg/animal males females males females males females IL-6 6 h 2x 2x 3x 7x** 7x** 8x** Il-10 6 h No 2x No 4x** No 4x** IP-10 6 h 17x** 11x** 22x** 17x** 29x** 17x** 24 h 4x** 4x** 4x** 4x** 5x** 5x** IFN-γ 6 h No No No No 6x** 2x TNF-α 6 h 1x 2x* 1x 2x** 1x 2x** 24 h No No No 1x No 2x**

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

For cytokine determination in LPT study #30283, serum samples were collected at 6 and 24 h after the 5th administration. Increases in serum levels of IL-2, IL-6, IP-10, IFN-α and IFN-γ were found (Table 18). A clear dose-dependent induction was observed for IL-6. For IFNγ, induction was observed after high dose treatment (statistically significant at p ≤ 0.01), being more pronounced in male animals. For IFNα, induction was observed after low dose treatment and after high dose treatment (statistically significant at p ≤ 0.01 or p ≤ 0.05), being more pronounced in female animals in group 5 (RNA set 2, high dose). Induction of all the above cytokines was reduced at 24 h after administration.

A relatively low but dose-dependent induction of IL-2 was observed in male and female animals at 6 and 24 h after treatment with low or high doses.

A significant dose-dependent effect was found for IP10 at all dose levels in male and female animals compared to controls 6 hours after high dose treatment. IP10 levels were still elevated 24 hours after administration compared to controls at all dose levels. This induction of IP-10 reflects the intended pharmacological effect and is not considered an adverse immunotoxicological event.

Table 18. Plasma cytokine levels (LPT #30283)

Samples for cytokine determination were collected 6 and 24 h after the 5th injection.

Test item-related changes in cytokine levels (mean values), expressed as x-fold increase over control group level, if applicable, or as increase only Cytokine Time Group 2:
5 µg/animal (RNA set 1)
+ 9 mcg/animal (liposomes) Group 3:
50 µg/animal (RNA set 1)
+ 90 mcg/animal (liposomes) Group 4:
5 µg/animal (RNA kit 2)
+ 9 mcg/animal (liposomes) Group 5:
50 µg/animal (RNA kit 2)
+ 90 mcg/animal (liposomes) Males females males females males females males females IL-2 6 h increase. 1x increase. 2x No 1x increase ** 31x 24 h No increase. increase * increase. increase. increase. increase. increase. IL-6 6 h 2x increase. 22x** increase ** 2x increase. 21x** increase ** 24 h No No No 2x 1x 3x 1x 2x IP-10 6 h 18x* 11x** 41x** 25x** 20x** 14x** 34x** 22x** 24 h 4x* 3x** 6x** 5x** 3x** 3x* 4x** 4x** IFN-α 6 h 5x* 8x* 15x** 28x** 8x* 9x 13x** 55x** 24 h No No No No No No No No IFN-γ 6 h 4x increase. 176x** increase ** 2x increase. 49x** increase ** 24 h No No No No No No No No

increase: An obvious 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.

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

Cytokine determination was also performed in a study comparing L1 and L2 liposomes (LPT #30586). Here, cytokines were analyzed 6 and 24 hours after the 4th immunization. No task-related changes were observed between groups.

Discussion and conclusion

Treatment with RNA-(LIP) is well tolerated in mice as demonstrated for a number of antigen-coding RNAs evaluated in three different repeat dose toxicity studies (LPT #28864, 30283, and 30586). Overall, treatment with up to eight intravenous injections was well tolerated, also in animals in the high dose groups. No test subject-related premature deaths were observed in Studies #30283 and 30586. One animal died prematurely following test subject administration in Study #28864. Since no results were observed in the low dose group of Study #30283, the NOAEL was achieved at a dose of 5 μg total RNA per animal (i.e., approximately 0.2 mg/kg body weight in mice). In addition, vaccination with RNA products (LIP) was also very well tolerated in a non-GLP pharmacological study in twelve cynomolgus macaques (no clinical observation data).

Toxicological evaluation of RNA-(LIP) in repeated dose toxicity studies in mice revealed effects including transient cytokine induction, hematological changes, and liver enzyme elevations that could be attributed to the test subject. The observed effects were primarily induction of the cytokines IP-10, IFN-α, IFN-γ, and IL-6 in the in vivo studies described herein and in the in vitro studies described and discussed above.

Notably, none of the proinflammatory cytokines such as TNF-α, IFN-γ or IL-2 were excessively elevated in mice. However, in the cynomolgus monkey study, at least one animal showed a high transient induction of IL-6 (1076 pg/mL). IL-6 induction was also observed in mice in a dose-dependent manner, but to a lesser extent. IL-6, along with other cytokines, will be closely monitored throughout the clinical trial and directly assayed in patients.

Effects such as lymphopenia and elevated liver enzymes have also been observed following treatment with plasmid lipoplexes and TLR activation in mice and monkeys and are usually observed as secondary effects caused by IFN-α secretion, which are commonly described for patients treated with recombinant IFN-α, which has been on the market for many years for the treatment of a number of oncological and non-oncological diseases.

The observed changes in liver parameters in the high dose group of mice suggest that the liver may be a target of toxicity at higher doses of liposome-containing RNA. Changes include increased liver weight, increased plasma GLDH, LDH, ASAT and ALAT levels. These changes are considered minor and were not observed in animals in the recovery group, suggesting full recovery of effects within at least three weeks. Furthermore, histopathology did not reveal liver toxicity. In the cynomolgus monkey, biochemical parameters of liposome-treated and test-subject-treated animals compared to controls were considered to be within the range of normal biological variability. Increases in CK observed for individual animals in groups 4, 5 or 6 compared to controls on test days 9, 16 or 23 were primarily explained by the CK-MM fraction and were considered stress-related.

The modest increase in liver parameters in mice may be a response to immunomodulatory effects that may be induced by phagocytosis of RNA-(LIP) by liver target cells such as Kupffer cells. Unlike the effects observed in the mouse spleen (lymphoid hyperplasia), this does not result in leukocyte recruitment to the liver, suggesting that the sought-after pharmacological effects such as TLR activation and lymphocyte trafficking are restricted to the lymphoid organ.

Complement activation has been previously reported for liposome-containing substances. For RNA-(LIP) vaccination, slightly elevated C5a levels were observed in female mice, but this was considered an event of low biological significance. Furthermore, mice are not considered a good model for extrapolating complement effects to humans.

Overall, the immunological responses observed in all three RNA-(LIP) repeated dose toxicity studies (LPT# 28864, 30283 and 30586) suggest a consistent pattern of increased spleen weight, cytokine/chemokine activation and lymphocyte trafficking. This reflects the induction of the intended pharmacological events and highlights the importance of mice as a valid test model for toxicity studies. In the interim study LPT# 30583 evaluating the toxicity of a pH-adapted liposomal formulation of L2, no significant differences were observed between the two liposomal formulations. This trial will use pH-adapted L4 liposomes, which are semi-concentrated L2 liposomes. No further binding studies were planned as the same characteristics were given as for the L2 liposomes.

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

On days 1, 8, 15 and 22, A2/DR1 mice were immunized with 200 μl of RNA(Lip) solution at a concentration of 0.15 mg/ml, which corresponds to 30 μg of RNA(Lip).

Splenocytes were obtained from immunized A2/DR1 mice five days after the last of four RNA (Lip) injections, and the induction of antigen-specific T cells was determined in vivo by ELISPOT assay. To test immunogenicity, isolated splenocytes were restimulated with a peptide pool (15 mers overlapping by 11 amino acids) covering the corresponding human proteins, KLK2, KLK3 (PSA), ACPP (PAP), NKX3-1, or HOXB13. The results are shown in Fig. 20. The number of IFN-γ+ spots induced by KLK2-, ACPP-, and HOXB13-encoding RNA (Lip) was statistically increased compared with restimulation with the control peptide CMV pp65 according to an unpaired t-test (P = 0.0056; P > 0.0001; P = 0.0095). In all immunized mice, restimulation of splenocytes with control peptides P2/P16/P17 also resulted in a large number of spots, indicating successful immunization. In all ELISPOT plates, the negative control medium induced only minimal spots, regardless of the animal from which the splenocytes were obtained.

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LIST OF SEQUENCES

<110> BIONTECH RNA PHARMACEUTICALS GMBH

<120> THERAPEUTIC RNA FOR PROSTATE CANCER

<130> 674-273 PCT2

<150> PCT/EP2019/056185

<151> 2019-03-12

<160> 31

<170> PatentIn version 3.5

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<213> Artificial Sequence

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gugugcuaga gcuuacucug agaaggugac agaguucaug uugugugcug ggcucuggac 660

aggugguaaa gacacuugug ggggugauuc ugggggucca cuugucugua auggugugcu 720

ucaagguauc acaucauggg gcccugagcc augugcccug ccugaaaagc cugcugugua 780

caccaaggug gugcauuacc ggaaguggau caaggacacc aucgcagcca accccggagg 840

auccgguggu ggcggcagcg gcggcaagaa gcaguacauc aaggccaaca gcaaguucau 900

cggcaucacc gagcugaaga agcugggagg gggcaaacgg ggaggcggca aaaagaugac 960

caacagcgug gacgacgccc ugaucaacag caccaagauc uacagcuacu uccccagcgu 1020

gaucagcaaa gugaaccagg gcgcucaggg caagaaacug ggcucuagcg gagggggagg 1080

cucuccuggc gggggaucua gcaucguggg aauuguggca ggacuggcag ugcuggccgu 1140

gguggugauc ggagccgugg uggcuaccgu gaugugcaga cggaagucca gcggaggcaa 1200

gggcggcagc uacagccagg ccgccagcuc ugauagcgcc cagggcagcg acgugucacu 1260

gacagccuag uaacucgagc ugguacugca ugcacgcaau gcuagcugcc ccuuucccgu 1320

ccuggguacc ccgagucucc cccgaccucg ggucccaggu augcucccac cuccaccugc 1380

cccacucacc accucugcua guuccagaca ccucccaagc acgcagcaau gcagcucaaa 1440

acgcuuagcc uagccacacc cccacgggaa acagcaguga uuaaccuuua gcaauaaacg 1500

aaaguuuaac uaagcuauac uaaccccagg guuggucaau uucgugccag ccacaccgag 1560

accuggucca gagucgcuag ccgcgucgcu aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620

gcauaugacu aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa aaaaaaaaaa 1700

<210> 5

<211> 261

<212> PRT

<213> Artificial Sequence

<220>

<223>PSA

<400> 5

Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly

1 5 10 15

Ala Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys Glu

20 25 30

Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg Gly Arg Ala

35 40 45

Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala

50 55 60

His Cys Ile Arg Asn Lys Ser Val Ile Leu Leu Gly Arg His Ser Leu

65 70 75 80

Phe His Pro Glu Asp Thr Gly Gln Val Phe Gln Val Ser His Ser Phe

85 90 95

Pro His Pro Leu Tyr Asp Met Ser Leu Leu Lys Asn Arg Phe Leu Arg

100 105 110

Pro Gly Asp Asp Ser Ser His Asp Leu Met Leu Leu Arg Leu Ser Glu

115 120 125

Pro Ala Glu Leu Thr Asp Ala Val Lys Val Met Asp Leu Pro Thr Gln

130 135 140

Glu Pro Ala Leu Gly Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile

145 150 155 160

Glu Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu

165 170 175

His Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val

180 185 190

Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly Gly Lys Ser Thr

195 200 205

Cys Ser Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln

210 215 220

Gly Ile Thr Ser Trp Gly Ser Glu Pro Cys Ala Leu Pro Glu Arg Pro

225 230 235 240

Ser Leu Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp Thr

245 250 255

Ile Val Ala Asn Pro

260

<210> 6

<211> 405

<212> PRT

<213> Artificial Sequence

<220>

<223>PSA Fusion

<400> 6

Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly

1 5 10 15

Ala Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys Glu

20 25 30

Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg Gly Arg Ala

35 40 45

Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala

50 55 60

His Cys Ile Arg Asn Lys Ser Val Ile Leu Leu Gly Arg His Ser Leu

65 70 75 80

Phe His Pro Glu Asp Thr Gly Gln Val Phe Gln Val Ser His Ser Phe

85 90 95

Pro His Pro Leu Tyr Asp Met Ser Leu Leu Lys Asn Arg Phe Leu Arg

100 105 110

Pro Gly Asp Asp Ser Ser His Asp Leu Met Leu Leu Arg Leu Ser Glu

115 120 125

Pro Ala Glu Leu Thr Asp Ala Val Lys Val Met Asp Leu Pro Thr Gln

130 135 140

Glu Pro Ala Leu Gly Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile

145 150 155 160

Glu Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu

165 170 175

His Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val

180 185 190

Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly Gly Lys Ser Thr

195 200 205

Cys Ser Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln

210 215 220

Gly Ile Thr Ser Trp Gly Ser Glu Pro Cys Ala Leu Pro Glu Arg Pro

225 230 235 240

Ser Leu Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp Thr

245 250 255

Ile Val Ala Asn Pro Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys

260 265 270

Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu

275 280 285

Lys Lys Leu Gly Gly Gly Lys Arg Gly Gly Gly Lys Lys Met Thr Asn

290 295 300

Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr Ser Tyr Phe

305 310 315 320

Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln Gly Lys Lys Leu

325 330 335

Gly Ser Ser Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Ser Ile Val

340 345 350

Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly Ala

355 360 365

Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly

370 375 380

Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp

385 390 395 400

Val Ser Leu Thr Ala

405

<210> 7

<211> 783

<212> RNA

<213> Artificial Sequence

<220>

<223>PSA CDS

<400> 7

augugggucc cgguugucuu ccucacccug uccgugacgu ggauuggugc ugcaccccuc 60

auccugucuc ggauuguggg aggcugggag ugcgagaagc auucccaacc cuggcaggug 120

cuuguggccu cucguggcag ggcagucugc ggcgguguuc uggugcaccc ccaguggguc 180

cucacagcug cccacugcau caggaacaaa agcgugaucu ugcugggucg gcacagccug 240

uuucauccug aagacacagg ccagguauuu caggucagcc acagcuuccc acacccgcuc 300

uacgauauga gccuccugaa gaaucgauuc cucaggccag gugaugacuc cagccacgac 360

cucaugcugc uccgccuguc agagccugcc gagcucacgg augcugugaa ggucauggac 420

cugcccaccc aggagccagc acuggggacc accugcuacg ccucaggcug gggcagcauu 480

gaaccagagg aguucuugac cccaaagaaa cuucagugug uggaccucca uguuauuucc 540

aaugacgugu gugcgcaagu ucacccucag aaggugacca aguucaugcu gugugcugga 600

cgcuggacag ggggcaaaag caccugcucg ggugauucug ggggcccacu ugucuguaau 660

ggugugcuuc aagguaucac gucauggggc agugaaccau gugcccugcc cgaaaggccu 720

ucccuguaca ccaagguggu gcauuaccgg aaguggauca aggacaccau cguggccaac 780

ccc 783

<210> 8

<211> 1700

<212> RNA

<213> Artificial Sequence

<220>

<223>PSA RNA

<400> 8

gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccaugugggu 60

cccgguuguc uuccucaccc uguccgugac guggauuggu gcugcacccc ucauccuguc 120

ucggauugug ggaggcuggg agugcgagaa gcauucccaa cccuggcagg ugcuuguggc 180

cucucguggc agggcagucu gcggcggugu ucuggugcac ccccaguggg uccucacagc 240

ugcccacugc aucaggaaca aaagcgugau cuugcugggu cggcacagcc uguuucaucc 300

ugaagacaca ggccagguau uucaggucag ccacagcuuc ccacacccgc ucuacgauau 360

gagccuccug aagaaucgau uccucaggcc aggugaugac uccagccacg accucaugcu 420

gcuccgccug ucagagccug ccgagcucac ggaugcugug aaggucaugg accugcccac 480

ccaggagcca gcacugggga ccaccugcua cgccucaggc uggggcagca uugaaccaga 540

ggaguucuug accccaaaga aacuucagug uguggaccuc cauguuauuu ccaaugacgu 600

gugugcgcaa guucacccuc agaaggugac caaguucaug cugugugcug gacgcuggac 660

aggggggcaaa agcaccugcu cgggugauuc ugggggccca cuugucugua auggugugcu 720

ucaagguauc acgucauggg gcagugaacc augugcccug cccgaaaggc cuucccugua 780

caccaaggug gugcauuacc ggaaguggau caaggacacc aucguggcca accccggagg 840

auccgguggu ggcggcagcg gcggcaagaa gcaguacauc aaggccaaca gcaaguucau 900

cggcaucacc gagcugaaga agcugggagg gggcaaacgg ggaggcggca aaaagaugac 960

caacagcgug gacgacgccc ugaucaacag caccaagauc uacagcuacu uccccagcgu 1020

gaucagcaaa gugaaccagg gcgcucaggg caagaaacug ggcucuagcg gagggggagg 1080

cucuccuggc gggggaucua gcaucguggg aauuguggca ggacuggcag ugcuggccgu 1140

gguggugauc ggagccgugg uggcuaccgu gaugugcaga cggaagucca gcggaggcaa 1200

gggcggcagc uacagccagg ccgccagcuc ugauagcgcc cagggcagcg acgugucacu 1260

gacagccuag uaacucgagc ugguacugca ugcacgcaau gcuagcugcc ccuuucccgu 1320

ccuggguacc ccgagucucc cccgaccucg ggucccaggu augcucccac cuccaccugc 1380

cccacucacc accucugcua guuccagaca ccucccaagc acgcagcaau gcagcucaaa 1440

acgcuuagcc uagccacacc cccacgggaa acagcaguga uuaaccuuua gcaauaaacg 1500

aaaguuuaac uaagcuauac uaaccccagg guuggucaau uucgugccag ccacaccgag 1560

accuggucca gagucgcuag ccgcgucgcu aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620

gcauaugacu aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa aaaaaaaaaa 1700

<210> 9

<211> 418

<212> PRT

<213> Artificial Sequence

<220>

<223> PAP

<400> 9

Met Arg Ala Ala Pro Leu Leu Leu Ala Arg Ala Ala Ser Leu Ser Leu

1 5 10 15

Gly Phe Leu Phe Leu Leu Phe Phe Trp Leu Asp Arg Ser Val Leu Ala

20 25 30

Lys Glu Leu Lys Phe Val Thr Leu Val Phe Arg His Gly Asp Arg Ser

35 40 45

Pro Ile Asp Thr Phe Pro Thr Asp Pro Ile Lys Glu Ser Ser Trp Pro

50 55 60

Gln Gly Phe Gly Gln Leu Thr Gln Leu Gly Met Glu Gln His Tyr Glu

65 70 75 80

Leu Gly Glu Tyr Ile Arg Lys Arg Tyr Arg Lys Phe Leu Asn Glu Ser

85 90 95

Tyr Lys His Glu Gln Val Tyr Ile Arg Ser Thr Asp Val Asp Arg Thr

100 105 110

Leu Met Ser Ala Met Thr Asn Leu Ala Ala Leu Phe Pro Pro Glu Gly

115 120 125

Val Ser Ile Trp Asn Pro Ile Leu Leu Trp Gln Pro Ile Pro Val His

130 135 140

Thr Val Pro Leu Ser Glu Asp Gln Leu Leu Tyr Leu Pro Phe Arg Asn

145 150 155 160

Cys Pro Arg Phe Gln Glu Leu Glu Ser Glu Thr Leu Lys Ser Glu Glu

165 170 175

Phe Gln Lys Arg Leu His Pro Tyr Lys Asp Phe Ile Ala Thr Leu Gly

180 185 190

Lys Leu Ser Gly Leu His Gly Gln Asp Leu Phe Gly Ile Trp Ser Lys

195 200 205

Val Tyr Asp Pro Leu Tyr Cys Glu Ser Val His Asn Phe Thr Leu Pro

210 215 220

Ser Trp Ala Thr Glu Asp Thr Met Thr Lys Leu Arg Glu Leu Ser Glu

225 230 235 240

Leu Ser Leu Leu Ser Leu Tyr Gly Ile His Lys Gln Lys Glu Lys Ser

245 250 255

Arg Leu Gln Gly Gly Val Leu Val Asn Glu Ile Leu Asn His Met Lys

260 265 270

Arg Ala Thr Gln Ile Pro Ser Tyr Lys Lys Leu Ile Met Tyr Ser Ala

275 280 285

His Asp Thr Thr Val Ser Gly Leu Gln Met Ala Leu Asp Val Tyr Asn

290 295 300

Gly Leu Leu Pro Pro Tyr Ala Ser Cys His Leu Thr Glu Leu Tyr Phe

305 310 315 320

Glu Lys Gly Glu Tyr Phe Val Glu Met Tyr Tyr Arg Asn Glu Thr Gln

325 330 335

His Glu Pro Tyr Pro Leu Met Leu Pro Gly Cys Ser Pro Ser Cys Pro

340 345 350

Leu Glu Arg Phe Ala Glu Leu Val Gly Pro Val Ile Pro Gln Asp Trp

355 360 365

Ser Thr Glu Cys Met Thr Thr Asn Ser His Gln Val Leu Lys Val Ile

370 375 380

Phe Ala Val Ala Phe Cys Leu Ile Ser Ala Val Leu Met Val Leu Leu

385 390 395 400

Phe Ile His Ile Arg Arg Gly Leu Cys Trp Gln Arg Glu Ser Tyr Gly

405 410 415

Asn Ile

<210> 10

<211> 562

<212> PRT

<213> Artificial Sequence

<220>

<223> PAP fusion

<400> 10

Met Arg Ala Ala Pro Leu Leu Leu Ala Arg Ala Ala Ser Leu Ser Leu

1 5 10 15

Gly Phe Leu Phe Leu Leu Phe Phe Trp Leu Asp Arg Ser Val Leu Ala

20 25 30

Lys Glu Leu Lys Phe Val Thr Leu Val Phe Arg His Gly Asp Arg Ser

35 40 45

Pro Ile Asp Thr Phe Pro Thr Asp Pro Ile Lys Glu Ser Ser Trp Pro

50 55 60

Gln Gly Phe Gly Gln Leu Thr Gln Leu Gly Met Glu Gln His Tyr Glu

65 70 75 80

Leu Gly Glu Tyr Ile Arg Lys Arg Tyr Arg Lys Phe Leu Asn Glu Ser

85 90 95

Tyr Lys His Glu Gln Val Tyr Ile Arg Ser Thr Asp Val Asp Arg Thr

100 105 110

Leu Met Ser Ala Met Thr Asn Leu Ala Ala Leu Phe Pro Pro Glu Gly

115 120 125

Val Ser Ile Trp Asn Pro Ile Leu Leu Trp Gln Pro Ile Pro Val His

130 135 140

Thr Val Pro Leu Ser Glu Asp Gln Leu Leu Tyr Leu Pro Phe Arg Asn

145 150 155 160

Cys Pro Arg Phe Gln Glu Leu Glu Ser Glu Thr Leu Lys Ser Glu Glu

165 170 175

Phe Gln Lys Arg Leu His Pro Tyr Lys Asp Phe Ile Ala Thr Leu Gly

180 185 190

Lys Leu Ser Gly Leu His Gly Gln Asp Leu Phe Gly Ile Trp Ser Lys

195 200 205

Val Tyr Asp Pro Leu Tyr Cys Glu Ser Val His Asn Phe Thr Leu Pro

210 215 220

Ser Trp Ala Thr Glu Asp Thr Met Thr Lys Leu Arg Glu Leu Ser Glu

225 230 235 240

Leu Ser Leu Leu Ser Leu Tyr Gly Ile His Lys Gln Lys Glu Lys Ser

245 250 255

Arg Leu Gln Gly Gly Val Leu Val Asn Glu Ile Leu Asn His Met Lys

260 265 270

Arg Ala Thr Gln Ile Pro Ser Tyr Lys Lys Leu Ile Met Tyr Ser Ala

275 280 285

His Asp Thr Thr Val Ser Gly Leu Gln Met Ala Leu Asp Val Tyr Asn

290 295 300

Gly Leu Leu Pro Pro Tyr Ala Ser Cys His Leu Thr Glu Leu Tyr Phe

305 310 315 320

Glu Lys Gly Glu Tyr Phe Val Glu Met Tyr Tyr Arg Asn Glu Thr Gln

325 330 335

His Glu Pro Tyr Pro Leu Met Leu Pro Gly Cys Ser Pro Ser Cys Pro

340 345 350

Leu Glu Arg Phe Ala Glu Leu Val Gly Pro Val Ile Pro Gln Asp Trp

355 360 365

Ser Thr Glu Cys Met Thr Thr Asn Ser His Gln Val Leu Lys Val Ile

370 375 380

Phe Ala Val Ala Phe Cys Leu Ile Ser Ala Val Leu Met Val Leu Leu

385 390 395 400

Phe Ile His Ile Arg Arg Gly Leu Cys Trp Gln Arg Glu Ser Tyr Gly

405 410 415

Asn Ile Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys Lys Gln Tyr

420 425 430

Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu

435 440 445

Gly Gly Gly Lys Arg Gly Gly Gly Lys Lys Met Thr Asn Ser Val Asp

450 455 460

Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr Ser Tyr Phe Pro Ser Val

465 470 475 480

Ile Ser Lys Val Asn Gln Gly Ala Gln Gly Lys Lys Leu Gly Ser Ser

485 490 495

Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Ser Ile Val Gly Ile Val

500 505 510

Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val Val Ala

515 520 525

Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr

530 535 540

Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser Leu

545 550 555 560

Thru Ala

<210> 11

<211> 1254

<212> RNA

<213> Artificial Sequence

<220>

<223> PAP CDS

<400> 11

augagagcug cuccccugcu gcuggcaaga gcugcuucuc ugucucuggg auuucuguuu 60

cugcuguuuu ucuggcugga uagaucugug cuggcaaaag aacugaaauu ugugacccug 120

guguuuagac acggagacag aucucccauu gauaccuuuc ccaccgaucc caucaaagaa 180

ucuucuuggc cacagggauu uggacagcug acccagcugg gaauggagca gcacuacgaa 240

cugggagaau acaucagaaa aagauacaga aaauuucuga augaaucuua caaacacgaa 300

cagguguaca ucagaucuac cgauguggau agaacccuga ugucugcaau gaccaaucug 360

gcugcucugu uucccccaga aggagugagc aucuggaauc ccauccugcu guggcagccc 420

aucccugugc acaccgugcc ucugucugaa gaucagcugc uguaccugcc uuuuagaaau 480

ugcccaagau uucaggaacu ugaaucugaa acccugaaau cugaagaauu ucagaaaaga 540

cugcacccuu acaaagauuu uaucgcaacc cugggaaaac ugucuggacu gcacggacag 600

gaucucuuug gaauuugguc aaaaguguac gauccucugu acugugaauc ugugcacaau 660

uuuacccugc ccucuugggc aaccgaagau accaugacca aacugagaga acugucugaa 720

cugucucugc ugucucugua cggaauccac aaacagaaag aaaaaucaag acugcaggga 780

ggagugcugg ugaaugaaau ccugaaucac augaaaagag caacccagau cccaucuuac 840

aagaagcuga ucauguacuc ugcucacgau accacagugu cuggacugca gauggcucug 900

gauguguaca auggacugcu gccucccuac gcuucuugcc accugaccga acuguacuuu 960

gaaaaaggag aauacuuugu ggaaauguac uacagaaaug aaacccagca cgaaccuuac 1020

ccccugaugc ugccuggaug cucucccucu ugcccucugg aaagauuugc ugaacuggug 1080

ggaccuguga ucccucagga uugguccaca gaaugcauga ccaccaauuc ucaccaggug 1140

cugaaaguga ucuuugcugu ggcuuuuugc cugaucucug cugugcugau ggugcugcug 1200

uuuauccaca ucaggagagg acugugcugg cagagagaau cuuacggaaa uauc 1254

<210> 12

<211> 2171

<212> RNA

<213> Artificial Sequence

<220>

<223> PAP RNA

<400> 12

gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccaugagagc 60

ugcuccccug cugcuggcaa gagcugcuuc ucugucucug ggauuucugu uucugcuguu 120

uuucuggcug gauagaucug ugcuggcaaa agaacugaaa uuugugaccc ugguguuuag 180

acacggagac agaucuccca uugauaccuu ucccaccgau cccaucaaag aaucuucuug 240

gccacaggga uuuggacagc ugacccagcu gggaauggag cagcacuacg aacugggaga 300

auacaucaga aaaagauaca gaaaauuucu gaaugaaucu uacaaacacg aacaggugua 360

caucagaucu accgaugugg auagaacccu gaugucugca augaccaauc uggcugcucu 420

guuuccccca gaaggaguga gcaucuggaa ucccauccug cuguggcagc ccaucccugu 480

gcacaccgug ccucugucug aagaucagcu gcuguaccug ccuuuuagaa auugcccaag 540

auuucaggaa cuugaaucug aaacccugaa aucugaagaa uuucagaaaa gacugcaccc 600

uuacaaagau uuuaucgcaa cccugggaaa acugucugga cugcacggac aggaucucuu 660

uggaauuugg ucaaaagugu acgauccucu guacugugaa ucugugcaca auuuuacccu 720

gcccucuugg gcaaccgaag auaccaugac caaacugaga gaacugucug aacugucucu 780

gcugucucug uacggaaucc acaaacagaa agaaaaauca agacugcagg gaggagugcu 840

ggugaaugaa auccugaauc acaugaaaag agcaacccag aucccaucuu acaagaagcu 900

gaucauguac ucugcucacg auaccacagu gucuggacug cagauggcuc uggaugugua 960

caauggacug cugccucccu acgcuucuug ccaccugacc gaacuguacu uugaaaaagg 1020

agaauacuuu guggaaaugu acuacagaaa ugaaacccag cacgaaccuu acccccugau 1080

gcugccugga ugcucucccu cuugcccucu ggaaagauuu gcugaacugg ugggaccugu 1140

gaucccucag gauuggucca cagaaugcau gaccaccaau ucucaccagg ugcugaaagu 1200

gaucuuugcu guggcuuuuu gccugaucuc ugcugugcug auggugcugc uguuuaucca 1260

caucaggaga ggacugugcu ggcagagaga aucuuacgga aauaucggag gauccggugg 1320

uggcggcagc ggcggcaaga agcaguacau caaggccaac agcaaguuca ucggcaucac 1380

cgagcugaag aagcugggag ggggcaaacg gggaggcggc aaaaagauga ccaacagcgu 1440

ggacgacgcc cugaucaaca gcaccaagau cuacagcuac uuccccagcg ugaucagcaa 1500

agugaaccag ggcgcucagg gcaagaaacu gggcucuagc ggagggggag gcucuccugg 1560

cggggggaucu agcaucgugg gaauuguggc aggacuggca gugcuggccg ugguggugau 1620

cggagccgug guggcuaccg ugaugugcag acggaagucc agcggaggca agggcggcag 1680

cuacagccag gccgccagcu cugauagcgc ccagggcagc gacgugucac ugacagccua 1740

guaacucgag cugguacugc augcacgcaa ugcuagcugc cccuuucccg uccuggguac 1800

cccgagucuc ccccgaccuc gggucccagg uaugcuccca ccuccaccug cccccacucac 1860

caccucugcu aguuccagac accucccaag cacgcagcaa ugcagcucaa aacgcuuagc 1920

cuagccacac ccccacggga aacagcagug auuaaccuuu agcaauaaac gaaaguuuaa 1980

cuaagcuaua cuaaccccag gguuggucaa uuucgugcca gccacaccga gaccuggucc 2040

agagucgcua gccgcgucgc uaaaaaaaaa aaaaaaaaaa aaaaaaaaaa agcauaugac 2100

uaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2160

aaaaaaaaaa 2171

<210> 13

<211> 284

<212> PRT

<213> Artificial Sequence

<220>

<223> HOXB13

<400> 13

Met Glu Pro Gly Asn Tyr Ala Thr Leu Asp Gly Ala Lys Asp Ile Glu

1 5 10 15

Gly Leu Leu Gly Ala Gly Gly Gly Arg Asn Leu Val Ala His Ser Pro

20 25 30

Leu Thr Ser His Pro Ala Ala Pro Thr Leu Met Pro Ala Val Asn Tyr

35 40 45

Ala Pro Leu Asp Leu Pro Gly Ser Ala Glu Pro Pro Lys Gln Cys His

50 55 60

Pro Cys Pro Gly Val Pro Gln Gly Thr Ser Pro Ala Pro Val Pro Tyr

65 70 75 80

Gly Tyr Phe Gly Gly Gly Gly Tyr Tyr Ser Cys Arg Val Ser Arg Ser Ser

85 90 95

Leu Lys Pro Cys Ala Gln Ala Ala Thr Leu Ala Ala Tyr Pro Ala Glu

100 105 110

Thr Pro Thr Ala Gly Glu Glu Tyr Pro Ser Arg Pro Thr Glu Phe Ala

115 120 125

Phe Tyr Pro Gly Tyr Pro Gly Thr Tyr Gln Pro Met Ala Ser Tyr Leu

130 135 140

Asp Val Ser Val Val Gln Thr Leu Gly Ala Pro Gly Glu Pro Arg His

145 150 155 160

Asp Ser Leu Leu Pro Val Asp Ser Tyr Gln Ser Trp Ala Leu Ala Gly

165 170 175

Gly Trp Asn Ser Gln Met Cys Cys Gln Gly Glu Gln Asn Pro Pro Gly

180 185 190

Pro Phe Trp Lys Ala Ala Phe Ala Asp Ser Ser Gly Gln His Pro Pro

195 200 205

Asp Ala Cys Ala Phe Arg Arg Gly Arg Lys Lys Arg Ile Pro Tyr Ser

210 215 220

Lys Gly Gln Leu Arg Glu Leu Glu Arg Glu Tyr Ala Ala Asn Lys Phe

225 230 235 240

Ile Thr Lys Asp Lys Arg Arg Lys Ile Ser Ala Ala Thr Ser Leu Ser

245 250 255

Glu Arg Gln Ile Thr Ile Trp Phe Gln Asn Arg Arg Val Lys Glu Lys

260 265 270

Lys Val Leu Ala Lys Val Lys Asn Ser Ala Thr Pro

275 280

<210> 14

<211> 464

<212> PRT

<213> Artificial Sequence

<220>

<223> HOXB13 fusion

<400> 14

Met Arg Val Met Ala Pro Arg Thr Leu Ile Leu Leu Leu Ser Gly Ala

1 5 10 15

Leu Ala Leu Thr Glu Thr Trp Ala Gly Ser Gly Gly Ser Gly Gly Gly

20 25 30

Gly Ser Gly Gly Met Glu Pro Gly Asn Tyr Ala Thr Leu Asp Gly Ala

35 40 45

Lys Asp Ile Glu Gly Leu Leu Gly Ala Gly Gly Gly Arg Asn Leu Val

50 55 60

Ala His Ser Pro Leu Thr Ser His Pro Ala Ala Pro Thr Leu Met Pro

65 70 75 80

Ala Val Asn Tyr Ala Pro Leu Asp Leu Pro Gly Ser Ala Glu Pro Pro

85 90 95

Lys Gln Cys His Pro Cys Pro Gly Val Pro Gln Gly Thr Ser Pro Ala

100 105 110

Pro Val Pro Tyr Gly Tyr Phe Gly Gly Gly Tyr Tyr Ser Cys Arg Val

115 120 125

Ser Arg Ser Ser Leu Lys Pro Cys Ala Gln Ala Ala Thr Leu Ala Ala

130 135 140

Tyr Pro Ala Glu Thr Pro Thr Ala Gly Glu Glu Tyr Pro Ser Arg Pro

145 150 155 160

Thr Glu Phe Ala Phe Tyr Pro Gly Tyr Pro Gly Thr Tyr Gln Pro Met

165 170 175

Ala Ser Tyr Leu Asp Val Ser Val Val Gln Thr Leu Gly Ala Pro Gly

180 185 190

Glu Pro Arg His Asp Ser Leu Leu Pro Val Asp Ser Tyr Gln Ser Trp

195 200 205

Ala Leu Ala Gly Gly Trp Asn Ser Gln Met Cys Cys Gln Gly Glu Gln

210 215 220

Asn Pro Pro Gly Pro Phe Trp Lys Ala Ala Phe Ala Asp Ser Ser Gly

225 230 235 240

Gln His Pro Pro Asp Ala Cys Ala Phe Arg Arg Gly Arg Lys Lys Arg

245 250 255

Ile Pro Tyr Ser Lys Gly Gln Leu Arg Glu Leu Glu Arg Glu Tyr Ala

260 265 270

Ala Asn Lys Phe Ile Thr Lys Asp Lys Arg Arg Lys Ile Ser Ala Ala

275 280 285

Thr Ser Leu Ser Glu Arg Gln Ile Thr Ile Trp Phe Gln Asn Arg Arg

290 295 300

Val Lys Glu Lys Lys Val Leu Ala Lys Val Lys Asn Ser Ala Thr Pro

305 310 315 320

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys Lys Gln Tyr Ile Lys

325 330 335

Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Gly Gly

340 345 350

Gly Lys Arg Gly Gly Gly Lys Lys Met Thr Asn Ser Val Asp Asp Ala

355 360 365

Leu Ile Asn Ser Thr Lys Ile Tyr Ser Tyr Phe Pro Ser Val Ile Ser

370 375 380

Lys Val Asn Gln Gly Ala Gln Gly Lys Lys Leu Gly Ser Ser Gly Gly

385 390 395 400

Gly Gly Ser Pro Gly Gly Gly Ser Ser Ile Val Gly Ile Val Ala Gly

405 410 415

Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val Val Ala Thr Val

420 425 430

Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser Gln

435 440 445

Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala

450 455 460

<210> 15

<211> 852

<212> RNA

<213> Artificial Sequence

<220>

<223> HOXB13 CDS

<400> 15

auggagcccg gcaauuaugc caccuuggau ggagccaagg auaucgaagg cuugcuggga 60

gcgggagggg ggcggaaucu ggucgcccac uccccuuga ccagccaccc agcggcgccu 120

acgcugaugc cugcugucaa cuaugccccc uuggaucugc caggcucggc ggagccgcca 180

aagcaaugcc acccaugccc uggggugccc caggggacgu ccccagcucc cgugccuuau 240

gguuacuuug gaggcgggua cuacuccugc cgaguguccc ggaguucgcu gaaacccugu 300

gcccaggcag ccacccuggc cgcguacccc gcggagacuc ccacggccgg ggaggaguac 360

cccagccgcc ccacugaguu ugccuucuau ccgggauauc cgggaaccua ccagccuaug 420

gccaguuacc uggacguguc guggugcag acucugggug cuccuggaga accgcgacau 480

gacucccugu ugccugugga caguuaccag ucuugggcuc ucgcuggugg cuggaacagc 540

cagauguguu gccagggaga acagaaccca ccaggucccu uuuggaaggc agcauuugca 600

gacuccagcg ggcagcaccc uccugacgcc ugcgccuuuc gucgcggccg caagaaacgc 660

auuccguaca gcaaggggca guugcgggag cuggagcggg aguaugcggc uaacaaguuc 720

aucaccaagg acaagaggcg caagaucucg gcagccacca gccucucgga gcgccagauu 780

accaucuggu uucagaaccg ccgggucaaa gagaagaagg uucucgccaa ggugaagaac 840

agcgcuaccccu 852

<210> 16

<211> 1877

<212> RNA

<213> Artificial Sequence

<220>

<223> HOXB13 RNA

<400> 16

gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccaugagagu 60

gauggccccc agaacccuga uccugcugcu gucuggcgcc cuggcccuga cagagacaug 120

ggccggaagc ggcggcucug gaggaggcgg cuccggaggc auggagcccg gcaauuaugc 180

caccuuggau ggagccaagg auaucgaagg cuugcuggga gcgggagggg ggcggaaucu 240

ggucgcccac uccccucuga ccagccaccc agcggcgccu acgcugaugc cugcugucaa 300

cuaugccccc uuggaucugc caggcucggc ggagccgcca aagcaaugcc acccaugccc 360

uggggugccc caggggacgu ccccagcucc cgugccuuau gguuacuuug gaggcgggua 420

cuacuccugc cgaguguccc ggaguucgcu gaaacccugu gcccaggcag ccacccuggc 480

cgcguacccc gcggagacuc ccacggccgg ggaggaguac cccagccgcc ccacugaguu 540

ugccuucuau ccgggauauc cgggaaccua ccagccuaug gccaguuacc uggacguguc 600

uguggugcag acucugggug cuccuggaga accgcgacau gacucccugu ugccugugga 660

caguuaccag ucuugggcuc ucgcuggugg cuggaacagc cagauguguu gccagggaga 720

acagaaccca ccaggucccu uuuggaaggc agcauuugca gaccaccagcg ggcagcaccc 780

uccugacgcc ugcgccuuuc gucgcggccg caagaaacgc auuccguaca gcaaggggca 840

guugcgggag cuggagcggg aguaugcggc uaacaaguuc aucaccaagg acaagaggcg 900

caagaucucg gcagccacca gccucucgga gcgccagauu accaucuggu uucagaaccg 960

ccgggucaaa gagaagaagg uucucgccaa ggugaagaac agcgcuaccc cuggaggauc 1020

cggugguggc ggcagcggcg gcaagaagca guacaucaag gccaacagca aguucaucgg 1080

caucaccgag cugaagaagc ugggaggggg caaacgggga ggcggcaaaa agaugaccaa 1140

cagcguggac gacgcccuga ucaacagcac caagaucuac agcuacuucc ccagcgugau 1200

cagcaaagug aaccagggcg cucagggcaa gaaacugggc ucuagcggag ggggaggcuc 1260

uccuggcggg ggaucuagca ucgugggaau uguggcagga cuggcagugc uggccguggu 1320

ggugaucgga gccguggugg cuaccgugau gugcagacgg aaguccagcg gaggcaaggg 1380

cggcagcuac agccaggccg ccagcucuga uagcgcccag ggcagcgacg ugucacugac 1440

agccuaguaa cucgagcugg uacugcaugc acgcaaugcu agcugccccu uucccguccu 1500

ggguaccccg agucuccccc gaccucgggu cccagguaug cucccaccuc caccugcccc 1560

acucaccacc ucugcuaguu cgacacaccu cccaagcacg cagcaaugca gcucaaaacg 1620

cuuagccuag ccacaccccc acgggaaaca gcagugauua accuuuagca auaaacgaaa 1680

guuuaacuaa gcuauacuaa ccccaggguu ggucaauuuc gugccagcca caccgagacc 1740

ugguccagag ucgcuagccg cgucgcuaaa aaaaaaaaaa aaaaaaaaaa aaaaaaagca 1800

uaugacuaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860

aaaaaaaaaa aaaaaaa 1877

<210> 17

<211> 234

<212> PRT

<213> Artificial Sequence

<220>

<223> NKX3-1

<400> 17

Met Leu Arg Val Pro Glu Pro Arg Pro Gly Glu Ala Lys Ala Glu Gly

1 5 10 15

Ala Ala Pro Pro Thr Pro Ser Lys Pro Leu Thr Ser Phe Leu Ile Gln

20 25 30

Asp Ile Leu Arg Asp Gly Ala Gln Arg Gln Gly Gly Arg Thr Ser Ser

35 40 45

Gln Arg Gln Arg Asp Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Gly

50 55 60

Gly Arg Ser Arg Ala Gly Ala Gln Asn Asp Gln Leu Ser Thr Gly Pro

65 70 75 80

Arg Ala Ala Pro Glu Glu Ala Glu Thr Leu Ala Glu Thr Glu Pro Glu

85 90 95

Arg His Leu Gly Ser Tyr Leu Leu Asp Ser Glu Asn Thr Ser Gly Ala

100 105 110

Leu Pro Arg Leu Pro Gln Thr Pro Lys Gln Pro Gln Lys Arg Ser Arg

115 120 125

Ala Ala Phe Ser His Thr Gln Val Ile Glu Leu Glu Arg Lys Phe Ser

130 135 140

His Gln Lys Tyr Leu Ser Ala Pro Glu Arg Ala His Leu Ala Lys Asn

145 150 155 160

Leu Lys Leu Thr Glu Thr Gln Val Lys Ile Trp Phe Gln Asn Arg Arg

165 170 175

Tyr Lys Thr Lys Arg Lys Gln Leu Ser Ser Glu Leu Gly Asp Leu Glu

180 185 190

Lys His Ser Ser Leu Pro Ala Leu Lys Glu Glu Ala Phe Ser Arg Ala

195 200 205

Ser Leu Val Ser Val Tyr Asn Ser Tyr Pro Tyr Tyr Pro Tyr Leu Tyr

210 215 220

Cys Val Gly Ser Trp Ser Pro Ala Phe Trp

225 230

<210> 18

<211> 414

<212> PRT

<213> Artificial Sequence

<220>

<223>NKX3-1 fusion

<400> 18

Met Arg Val Met Ala Pro Arg Thr Leu Ile Leu Leu Leu Ser Gly Ala

1 5 10 15

Leu Ala Leu Thr Glu Thr Trp Ala Gly Ser Gly Gly Ser Gly Gly Gly

20 25 30

Gly Ser Gly Gly Met Leu Arg Val Pro Glu Pro Arg Pro Gly Glu Ala

35 40 45

Lys Ala Glu Gly Ala Ala Pro Pro Thr Pro Ser Lys Pro Leu Thr Ser

50 55 60

Phe Leu Ile Gln Asp Ile Leu Arg Asp Gly Ala Gln Arg Gln Gly Gly

65 70 75 80

Arg Thr Ser Ser Gln Arg Gln Arg Asp Pro Glu Pro Glu Pro Glu Pro

85 90 95

Glu Pro Glu Gly Gly Arg Ser Arg Ala Gly Ala Gln Asn Asp Gln Leu

100 105 110

Ser Thr Gly Pro Arg Ala Ala Pro Glu Glu Ala Glu Thr Leu Ala Glu

115 120 125

Thr Glu Pro Glu Arg His Leu Gly Ser Tyr Leu Leu Asp Ser Glu Asn

130 135 140

Thr Ser Gly Ala Leu Pro Arg Leu Pro Gln Thr Pro Lys Gln Pro Gln

145 150 155 160

Lys Arg Ser Arg Ala Ala Phe Ser His Thr Gln Val Ile Glu Leu Glu

165 170 175

Arg Lys Phe Ser His Gln Lys Tyr Leu Ser Ala Pro Glu Arg Ala His

180 185 190

Leu Ala Lys Asn Leu Lys Leu Thr Glu Thr Gln Val Lys Ile Trp Phe

195 200 205

Gln Asn Arg Arg Tyr Lys Thr Lys Arg Lys Gln Leu Ser Ser Glu Leu

210 215 220

Gly Asp Leu Glu Lys His Ser Ser Leu Pro Ala Leu Lys Glu Glu Ala

225 230 235 240

Phe Ser Arg Ala Ser Leu Val Ser Val Tyr Asn Ser Tyr Pro Tyr Tyr

245 250 255

Pro Tyr Leu Tyr Cys Val Gly Ser Trp Ser Pro Ala Phe Trp Gly Gly

260 265 270

Ser Gly Gly Gly Gly Ser Gly Gly Lys Lys Gln Tyr Ile Lys Ala Asn

275 280 285

Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Gly Gly Gly Lys

290 295 300

Arg Gly Gly Gly Lys Lys Met Thr Asn Ser Val Asp Asp Ala Leu Ile

305 310 315 320

Asn Ser Thr Lys Ile Tyr Ser Tyr Phe Pro Ser Val Ile Ser Lys Val

325 330 335

Asn Gln Gly Ala Gln Gly Lys Lys Leu Gly Ser Ser Gly Gly Gly Gly

340 345 350

Ser Pro Gly Gly Gly Ser Ser Ile Val Gly Ile Val Ala Gly Leu Ala

355 360 365

Val Leu Ala Val Val Val Ile Gly Ala Val Val Ala Thr Val Met Cys

370 375 380

Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser Gln Ala Ala

385 390 395 400

Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala

405 410

<210> 19

<211> 702

<212> RNA

<213> Artificial Sequence

<220>

<223> NKX3-1 CDS

<400> 19

augcugagag ugcccgaacc uagaccuggc gaggcuaaag cugaaggcgc ugcuccuccu 60

acaccuagca agccucugac cagcuuccug auccaagaca uccugagaga uggcgcccag 120

agacaaggcg gcagaacaag cucucagaga cagagagauc ccgagccuga gccugaacca 180

gaaccugaag gcggaagauc uagagccggc gcucagaacg aucagcugag cacaggaccu 240

agagccgcuc cugaggaagc cgaaacacug gccgaaaccg agccagagag acaccugggc 300

agcuaccugc uggacagcga gaauacuucu ggcgcccugc cuagacugcc cgacacaccu 360

aaacagccuc agaagcggag cagagccgcc uuuagccaca cacaagugau cgagcuggaa 420

cggaaguuca gccaccagaa guaccugagc gccccugaaa gagcccaccu ggccaagaau 480

cugaagcuga ccgagacuca agugaagauc ugguuccaga accggcggua caagaccaag 540

cggaagcagc ugucaagcga gcugggcgac cuggaaaagc acagcucucu gcccgcucug 600

aaagaggaag ccuucuccag agccagccug guguccgugu acaacagcua cccuuacuac 660

cccuaccugu acugcgucgg cucuuggagc ccugccuuuu gg 702

<210> 20

<211> 1727

<212> RNA

<213> Artificial Sequence

<220>

<223> NKX3-1 RNA

<400> 20

gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccaugagagu 60

gauggccccc agaacccuga uccugcugcu gucuggcgcc cuggcccuga cagagacaug 120

ggccggaagc ggcggcucug gaggaggcgg cuccggaggc augcugagag ugcccgaacc 180

uagaccuggc gaggcuaaag cugaaggcgc ugcuccuccu acaccuagca agccucugac 240

cagcuuccug auccaagaca uccugagaga uggcgcccag agacaaggcg gcagaacaag 300

cucucagaga cagagagauc ccgagccuga gccugaacca gaaccugaag gcggaagauc 360

uagagccggc gcucagaacg aucagcugag cacaggaccu agagccgcuc cugaggaagc 420

cgaaacacug gccgaaaccg agccagagag acaccugggc agcuaccugc uggacagcga 480

gaauacuucu ggcgcccugc cuagacugcc cgacacaccu aaacagccuc agaagcggag 540

cagagccgcc uuuagccaca cacaagugau cgagcuggaa cggaaguuca gccaccagaa 600

guaccugagc gccccugaaa gagcccaccu ggccaagaau cugaagcuga ccgagacuca 660

agugaagauc ugguuccaga accggcggua caagaccaag cggaagcagc ugucaagcga 720

gcugggcgac cuggaaaagc acagcucucu gcccgcucug aaagaggaag ccuucucccag 780

agccagccug guguccgugu acaacagcua cccuuacuac cccuaccugu acugcgucgg 840

cucuuggagc ccugccuuuu ggggaggauc cggugguggc ggcagcggcg gcaagaagca 900

guacaucaag gccaacagca aguucaucgg caucaccgag cugaagaagc ugggagggg 960

caaacgggga ggcggcaaaa agaugaccaa cagcguggac gacgcccuga ucaacagcac 1020

caagaucuac agcuacuucc ccagcgugau cagcaaagug aaccagggcg cucagggcaa 1080

gaaacugggc ucuagcggag ggggaggcuc uccuggcggg ggaucuagca ucgugggaau 1140

uguggcagga cuggcagugc uggccguggu ggugaucgga gccguggugg cuaccgugau 1200

gugcagacgg aaguccagcg gaggcaaggg cggcagcuac agccaggccg ccagcucuga 1260

uagcgcccag ggcagcgacg ugucacugac agccuaguaa cucgagcugg uacugcaugc 1320

acgcaaugcu agcugccccu uucccguccu ggguaccccg agucuccccc gaccucgggu 1380

cccagguaug cucccaccuc caccugcccc acucaccacc ucugcuaguu cgacacaccu 1440

cccaagcacg cagcaaugca gcucaaaacg cuuagccuag cccaccccc acgggaaaca 1500

gcagugauua accuuuagca auaaacgaaa guuuaacuaa gcuauacuaa ccccaggguu 1560

ggucaauuuc gugccagcca caccgagacc ugguccagag ucgcuagccg cgucgcuaaa 1620

aaaaaaaaaa aaaaaaaaaa aaaaaaagca uaugacuaaa aaaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 1727

<210> 21

<211> 52

<212> RNA

<213> Artificial Sequence

<220>

<223> 5' UTR

<400> 21

gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca cc 52

<210> 22

<211> 26

<212> PRT

<213> Artificial Sequence

<220>

<223> Secret signal

<400> 22

Met Arg Val Met Ala Pro Arg Thr Leu Ile Leu Leu Leu Ser Gly Ala

1 5 10 15

Leu Ala Leu Thr Glu Thr Trp Ala Gly Ser

20 25

<210> 23

<211> 78

<212> RNA

<213> Artificial Sequence

<220>

<223> Secret signal

<400> 23

augagaguga uggcccccag aacccugauc cugcugcugu cuggcgcccu ggcccugaca 60

gagacauggg ccggaagc 78

<210> 24

<211> 55

<212> PRT

<213> Artificial Sequence

<220>

<223> MITD

<400> 24

Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile

1 5 10 15

Gly Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly

20 25 30

Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly

35 40 45

Ser Asp Val Ser Leu Thr Ala

50 55

<210> 25

<211> 171

<212> RNA

<213> Artificial Sequence

<220>

<223> MITD

<400> 25

aucgugggaa uuguggcagg acuggcagug cuggccgugg uggugaucgg agccguggug 60

gcuaccguga ugugcagacg gaaguccagc ggaggcaagg gcggcagcua cagccaggcc 120

gccagcucug auagcgccca gggcagcgac gugucacuga cagccuagua a 171

<210> 26

<211> 65

<212> PRT

<213> Artificial Sequence

<220>

<223> P2P16

<400> 26

Lys Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu

1 5 10 15

Leu Lys Lys Leu Gly Gly Gly Lys Arg Gly Gly Gly Lys Lys Met Thr

20 25 30

Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr Ser Tyr

35 40 45

Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln Gly Lys Lys

50 55 60

Leu

65

<210> 27

<211> 195

<212> RNA

<213> Artificial Sequence

<220>

<223> P2P16

<400> 27

aagaagcagu acaucaaggc caacagcaag uucaucggca ucaccgagcu gaagaagcug 60

ggagggggca aacggggagg cggcaaaaag augaccaaca gcguggacga cgcccugauc 120

aacagcacca agaucuacag cuacuucccc agcgugauca gcaaagugaa ccagggcgcu 180

cagggcaaga aacug 195

<210> 28

<211> 317

<212> RNA

<213> Artificial Sequence

<220>

<223> 3' UTR

<400> 28

cucgagcugg uacugcaugc acgcaaugcu agcugccccu uucccguccu ggguaccccg 60

agucuccccc gaccucgggu cccagguaug cucccaccuc caccugcccc acucaccacc 120

ucugcuaguu cgacacaccu cccaagcacg cagcaaugca gcucaaaacg cuuagccuag 180

cccaccccc acgggaaaca gcagugauua accuuuagca auaaacgaaa guuuaacuaa 240

gcuauacuaa ccccaggguu ggucaauuuc gugccagcca caccgagacc ugguccagag 300

ucgcuagccg cgucgcu 317

<210> 29

<211> 110

<212> RNA

<213> Artificial Sequence

<220>

<223> A30L70

<400> 29

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gcauaugacu aaaaaaaaaa aaaaaaaaaa 60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 110

<210> 30

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> P2

<400> 30

Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu

1 5 10 15

<210> 31

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

<223> P16

<400> 31

Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr

1 5 10 15

Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln Gly

20 25 30

<---

Claims (143)

1. A pharmaceutical composition for the treatment or prevention of prostate cancer, comprising at least one RNA, wherein 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 a 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, wherein (a) the amino acid sequence of (i) comprises the amino acid sequence of SEQ ID NO: 1 or 2 or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 or 2; b) the amino acid sequence according to (ii) comprises the amino acid sequence of SEQ ID NO: 5 or 6 or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 5 or 6; c) the amino acid sequence according to (iii) comprises the amino acid sequence of SEQ ID NO: 9 or 10 or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 9 or 10; d) the amino acid sequence of (iv) comprises the amino acid sequence of SEQ ID NO: 13 or 14 or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 13 or 14; e) the amino acid sequence of (v) comprises the amino acid sequence of SEQ ID NO: 17 or 18 or an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 17 or 18, and the immunogenic fragment or immunogenic variant is an immunological equivalent of KLK2, PSA, PAP, HOXB13 or NKX3-1, respectively, wherein the pharmaceutical composition also comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients. 2. A kit for the treatment or prevention of prostate cancer, containing an effective amount of RNA, wherein the RNA encodes the following amino acid sequences and is in separate vials: (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 a 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, wherein (a) the amino acid sequence of (i) comprises the amino acid sequence of SEQ ID NO: 1 or 2 or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 or 2; b) the amino acid sequence according to (ii) comprises the amino acid sequence of SEQ ID NO: 5 or 6 or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 5 or 6; c) the amino acid sequence according to (iii) comprises the amino acid sequence of SEQ ID NO: 9 or 10 or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 9 or 10; d) the amino acid sequence of (iv) comprises the amino acid sequence of SEQ ID NO: 13 or 14 or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 13 or 14; e) the amino acid sequence of (v) comprises the amino acid sequence of SEQ ID NO: 17 or 18 or an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 17 or 18, and the immunogenic fragment or immunogenic variant is an immunological equivalent of KLK2, PSA, PAP, HOXB13, or NKX3-1, respectively. 3. The pharmaceutical composition according to claim 1 or the kit according to claim 2, characterized in that each of the amino acid sequences according to (i), (ii), (iii), (iv) or (v) is encoded by a separate RNA. 4. The pharmaceutical composition of claim 1 or 3 or the kit of claim 2 or 3, wherein the RNA encoding the amino acid sequence of (i) comprises the nucleotide sequence of SEQ ID NO: 3 or 4 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: 3 or 4; and/or the amino acid sequence of (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% or 85% identical to the amino acid sequence of SEQ ID NO: 1 or 2. 5. A pharmaceutical composition according to any one of claims 1, 3 or 4 or a kit according to any one of claims 2-4, where RNA encoding the amino acid sequence of (ii) comprises 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 the amino acid sequence of (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% or 85% identical to the amino acid sequence of SEQ ID NO: 5 or 6. 6. A pharmaceutical composition according to any one of claims 1 or 3-5 or a kit according to any one of claims 2-5, where An RNA encoding the amino acid sequence of (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 the amino acid sequence according to (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% or 85% identical to the amino acid sequence of SEQ ID NO: 9 or 10. 7. A pharmaceutical composition according to any one of claims 1 or 3-6 or a kit according to any one of claims 2-6, where RNA encoding the amino acid sequence of (iv) comprises 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 the amino acid sequence of paragraph (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% or 85% identical to the amino acid sequence of SEQ ID NO: 13 or 14. 8. A pharmaceutical composition according to any one of claims 1 or 3-7 or a kit according to any one of claims 2-7, where An RNA encoding the amino acid sequence of (v) comprises the nucleotide sequence of SEQ ID NO: 19 or 20 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: 19 or 20; and/or the amino acid sequence of (v) 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% or 85% identical to the amino acid sequence of SEQ ID NO: 17 or 18. 9. A pharmaceutical composition according to any one of claims 1 or 3-8 or a kit according to any one of claims 2-8, wherein at least one of the amino acid sequences of (i), (ii), (iii), (iv) or (v) is encoded by a sequence that is codon optimized and/or has an increased G/C content compared to the wild-type coding sequence, wherein the codon optimization and/or the increase in G/C content preferably does not alter the sequence of the encoded amino acid sequence. 10. A pharmaceutical composition according to any one of claims 1 or 3-9 or a kit according to any one of claims 2-9, wherein each of the amino acid sequences of (i), (ii), (iii), (iv) or (v) is encoded by a sequence that is codon optimized and/or has an increased G/C content compared to the wild-type coding sequence, wherein the codon optimization and/or the increase in G/C content preferably does not alter the sequence of the encoded amino acid sequence. 11. A pharmaceutical composition according to any one of claims 1 or 3-10 or a kit according to any one of claims 2-10, wherein at least one RNA comprises a 5'-cap m ₂ ^7,2' - ^O Gpp _s p(5')G. 12. A pharmaceutical composition according to any one of claims 1 or 3-11 or a kit according to any one of claims 2-11, wherein each RNA contains a 5'-cap m ₂ ^7,2' - ^O Gpp _s p(5')G. 13. The pharmaceutical composition according to any one of claims 1 or 3-12 or the kit according to any one of claims 2-12, wherein 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. 14. The pharmaceutical composition according to any one of claims 1 or 3-13 or the kit according to any one of claims 2-13, wherein 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. 15. A pharmaceutical composition according to any one of claims 1 or 3-14 or a kit according to any one of claims 2-14, wherein at least one amino acid sequence according to (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that enhances antigen processing and/or presentation. 16. A pharmaceutical composition according to any one of claims 1 or 3-15 or a kit according to any one of claims 2-15, wherein each amino acid sequence according to (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that enhances antigen processing and/or presentation. 17. A pharmaceutical composition or kit according to claim 15 or 16, wherein the amino acid sequence that enhances the processing and/or presentation of an antigen comprises an amino acid sequence corresponding to the transmembrane and cytoplasmic domain of an MHC molecule, preferably an MHC class I molecule. 18. A pharmaceutical composition or kit according to any of paragraphs 15-17, characterized in that An RNA encoding an 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 an 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. 19. A pharmaceutical composition according to any one of claims 1 or 3-18 or a kit according to any one of claims 2-18, wherein at least one amino acid sequence according to (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that breaks immunological tolerance. 20. A pharmaceutical composition according to any one of claims 1 or 3-19 or a kit according to any one of claims 2-19, wherein each amino acid sequence according to (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that breaks immunological tolerance. 21. A pharmaceutical composition or kit according to claim 19 or 20, wherein the amino acid sequence that disrupts immunological tolerance comprises helper epitopes, preferably helper epitopes derived from tetanus toxoid. 22. A pharmaceutical composition or kit according to any one of paragraphs 19-21, where An RNA encoding an amino acid sequence that breaks immunological 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 an amino acid sequence that breaks immunological 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 nucleotide sequence of SEQ ID NO: 26. 23. The pharmaceutical composition according to any one of claims 1 or 3-22 or the kit according to any one of claims 2-22, wherein 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. 24. The pharmaceutical composition according to any one of claims 1 or 3-23 or the kit according to any one of claims 2-23, wherein 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. 25. The pharmaceutical composition according to any one of claims 1 or 3-24 or the kit according to any one of claims 2-24, wherein at least one RNA comprises a poly-A sequence. 26. The pharmaceutical composition according to any one of claims 1 or 3-25 or the kit according to any one of claims 1-25, wherein each RNA comprises a poly-A sequence. 27. The pharmaceutical composition or kit according to claim 25 or 26, wherein the poly-A sequence comprises at least 100 nucleotides. 28. A pharmaceutical composition or kit according to any one of claims 25-27, wherein the poly-A sequence comprises or consists of the nucleotide sequence SEQ ID NO: 29. 29. A pharmaceutical composition according to any one of claims 1 or 3-28 or a kit according to any one of claims 1-28, wherein the RNA is included in a composition or formulation that is in the form of a liquid, a solid, or a combination thereof. 30. A pharmaceutical composition according to any one of claims 1 or 3-29 or a kit according to any one of claims 2-29, wherein the RNA is included in a composition or formulation for injection. 31. A pharmaceutical composition according to any one of claims 1 or 3-30 or a kit according to any one of claims 2-30, wherein the RNA is included in a composition or formulation for intravenous administration. 32. A pharmaceutical composition according to any one of claims 1 or 3-31 or a kit according to any one of claims 2-31, wherein the RNA is formulated in the form of lipoplex particles. 33. The pharmaceutical composition or kit according to claim 32, wherein the RNA lipoplex particles can be obtained by mixing RNA with liposomes. 34. The kit according to item 33, wherein the RNA and liposomes are in separate vials. 35. The kit of claim 34, further comprising instructions for using the RNA and, optionally, liposomes for treating or preventing prostate cancer. 36. Use of a pharmaceutical composition according to any one of claims 1 or 3-33 as a medicinal product for the treatment or prevention of prostate cancer. 37. Use of the kit according to any of paragraphs 2-35 as a medicinal product for the treatment or prevention of prostate cancer. 38. A pharmaceutical composition according to any one of claims 1 or 3-33 or a kit according to any one of claims 2-35, intended for administration to humans. 39. The use according to any of paragraphs 36 or 37, where the medicinal product is intended for administration to a human. 40. The use according to any of paragraphs 38 or 39, wherein the therapeutic or prophylactic treatment also includes additional therapy. 41. The use according to claim 40, wherein the additional therapy comprises one or more agents selected from the group consisting of: (a) surgical intervention for excision, resection or removal of the tumor, (b) radiation therapy and (c) chemotherapy. 42. The use according to claim 40 or 41, wherein the additional therapy comprises the administration of a therapeutic agent. 43. The use according to claim 42, wherein the additional therapeutic agent is an anticancer therapeutic agent. 44. The use according to claim 42 or 43, wherein the additional therapeutic agent is a checkpoint modulator. 45. The use according to claim 44, wherein the checkpoint modulator is an anti-PD1 antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD1 antibody and an anti-CTLA-4 antibody. 46. A method 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 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 a 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, wherein (a) the amino acid sequence of (i) comprises the amino acid sequence of SEQ ID NO: 1 or 2 or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 or 2; b) the amino acid sequence according to (ii) comprises the amino acid sequence of SEQ ID NO: 5 or 6 or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 5 or 6; c) the amino acid sequence according to (iii) comprises the amino acid sequence of SEQ ID NO: 9 or 10 or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 9 or 10; d) the amino acid sequence of (iv) comprises the amino acid sequence of SEQ ID NO: 13 or 14 or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 13 or 14; e) the amino acid sequence of (v) comprises the amino acid sequence of SEQ ID NO: 17 or 18 or an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 17 or 18, and the immunogenic fragment or immunogenic variant is an immunological equivalent of KLK2, PSA, PAP, HOXB13, or NKX3-1, respectively. 47. The method of claim 46, wherein each of the amino acid sequences of (i), (ii), (iii), (iv) or (v) is encoded by a separate RNA. 48. The method according to item 46 or 47, where An RNA encoding the amino acid sequence of (i) comprises the nucleotide sequence of SEQ ID NO: 3 or 4 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: 3 or 4; and/or the amino acid sequence of (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% or 85% identical to the amino acid sequence of SEQ ID NO: 1 or 2. 49. The method according to any of paragraphs 46-48, where RNA encoding the amino acid sequence of (ii) comprises 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 the amino acid sequence of (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% or 85% identical to the amino acid sequence of SEQ ID NO: 5 or 6. 50. The method according to any of paragraphs 46-49, where An RNA encoding the amino acid sequence of (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 the amino acid sequence according to (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% or 85% identical to the amino acid sequence of SEQ ID NO: 9 or 10. 51. The method according to any of paragraphs 46-50, where RNA encoding the amino acid sequence of (iv) comprises 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 the amino acid sequence according to (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% or 85% identical to the amino acid sequence of SEQ ID NO: 13 or 14. 52. The method according to any of paragraphs 46-51, where An RNA encoding the amino acid sequence of (v) comprises the nucleotide sequence of SEQ ID NO: 19 or 20 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: 19 or 20; and/or the amino acid sequence of (v) 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% or 85% identical to the amino acid sequence of SEQ ID NO: 17 or 18. 53. The method according to any one of claims 46-52, wherein at least one of the amino acid sequences of (i), (ii), (iii), (iv) or (v) is encoded by a sequence that is codon optimized and/or has an increased G/C content compared to the wild-type coding sequence, wherein the codon optimization and/or the increase in G/C content preferably does not alter the sequence of the encoded amino acid sequence. 54. The method according to any one of claims 46-53, wherein each of the amino acid sequences of (i), (ii), (iii), (iv) or (v) is encoded by a sequence that is codon optimized and/or has an increased G/C content compared to the wild-type coding sequence, wherein the codon optimization and/or the increase in G/C content preferably does not alter the sequence of the encoded amino acid sequence. 55. The method according to any one of claims 46-54, wherein at least one RNA comprises a 5'-cap m ₂ ^7,2' - ^O Gpp _s p(5')G. 56. The method according to any one of claims 46-55, wherein each RNA comprises a 5'-cap m ₂ ^7,2' - ^O Gpp _s p(5')G. 57. The method according to any one of claims 46-56, wherein 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. 58. The method according to any one of claims 46-57, wherein 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. 59. The method according to any one of claims 56-58, wherein at least one amino acid sequence according to (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that enhances antigen processing and/or presentation. 60. The method according to any one of claims 46-59, wherein each amino acid sequence of (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that enhances antigen processing and/or presentation. 61. The method according to claim 59 or 60, wherein the amino acid sequence that enhances the processing and/or presentation of an antigen comprises an amino acid sequence corresponding to the transmembrane and cytoplasmic domain of an MHC molecule, preferably an MHC class I molecule. 62. The method according to any of paragraphs 59-61, where An RNA encoding an 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 an 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. 63. The method according to any one of claims 46-62, wherein at least one amino acid sequence according to (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that breaks immunological tolerance. 64. The method according to any one of claims 46-63, wherein each amino acid sequence of (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that breaks immunological tolerance. 65. The method according to claim 63 or 64, wherein the amino acid sequence that breaks immunological tolerance comprises helper epitopes, preferably helper epitopes obtained from tetanus toxoid. 66. The method according to any of paragraphs 63-65, where An RNA encoding an amino acid sequence that breaks immunological 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 an amino acid sequence that breaks immunological 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 nucleotide sequence of SEQ ID NO: 26. 67. The method according to any one of claims 46-66, wherein 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. 68. The method according to any one of claims 46-67, wherein 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. 69. The method according to any one of claims 46-68, wherein at least one RNA comprises a poly-A sequence. 70. The method according to any one of claims 46-69, wherein each RNA comprises a poly-A sequence. 71. The method according to claim 69 or 70, wherein the poly-A sequence comprises at least 100 nucleotides. 72. The method according to any one of claims 69-71, wherein the poly-A sequence comprises or consists of the nucleotide sequence SEQ ID NO: 29. 73. The method according to any one of claims 46-72, wherein the RNA is administered by injection. 74. The method according to any one of paragraphs 46-73, wherein the RNA is administered intravenously. 75. The method according to any one of paragraphs 46-74, wherein the RNA is formulated in the form of lipoplex particles. 76. The method according to any one of claims 46-75, wherein the RNA lipoplex particles can be obtained by mixing RNA with liposomes. 77. The method according to any of paragraphs 46-76, wherein the subject is a human. 78. The method according to any one of paragraphs 46-77, which also includes carrying out additional therapy. 79. The method of claim 78, wherein the additional therapy comprises one or more agents selected from the group consisting of: (a) surgical intervention to excise, resect or remove the tumor, (b) radiation therapy, and (c) chemotherapy. 80. The method according to claim 78 or 79, wherein the additional therapy comprises administering a therapeutic agent. 81. The method of claim 80, wherein the additional therapeutic agent is an anticancer therapeutic agent. 82. The method according to claim 80 or 81, wherein the additional therapeutic agent is a checkpoint modulator. 83. The method of claim 82, wherein the checkpoint modulator is an anti-PD1 antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD1 antibody and an anti-CTLA-4 antibody.