Therapeutic RNA for prostate cancer

文档序号：310046 发布日期：2021-11-26 浏览：13次中文

阅读说明：本技术 用于前列腺癌的治疗性rna (Therapeutic RNA for prostate cancer ) 是由大卫·韦伯卡里纳·韦尔特戴安娜·巴雷亚罗尔丹鲁普雷希特·库纳叶利夫·迪肯马丁·苏于 2020-03-11 设计创作，主要内容包括：本文中公开了用于治疗前列腺癌的组合物、用途和方法。在一个方面中,本文中公开了包含至少一种RNA的组合物或药物制剂,其中所述至少一种RNA编码以下氨基酸序列：(i)包含激肽释放酶-2(KLK2)、其免疫原性变体、或者KLK2的免疫原性片段或其免疫原性变体的氨基酸序列；(ii)包含前列腺特异性抗原(PSA)、其免疫原性变体、或者PSA的免疫原性片段或其免疫原性变体的氨基酸序列；(iii)包含前列腺酸性磷酸酶(PAP)、其免疫原性变体、或者PAP的免疫原性片段或其免疫原性变体的氨基酸序列；(iv)包含同源框B13(HOXB13)、其免疫原性变体、或者HOXB13的免疫原性片段或其免疫原性变体的氨基酸序列；以及(v)包含NK3同源框1(NKX3-1)、其免疫原性变体、或者NKX3-1的免疫原性片段或其免疫原性变体的氨基酸序列。(Disclosed herein are compositions, uses and methods for treating prostate cancer. In one aspect, disclosed herein is a composition or pharmaceutical formulation comprising at least one RNA encoding the amino acid sequence: (i) an amino acid sequence comprising kallikrein-2 (KLK2), an immunogenic variant thereof, or an immunogenic fragment of KLK2 or an immunogenic variant thereof; (ii) an amino acid sequence comprising Prostate Specific Antigen (PSA), an immunogenic variant thereof, or an immunogenic fragment of PSA or an immunogenic variant thereof; (iii) an amino acid sequence comprising Prostatic Acid Phosphatase (PAP), an immunogenic variant thereof, or an immunogenic fragment of PAP or an immunogenic variant thereof; (iv) an amino acid sequence comprising homology box 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.)

1. A composition or pharmaceutical formulation comprising at least one RNA, wherein the at least one RNA encodes the amino acid sequence:

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

(ii) an amino acid sequence comprising Prostate Specific Antigen (PSA), an immunogenic variant thereof, or an immunogenic fragment of said 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 said PAP or an immunogenic variant thereof;

(iv) an amino acid sequence comprising homology box B13(HOXB13), an immunogenic variant thereof, or an immunogenic fragment of said HOXB13 or an immunogenic variant thereof; and

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

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

3. The composition or pharmaceutical formulation of claim 1 or 2, wherein:

(i) (ii) the RNA encoding the amino acid sequence set forth in (i) comprises the amino acid sequence set forth in SEQ ID NO: 3 or 4, or a nucleotide sequence identical to SEQ ID NO: 3 or 4, having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity; and/or

(ii) (ii) the amino acid sequence described in (i) comprises SEQ ID NO: 1 or 2, or an amino acid sequence substantially identical to SEQ ID NO: 1 or 2, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity.

4. The composition or pharmaceutical formulation of any one of claims 1 to 3, wherein:

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

(ii) (ii) the amino acid sequence comprises SEQ ID NO: 5 or 6, or an amino acid sequence substantially identical to SEQ ID NO: 5 or 6, having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity.

5. The composition or pharmaceutical formulation of any one of claims 1 to 4, wherein:

(ii) (iii) the amino acid sequence comprises SEQ ID NO: 9 or 10, or an amino acid sequence substantially identical to SEQ ID NO: 9 or 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity.

6. The composition or pharmaceutical formulation of any one of claims 1 to 5, wherein:

(i) (iii) the RNA encoding the amino acid sequence in (iv) comprises SEQ ID NO: 15 or 16, or a nucleotide sequence identical to SEQ ID NO: 15 or 16 has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity; and/or

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

7. The composition or pharmaceutical formulation of any one of claims 1 to 6, wherein:

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

(ii) (v) the amino acid sequence comprises SEQ ID NO: 17 or 18, or an amino acid sequence identical to SEQ ID NO: 17 or 18, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity.

8. The composition or pharmaceutical preparation of any one of claims 1 to 7, wherein at least one of the amino acid sequences in (i), (ii), (iii), (iv) or (v) is encoded by a coding sequence that: increased G/C content compared to the wild type coding sequence, and/or which is codon optimized, wherein said codon optimization and/or said increased G/C content preferably does not alter the sequence of the encoded amino acid sequence.

9. A composition or pharmaceutical formulation as claimed in any one of claims 1 to 8, wherein (i), (ii) already exists,

(iii) (vi) each of the amino acid sequences set forth in (iv) or (v) is encoded by a coding sequence that: increased G/C content compared to the wild type coding sequence, and/or which is codon optimized, wherein said codon optimization and/or said increased G/C content preferably does not alter the sequence of the encoded amino acid sequence.

10. The composition or pharmaceutical preparation of any one of claims 1 to 9, wherein at least one RNA comprises a 5' cap m ₂ ⁷ ^，2’-OGpp_sp(5’)G。

11. The composition or pharmaceutical preparation of any one of claims 1 to 10, wherein each RNA comprises a 5' cap m₂ ^7，2’- ^OGpp_sp(5’)G。

12. The composition or pharmaceutical preparation of any one of claims 1 to 11, wherein at least one RNA comprises a 5' UTR comprising: SEQ ID NO: 21, or a nucleotide sequence identical to SEQ ID NO: 21, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity.

13. The composition or pharmaceutical preparation of any one of claims 1 to 12, wherein each RNA comprises a 5' UTR comprising: SEQ ID NO: 21, or a nucleotide sequence identical to SEQ ID NO: 21, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity.

14. The composition or pharmaceutical preparation of any one of claims 1 to 13, wherein at least one amino acid sequence of (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that enhances antigen processing and/or presentation.

15. The composition or pharmaceutical preparation of any one of claims 1 to 14, wherein each amino acid sequence of (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that enhances antigen processing and/or presentation.

16. The composition or pharmaceutical preparation of claim 14 or 15, wherein said amino acid sequence that enhances antigen processing and/or presentation comprises an amino acid sequence corresponding to the transmembrane and cytoplasmic domains of an MHC molecule, preferably an MHC class I molecule.

17. The composition or pharmaceutical formulation of any one of claims 14 to 16, wherein:

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

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

18. The composition or pharmaceutical preparation of any one of claims 1 to 17, wherein at least one amino acid sequence of (i), (ii), (iii), (iv), or (v) comprises an amino acid sequence that disrupts immune tolerance.

19. The composition or pharmaceutical preparation of any one of claims 1 to 18, wherein each amino acid sequence of (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that disrupts immune tolerance.

20. The composition or pharmaceutical preparation of claim 18 or 19, wherein the amino acid sequence that disrupts immune tolerance comprises a helper epitope, preferably a tetanus toxoid-derived helper epitope.

21. The composition or pharmaceutical formulation of any one of claims 18 to 20, wherein:

(i) the RNA encoding the immune tolerance-disrupting amino acid sequence comprises SEQ ID NO: 27, or a nucleotide sequence identical to SEQ ID NO: 27 has a nucleotide sequence of at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity; and/or

(ii) The amino acid sequence that disrupts immune tolerance comprises SEQ ID NO: 26, or an amino acid sequence identical to SEQ ID NO: 26, has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity.

22. The composition or pharmaceutical preparation of any one of claims 1 to 21, wherein at least one RNA comprises a 3' UTR comprising: SEQ ID NO: 28, or a nucleotide sequence identical to SEQ ID NO: 28, has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity.

23. The composition or pharmaceutical preparation of any one of claims 1 to 22, wherein each RNA comprises a 3' UTR comprising: SEQ ID NO: 28, or a nucleotide sequence identical to SEQ ID NO: 28, has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity.

24. The composition or pharmaceutical preparation of any one of claims 1 to 23, wherein at least one RNA comprises a poly-a sequence.

25. The composition or pharmaceutical preparation of any one of claims 1 to 24, wherein each RNA comprises a poly-a sequence.

26. The composition or pharmaceutical preparation of claim 24 or 25, wherein said poly-a sequence comprises at least 100 nucleotides.

27. The composition or pharmaceutical formulation of any one of claims 24 to 26, wherein the poly-a sequence comprises SEQ ID NO: 29 or consisting of the nucleotide sequence of seq id no.

28. The composition or pharmaceutical preparation of any one of claims 1 to 27, wherein said RNA is formulated as a liquid, as a solid, or a combination thereof.

29. The composition or pharmaceutical preparation of any one of claims 1 to 28, wherein said RNA is formulated for injection.

30. The composition or pharmaceutical formulation of any one of claims 1 to 29, wherein the RNA is formulated for intravenous administration.

31. The composition or pharmaceutical preparation of any one of claims 1 to 30, wherein said RNA is formulated or to be formulated as a lipid complex particle.

32. The composition or pharmaceutical preparation of any one of claims 1 to 31, wherein said RNA lipid complex particles are obtainable by mixing said RNA with liposomes.

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

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

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

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

37. The composition or pharmaceutical preparation of claim 35 or 36, further comprising instructions for use of said RNA and optionally said liposomes for treating or preventing prostate cancer.

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

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

40. The composition or pharmaceutical preparation of claim 39, wherein therapeutic or prophylactic treatment of the disease or condition comprises treatment or prevention of prostate cancer.

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

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

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

44. The composition or pharmaceutical formulation of claim 42 or 43, wherein the additional treatment comprises administration of an additional therapeutic agent.

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

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

47. The composition or pharmaceutical formulation of claim 46, wherein the checkpoint modulator is an anti-PD 1 antibody, an anti-CTLA-4 antibody or an anti-PD 1 antibody in combination with an anti-CTLA-4 antibody.

48. 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 amino acid sequence:

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

(ii) an amino acid sequence comprising Prostate Specific Antigen (PSA), an immunogenic variant thereof, or an immunogenic fragment of said 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 said PAP or an immunogenic variant thereof;

(iv) an amino acid sequence comprising homology box B13(HOXB13), an immunogenic variant thereof, or an immunogenic fragment of said HOXB13 or an immunogenic variant thereof; and

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

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

50. The method of claim 48 or 49, wherein:

51. The method of any one of claims 48 to 50, wherein:

52. The method of any one of claims 48 to 51, wherein:

53. The method of any one of claims 48-52, wherein:

54. The method of any one of claims 48 to 53, wherein:

55. The method of any one of claims 48 to 54, wherein at least one of the amino acid sequences in (i), (ii), (iii), (iv) or (v) is encoded by a coding sequence that: increased G/C content compared to the wild type coding sequence, and/or which is codon optimized, wherein said codon optimization and/or said increased G/C content preferably does not alter the sequence of the encoded amino acid sequence.

56. The method of any one of claims 48 to 55, wherein each of the amino acid sequences in (i), (ii), (iii), (iv) or (v) is encoded by a coding sequence that: increased G/C content compared to the wild type coding sequence, and/or which is codon optimized, wherein said codon optimization and/or said increased G/C content preferably does not alter the sequence of the encoded amino acid sequence.

57. The method of any one of claims 48 to 56, wherein at least one RNA comprises a 5' cap m₂ ^7，2’-OGpp_sp(5’)G。

58. The method of any one of claims 48 to 57, wherein each RNA comprises a 5' cap m₂ ^7，2’-OGpp_sp(5’)G。

59. The method of any one of claims 48 to 58, wherein at least one RNA comprises a 5' UTR comprising: SEQ ID NO: 21, or a nucleotide sequence identical to SEQ ID NO: 21, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity.

60. The method of any one of claims 48 to 59, wherein each RNA comprises a 5' UTR comprising: SEQ ID NO: 21, or a nucleotide sequence identical to SEQ ID NO: 21, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity.

61. The method of any one of claims 48 to 60, wherein at least one amino acid sequence of (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that enhances antigen processing and/or presentation.

62. The method of any one of claims 48 to 61, wherein each amino acid sequence of (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that enhances antigen processing and/or presentation.

63. The method of claim 61 or 62, wherein the amino acid sequence that enhances antigen processing and/or presentation comprises an amino acid sequence corresponding to the transmembrane and cytoplasmic domains of an MHC molecule, preferably an MHC class I molecule.

64. The method of any one of claims 61 to 63, wherein:

65. The method of any one of claims 48 to 64, wherein at least one amino acid sequence of (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that disrupts immune tolerance.

66. The method of any one of claims 48 to 65, wherein each amino acid sequence of (i), (ii), (iii), (iv) or (v) comprises an amino acid sequence that disrupts immune tolerance.

67. The method of claim 65 or 66, wherein the amino acid sequence that disrupts immune tolerance comprises a helper epitope, preferably a tetanus toxoid-derived helper epitope.

68. The method of any one of claims 65 to 67, wherein:

(i) the RNA encoding the immune tolerance-disrupting amino acid sequence comprises SEQ ID NO: 27, or a sequence identical to SEQ ID NO: 27, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity; and/or

69. The method of any one of claims 48 to 68, wherein at least one RNA comprises a 3' UTR comprising: SEQ ID NO: 28, or a nucleotide sequence identical to SEQ ID NO: 28, has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity.

70. The method of any one of claims 48 to 69, wherein each RNA comprises a 3' UTR comprising: SEQ ID NO: 28, or a nucleotide sequence identical to SEQ ID NO: 28, has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity.

71. The method of any one of claims 48 to 70, wherein at least one RNA comprises a poly-A sequence.

72. The method of any one of claims 48 to 71, wherein each RNA comprises a poly-A sequence.

73. The method of claim 71 or 72, wherein the poly-A sequence comprises at least 100 nucleotides.

74. The method of any one of claims 71-73, wherein the poly-A sequence comprises SEQ ID NO: 29 or consisting of the nucleotide sequence of seq id no.

75. The method of any one of claims 48 to 74, wherein the RNA is administered by injection.

76. The method of any one of claims 48 to 75, wherein the RNA is administered by intravenous administration.

77. The method of any one of claims 48 to 76, wherein the RNA is formulated as a lipid complex particle.

78. The method of any one of claims 48 to 77, wherein said RNA lipid complex particles are obtainable by mixing said RNA with liposomes.

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

80. The method of any one of claims 48 to 79, further comprising administering an additional treatment.

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

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

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

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

85. The method of claim 84, wherein the checkpoint modulator is an anti-PD 1 antibody, an anti-CTLA-4 antibody or an anti-PD 1 antibody in combination with an anti-CTLA-4 antibody.

Summary of The Invention

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

(i) an amino acid sequence comprising Kallikrein-2 (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 Prostate Acid Phosphatase (PAP), an immunogenic variant thereof, or an immunogenic fragment of PAP or an immunogenic variant thereof;

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

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

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

In one embodiment of the process of the present invention,

In one embodiment, at least one of the amino acid sequences recited in (i), (ii), (iii), (iv) or (v) is encoded by a coding sequence that: increased G/C content compared to the wild type coding sequence, and/or which is codon optimized, wherein codon optimization and/or increased G/C content preferably does not alter the sequence of the encoded amino acid sequence. In one embodiment, each of the amino acid sequences recited in (i), (ii), (iii), (iv) or (v) is encoded by a coding sequence that: increased G/C content compared to the wild type coding sequence, and/or which is codon optimized, wherein codon optimization and/or increased 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 stable 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 modified nucleosides 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 (m1 ψ), and 5-methyl-uridine (m 5U).

In one embodiment, at least one RNA comprises a 5' -cap m₂ ^7，2’-OGpp_sp (5') G. In one embodiment, each RNA comprises a 5' -cap m₂ ^7，2’-OGpp_sp(5’)G。

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

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

In one embodiment of the process of the present invention,

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 embodiment of the process of the present invention,

(i) the RNA encoding the secretion signal peptide comprises SEQ ID NO: 23, or a nucleotide sequence identical to SEQ ID NO: 23, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity; and/or

(ii) The secretion signal peptide comprises SEQ ID NO: 22, or an amino acid sequence identical to SEQ ID NO: 22, having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity.

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

In one embodiment of the process of the present invention,

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

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 SEQ ID NO: 29 or consisting of the nucleotide sequence of seq id no.

In one embodiment, the RNA is formulated as a liquid, as a solid, or a combination thereof. In one embodiment, the RNA is formulated for injection. In one embodiment, the RNA is formulated for intravenous administration. In one embodiment, the RNA is formulated or to be formulated as a lipid complex particle (lipoplex particle). In one embodiment, the RNA lipid complex particle may be obtained by mixing RNA with a liposome.

In one embodiment, the composition or pharmaceutical 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 pharmaceutical formulation is a kit (kit). In one embodiment, the RNA and optionally the liposome are in separate vials.

In one embodiment, the composition or pharmaceutical preparation further comprises RNA and optionally instructions for the use of liposomes for the treatment or prevention of prostate cancer (instructions).

In one aspect, provided herein are compositions or pharmaceutical formulations described herein for pharmaceutical use. In one embodiment, the pharmaceutical use comprises a therapeutic or prophylactic treatment of a disease or disorder. In one embodiment, therapeutic or prophylactic treatment of a disease or condition includes treatment or prevention of prostate cancer. In one embodiment, the composition or pharmaceutical formulation described herein is for administration to a human.

In one embodiment, the therapeutic or prophylactic treatment of a disease or disorder further comprises administering an additional treatment. In one embodiment, the additional treatment comprises one or more selected from the group consisting of: (i) surgery to resect, resect or debulk a tumor, (ii) radiation therapy, and (iii) chemotherapy. In one embodiment, the additional treatment comprises administration of an additional therapeutic agent. In one embodiment, the additional therapeutic agent comprises an anti-cancer therapeutic agent. In one embodiment, the additional therapeutic agent is a checkpoint modulator. In one embodiment, the checkpoint modulator is an anti-PD 1 antibody, an anti-CTLA-4 antibody, or an anti-PD 1 antibody in combination with an anti-CTLA-4 antibody.

In one aspect, provided herein is 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 amino acid sequence:

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

(ii) an amino acid sequence comprising Prostate Specific Antigen (PSA), an immunogenic variant thereof, or an immunogenic fragment of PSA or an immunogenic variant thereof;

(iii) an amino acid sequence comprising Prostatic Acid Phosphatase (PAP), an immunogenic variant thereof, or an immunogenic fragment of PAP or an immunogenic variant thereof;

(iv) an amino acid sequence comprising homology box B13(HOXB13), an immunogenic variant thereof, or an immunogenic fragment of HOXB13 or an immunogenic variant thereof; and

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

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

In one embodiment of the process of the present invention,

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

In one embodiment of the process of the present invention,

In one embodiment, at least one RNA comprises a 5' -cap m₂ ^7，2’-OGpp_sp (5') G. In one embodiment, each RNA comprises a 5' -cap m₂ ^7，2’-OGpp_sp(5’)G。

In one embodiment of the process of the present invention,

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 embodiment of the process of the present invention,

(ii) The secretion signal peptide comprises SEQ ID NO: 22, or an amino acid sequence identical to SEQ ID NO: 22, having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity.

In one embodiment of the process of the present invention,

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

In one embodiment, the RNA is formulated as a lipid complex particle. In one embodiment, the RNA lipid complex particle may be obtained by mixing RNA with a liposome.

In one embodiment, the subject is a human.

In one embodiment, the methods described herein further comprise administering an additional treatment. In one embodiment, the additional treatment comprises one or more selected from the group consisting of: (i) surgery to resect, resect or debulk a tumor, (ii) radiation therapy, and (iii) chemotherapy. In one embodiment, the additional treatment comprises administration of an additional therapeutic agent. In one embodiment, the additional therapeutic agent comprises an anti-cancer therapeutic agent. In one embodiment, the additional therapeutic agent is a checkpoint modulator. In one embodiment, the checkpoint modulator is an anti-PD 1 antibody, an anti-CTLA-4 antibody, or an anti-PD 1 antibody in combination with an anti-CTLA-4 antibody.

In one aspect, provided herein are RNAs described herein, e.g., for use in the methods described herein:

(i) an RNA encoding 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 RNA encoding an amino acid sequence comprising Prostate Specific Antigen (PSA), an immunogenic variant thereof, or an immunogenic fragment of PSA or an immunogenic variant thereof;

(iii) an RNA encoding an amino acid sequence comprising Prostatic Acid Phosphatase (PAP), an immunogenic variant thereof, or an immunogenic fragment of PAP or an immunogenic variant thereof;

(iv) an RNA encoding an amino acid sequence comprising homology box B13(HOXB13), an immunogenic variant thereof, or an immunogenic fragment of HOXB13 or an immunogenic variant thereof; and/or

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

Brief Description of Drawings

FIG. 1: general structure of RNA RBL038.1, RBL039.1, RBL040.1, RBL041.1 and RBL 045.1.

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

FIG. 2: 5' -capping structure beta-S-ARCA (D1) (m) ₂ ^7，2`-OGppSpG)。

Shown in red are β -S-ARCA (D1) and the basic cap analog m⁷Differences between gppppg: building block m⁷-OCH at C2' position of G₃Radical and sulfur substitution of the non-bridging oxygen at the beta-phosphate. The phosphorothioate cap analog β -S-ARCA exists in two diastereomers due to the presence of a stereogenic P-center (marked with an asterisk). These have been named D1 and D2 based on their elution order in reverse phase HPLC.

FIG. 3: vector map of plasmid pST4-hAg-Kozak-KLK2-GS-P2P16-GS-MITD-FI-A30L70 for RBL038.1 production.

Inserts with sequence elements such as labels are shown in different colors. Eam1104I represents a recognition site for a linearized restriction endonuclease. Kanamycin (Kanamycin) resistance gene is shown in black.

FIG. 4: vector map of plasmid pST4-hAg-Kozak-KLK3-GS-P2P16-GS-MITD-FI-A30L70 for RBL039.1 production.

Inserts with sequence elements such as labels are shown in different colors. Eam1104I represents a recognition site for a linearized restriction endonuclease. Kanamycin resistance gene is shown in black.

FIG. 5: vector mapping of plasmid pST4-hAg-Kozak-ACPP-GS-P2P16-GS-MITD-FI-A30L70 for RBLA0.1 production.

FIG. 6: vector map of plasmid pST4-hAg-Kozak-sec-GS-HOXB13-GS-P2P16-GS-MITD-FI-A30L70 for RBL041.1 production.

FIG. 7: vector map of plasmid pST4-hAg-Kozak-sec-GS-NKX3-1-GS-P2P16-GS-MITD-FI-A30L70 for RBL045.1 production.

FIG. 8: chemical structures of selected cationic lipids and co-lipids (co-lipids) tested during formulation development.

FIG. 9: organ selectivity of RNA lipid complexes with different charge ratios.

The positively charged luc-RNA lipid complexes showed high luciferase expression in the lung, while the negatively charged RNA lipid complexes showed highly selective luciferase expression in the spleen.

FIG. 10: the biological activity of the RNA lipid complex depends on the particle size and the size of the liposome used for the formulation.

Mu.g luc-RNA was condensed with small (198nm) and large (381nm) liposomes for reconstitution of RNA lipid complexes (RNA lipoplexe, RNA (LIP)) and injected into BALB/c mice (n-5). Luciferase expression (mean ± SD) in the spleen was analyzed 6 hours after luc-rna (lip) administration.

FIG. 11: particle size of RNA lipid complexes reconstituted according to clinical formulation protocol.

Particle size of RNA lipid complexes reconstituted by different experimenters, in different laboratories and with different RNA constructs was analyzed by PCS measurements. For the experiments numbered 3 and 10, two independent preparations were performed.

FIG. 12: size and polydispersity index of RNA lipid complexes with different charge ratios.

Particle size (z-average) and polydispersity index of RNA lipid complexes with different charge ratios (DOTMA: RNA) were measured 10 min, 2 h and 24 h after preparation.

FIG. 13: size and biological activity of RNA lipid complexes with different charge ratios.

(A) The particle size (z-average) and polydispersity index of RNA lipid complexes with different charge ratios (DOTMA: RNA) were measured directly after preparation (10 minutes). (B) Luciferase expression in the spleen was analyzed 6 hours after luc-RNA (lip) (20 μ g RNA) administration in BALB/c mice (n ═ 4 to 5).

FIG. 14: localization of bioluminescent signal following IV administration of luciferase rna (lip).

Bioluminescence imaging of (a) and ex vivo transplanted spleen, liver and lung (B) 6 hours after intravenous injection of luc-RNA (lip) (20 μ g RNA) into BALB/c mice (n ═ 3) in vivo. One representative mouse is shown.

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

BALB/c mice (n ═ 3) were injected intravenously with Cy5-RNA (40. mu.g (HED: 9.48mg)) formulated with rhodamine-labeled liposomes. Uptake of Cy 5-labeled RNA (lower panel) or rhodamine-labeled liposomes (upper panel) by cell populations in the spleen was assessed by flow cytometry 1 hour after lipid complex injection. Representative dot plots are shown.

FIG. 16: disruption of tolerance and antigen-specific in vivo cytotoxicity following immunization with AH5-RNA (LIP).

BALB/c mice (n ═ 5) were immunized intravenously with AH5-rna (lip) (40 μ gRNA) on days 0, 3, 8 and 15 (green). The frequency of antigen-specific CD8+ T cells was monitored in blood by gp70-MHC tetramer staining (grey). The line represents the average (A) of tetramer frequencies. BALB/c mice (n-5) were immunized intravenously with AH5-RNA (lip) (40 μ g RNA) on days 0, 3, and 8 or left untreated. On day 12, in vivo cytotoxicity assays were performed by administering a mixture of CFSEhigh (loaded with AH-5 peptide) and CFSElow (loaded with Inf-HA peptide) splenocytes from naive BALB/c mice. AH5 specific lysis is shown for one representative mouse. All AH5-rna (lip) immunized mice showed more than 90% antigen specific lytic activity (B).

FIG. 17: a transient increase in IFN- α following RNA (LIP) vaccination.

(A) C57BL/6 mice (n ═ 3) were injected with HA-RNA (lip) (40 μ g RNA), liposomes alone or PBS as control. Serum concentrations of IFN- α and TNF- α (mean. + -. SD) were assessed by ELISA 6 hours and 24 hours after treatment. (B) Uncontacted or splenectomized C57BL/6 mice (n-2) were injected intravenously with HA-RNA (lip) (40 μ g RNA). Serum concentrations of IFN- α (mean. + -. SD) were assessed by ELISA 6 hours after treatment.

FIG. 18: vaccination with W _ pro1 antigen RNA resulted in antigen-specific T cell responses.

Splenocytes from intravenous vaccinated a2/DR1 mice (n-4 to 5/group) were restimulated with BMDCs electroporated with the corresponding mRNA as indicated for 20 hours. BMDCs electroporated with irrelevant mRNA were used as controls (open symbols, gray bars). Effector function was measured using an IFN- γ ELISPOT assay. Symbols represent the average of triplicate wells from individual animals. Bars represent median of all animals per group.

FIG. 19: average levels of IFN-alpha (black bars) and IL-6 (gray bars) in the high dose group animals.

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

FIG. 20: induction of antigen-specific T cells in the spleen by KLK2-, KLK3-, ACPP-, NKX 3-1-and HOXB 13-encoding RNA.

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

Description of sequences

The following table provides a list of certain sequences referenced herein.

Detailed Description

Although the present disclosure is described in detail below, it is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described herein as these 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 disclosure 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.

Preferably, the terms used herein are defined as "A multilingual collaboration of biotechnology terms: (IUPAC Recommendations), "h.g.w.leuenberger, b.nagel and H.Eds, Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

Unless otherwise indicated, the practice of the present disclosure will employ conventional methods of chemical, biochemical, cell biological, immunological and recombinant DNA techniques as set forth in the literature of the art (see, e.g., Molecular Cloning: A Laboratory Manual, second edition, J.Sambrook et al, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

Hereinafter, elements of the present disclosure will be described. These elements are listed with some specific embodiments, however, it should be understood that they may be combined in any manner and in any number to produce additional embodiments. The various described examples and embodiments should not be construed as limiting the disclosure to only some of the embodiments explicitly described. This description should be understood to disclose and cover embodiments that combine the explicitly described embodiments with any number of the disclosed elements. Moreover, any arrangement or combination of all described elements is deemed to be disclosed by the specification unless otherwise indicated by the context.

The term "about" means about or near, and in one embodiment in the context of a numerical value or range recited herein means ± 20%, ± 10%, ± 5%, or ± 3% of the numerical value or range recited or claimed.

The use of nouns without quantitative modification and similar references in the context of describing the disclosure (especially in the context of the claims) is to be construed to cover one and/or more unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

The term "comprising" is used in the context of this document to indicate that there may optionally be additional members other than the members of the list introduced by "comprising" unless explicitly stated otherwise. However, the term "comprising" is contemplated as a specific embodiment of the disclosure to cover the possibility that no other member is present, i.e., for this purpose, the embodiment "comprising" is to be understood as having the meaning of "consisting of … …".

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

Definition of

The following definitions will be provided for all aspects of the present disclosure. Unless otherwise indicated, the following terms have the following meanings. Any undefined term has its art-recognized meaning.

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

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

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

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

where c is the molar concentration of the particular ionic species and z is the absolute value of its charge. The sum sigma is taken from all the different species of ions (i) in the solution.

In accordance with the present disclosure, in one embodiment, the term "ionic strength" relates to the presence of monovalent ions. With respect to the presence of divalent ions, particularly divalent cations, in one embodiment, the concentration or effective concentration thereof (presence of free ions) is low enough to prevent degradation of the RNA due to the presence of the chelating agent. In one embodiment, the concentration or effective concentration of the divalent ion is below the catalytic level for hydrolyzing phosphodiester bonds between RNA nucleotides. In one embodiment, the concentration of free divalent ions is 20 μ M or less. In one embodiment, free divalent ions are absent or substantially absent.

The term "freezing" relates to solidification of a liquid, usually accompanied by removal of heat.

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

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

The term "cryoprotectant" relates to a substance added to a formulation to protect an active ingredient during the freezing phase.

The term "lyoprotectant" relates to a substance added to a formulation to protect an active ingredient during the drying phase.

The term "reconstituting" relates to adding a solvent (e.g. water) to a dry product to return it to a liquid state, e.g. its original liquid state.

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

The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, peptides or nucleic acids that are present in organisms (including viruses) and that can be isolated from sources in nature and that have not been intentionally modified by man in the laboratory are naturally occurring. The term "found in nature" means "occurring in nature" and includes known objects as well as objects that have not been found and/or isolated from nature but may be found and/or isolated from natural sources in the future.

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

In the context of the present disclosure, the term "RNA lipid complex particle" relates to a particle comprising 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-lipid complex particles. Positively charged liposomes can generally be synthesized using cationic lipids (e.g., DOTMA) and additional lipids (e.g., DOPE). In one embodiment, the RNA lipid complex particle is a nanoparticle.

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

The term "mean diameter" refers to the average hydrodynamic diameter of a particle, as measured by Dynamic Light Scattering (DLS) and data analysis using the so-called cumulant algorithm (cumulant algorithm), which provides a so-called Z with a length dimension _{Mean value of}And dimensionless Polydispersity Index (PI) (Koppel, d., j.chem.phys.57, 1972, pages 4814 to 4820, ISO 13321). Here, the "average diameter", "diameter" or "size" of the particles and the Z_{Mean value of}The values of (A) are used synonymously.

The term "polydispersity index" is used herein as a measure of the size distribution of a particle (e.g., nanoparticle) ensemble (ensemble). The polydispersity index is calculated by so-called cumulant analysis based on dynamic light scattering measurements.

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 lipids throughout the solution and promotes lipid structure formation, e.g., lipid vesicle formation such as liposome formation. In general, the RNA lipid complex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. In one embodiment, such colloidal liposome dispersions are formed using ethanol injection techniques as follows: an ethanol solution comprising a lipid, for example a cationic lipid (such as DOTMA) and a further lipid, is injected into the aqueous solution under stirring. In one embodiment, the RNA lipid complex particles described herein are obtainable without an extrusion step.

The term "extrusion" and variations thereof refers to the production of particles having a fixed cross-sectional profile. In particular, it refers to the miniaturization of particles, whereby the particles are forced through a filter with defined pores.

The prostate is a small gland present in the lower abdomen of men. It is located below the bladder and around the urethra. The prostacyclin is regulated by testosterone and produces seminal fluid, also known as semen (semen). Semen is a substance containing sperm that exits the urinary tract during ejaculation.

As used herein, "prostate cancer" is cancer in the prostate. When abnormal, malignant growth of cells, called tumors, forms in the prostate, this is called prostate cancer. Most prostate cancers grow slowly; however, some grow relatively fast. Cancer cells can spread from the prostate to other areas of the body, particularly the bones and lymph nodes. It may not initially cause symptoms. In subsequent stages, it can lead to difficulty in 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. Many cases are managed by active monitoring (active reporting) or watchful waiting (watchful waiting). Other treatments may include a combination of surgery, radiation therapy, hormonal therapy, or chemotherapy. It may be curable when it occurs only within the prostate. Analgesics, bisphosphonates, targeted therapies, and the like may be useful in patients where the disease has spread to bone. The results depend on the age and other health issues of the person, and how aggressive and widespread the cancer is. Globally, it is the second most common cancer type and the fifth leading cause of cancer-related death in men.

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

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

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

The terms "identical (%)," identity (%) "or similar terms are particularly intended to refer to the percentage of nucleotides or amino acids that are identical in the best alignment between the sequences to be compared. The percentages are purely statistical and the differences between the two sequences may (but need not) be randomly distributed over the entire length of the sequences to be compared. Comparison of two sequences is typically performed by comparing the sequences after optimal alignment with respect to the segments or "comparison windows" to identify local regions of the corresponding sequences. The optimal alignment for comparison can be performed manually, or by means of the local homology algorithm of Smith and Waterman, 1981, Ads App.Math.2, 482, by means of the local homology algorithm of Needleman and Wunsch, 1970, J.mol.biol.48, 443, by means of the similarity search algorithm of Pearson and Lipman, 1988, Proc.Natl Acad.Sci.USA 85, 2444, or by means of Computer programs using said algorithms (BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm available on the National Center for Biotechnology Information (NCBI) website (e.g., BLAST. NCBI. nlm. nih. gov/BLAST. cgipap _ TYPE ═ BLAST search & BLAST _ SPEC ═ BLAST2seq & LINK _ LOC ═ align2 seq). In some embodiments, the algorithm parameters for the BLASTN algorithm on the NCBI website include: (i) the expected threshold is set to 10; (ii) the word size is set to 28; (iii) the maximum match within the query range is set to 0; (iv) match/no match scores are set to 1, -2; (v) the Gap Cost (Gap Cost) is set to be linear; and (vi) the filter of the low complexity area being used. In some embodiments, the algorithm parameters for the BLASTP algorithm on the NCBI website include: (i) the expected threshold is set to 10; (ii) the word length is set to 3; (iii) the maximum match within the query range is set to 0; (iv) the matrix is set to BLOSUM 62; (v) the notch cost is set to exist 11: extension: 1; and (vi) conditioning composition scoring matrix adjustment.

Percent identity is obtained by determining the number of identical positions to which the sequences to be compared correspond, dividing that number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying the 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 full length of the reference sequence. For example, in some embodiments, if a 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 consecutive nucleotides. In some embodiments, the degree of identity is given over the full length of the reference sequence.

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

RNA

In the present disclosure, the term "RNA" relates to a nucleic acid molecule comprising ribonucleotide residues. In some preferred embodiments, the RNA comprises all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' -position of the β -D-ribofuranosyl group. RNA encompasses, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA), substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such alteration may refer to the addition of non-nucleotide species to internal RNA nucleotides or to the RNA ends. It is also contemplated herein that the nucleotides in the RNA can be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the purposes of this disclosure, these altered RNAs are considered analogs of naturally occurring RNAs.

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

In one embodiment, the RNA is an in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter used to control transcription may be any promoter of any RNA polymerase. DNA templates for in vitro transcription can be obtained by cloning nucleic acids, in particular cDNA, and introducing them into suitable vectors for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

In one embodiment, the RNA can have modified nucleosides. In some embodiments, the RNA comprises modified nucleosides in place of at least one (e.g., each) uridine.

The term "uracil" as used herein describes one of the nucleobases that may be present in an RNA nucleic acid. Uracil has the structure:

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

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

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

"pseudouridine" is an example of a modified nucleoside that is an isomer of uridine, in which uracil is attached to the pentose ring by a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond.

Another exemplary modified nucleoside is N1-methyl-pseudouridine (m1 Ψ), having the following structure:

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

another exemplary modified nucleoside is 5-methyl-uridine (m5U), which has the following structure:

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

In some embodiments, the modified uridine instead of uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m1 ψ), or 5-methyl-uridine (m 5U).

In some embodiments, the modified nucleoside that replaces one or more uridines in the RNA may be any one or more of: 3-methyl-uridine (m)³U), 5-methoxy-uridine (mo)⁵U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine(s)²U), 4-thio-uridine(s)⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho)⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-glycolate (cmo)⁵U), uridine 5-glycolate (mcmo)⁵U), 5-carboxymethyl-uridine (cm) ⁵U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm)⁵U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm)⁵U), 5-methoxycarbonylmethyl-uridine (mcm)⁵U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm)⁵s²U), 5-aminomethyl-2-thio-uridine (nm)⁵s²U), 5-methylaminomethyl-uridine (mnm)⁵U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm)⁵s²U), 5-methylaminomethyl-2-seleno-uridine (mnm)⁵se²U), 5-carbamoylmethyl-uridine (ncm)⁵U), 5-Carboxymethylaminomethyl-uridine (cmnm)⁵U), 5-Carboxymethylaminomethyl-2-thio-uridine (cmnm)⁵s²U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taunomethyl-uridine (. tau.m)⁵U), 1-taunomethyl-pseudouridine, 5-taunomethyl-2-thio-uridine (. tau.m.sup.5s2U), 1-taunomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m.sup.m)⁵s²U), 1-methyl-4-thio-pseudouridine (m)¹s⁴Psi), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m)³Psi), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5, 6-dihydrouridine, 5-methyl-dihydrouridine (m)⁵D) 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3- (3-amino-3-carboxypropyl) uridine (acp)³U), 1-methyl-3- (3-amino-3-carboxypropyl) pseudouridine (acp)³Psi), 5- (isopentenylaminomethyl) uridine (inm)⁵U), 5- (isopentenylaminomethyl) -2-thio-uridine (inm)⁵s²U), α -thio-uridine, 2 '-O-methyl-uridine (Um), 5, 2' -O-dimethyl-uridine (m)⁵Um), 2 '-O-methyl-pseudouridine (ψ m), 2-thio-2' -O-methyl-uridine(s)²Um), 5-methoxycarbonylmethyl-2' -O-methyl-uridine (mcm)⁵Um), 5-carbamoylmethyl-2' -O-methyl-uridine (ncm)⁵Um), 5-carboxymethylaminomethyl-2' -O-methyl-uridine (cmnm)⁵Um), 3, 2' -O-dimethyl-uridine (m)³Um), 5- (isopentenylaminomethyl) -2' -O-methyl-uridine (inm)⁵Um), 1-thio-uridine, deoxythymidine, 2 ' -F-arabino-uridine, 2 ' -F-uridine, 2 ' -OH-arabino-uridine, 5- (2-methoxycarbonylethenyl) uridine, 5- [3- (1-E-propenylamino) uridine, or any other modified uridine known in the art.

In some embodiments, the at least one RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, the at least one RNA comprises modified nucleosides 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-methyl-pseudouridine (m1 ψ), and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1 ψ). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m 5U). In some embodiments, at least one RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1 ψ), and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ) and N1-methyl-pseudouridine (m1 ψ). In some embodiments, the modified nucleoside comprises pseudouridine (ψ) and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1 ψ) and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ), N1-methyl-pseudouridine (m1 ψ), and 5-methyl-uridine (m 5U).

In some embodiments, an RNA according to the present disclosure comprises a 5' -cap. In one embodiment, the RNA of the present disclosure does not have uncapped 5' -triphosphates. In one embodiment, the RNA may be modified with a 5' -cap analog. The term "5 '-cap" refers to a structure present on the 5' end of an mRNA molecule and typically consists of guanosine nucleotides linked to the mRNA by 5 '-to 5' -triphosphate linkages. In one embodiment, the guanosine is methylated at position 7. Providing RNA with a 5 ' -cap or 5 ' -cap analog can be achieved by in vitro transcription, where the 5 ' -cap is co-transcribed into the RNA strand, or can be linked to the RNA post-transcription using a capping enzyme.

In some embodiments, the building block cap of the RNA is m₂ ^7，3’-OGppp(m₁ ^2’-O) ApG (sometimes also referred to as m)₂ ^7，3’ ^OG(5’)ppp(5’)m^2’-OApG) having the following structure:

below is an exemplary Cap 1(Cap1) RNA comprising RNA and m₂ ^7，3`OG(5’)PPP(5’)m^2’-OApG：

The following is another exemplary cap 1RNA (capless analog):

in some embodiments, in one embodiment, a cap analog anti-inversion cap (ARCA cap (m) having the structure below is used₂ ^7’3`OG (5 ') ppp (5') G)) modified RNA with a "Cap 0(Cap 0)" structure:

the following is a DNA fragment containing RNA and m₂ ^7，3`OExemplary cap 0RNA for G (5 ') ppp (5') G:

in some embodiments, a cap analog having the structure β -S-ARCA (m) is used₂ ^7，2`OG (5 ') ppSp (5') G) to produce a "cap 0" structure:

the following are compositions comprising beta-S-ARCA (m)₂ ^7，2`OG (5 ') ppSp (5') G) and an exemplary cap of RNA 0 RNA:

particularly preferred caps comprise a 5' -cap m₂ ^7，2`OG (5 ') ppSp (5') G. In some embodiments, at least one RNA described herein comprises a 5' -cap m₂ ^7，2`OG (5 ') ppSp (5') G. In some embodiments, each RNA described herein comprises a 5' cap m₂ ^7，2`OG(5’)ppSp(5’)G。

In some embodiments, an RNA according to the present disclosure comprises a 5 '-UTR and/or a 3' -UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to a corresponding region in an RNA molecule (e.g., an mRNA molecule). Untranslated regions (UTRs) may be present 5 '(upstream) (5' -UTR) and/or 3 '(downstream) (3' -UTR) of the open reading frame. The 5 '-UTR (if present) is located at the 5' end, upstream of the start codon of the protein coding region. The 5 ' -UTR is located downstream of the 5 ' -cap (if present), e.g., directly adjacent to the 5 ' -cap. The 3 ' -UTR, if present, is located at the 3 ' end, downstream of the stop codon of the protein coding region, but the term "3 ' -UTR" preferably does not comprise a poly-A sequence. Thus, the 3' -UTR is located upstream of the poly-A sequence (if present), e.g., immediately adjacent to the poly-A sequence.

A particularly preferred 5' -UTR comprises SEQ ID NO: 21. A particularly preferred 3' -UTR comprises SEQ ID NO: 28.

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

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

The term "poly-A tail" or "poly-A sequence" as used herein refers to an uninterrupted or interrupted sequence of adenylate residues typically located at the 3' end of an RNA molecule. The poly-A tail or poly-A sequence is known to those skilled in the art and may follow the 3' -UTR in the RNA described herein. The uninterrupted poly-A tail is characterized by continuous adenylate residues. In practice, uninterrupted poly-A tails are typical. The RNAs disclosed herein may have a poly-a tail that is linked to the free 3' end of the RNA after transcription by a template-independent RNA polymerase or a poly-a tail that is encoded by DNA and transcribed by a template-dependent RNA polymerase.

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

The poly-A tail may be of any length. In some embodiments, the poly-a tail comprises, consists essentially of, or consists of: at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 a nucleotides, and particularly about 120 a nucleotides. In the present context, "consisting essentially of … …" means that the majority of 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% by number of nucleotides in the poly-A tail, are A nucleotides, but that the remaining nucleotides are allowed to be nucleotides other than A nucleotides, such as U nucleotides (uridines), G nucleotides (guandines), or C nucleotides (cytidines). In the present context, "consisting of … …" means that all nucleotides in the poly-A tail, i.e. 100% by number of nucleotides in the poly-A tail are A nucleotides. The term "A nucleotide" or "A" refers to an adenosine.

In some embodiments, a poly-a tail is ligated during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence (coding strand) encoding the poly-A tail is referred to as the poly (A) cassette.

In some embodiments, the poly (a) cassette present in the DNA coding strand consists essentially of dA nucleotides, but is interrupted by a random sequence of four nucleotides (dA, dC, dG, and dT). Such random sequences may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016/005324 a1, which is incorporated herein by reference. Any of the poly (A) cassettes disclosed in WO 2016/005324A 1 may be used in the present invention. The following are contemplated: a poly (a) cassette consisting essentially of dA nucleotides but interrupted by a random sequence with four nucleotides (dA, dC, dG, dT) in equal distribution and a length of e.g. 5 to 50 nucleotides shows a constant proliferation of plasmid DNA in e.coli (e.coli) at the DNA level, while still being associated with beneficial properties for supporting RNA stability and translation efficiency at the RNA level. Thus, in some embodiments, the poly-a tail comprised in an RNA molecule described herein consists essentially of a nucleotides, but is interrupted by a random sequence of four nucleotides (A, C, G, U). Such random sequences may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

In some embodiments, no nucleotide other than an a nucleotide is flanked on its 3 'end by a poly-a tail, i.e., the poly-a tail is not masked or followed at its 3' end by a nucleotide other than a.

In some embodiments, the poly-A tail comprises 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 consist of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may comprise SEQ ID NO: 29, poly-A tail as shown in. 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 disclosure, the term "transcription" relates to a process in which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into a peptide or protein.

With respect to RNA, the terms "expression" or "translation" relate to the process in the ribosome of a cell by which a strand of mRNA directs the assembly of an amino acid sequence to produce a peptide or protein.

In one embodiment, at least a portion of the RNA is delivered to the target cell after administration of the RNA described herein, e.g., formulated as an RNA lipid complex particle. In one embodiment, at least a portion of the RNA is delivered to the cytosol of the target cell. In one embodiment, the RNA is translated by the target cell to produce the peptide or protein encoded thereby. 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 macrophage. The RNA lipid complex particles described herein can be used to deliver RNA to such target cells. Accordingly, the present disclosure also relates to a method for delivering RNA to a target cell in a subject, the method comprising administering to the subject an RNA lipid complex particle as described herein. In one embodiment, the RNA is delivered to the cytosol of the target cell. In one embodiment, the RNA is translated by a target cell to produce a peptide or protein encoded by the RNA.

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

According to the present disclosure, the term "peptide" encompasses oligopeptides and polypeptides, and refers to a substance comprising about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked to each other by peptide bonds. The term "protein" refers to large peptides, particularly peptides having at least about 151 amino acids, although the terms "peptide" and "protein" are generally used herein as synonyms.

The term "antigen" relates to a substance comprising such an epitope: an immune response can be generated against the epitope. In particular, the term "antigen" includes proteins and peptides. In one embodiment, the antigen is presented by a cell of the immune system (e.g., an antigen presenting cell such as a dendritic cell or macrophage). In one embodiment, the antigen or processed product thereof, e.g., a T cell epitope, is bound by a T or B cell receptor or by an immunoglobulin molecule, e.g., an antibody. Thus, an antigen or its processed product can specifically react with an antibody or a T lymphocyte (T cell). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, and the epitope is derived from such an antigen.

The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. Disease-associated antigens are molecules that: which comprises epitopes that will stimulate the immune system of the host to generate a cellular antigen-specific immune response and/or a humoral antibody response against the disease. Thus, the disease-associated antigen or epitope thereof can be used for therapeutic purposes. The disease-associated antigen may be associated with a cancer (typically a tumour).

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

The term "epitope" refers to a portion or fragment of a molecule (e.g., an antigen) that is recognized by the immune system. For example, the epitope may be recognized by a T cell, B cell, or antibody. An epitope of an antigen may comprise a continuous or discontinuous portion of the antigen and may be about 5 to about 100 amino acids in length. In one embodiment, the epitope is 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 T cells when present in the context of MHC molecules. The terms "major histocompatibility complex" and the abbreviation "MHC" include MHC class I and MHC class II molecules and relate to the gene complex present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting or diseased cells in immune responses, where they bind peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the cell surface and display to T cells both self-antigens (peptide fragments from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms). In the case of 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. In the case of MHC class II/peptide complexes, the binding peptides are generally about 10 to about 25 amino acids long, and in particular about 13 to about 18 amino acids long, although longer and shorter peptides may be effective.

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

The RNA administered

In some embodiments, the compositions described herein comprise RNA encoding kallikrein-2 (KLK2) protein, RNA encoding Prostate Specific Antigen (PSA) protein, RNA encoding Prostate Acid Phosphatase (PAP) protein, RNA encoding homeobox B13(HOXB13) protein, and RNA encoding NK3 homeobox 1(NKX3-1) protein. Likewise, the methods described herein comprise administering RNA encoding kallikrein-2 (KLK2) protein, RNA encoding Prostate Specific Antigen (PSA) protein, RNA encoding Prostate Acid Phosphatase (PAP) protein, RNA encoding homeobox B13(HOXB13) protein, and RNA encoding NK3 homeobox 1(NKX3-1) protein.

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

A Prostate Specific Antigen (PSA) protein comprises an amino acid sequence comprising PSA, an immunogenic variant thereof, or an immunogenic fragment of PSA or an immunogenic variant thereof, and may have an amino acid sequence comprising: SEQ ID NO: 5 or 6, or an amino acid sequence substantially identical to SEQ ID NO: 5 or 6, having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity. Rna encoding a PSA protein (i) may comprise SEQ ID NO: 7 or 8, or a nucleotide sequence identical to SEQ ID NO: 7 or 8, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity; and/or (ii) may encode an amino acid sequence comprising: SEQ ID NO: 5 or 6, or an amino acid sequence substantially identical to SEQ ID NO: 5 or 6, having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity.

The Prostatic Acid Phosphatase (PAP) protein comprises an amino acid sequence comprising PAP, an immunogenic variant thereof, or an immunogenic fragment of PAP or an immunogenic variant thereof, and may have an amino acid sequence comprising: SEQ ID NO: 9 or 10, or an amino acid sequence substantially identical to SEQ ID NO: 9 or 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity. Rna encoding a PAP protein (i) may comprise SEQ ID NO: 11 or 12, or a nucleotide sequence identical to SEQ ID NO: 11 or 12, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity; and/or (ii) may encode an amino acid sequence comprising: SEQ ID NO: 9 or 10, or an amino acid sequence substantially identical to SEQ ID NO: 9 or 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity.

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

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

By "variant" 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 naturally occurring or Wild Type (WT) amino acid sequence, or may be a modified form of a wild type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification as compared to the parent amino acid sequence, e.g., from 1 to about 20 amino acid modifications as compared to the parent, and preferably from 1 to about 10 or from 1 to about 5 amino acid modifications.

"wild-type" or "WT" or "native" herein means an amino acid sequence that occurs in nature, including allelic variations. The wild-type amino acid sequence, peptide or protein has an amino acid sequence which has not been intentionally modified.

For the purposes of the present disclosure, "variants" of an amino acid sequence (peptide, protein, or polypeptide) include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and/or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformers (conformations), isoforms, allelic variants, species variants and species homologues, in particular those occurring in nature.

Amino acid insertion variants include insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a particular site in the amino acid sequence, although random insertion and appropriate screening of the resulting product are also possible. Amino acid addition variants comprise amino and/or carboxy terminal fusions of one or more amino acids, e.g., 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, e.g., the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may be in any position of the protein. Deletion variants comprising a deletion of an amino acid at the N-terminal and/or C-terminal end of the protein are also referred to as N-terminal and/or C-terminal truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Preference is given to modifications in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and/or to replacing amino acids with further amino acids having similar properties. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions that resemble charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one of the related families of amino acids in its side chain. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid); basic amino acids (lysine, arginine, histidine); nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups:

Glycine, alanine;

valine, isoleucine, leucine;

aspartic acid, glutamic acid;

asparagine, glutamine;

serine, threonine;

lysine, arginine; and

phenylalanine, tyrosine.

Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that 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 a region of amino acids that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the full length of the reference amino acid sequence. For example, if a 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 consecutive amino acids. In some preferred embodiments, the degree of similarity or identity is given over the full length of the reference amino acid sequence.

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

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

When peptide and protein antigens (KLK2 protein, PSA protein, PAP protein, HOXB13 protein, and NKX3-1 protein) as described herein are provided to a subject by administering RNA encoding the antigen (i.e., vaccine antigen), stimulation, sensitization, and/or expansion of T cells is preferably caused in the subject. The 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, a vaccine antigen may comprise a disease-associated antigen, or a fragment or variant thereof. In one embodiment, such a fragment or variant is immunologically equivalent to a disease-associated antigen. In the context of the present disclosure, the term "fragment of an antigen" or "variant of an antigen" means a substance that results in stimulation, priming and/or expansion of T cells that target a disease-associated antigen, particularly when expressed on the surface of a diseased cell, tissue and/or organ. Thus, a vaccine antigen administered according to the present disclosure may correspond to or may comprise a disease-associated antigen, may correspond to or may comprise a fragment of a disease-associated antigen, or may correspond to or may comprise an antigen that is homologous to a disease-associated antigen or a fragment thereof. If a vaccine antigen administered according to the present disclosure comprises a fragment of a disease-associated antigen or an amino acid sequence homologous to a fragment of a disease-associated antigen, the fragment or amino acid sequence may comprise an epitope of a disease-associated antigen or a sequence homologous to an epitope of a disease-associated antigen to which T cells bind. Thus, according to the present disclosure, an antigen may comprise an immunogenic fragment of a disease-associated antigen or an amino acid sequence homologous to an immunogenic fragment of a disease-associated antigen. An "immunogenic fragment of an antigen" according to the present disclosure preferably relates to a fragment of an antigen capable of stimulating, priming and/or expanding T cells. Preferably, the vaccine antigen (similar to a disease-associated antigen) provides an associated epitope to be bound by T cells. Also preferably, vaccine antigens (similar to disease-associated antigens) are expressed on the surface of cells, such as antigen presenting cells, to provide the relevant epitopes for binding by T cells. The vaccine antigen according to the invention may be a recombinant antigen.

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

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

The term "priming" refers to a process in which T cells first come into contact with their specific antigen and lead to differentiation into effector T cells.

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

Lipid complex particles

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

Spleen-targeting RNA lipid complex particles are described in WO 2013/143683, which is incorporated herein by reference. It has been found that RNA lipid complex 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. Thus, following administration of the RNA lipid complex particles, RNA accumulation and/or RNA expression occurs in the spleen. Thus, the RNA lipid complex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, no or substantially no RNA accumulation and/or RNA expression occurs in the lung and/or liver after administration of the RNA lipid complex particle. In one embodiment, RNA accumulation and/or RNA expression occurs in antigen presenting cells, e.g., professional antigen presenting cells in the spleen, after administration of the RNA lipid complex particles. Thus, the RNA lipid complex particles of the present disclosure can be used to express RNA in such antigen presenting cells. In one embodiment, the antigen presenting cell is a dendritic cell and/or a macrophage.

RNA lipid Complex particle diameter

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

In one embodiment, the RNA lipid complex particles described herein exhibit a polydispersity index of less than about 0.5, less than about 0.4, or less than about 0.3. For example, the RNA lipid complex particles can exhibit a polydispersity index of about 0.1 to about 0.3.

Lipid

In one embodiment, the lipid solution, liposome, and RNA lipid complex particles described herein comprise a cationic lipid. As used herein, "cationic lipid" refers to a lipid having a net positive charge. Cationic lipids bind negatively charged RNA through electrostatic interactions with the lipid matrix. Generally, cationic lipids have a lipophilic moiety, such as a sterol, acyl, or diacyl chain, and the head group of the lipid typically carries a positive charge. Some 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 propane, 1, 2-dialkoxy-3-dimethylammonium propane, dioctadecyldimethylammonium chloride (DODAC), 2, 3-ditetradecyloxy-propyl- (2-hydroxyethyl) -dimethylammonium (2, 3-di (tetracoxy) propyl- (2-hydroxyethoxyl) -Dimethylammonium (DMRIE), 1, 2-dimyristoyl-sn-glycero-3-ethylphosphonic acid choline (DMEPC), 1, 2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1, 2-dioleoxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2, 3-dioleoyloxy-N- [2 (spermine carboxamide) ethyl ] -N, N-dimethyl-1-trifluoroacetate propylamine (DOSPA). Preferred are DOTMA, DOTAP, DODAC and DOSPA. In some embodiments, the cationic lipid is DOTMA and/or DOTAP.

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

In certain embodiments, the RNA lipid complex particle comprises both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE. Without wishing to be bound by theory, the amount of the at least one cationic lipid compared to the amount of the at least one additional lipid may affect important RNA lipid complex particle characteristics, such as charge, particle size, stability, tissue selectivity, and biological activity of RNA. Thus, in some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10: 0 to about 1: 9, from about 4: 1 to about 1: 2, or from about 3: 1 to about 1: 1. In some embodiments, the molar ratio may be about 3: 1, about 2.75: 1, about 2.5: 1, about 2.25: 1, about 2: 1, about 1.75: 1, about 1.5: 1, about 1.25: 1, or about 1: 1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2: 1.

Charge ratio

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

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

It has been found that RNA lipid complex particles having such charge ratios are useful for preferentially targeting spleen tissue or spleen cells, such as antigen presenting cells, in particular dendritic cells. Thus, in one embodiment, RNA accumulation and/or RNA expression occurs in the spleen after administration of the RNA lipid complex particle. Thus, the RNA lipid complex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, no or substantially no RNA accumulation and/or RNA expression occurs in the lung and/or liver after administration of the RNA lipid complex particle. In one embodiment, RNA accumulation and/or RNA expression occurs in antigen presenting cells, e.g., professional antigen presenting cells in the spleen, after administration of the RNA lipid complex particles. Thus, the RNA lipid complex particles of the present disclosure can be used to express RNA in such antigen presenting cells. In one embodiment, the antigen presenting cell is a dendritic cell and/or a macrophage.

A. Salt and ionic strength

In accordance with the present disclosure, the compositions described herein may comprise a salt, such as sodium chloride. Without wishing to be bound by theory, sodium chloride is used as an ionic osmolyte agent (ionic osmolyte agent) for pre-treating RNA prior to mixing with the at least one cationic lipid. In the present disclosure, certain embodiments contemplate alternative organic or inorganic salts to sodium chloride. Alternative salts include, but are not limited to: potassium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium acetate, potassium hydrogen carbonate, potassium sulfate, potassium acetate, disodium phosphate, sodium dihydrogen phosphate, sodium acetate, sodium hydrogen carbonate, sodium sulfate, sodium acetate, lithium chloride, magnesium phosphate, calcium chloride, and Ethylene Diamine Tetraacetic Acid (EDTA) sodium salt.

In general, compositions comprising the RNA lipid complex particles described herein comprise sodium chloride in a concentration preferably from 0mM to about 500mM, from about 5mM to about 400mM, or from about 10mM to about 300 mM. In one embodiment, the composition comprising RNA lipid complex particles comprises an ionic strength corresponding to such a sodium chloride concentration.

B. Stabilizer

The compositions described herein may comprise a stabilizer to avoid substantial loss of product quality, and in particular to avoid substantial loss of RNA activity, during freezing, lyophilization, spray drying, or storage, e.g., storage of the frozen, lyophilized, or spray dried compositions.

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

In some embodiments of the present disclosure, the stabilizing agent is mannose, glucose, sucrose, or trehalose.

In accordance with the present disclosure, the RNA lipid complex particle compositions described herein have a stabilizer concentration suitable for the stability of the composition, in particular the stability of the RNA lipid complex particles and the stability of the RNA.

pH and buffer

In accordance with the present disclosure, the RNA lipid complex particle compositions described herein have a pH suitable for the stability of the RNA lipid complex particles, and in particular for the stability of RNA. In one embodiment, the pH of the RNA lipid complex particle composition described herein is from about 5.5 to about 7.5.

In accordance with the present disclosure, compositions comprising a buffering agent are provided. Without wishing to be bound by theory, the use of a buffering agent maintains the pH of the composition during manufacture, storage, and use of the composition. In certain embodiments of the present disclosure, the buffering agent may be sodium bicarbonate, sodium dihydrogen phosphate, disodium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, [ Tris (hydroxymethyl) methylamino ] propanesulfonic acid (TAPS), 2- (bis (2-hydroxyethyl) amino) acetic acid (Bicine), 2-amino 2- (hydroxymethyl) propane-1, 3-diol (Tris), N- (2-hydroxy-1, 1-bis (hydroxymethyl) ethyl) glycine (Tricine), 3- [ [1, 3-dihydroxy-2- (hydroxymethyl) 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-piperazinediethanesulfonic acid (PIPES), dimethylarsinic acid, 2-morpholin-4-ylethanesulfonic acid (MES), 3-morpholin-2-hydroxypropanesulfonic acid (MOPSO) or Phosphate Buffered Saline (PBS). Other suitable buffers may be acetates, citrates, borates and phosphates.

In one embodiment, the buffer is HEPES.

In one embodiment, the buffer is at a concentration of about 2.5mM to about 15 mM.

D. Chelating agents

Certain embodiments of the present disclosure contemplate the use of a chelating agent. Chelating agents refer to chemical compounds capable of forming at least two coordinate covalent bonds with a metal ion, thereby producing a stable water-soluble complex. Without wishing to be bound by theory, the chelating agent reduces the concentration of free divalent ions, which in the present disclosure may additionally induce accelerated RNA degradation. Some examples of suitable chelating agents include, but are not limited to: ethylenediaminetetraacetic acid (EDTA), EDTA salts, desferrioxamine b (desferrioxamine b), deferoxamine (deferoxamine), sodium dithiocarbaminate (dithiocarb sodium), penicillamine, calcium pentanate, valeric acid sodium salt, succinic acid (succimer), trientine (trientine), nitrilotriacetic acid (nitrilotriacetic acid), trans-diaminocyclohexane tetraacetic acid (DCTA), diethylenetriaminepentaacetic acid (DTPA), bis (aminoethyl) glycolether-N, N' -tetraacetic acid, iminodiacetic acid, citric acid, tartaric acid, fumaric acid, or salts thereof. In certain embodiments, the chelating agent is EDTA or an EDTA salt. In an exemplary embodiment, the chelating agent is disodium EDTA dihydrate.

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

E. Physical state of the compositions of the present disclosure

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

Pharmaceutical compositions of the present disclosure

The RNA as described herein, e.g. formulated as RNA lipid complex particles, may be used as or for the preparation of a pharmaceutical composition or medicament for therapeutic or prophylactic treatment.

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

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

The pharmaceutical compositions of the present disclosure preferably comprise or can be administered with one or more adjuvants. The term "adjuvant" relates to compounds that prolong, enhance or accelerate the immune response. Adjuvants include compounds such as oil emulsions (e.g., Freund's adjuvant), mineral compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), orA heterogeneous group of immunostimulatory complexes. Some examples of adjuvants include, but are not limited to: LPS, GP96, CpG oligodeoxynucleotides, growth factors and cytokines, such as monokines, lymphokines, interleukins, chemokines. The chemokine can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INFa, INF-gamma, GM-CSF, LT-a. Other known adjuvants are aluminium hydroxide, Freund's adjuvant or oils, e.g.ISA 51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3 Cys.

The pharmaceutical compositions according to the present disclosure are typically applied in "pharmaceutically effective amounts" and in "pharmaceutically acceptable formulations".

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

The term "pharmaceutically effective amount" refers to an amount that alone or in combination with another dose achieves a desired response or desired effect. In the case of treatment of a particular disease, the desired response preferably involves inhibition of the disease process. This includes slowing the progression of the disease and in particular interrupting or reversing the progression of the disease. The desired response in the treatment of a disease may also be the delay in onset or prevention of the onset of the disease or the condition. The effective amount of the compositions described herein will depend on: the condition to be treated, the severity of the disease, individual parameters of the patient including age, physiological condition, size and weight, duration of treatment, type of concomitant therapy (if any), specific route of administration and the like. Thus, the dosage of administration of the compositions described herein may depend on a variety of such parameters. In the event that the response in the patient is inadequate at the initial dose, higher doses may be used (or an effectively higher dose achieved by a different, more topical route of administration).

In some embodiments, an effective amount comprises an amount sufficient to cause tumor/lesion shrinkage. In some embodiments, an effective amount is an amount sufficient to reduce the rate of tumor growth (e.g., inhibit tumor growth). In some embodiments, an effective amount is an amount sufficient to delay tumor 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 increase the immune response of a subject to a tumor such that tumor growth and/or size and/or metastasis is reduced, delayed, improved and/or prevented. An effective amount may be administered in one or more administrations. In some embodiments, administration of an effective amount (e.g., a composition comprising mRNA) can: (i) reducing the number of cancer cells; (ii) reducing tumor size; (iii) inhibit, delay, slow down and prevent cancer cell infiltration into peripheral organs to some extent; (iv) inhibit (e.g., slow and/or block or prevent to some extent) metastasis; (v) inhibiting tumor growth; (vi) preventing or delaying the occurrence and/or recurrence of a tumor; and/or (vii) alleviate one or more symptoms associated with cancer to some extent.

The pharmaceutical compositions of the present disclosure may comprise a salt, a buffer, a preservative, and optionally other therapeutic agents. In one embodiment, the pharmaceutical composition of the present disclosure comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.

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

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

The term "diluent" relates to a diluent (diluting agent) and/or a thinning agent (thining agent). Further, the term "diluent" includes any one or more of a fluid, liquid or solid suspension, and/or a mixing medium. Some examples of suitable diluents include ethanol, glycerol, and water.

The term "carrier" refers to a component that can be natural, synthetic, organic, inorganic, in which the active components are combined to facilitate, enhance, or effect administration of the pharmaceutical composition. The carrier used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a subject. Suitable vectors include, but are not limited to: sterile water, Ringer's solution (Ringer), lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycol, hydrogenated naphtho and in particular biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure comprises isotonic saline.

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 editors 1985).

The choice of pharmaceutically acceptable carrier, excipient or diluent can be made according to the intended route of administration and standard pharmaceutical practice.

Route of administration of pharmaceutical compositions of the present disclosure

In one embodiment, the pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally, intratubercularly, or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to administration in any manner other than through the gastrointestinal tract, for example, by intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for systemic administration. In another preferred embodiment, the systemic administration is by intravenous administration.

Use of pharmaceutical compositions of the present disclosure

The RNA, e.g., formulated as RNA lipid complex particles, described herein can be used in the therapeutic or prophylactic treatment of a disease, wherein providing an amino acid sequence encoded by the RNA to a subject results in a therapeutic or prophylactic effect.

The term "disease" refers to an abnormal condition affecting the body of an individual. A disease is generally interpreted as a medical condition associated with specific symptoms and signs. The disease may be caused by factors originally from an external source, such as an infectious disease, or the disease may be caused by internal dysfunction, such as an autoimmune disease. In humans, "disease" is generally used more broadly to refer to any condition that causes: pain, dysfunction, confusion, social problems, or death of the afflicted individual, or problems similar to those that contact the individual. In a broad sense, diseases sometimes include injuries, disabilities, disorders, syndromes, infections, isolated symptoms, abnormal behavior, and atypical changes in structure and function, while in other contexts and for other purposes, these may be considered distinguishable categories. Diseases often affect individuals not only physically but also emotionally, as infection with multiple diseases and living in the presence of multiple diseases can change a person's opinion of life and the person's personality.

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

The term "therapeutic treatment" relates to any treatment that improves a health condition and/or extends (enhances) the longevity of an individual. The treatment can eliminate the disease in the subject, arrest or slow the progression of the disease in the subject, inhibit or slow the progression of the disease in the subject, reduce the frequency or severity of symptoms in the subject, and/or reduce relapse in a subject currently suffering from or previously suffering from the disease.

The term "prophylactic treatment" or "prophylactic treatment" relates to any treatment intended to prevent the occurrence of a disease in an individual. The terms "prophylactic treatment" or "prophylactic treatment" are used interchangeably herein.

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

The term "patient" means an individual or subject to be treated, particularly an individual or subject suffering from a disease.

In one embodiment of the present disclosure, it is an object to provide an immune response against cancer cells expressing one or more tumor antigens and to treat cancer diseases involving cells expressing one or more tumor antigens. In one embodiment, the cancer is prostate cancer. In one embodiment, the tumor antigen is KLK2, PSA, PAP, HOXB13 and/or NKX 3-1.

A pharmaceutical composition comprising an RNA can be administered to a subject to elicit an immune response in the subject against one or more antigens or one or more epitopes encoded by the RNA, which can be therapeutic or partially or fully protective. One skilled in the art will appreciate that one of the principles of immunotherapy and vaccination is based on the following facts: an immunoprotective response to a disease is generated by immunizing a subject with an antigen or epitope that is immunologically related to the disease to be treated. Thus, the pharmaceutical compositions described herein may be applied to induce or enhance an immune response. Accordingly, the pharmaceutical compositions described herein may be used in the prophylactic and/or therapeutic treatment of diseases involving antigens or epitopes, in particular prostate cancer.

As used herein, "immune response" refers to an integrated bodily response to an antigen or cell expressing an antigen, and refers to a cellular immune response and/or a humoral immune response. Cellular immune responses include, but are not limited to, cellular responses against cells that express an antigen and are characterized by presentation of the antigen with MHC class I or class II molecules. The cellular response is associated with T lymphocytes, which can be classified as helper T cells (also known as CD4+ T cells) by modulating the immune response or killing cells (also known as cytotoxic T cells, CD 8)⁺T cells or CTLs) to induce apoptosis in infected or cancer cells. In one embodiment, administration of the pharmaceutical composition of the present disclosure involves stimulating anti-tumor CD8 against cancer cells expressing one or more tumor antigens⁺T cell response. In a specific embodiment, the tumor antigen is presented together with an MHC class I molecule.

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

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

The term "immunization" or "vaccination" describes the process of administering an antigen to an individual for the purpose of inducing an immune response, e.g. for therapeutic or prophylactic reasons.

In one embodiment, the present disclosure contemplates embodiments wherein: wherein an RNA lipid complex particle as described herein that targets spleen tissue is administered. The RNA encodes a peptide or protein comprising an antigen or epitope, e.g., as described herein. The RNA is taken up by antigen presenting cells (e.g., dendritic cells) in the spleen to express the peptide or protein. Following optional processing and presentation by the antigen presenting cells, an immune response may be generated against the antigen or epitope, resulting in prophylactic and/or therapeutic treatment of a disease in which the antigen or epitope is involved. In one embodiment, the immune response induced by the RNA lipid complex particles described herein includes presentation of an antigen or fragment thereof, e.g., an epitope, by an antigen presenting cell, e.g., a dendritic cell and/or macrophage, and activation of cytotoxic T cells due to the presentation. For example, a peptide encoded by an RNA or a processed product or protein thereof may be presented by a Major Histocompatibility Complex (MHC) protein expressed on an antigen presenting cell. The MHC peptide complexes can then be recognized by immune cells (e.g. T cells or B cells) resulting in their activation.

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

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

In one embodiment, systemic administration of an RNA lipid complex particle or a pharmaceutical composition comprising an RNA lipid complex particle as described herein results in targeting and/or accumulation of the RNA lipid complex particle or RNA in the spleen but not in the lung and/or liver. In one embodiment, the RNA lipid complex particle releases RNA in the spleen and/or enters cells in the spleen. In one embodiment, systemic administration of an RNA lipid complex particle or a pharmaceutical composition comprising an RNA lipid complex particle as described 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 produced by differentiation of monocytes. Macrophages activated by inflammation, immune cytokines, or microbial products nonspecifically phagocytose and kill foreign pathogens within the macrophage by hydrolytic and oxidative attack, resulting in degradation of the pathogen. Peptides from degraded proteins are displayed on the macrophage surface where they can be recognized by T cells and they can interact directly with antibodies on the B cell surface, resulting in T and B cell activation and further stimulation of the immune response. Macrophages belong to the class of antigen presenting cells. In one embodiment, the macrophage is a spleen macrophage.

The term "dendritic cell" (DC) refers to another subset of phagocytic cells that belongs to the class of antigen presenting cells. In one embodiment, the dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells are initially transformed into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation potential. Immature dendritic cells are constantly sampling the surrounding environment for pathogens such as viruses and bacteria. Once they are contacted with presentable antigens, they are activated into mature dendritic cells and begin to migrate to the spleen or to lymph nodes. Immature dendritic cells phagocytose pathogens and degrade their proteins into small pieces (pieces), and after maturation, present these fragments at their cell surface using MHC molecules. At the same time, they upregulate cell surface receptors that act as co-receptors in T cell activation, such as CD80, CD86, and CD40, greatly enhancing their ability to activate T cells. They also up-regulate CCR7, a chemotactic receptor that induces dendritic cells to reach the spleen through the bloodstream, or to reach lymph nodes through the lymphatic system. Where they act as antigen presenting cells and activate helper and killer T cells as well as B cells by presenting their antigens together with non-antigen specific costimulatory signals. Thus, 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) is a cell of a variety of cells that is capable of displaying, capturing and/or presenting at least one antigen or antigenic fragment on (or at) its cell surface. Antigen presenting cells may be distinguished between professional and non-professional antigen presenting cells.

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

The term "non-professional antigen presenting cell" refers to an antigen presenting cell that does not constitutively express MHC class II molecules, but constitutively expresses MHC class II molecules after stimulation by certain cytokines, such as interferon-gamma. Some exemplary 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 degradation of an antigen into a processed product that is a fragment of the antigen (e.g., the degradation of a protein into a peptide), and refers to the association of one or more of these fragments (e.g., by binding) with an MHC molecule for presentation by a cell, e.g., an antigen presenting cell, to a particular T cell.

The term "disease in which an antigen is involved" or "disease in which an epitope is involved" refers to any disease in which an antigen or epitope is involved, for example, a disease characterized by the presence of an antigen or epitope. The disease in which the antigen or epitope is involved may be a cancer disease or simply a cancer. As described 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 term "cancer disease" or "cancer" refers to or describes a physiological condition in an individual that is typically characterized by unregulated cell growth. Some examples of cancer include, but are not limited to: carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More particularly, some examples of such cancers include bone cancer, leukemia lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, gastric cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, cancer of the sexual and reproductive organs, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, cancer of the renal cell, cancer of the renal pelvis, neoplasms of the Central Nervous System (CNS), cancer of the neuroectodermal, tumor of the spinal axis (spinal axis tumors), glioma, meningioma and pituitary adenoma. One particular form of cancer that may be treated by the compositions and methods described herein is prostate cancer. The term "cancer" according to the present disclosure also includes cancer metastasis.

Due to the resulting synergy, a combination strategy in cancer treatment may be desirable, which may have a much stronger impact than monotherapy approaches. In one embodiment, the pharmaceutical composition is administered with an immunotherapeutic agent. As used herein, "immunotherapeutic agent" relates to any agent that may be involved in the activation of a specific immune response and/or immune effector function. The present disclosure contemplates the use of antibodies as immunotherapeutic agents. Without wishing to be bound by theory, antibodies can achieve therapeutic effects against cancer cells through a variety of mechanisms, including inducing apoptosis, blocking components of signal transduction pathways, or inhibiting proliferation of tumor cells. In certain embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies can induce cell death by antibody-dependent cell mediated cytotoxicity (ADCC), or bind complement proteins, resulting in direct cytotoxicity, known as Complement Dependent Cytotoxicity (CDC). Some non-limiting examples of anti-cancer antibodies and potential antibody targets (in parentheses) that may be used in combination with the present disclosure include: abamectin (CA-125), abciximab (CD41), adalimumab (EpCAM), aftuzumab (Aftuzumab) (CD20), pertuzumab (Alacizumab pegol) (VEGFR2), pentoxyzumab (CEA), Amatuximab (Amatuximab) (MORAB-009), Maana momab (TAG-72), aprepizumab (HLA-DR), aximumab (CEA), atezumab (Atezolizumab) (PD-L1), bazedoximab (phosphatidylserine), betuzumab (CD22), Belimumab (BAFF), bevacizumab (VEGF-A), mobitumumab (Bivatuzumab mertansine) (CD44v6), bornatemumab (Blinatomab) (CD 19), TNFatuzumab (CD30), Rituximab (RSF), Mutuzumab (MUVATUB 8), and prostate cancer (CAC 1), and trastuzumab (Canotuzumab), and, Carluzumab (Carlumab) (CNT0888), Rituzumab (EpCAM, CD3), Cetuximab (EGFR), Posituzumab (Cituzumab bogatox) (EpCAM), Cetuzumab (IGF-1 receptor), Clatuximab (Claudiximab) (claudin), Titanketuzumab (Clivatuzumab tetatan) (MUC1), Cetuzumab (TRAIL-2), Daxizumab (CD40), Dalutuzumab (Dalotuzumab) (insulin-like growth factor I receptor), dinomab (RANKL), dimuzumab (B-lymphoma cells), Dozizumab (Drozitumumab) (DR5), Emetuzumab (GD3 ganglioside), Epjuzumab (EpCAM), Epotuzumab (SLE 7), Erituzumab (PDL 2), Epitumab (PDL 2), Epittuzumab (NPc 22/E2), Epittuzumab (NP5634/E) (Epitumab) Ibritumumab (integrin α v β 3), Farletuzumab (farlettuzumab) (folate receptor 1), FBTA05(CD20), fenkratuzumab (Ficlatuzumab) (SCH 900105), fintuzumab (filtuzumab) (IGF-1 receptor), fravatuzumab (Flanvotumab) (glycoprotein 75), Fresolimumab (Fresolimumab) (TGF- β), Galiximab (Galiximab) (CD80), icganeitab (IGF-I), gemtuzumab ozolomide (CD33), gemovazumab (Gevokizumab) (IL-I β), gemtuximab (Girentuximab) (carbonic anhydrase 9(CA-IX)), gemtuzumab-vevimab (glentuzumab) (gmb), ibritumomab (nmb), ibritumomab (sdcu 2), ecub (VEGFR-125), gemtuzumab-ivumab (VEGFR-64) (VEGFR-1), gemtuzumab (VEGFR-I-1 (VEGFR-I), gemtuzumab-ivotuzumab (VEGFR-ii) (nmb-I), gemtuzumab-ivotuzumab (VEGFR-I) (gpb-l-I (VEGFR-l), gemtuzumab-ii) (nmb-l (gptamb), gemtut-l (gptamb-l) and VEGFR-2), gemtut-l (VEGFR-d-l (gptamb) and VEGFR-l) and (gptam-l) and (c-l) for example, and other, Ontotuzumab (CD22), ipilimumab (CD 152), rituximab (Iratumumab) (CD30), trastuzumab (CEA), lexatuzumab (TRAIL-R2), ribavirin (hepatitis B surface antigen), lintuzumab (CD33), rituzumab (Lorvotuzumab mertansine) (CD56), lucatuzumab (CD40), lucitumumab (CD23), mapitumumab (TRAIL-R1), matuzumab (EGFR), merilizumab (IL-5), milnaclizumab (Milatuzumab) (CD74), mituzumab (GD3 ganglioside), moguzumab (Mogalizumab) (CCR4), mototuzumab (Moxetuzumab) (CD22), natalizumab (C242), namomab (T4), Negalizumab (Netuzumab) (EGFR), Netuzumab (R595925), and EGFR (Rotuzumab (R4), Ofatumumab (CD20), Olaratumab (Olaratumab) (PDGF-Ra), Onartuzumab (Onartuzumab) (human scatter factor receptor kinase), moelcuzumab (Oportuzumab monatox) (EpCAM), ogovazumab (CA-125), eculizumab (Oxelumab) (OX-40), panitumumab (EGFR), pertuzumab (Patritumab) (HER3), pemetrexezumab (pemteumab) (MUC1), pertuzumab (HER2/neu), pertuzumab (adenocarcinoma antigen), pertuzumab (vimentin), ranituzumab (rasotumumab) (N-glycolyl neuraminic acid), ranituzumab (radretumamab) (fibronectin extra domain-B), ranibivir (rabies virus glycoprotein), ranituzumab (2), rituximab (ritumumab) (Rilotumumab), rituximab (riluzumab) (VEGFR 38), IGF-20 (VEGFR-361), rituximab (receptor (rocumab receptor) Salacilizumab (Samalizumab) (CD200), Cetuzumab (FAP), Setuximab (IL-6), Tabeuzumab (Tabalumab) (BAFF), Tabizumab (alphafetoprotein), Protecumab (CD 19), Cetuzumab (Tenitumomab) (tenascin C), Tetuzumab (Teprotimumab) (CD221), Cetuzumab (CTLA-4), Tegazumab tegafur (TRAIL-R2), TNX-650(IL-13), tositumomab (CD20), trastuzumab (HER2/neu), TRBS07(GD2), tiximumab (CTLA-4), simethicin cheuzumab (tucotuzumab celeukin) (EpCAM), ulituximab (Ublituximab) (MS4a1), ureuzumab (Urelumab) (4-1 BB), volvacizumab (integrin α 5 β 1), volitumumab (tumor antigen CTAA 16.88), tuzalimumab (EGFR), and zalimumab (CD 4).

In one embodiment, the immunotherapeutic agent is a PD-1 axis binding antagonist. PD-1 axis binding antagonists include, but are not limited to: PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists. Alternative names for "PD-1" include CD279 and SLEB 2. Alternative names for "PD-L1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PD-L2" include B7-DC, Btdc, and CD 273. In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a particular aspect, the PD-1 ligand binding partner is PD-L1 and/or PD-L2. In another embodiment, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a particular embodiment, the PD-L1 binding partner is PD-1 and/or B7-1. In another embodiment, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In a specific embodiment, the PD-L2 binding partner is PD-1. The PD-1 binding antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). Some examples of anti-PD-1 antibodies include, but are not limited to, MDX-1106 (nivolumab, OPDIVO), Merck 3475(MK-3475, pembrolizumab, KEYTRUDA), MEDI-0680(AMP-514), PDR001, REGN2810, BGB-108, and BGB-A317.

In one embodiment, the PD-1 binding antagonist is an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region. In one embodiment, the PD-1 binding antagonist is AMP-224 (also known as B7-DCIg, and is PD-L2-Fc), which is a fusion soluble receptor described in WO2010/027827 and WO 2011/066342.

In one embodiment, the PD-1 binding antagonist is an anti-PD-L1 antibody, including but not limited to yw243.55.s70, MPDL3280A (atelizumab), MEDI4736 (doxomab), MDX-1105, and MSB0010718C (avizumab).

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

Citation of documents and studies cited herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the contents of these documents.

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

Examples

Example 1: intravenous vaccine for treating prostate cancer

Described herein are second generation vaccines for Intravenous (IV) administration. It consists of RNAs targeting five antigens expressed in prostate cancer, which are each complexed with liposomes into serum stable RNA lipid complexes (RNA (lip)). Rna (lip) delivers the encoded vaccine antigen to Antigen Presenting Cells (APCs) in lymphoid organs, which results in efficient induction of an antigen-specific immune response.

The vaccine for IV injection consists of five different RNA drug products targeting five prostate cancer associated antigens. The RNA cancer vaccine for prostate cancer is composed of RBL038.1, RBL039.1, RBL040.1, RBL041.1 and RBL 045.1. Each RNA cancer vaccine consists of one RNA drug substance that encodes each of the following and is selected based on its selective expression in prostate cancer: the antigens Kallikrein-2 (KLK2), Kallikrein-3 (Kallikrein-3, KLK3, also known as Prostate Specific Antigen (PSA)), Acid phosphatase, prostate (Acid phosphatase, prostate, ACPP, also known as Prostatic Acid Phosphatase (PAP)), homeobox B13(HOXB13), and NK3 homeobox 1(NKX 3-1).

The RNA is reconstituted into RNA lipid complexes (RNA (lip)) and then administered.

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

Specialized materials for reconstitution, such as syringes and cannulae, and additional isotonic saline solution that allows further dilution of rna (lip) are available as clinical standards.

Drug substance:

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

the encoded antigen: human kallikrein-2 (corresponding Gene ID (HG 19): uc002ptu.3, uc002ptv.3, uc002ptt.3, uc010ycl.2, uc010ycm.2, uc010eog.3)

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

The encoded antigen: human prostate specific antigen, also known as human kallikrein-3 (corresponding gene ID (HG 19: uc002pts.1, uc002ptr.1, uc021uy.1, uc010eof.1)

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

The encoded antigen: human prostatic acid phosphatase, also known as human acid phosphatase, prostate (corresponding Gene ID (HG 19): uc003eop.4)

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

The encoded antigen: human homeobox B13 (corresponding Gene ID (HG 19): uc002ioa.3r)

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

The encoded antigen: NK3 homeobox 1 (corresponding to Gene ID (HG 19): uc011kzx.2).

The active ingredient in each drug substance is a single-stranded 5' capped mRNA that is translated into the corresponding protein upon entry into an Antigen Presenting Cell (APC).

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

The general sequence elements of the mRNA as depicted in figure 1 are given below.

KLK2, PSA (KLK3), pap (acpp), HOXB13, and NKX 3-1: (ii) a codon optimized sequence encoding the respective target protein.

hAg-Kozak: the 5 ' -UTR sequence of human α -globin mRNA with optimized ' Kozak sequence ' to improve translation efficiency.

sec/MITD: fusion protein tags derived from sequences encoding human MHC class I complexes (HLA-B51, haplotype A2, B27/B51, Cw2/Cw3), which have been shown to enhance antigen processing and presentation. sec corresponds to a 78bp fragment encoding a secretory signal peptide, which directs translocation of the nascent polypeptide chain into the endoplasmic reticulum. MITD corresponds to the transmembrane and cytoplasmic domains of MHC class I molecules, also known as MHC class I transport domains. Note that KLK2, PSA (KLK3) and pap (acpp) each have their own secretion signal peptide. Therefore, no sec fusion tag was added to these antigens.

GS/linker: sequences encoding short linker peptides consisting essentially of the amino acids glycine (G) and serine (S) as are commonly used in fusion proteins.

P2P 16: sequences encoding tetanus toxoid-derived helper epitopes to disrupt immune tolerance.

An FI element: the 3' -UTR is a combination of two sequence elements derived from the "amino terminal enhancer of split, AES, mRNA (referred to as F) and the mitochondrially encoded 12S ribosomal RNA (referred to as I). These were identified by an ex vivo selection process of sequences conferring RNA stability and increased total protein expression.

A30L 70: the poly (a) tail of 110 nucleotides in length was measured, consisting of the following regions: 30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues, are intended to enhance RNA stability and translation efficiency in dendritic cells.

The complete nucleotide sequence of the five RNA drug substances RBL038.1, RBL039.1, RBL040.1, RBL041.1 and RBL045.1 is given below:

nucleotide sequence of RBL 038.1.

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

Nucleotide sequence of RBL 039.1.

Nucleotide sequence of RBL 040.1.

Nucleotide sequence of RBL 041.1.

Nucleotide sequence of RBL 045.1.

Recombinant DNA synthesis techniques for generating 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(pST 040.1-040.1-Kozak-ACPP-GS-P2P 040.1-GS-MITD-FI-A30L 040.1), RBL040.1(pST 040.1-040.1-Kozak-sec-GS-HOXB 040.1-P2P 040.1-GS-MITD-A30L 040.1) and RBL040.1(pST 040.1-040.1-Kozak-sec-GS-X040.1-NKP 2P 040.1-MING-MITD-FI-A30L 040.1) were used alone to generate recombinant DNA. In addition to the sequence encoding the transcribed region, the plasmid DNA contains a promoter for T7RNA polymerase, a recognition sequence for a type II endonuclease for linearization, a kanamycin-resistant gene, and an origin of replication (ori).

Plasmid DNA pST4-hAg-Kozak-sec-GS-SIINFEKL-GS-Ova-GS-P2P16-GS-MITD-FI-A30L70 was used as the origin of the DNA template for the production of RBL038.1, RBL039.1, RBL040.1, RBL041.1 and RBL 045.1.

Vector maps are shown in figures 3 to 7.

The circular plasmid DNA is linearized with the appropriate restriction enzymes to obtain the starting material for RNA transcription. Here, the enzyme Eam1104I (Thermo Fisher Scientific Baltims UAB, Vilnius, Lithounia) was chosen, since linearization with such a class II restriction endonuclease allows transcription of RNA encoding a "poly (A) -free tail, i.e.without additional nucleotides at the 3' end. It can be shown that this provides higher protein expression.

The RNA (LIP) product may be prepared in a two-step procedure comprising: (i) diluting the RNA concentrate with a NaCl solution, and (ii) forming RNA lipid complexes by addition of liposomes. For RNA (lip) preparation, liposomes can be added to the diluted RNA. For lipids, the synthetic cationic lipid DOTMA and the naturally occurring phospholipid DOPE can be used. IV injectable products are formulations with pharmaceutical and physiological characteristics that allow RNA to be selectively targeted to APCs that are predominantly present in the spleen. RNA lipid complexes are formed by first condensing RNA in a suitable ionic environment and then incubating with positively charged liposomes.

For RNA condensation, various monovalent and divalent ions, peptides and buffers were applied at different concentrations. Monovalent ions such as sodium and ammonium were tested at concentrations up to 1.5M. Testing of divalent ions, especially Ca, at concentrations up to 50mM²⁺、Mg²⁺、Zn²⁺And Fe²⁺. In addition, a number of commercially available buffer solutions were also tested.

Liposomes containing cationic lipids and different co-lipids were extensively tested for rna (lip) formation. Liposomes that differ in charge, phase, size, lamellarity and surface functionalization were studied. Only lipid components that could be used for GMP grade and were previously tested in clinical trials or for commercially approved products were considered (figure 8).

Using the above liposome components, with different cationic lipids: RNA and different charge ratios assemble RNA lipid complexes, where the charge ratio is calculated from the number of positive charges from the lipid and negative charges from the RNA nucleotides (i.e. from the RNA phosphate group). More specifically, the charge ratio is calculated as follows:

it is assumed that the RNA consists of nucleotides with an average molar mass of 330Da, each carrying a phosphate group with a negative charge. Thus, a 1mg/mL RNA solution accounts for about 3mM of negative charge. In another aspect, one positive charge per monovalent cationic lipid is contemplated. For example, liposomes with cationic lipid DOTMA having a molar mass of 670Da and a DOTMA concentration of 2mg/mL provide a positive charge concentration of 3 mM. Thus, in this case, the (+/-) charge ratio is considered to be 1: 1. The concentration of uncharged co-lipids present in most cases does not contribute to this calculation.

The chemical and physicochemical properties of liposomes and the RNA lipid complexes formed on this basis (i.e. with respect to chemical composition, particle size, zeta potential) were thoroughly investigated. For regular control of product quality, chemical composition was determined by HPLC analysis and 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 flow fractionation, analytical ultracentrifugation and spectroscopic techniques were applied during formulation development. By this procedure, optimized formulations for further drug development were identified.

Suitable liposomal formulations were tested in vitro and in vivo. To optimize targeting of APCs present predominantly in the spleen, expression of luciferase as a reporter gene was observed in vivo. It can be shown that at a suitable charge ratio (negative or positive charge excess), colloidally stable nanoparticulate lipid complex formulations having discrete particle sizes can be formed. Furthermore, it was shown in vivo that the negatively charged luciferase-RNA lipid complex formulation showed high selectivity for the spleen which served as a reservoir for professional APC. By varying the charge ratio, the selectivity of luciferase expression in the spleen can be adjusted as desired, as shown in fig. 9, where organ selectivity of RNA lipid complexes from the same liposomes at different mixing ratios of cationic lipid to RNA is shown. The large number of lipid compositions can validate the observation that negatively charged lipid complexes target splenic APCs. Liposomes composed of the cationic lipid DOTMA and the helper phospholipid DOPE were identified as most suitable in terms of particle characteristics to form the appropriate RNA lipid complexes for the intended spleen APC targeting. Optimized selectivity and efficacy of spleen targeting was observed when the negative charge consisting of excess RNA was slightly excessive. RNA lipid complexes that are slightly more positively charged and show comparable efficacy are not suitable for the development of pharmaceutical products, since they are too colloidally unstable and there is a high risk of aggregation and precipitation under these conditions.

Furthermore, it can be shown that for a given RNA, the biological activity of the formulation increases with the particle size of the RNA lipid complex. More specifically, it can be shown that the RNA lipid complex itself formed by the larger liposome (e.g., about 400nm) is larger than the RNA lipid complex prepared with the smaller liposome (e.g., about 200nm) and shows higher bioactivity (fig. 10). Thus, liposomes larger than 200nm are preferably used for RNA (LIP) formation.

Based on the above findings (refining), a robust and reproducible protocol for rna (lip) preparation was developed. By using defined components and defined preparation protocols, RNA lipid complexes form the desired physicochemical characteristics and biological activities by self-assembly. As an example, particle sizes from multiple independently prepared RNA lipid complexes are given in fig. 11. The limited dispersion of the obtained RNA lipid complex particle size indicates the robustness of the reconstitution procedure.

To determine the limitation and robustness of RNA (LIP) preparation, particle sizes of different charge ratios (mixing ratio between cationic lipid and nucleotide) of 1.0: 2.0 to 1.9: 2.0 were measured. In fig. 12, results from size measurements of RNA lipid complexes after mixing liposomes with RNA in different ratios are shown. Particle size was measured at different time points after rna (lip) preparation. For a ratio of 1.0: 2.0 to 1.6: 2.0, comparable particle sizes stable over time were obtained. For ratios of 1.7: 2.0 and higher, the particle size of the RNA lipid complex increases both initially and over time. This finding was most pronounced after 24 hours.

Based on these data, charge ratios of 1.0: 2.0 to 1.6: 2.0 are considered suitable for obtaining acceptable particle characteristics for the RNA lipid complex product. At higher ratios (1.7: 2.0 and above 1.7: 2.0), the particle size increases, leading to potential product quality deviations. No change in particle characteristics was observed for lower charge ratios, however, lower ratios were not considered due to potentially lower activity in this range (data not shown). The experiment was repeated for 1.1: 2.0 to 1.6: 2.0 and in addition to the size measurement (FIG. 13A), the biological activity was studied (FIG. 13B). In agreement with previous experiments, the particle size was almost constant. The same applies to biological activity (luciferase expression). Taken together, all of the RNA lipid complexes of the charge ratios tested delivered RNA to APC without significant changes in physicochemical or biological properties. Thus, it is believed that 1.1: 2.0 to 1.6: 2.0 produces RNA lipid complexes of equivalent quality.

Example 2: non-clinical data

This example reviews non-clinical studies conducted to elucidate the mode of action, pharmacodynamics, anti-tumor activity, pharmacokinetics and potential toxicity of rna (lip) vaccines. Key survey results are summarized in table 1.

The first section of this section provides a brief overview of the scientific basis and preparation work for developing the vaccine platform itself (section 1) and a brief overview of the target characteristics of the W _ pro1 target.

The following section describes the study of primary pharmacodynamics (primary pharmacodynamic) of rna (lip), i.e. induction of antigen-specific T cells in vivo, and anti-tumor activity of rna (lip) vaccination (section 2).

Studies on secondary pharmacodynamics (secondary pharmacodynamic) showed the results of testing for rna (lip) -mediated induction of pro-inflammatory cytokines (section 3). Pharmacodynamic non-GLP studies were performed in cynomolgus monkeys (cynomolgus monkey) to refine cytokine kinetic data, as well as hematological changes that have been observed in mice. In this section, in vitro studies analyzing cytokine secretion in human and cynomolgus monkey blood cells after incubation with rna (lip) preparations are also summarized.

The safety pharmacology study for respiratory and nervous systems is summarized in section 4.

A brief overview of in vivo biodistribution, pharmacokinetics and metabolism is given in section 5.

GLP compliance repeat dose toxicity studies, incorporating immunotoxicity studies, were performed and are presented and discussed in section 6.

Table 1: summary of major pharmacological and toxicological characteristics of rna (lip) vaccines.

Section 1: scientific basis

Sequence features enhance RNA translation and intracellular stability

The RNA vaccine platform has been developed and systematically optimized for over 20 years for the encoded antigen to safely and efficiently induce antigen-specific CD8+ and CD4+ T cell responses.

As mentioned above, the active component (drug substance) is a single-stranded, capped messenger rna (mrna), which is translated into a protein antigen upon entry into an antigen presenting cell. The mRNA vaccine format was pharmacologically optimized by: (i) a modified cap analogue to stabilize translationally active RNA, (II) optimized 5 '-and 3' -UTRs to improve stability and RNA translation, (iii) signal peptides and MITD sequences to improve MHC class I and class II antigen processing, (iv) tetanus toxoid-derived helper epitopes to disrupt immune tolerance by providing non-specific CD4+ T cell help, and (v) an extended free-terminal poly (a) tail to further enhance RNA stability and translation efficiency.

Table 2: RNA structural elements for immunopharmacological optimization.

Targeting of lymphocyte-resident antigen-presenting cells by antigen-encoding RNA

For systemic delivery of RNA to dendritic cells, each individual RNA drug product of W _ pro1 was formulated to form an RNA lipid complex (RNA (lip)) that allowed IV administration. RNA (lip) preparations were engineered to protect RNA from plasma ribonuclease degradation and optimized to deliver formulated RNA DP selectively to Antigen Presenting Cells (APCs) residing primarily in the spleen (fig. 14) and other lymphoid organs, where RNA uptake by dendritic cells and macrophages has been shown to be selective (fig. 15).

Once APCs in the spleen have taken up the RNA lipid complex, mRNA is expressed, processed and presented by MHC molecules. This results in a highly efficient induction of antigen-specific CD8+ and CD4+ T cell responses and T cell memory, which is further supported by an immune stimulatory environment in the spleen induced by rna (lip) -mediated TLR signaling.

Induction of antigen-specific T cell responses

To explore the efficacy of rna (lip) immunization and investigate the ability of rna (lip) to break tolerance to endogenously expressed antigens, BALB/c mice were immunized with rna (lip) encoding the AH5 epitope of murine leukemia virus (murine leukemia virus, muLV) derived gp70 protein. Repeated immunizations with AH5-rna (lip) resulted in significant gp 70-specific CD8+ T cell expansion (fig. 16A). CD8+ T cells induced by vaccination were able to lyse the target in vivo in an antigen-specific manner (fig. 16B), which means that functional antigen-specific CD8+ T cells with lytic capacity could be induced by rna (lip) immunization.

Induction of cell activation Process

mRNA is a ligand for human Toll-like receptors (TLRs) and is therefore capable of eliciting an immunomodulatory effect. After cellular uptake of In Vitro Transcribed (IVT) -RNA, recognition of TLRs occurs in endosomal compartments where these receptors are predominantly located. This elicits a cascade of signaling events that ultimately lead to activation and maturation of DCs, as shown by the maturation of splenic DCs after intravenous administration of an rna (lip) vaccine into mice. Other consequences of these immunomodulatory effects are subsequent activation of spleen T, B, NK cells and macrophages and reversible induction of pro-inflammatory cytokines.

Most importantly, injection of model rna (lip) encoding influenza hemagglutinin HA showed strong induction of IFN- α in mice (fig. 17A), which in splenectomized mice were shown to be spleen derived (fig. 17B). Notably, only rna (lip) showed induction of IFN- α, whereas liposomes alone did not result in induction of IFN- α in mice.

The transient cellular activation and cytokine and RNA vaccines observed in mice treated with RNA (lip) vaccine were consistent with the finding that TLR could bind and trigger TLR. Others have also shown that RNA formulated as particles, as well as RNA formulated in aqueous solutions, can activate TLRs. TLR activation has been shown to induce lymphopenia, resulting in a type I interferon-dependent recycling event of leukocytes. In line with this, activation of dendritic cells and other spleen cell populations was severely hampered in the TLR 7-/-or IFNAR-/-mice. Thus, studies in IFNAR-/-mice administered with RNA (LIP) did show that the transient hematological changes observed after IV administration are mainly mediated by IFN-. alpha.downstream effects.

P2P16 tetanus-derived helper epitope in W _ pro1

Each W _ pro1RNA DP contains the so-called P2P16 amino acid sequence of the Tetanus Toxoid (TT) from Clostridium tetani (Clostridium tetani). These sequences support overcoming the self-tolerance mechanism to efficiently induce an immune response to self-antigens by providing tumor non-specific T cell help during sensitization.

Tetanus toxoid heavy chains comprise epitopes that promiscuously bind to MHC class II alleles and induce CD4+ memory T cells in nearly all tetanus vaccinated individuals. In addition, the combination of TT helper epitopes with tumor associated antigens is known to provide CD4 during priming by providing CD4 in comparison to the application of tumor associated antigens alone⁺Mediated T cell help to enhance immune stimulation. In order to reduce the irritation CD8⁺Risk of T cells, two peptide sequences known to contain promiscuous binding helper epitopes were chosen to ensure binding to as many MHC class II alleles as possible. Based on the data of the ex vivo studies cited above, the well-known epitopes P2 (QYIKANSKFIGITEL; TT positions 830 to 844) and P16 (MTNSVDDALINSTKIYSYFPSVISKVNQGAQG; TT positions 578 to 609) were selected.

Pharmacology of

The mode of action of RNA (lip) vaccination relies on (I) recruitment of antigen-specific T lymphocytes following presentation of peptides derived from RNA-encoded antigens by professional APCs, and (ii) TLR-mediated immune modulation, which results in cell activation and induction of pro-inflammatory cytokines (e.g., type I interferons), thereby enhancing vaccination.

In section 2, we report the activation and expansion of target antigen-specific T cells following immunization with cancer antigen-encoding RNA, and the anti-tumor effects of antigen RNA (lip) vaccination.

Extensive in vitro and in vivo studies were conducted to investigate the potential secondary effects (secondary effects) of the administration of rna (lip) vaccines, such as the induction of pro-inflammatory cytokines and hematological changes caused by the expected immunomodulatory effects of rna (lip).

In section 3, a study group evaluating the degree of cell activation of human peripheral blood cells (PBMCs) and blood cells in heparinized whole blood is discussed. Furthermore, the extent of cytokine induction and hematologic changes induced by vaccination in cynomolgus monkeys treated with doses higher than the highest expected clinical dose in humans is shown. Finally, in vitro cytokine induction in blood samples from human donors and cynomolgus monkeys were compared side-by-side and the data generated in these studies was used to support safe starting doses defining an ongoing clinical trial in malignant melanoma (Lipo-MERIT) and other trials investigating rna (lip) immunotherapy

An overview of a non-clinical study using human blood cells, mice and cynomolgus monkeys as the test system to assess secondary pharmacodynamics of rna (lip) is given in section 3.

We expect that the observed secondary pharmacodynamic effects observed for liposome-formulated RNA are not sequence-dependent, and therefore the study is equally applicable to other RNA drug products used.

Section 2: major pharmacodynamics

Several in vitro and in vivo experiments were performed to demonstrate the immunogenicity of vaccination with rna (lip) vaccines encoding a number of different mrnas with antigens specific for melanoma, breast, HPV + head and neck, ovarian and other cancer types. In vivo studies were performed in mice with R & D and GMP grade materials.

Induction of antigen-specific T cell response with W _ pro1 mRNA

To obtain more information about the induction of prostate specific antigens KLK2, PSA (KLK3), pap (acpp), HOXB13 and NKX3-1 in vivo against antigen specific T cells, RNA (lip) products were prepared using R & D quality RNA and GMP-like quality liposomes.

The rna (lip) product was injected intravenously into transgenic mice manipulated to express the human leukocyte antigens HLA-a 0201 and-DRB 1 x 01. Using these mice, sensitization and expansion of T cells specific for HLA-restricted epitopes can be examined in vivo. A2/DR1 mice were vaccinated four to five times by injecting 30 μ g of each antigenic RNA complexed to liposomes, followed by isolation of splenocytes (5 days after the last vaccination). The sensitization efficiency of the test items was evaluated by IFN- γ ELISPOT assay after restimulation with bone marrow-derived dendritic cells (BMDCs) electroporated with the corresponding mRNA or an irrelevant control mRNA.

Four vaccinations with rna (lip) preparations were sufficient to sensitize the treated animals to specific T cell responses for all antigens (fig. 18).

These results indicate that rna (lip) vaccines are effective in inducing T cell responses against W _ pro1 antigen in vivo.

The mRNA used in the study was produced under R & D conditions. Additional immunogenicity studies in A2/DR1 mice using RBL038.1, RBL039.1, RBL040.1, RBL041.1, and RBL045.1 made under GMP conditions will be performed. Immunogenicity was expected to be comparable to results from earlier studies.

In vivo anti-tumor Activity of antigen-specific T cells induced by model antigen RNA

In association with the known challenge of identifying murine tumor models, no additional studies were performed against the W _ pro1 antigen, due to the absence of murine homologues of these antigens. In contrast, we developed a suitable tumor model for the ovalbumin source SIINFEKL epitope; human papillomavirus-derived E6/E7 antigen and gp70 were used for vaccination as models of foreign and mouse autoantigens, respectively.

A summary of the in vivo anti-tumor effects induced by RNA (LIP) is given in Table 3.

Table 3: summary of in vivo anti-tumor effects of RNA (LIP).

Section 3: secondary pharmacodynamics

To investigate the potential secondary effects of administration of RNA lipid complex vaccines, such as induction of inflammatory cytokines and hematological changes induced by the expected immunomodulation, we conducted extensive in vitro and in vivo studies using human blood cells and cynomolgus monkeys as the test system. To our knowledge, the secondary pharmacodynamic effects triggered by TLRs and the innate immune activation by therapeutic mrnas are not sequence-dependent, and the studies presented in this chapter were performed with aliquots of ATM quality liposome-formulated RBL001.1, RBL002.2, RBL003.1 and RBL 004.1. These RNAs encode melanosome antigens and have been used in clinical trials. The study has not been repeated with W _ pro1 RNA DP encoding TAA.

The extent of cytokine induction, hematological changes, complement activation, and clinical chemistry induced by rna (lip) vaccination was studied in cynomolgus monkeys treated with doses corresponding to the expected doses in humans, as described below. Furthermore, in non-GLP and GLP studies, the extent of cytokine release in human and cynomolgus peripheral blood cells (PBMCs) and blood cells in response to RNA lipid complex treatment in heparinized whole blood was investigated.

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

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

In addition to its characteristics of encoding protein antigens, RNA also has an immunomodulatory effect, derived from its ability to trigger induction of cellular activation processes through TLRs. In one aspect, the immunomodulatory capacity of RNA vaccines enhances the induction of antigen-specific T cell responses, and this should be considered as the primary pharmacodynamic effect. On the other hand, too strong or non-specific immune cell activation may lead to undesired secondary effects and should have been addressed in preclinical studies.

To investigate the extent of cell activation of human blood cells, aliquots of heparinized whole blood and PBMCs (isolated from heparinized whole blood) from four healthy donors were incubated in vitro with ATM quality aliquots of liposome-formulated RNA (RBL001.1, RBL002.2, RBL003.1 and RBL004.1) encoding melanosome tumor-associated antigens. Since the activation of TLR by RNA is not sequence dependent, this study was not repeated with W _ pro1 RNA.

RNA (LIP) was prepared separately for each of the four RNA drug products according to the clinical formulation protocol. In this first study (study 1, STR-30207-. As a primary endpoint, activation of cells was determined by secretion of cytokines (IP-10, IFN- α, IFN- γ, TNF- α, IL-1 β, IL-2, IL-6, and IL-12) into cell culture medium (PBMC) or plasma (whole blood), respectively, after 6 hours and 24 hours.

Table 5: dose profiles for in vitro studies based on the expected clinical dose cohort.

The following values represent total RNA (. mu.g)/mL whole blood or culture medium, respectively.

^[1]Assume a mean total blood volume of 5L.

After incubation of PBMC with rna (lip) mixtures, there was detectable dose-dependent activation for all eight analytes tested, but high variation in concentration levels. The cytokine response is dominated by five of the eight selected markers, IP-10, IFN- γ, TNF- α, IL-1 β and IL-6 (see Table 4 for a summary). IFN-alpha, IL-2 and IL-12 in the highest test dose level showed only slight induction.

In contrast, no secretion of IFN-. gamma., TNF-. alpha., IL-1. beta., IL-2 and IL-12 was detected in the whole blood test system after incubation with RNA (LIP). Here, a dose-dependent increase in secretion of IP-10 and IL-6 was observed. For IFN- α, only low levels of baseline secretion were observed, which were comparable to the diluent control and did not increase further by incubation with rna (lip) (see table 4 for summary).

In conclusion, the findings in PBMC showed significant differences compared to whole blood, indicating a higher sensitivity of the tested systems with PBMC. Although increased cytokine levels were detected in PBMCs for all eight analytes tested, cytokine detection was limited to IFN-. alpha.IP-10 and IL-6 when whole blood samples were used as the test system.

Table 4: the results for PBMC and whole blood in all donors were summarized (study STR-30207-.

To further investigate cytokine release by human cells in vitro in response to rna (lip) and compare and classify in vivo data from mouse immunotoxicity studies (see below) and cynomolgus monkey studies (see below), an additional GLP compliance in vitro study was performed at the external CRO (LPT No. 31031). The main objective of this study was to determine (i) whether the findings in cynomolgus monkeys were comparable to humans, and (ii) which tested system better reflected the cytokine response pattern observed in cynomolgus monkeys. Study LPT No.31031 was designed as follows: in vitro induction of proinflammatory cytokines in healthy human donors and cynomolgus monkeys was tested in two tested systems (i.e., PBMC and whole blood). The same dose ranges and dose steps were tested as for RNA (LIP) in study No. STR-30207-. The test item was also a mixture of separately prepared liposomal formulated RBL001.1, RBL002.2, RBL003.1, and RBL004.1 RNA of ATM quality. As mentioned above, the data generated with these IVT-RNAs also explains the RNA DP encoding TAA, since TLR activation is RNA sequence independent. A total of four individual samples of each species were analyzed. As primary endpoints, activation of cells was determined by secretion of pro-inflammatory cytokines into cell culture medium (PBMC) or plasma (whole blood), after 6 hours, 24 hours and 48 hours, respectively.

Separately, the cytokine responses observed for the whole blood test system are summarized in table 6, and for the PBMC test system in table 7.

Table 6: summary of cytokine responses in whole blood test systems.

Table 7: summary of cytokine response in PBMC test systems (LPT No. 31031).

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

Most importantly, strong subject-related cytokine induction of these three pro-inflammatory cytokines was only observed at dose levels ≧ 5,500 μ g, which were higher than the highest expected dose level of 100 μ g in patients and > 100-fold higher than the planned dose (═ 50 μ g RNA) for the initial vaccination cycle. Notably, the results from healthy donors established the in vitro studies of STR-30207-013 and the cynomolgus cytokine response patterns observed in the whole blood test system were similar to those from the in vivo study of LPT No.29928, in which only IL-6 was detectable in cynomolgus monkeys treated with RNA (LIP) (see below).

Table 9 shows the data generated in the study LPT No.31031 in which PBMCs from four cynomolgus monkeys and four healthy donors were analyzed after 6 and 24 hours incubation with six different doses of rna (lip). In human and cynomolgus PBMC, the induction of IL-6 and TNF- α is comparable for: (i) absolute amounts of cytokines induced (differences between species are less than factor 2), (ii) kinetics (early induction of IL-6 and TNF-a after 6 hours), and (iii) dose levels of rna (lip) leading to cytokine induction. IFN- γ was detected in PBMCs from both species treated with only an intermediate dose of RNA (LIP), albeit to a greater extent in humans, 24 hours after RNA (LIP) stimulation. In conclusion, the cytokine profile induced in PBMCs by RNA (LIP) for IL-6 and TNF- α was comparable between species. The results obtained in this study indicate that cynomolgus monkeys are the relevant species for assessing rna (lip) -mediated cytokine induction, and that human PBMCs constitute a more sensitive system for capturing IFN- γ induction.

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

The table shows the data generated in study LPT No. 31031: cytokine levels (pg/mL) of IL-6 (upper), TNF- α (middle) and IFN- γ (lower) were detected after incubation of whole blood with different doses of RNA (LIP). The red code indicates the height of the cytokine level, with darker red indicating higher cytokine levels. The first column represents the total dose level applied in the clinical setting. The second column shows the amount of RNA used in the in vitro test system assuming a 5L blood volume. No data was collected.

^[1]Assume a mean total blood volume of 5L.

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

The table shows the data generated in study LPT No. 31031: cytokine levels (pg/mL) of IL-6 (upper), TNF- α (middle) and IFN- γ (lower) were detected after incubation of PBMCs with different doses of RNA (LIP). The red code indicates the height of the cytokine level, with darker red indicating higher cytokine levels. The first column represents the total dose level applied in the clinical setting. The second column shows the amount of RNA used in the in vitro test system assuming a 5L blood volume. No data was collected.

^[1]Assume a mean total blood volume of 5L.

When comparing the results found in whole blood and PBMC test systems, it is evident that the pro-inflammatory cytokines in PBMC test systems are generally more extensive, reach higher absolute values, and start at lower dose levels than in whole blood test systems.

In this most sensitive in vitro test system, a sharp increase in cytokine levels as measured after 24 hours was observed for dose ranges of 615 μ g to 1,850 μ g RNA for IL-6, 1,850 μ g to 5,550 μ g RNA for IFN- γ, and 5,550 μ g to 16,650 μ g RNA for TNF- α. Even for the most sensitive cytokine marker IL-6 in an in vitro system, the expected initial dose of 25. mu.g was 25-fold lower than the dose level at which the induction of strong in vitro cytokines was initiated.

In addition to GLP study LPT No.31031, a non-GLP in vitro study (report _ RB _14_001_ B) was also performed using a similar experimental setup, with samples from three individuals of each species tested, with similar observations made to determine results from the GLP study (data not shown). Taken together, the results of the study highlighted (i) the stimulation of cells after incubation with the test item, the similarity of the two species, cynomolgus monkey and human (compliance). Furthermore, these observations indicate that (ii) the whole blood test system more closely reflects the in vivo situation than PBMCs. In whole blood test systems cytokine induction was generally less pronounced and was only observed in the highest dose group, and the main induction of IL-6 was similar to the findings of the cynomolgus in vivo study (see below).

We acknowledge more significant findings in PBMC, which are considered artificial, but also more sensitive in vitro test systems, and thus integrate the results from this more sensitive test system in a strategy defining a safe initial starting dose.

In vivo testing of secondary pharmacology in cynomolgus monkeys

To more accurately understand the kinetics of rna (lip) and the correlation of secondary effects of rna (lip) with cytokine expression, non-GLP studies were performed in male cynomolgus monkeys (see table 10 for treatment regimens and dosages, and table 11 for detailed study design and amounts of all formulation components). Animals in groups 1 to 5 (2 males per dose) were treated with the four melanosome rna (lip) vaccines RBL001.1, RBL002.2, RBL003.1 and RBL004.1(ATM quality) and then the control solution was given as a slow bolus injection (about 10 seconds) with 30 minutes intervals between each injection (i.e. the last injection was given after 1.5 hours). The results of this study should also be applicable to the W _ pro1 injection, as secondary effects are not sequence dependent.

Doses up to 42-fold higher than the highest clinical dose were tested in the study. In addition, animals in dose group 6 received a single dose of 4X 88.6. mu.g RNA on day 1, followed by a single dose of 4X 3.6. mu.g RNA on day 22.

Table 10: study protocol and dose in relation to the expected dose in the patient (LPT No. 29928).

And (3) treatment: animals 1 to 10 (groups 1 to 5) were treated 5 times with four subsequent injections of NaCl (saline) (group 1), the same dose of liposomes (group 2) as the high dose animals and rna (lip)1 to 4(ATM quality, groups 3 to 5). Animals in group 6 received a single treatment with 4X 88.6. mu.g (total 354. mu.g RNA) on test day 1, followed by a single treatment with 4X 3.6. mu.g (total 14.4. mu.g RNA) on test day 22. Dosage: the doses are shown as total RNA dose (mg/kg body weight) and as total RNA dose (μ g/individual, estimated patient weight 70 kg).

Table 11: design of pharmacodynamic study in cynomolgus monkey (LPT No. 29928).

^[1]NaCl was considered the most suitable control group. The neat liposomes applied in group 2 were significantly different in physical characteristics (e.g., charge and structure) compared to the liposome formulated RNA forming RNA of defined size and charge (lip), resulting in different pharmacological properties and changes in vivo biodistribution.

Clinical observations

Overall, the treatment was very well tolerated. Abnormal signs of intolerance were not noted in any animal for both local and systemic tolerability observations (including behavior, appearance, stool, mortality, body weight, and food and water intake).

Cytokine analysis

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

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

Very low levels of IFN- α induction were observed only in the high dose group 6 animals, reaching a maximum level after 5 hours, which returned to pre-dose levels after 24 hours (fig. 19). In contrast to the observations in cultured human cells and in mice, no IP-10 induction was observed in monkeys. The reason for not observing IP-10 in this study remains open, since IP-10 induction was observed in monkeys after TLR activation agonists as reported by others.

Other test cytokines (IFN-. gamma., TNF-. alpha., IL-1. beta., IL-2, IL-10, and IL-12p70) were unchanged. Liposomes alone had no effect on cytokine release.

Hematology

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

Transient reduction of lymphocytes and transient increase of neutrophils were found in a dose-dependent manner as a result of a study related to the test item. In high dose animals, lymphocytes declined very rapidly up to 5-fold 5 hours after completion of treatment (the minimum in group 6 animals was about 1,000 lymphocytes/μ L). This effect is transient and recovers in about 48 hours. Notably, a lower degree of lymphocyte depletion was also observed in the liposome group animals, but not in the NaCl control group (table 12). No adaptation effect induced by IL-6 was observed.

Due to the treatment, neutrophil increases were also observed in the NaCl control group, however, significant differences were observed in groups 3 to 6 when compared to the control. The maximum effect was observed 10 hours after treatment and was 44%, 34%, 89% and 91% in groups 3, 4, 5 and 6, respectively, relative to the control.

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

Table 12: results of absolute lymphocyte count [1,000/. mu.L ] in cynomolgus monkeys (average value n ═ 2).

Complement activation

C3a was measured before dosing, 0.5, 2, 5, 9, 24 and 48 hours after completion of the 1 st and 5 th treatments, 1 week and 3 weeks after the last dose. No test item related changes were observed and all values were considered within the normal range of biological variability.

Clinical chemistry

Standard parameters were tested before dosing, 24 hours after each dose, and another 4 days after 3 and 4 doses and 1 and 3 weeks after the last dose.

The test item-related impact on biochemical parameters was not assessed for animals in the liposome-treated group and for animals subjected to the test item treatment, as compared to background data available at control animals and/or the CRO in which the study was conducted. In part, the data showed some dispersion due to the small number of animals used per group.

No test-item related changes were noted for serum levels of bile acids, bilirubin, cholesterol, creatinine, glucose, phosphate, total protein, triglycerides, urea, calcium, chloride, potassium, and sodium, and for serum proteins (albumin, globulin, and albumin/globulin ratio).

The seroenzymatic activities of alanine aminotransferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), Lactate Dehydrogenase (LDH), alpha-amylase, creatine kinase (CK, including isoforms CK-BB, CK-MB, and CK-MM), gamma-glutamyl transferase (gamma-GT), and glutamate dehydrogenase (GLDH) are all considered within the limits of normal biological variability.

Higher values were noted for LDH, α -amylase and CK enzyme activities in animal No. 11 treated with 4X 3.5 μ g RNA/animal on test day 22 at test day 23. However, these changes were considered stress-related (due to the confinement of monkeys in the infusion chair) and not test items.

Although assessed as being independent of the test item, slight changes in CK were evaluated in more detail. Differential analysis of the CK isozymes CK-BB, CK-MB and CK-MM revealed that the increase in CK activity noted for individual animals of groups 4, 5 or 6 was mainly due to an increase in the CK-MM fraction compared to control animals tested on day 9, 16 or 23. Generally, no increase was noted for CK-BB and CK-MB, thus establishing that an increase in total CK levels was associated with stress.

Cardiovascular examination

ECG and blood pressure measurements did not show any effect on the cardiovascular system.

Sequence homology screening between RNA DP encoding TAA and human proteome

All mRNA sequences used in the W _ pro1 method were fused in-frame with up to two glycine/serine (GS) -rich flanking linker sequences. These fused suture sites can generate novel antigen fusion proteins or peptides that, if homologous to human proteins, could potentially elicit an undesirable autoimmune response. Thus, a BLASTp-based homology search against an established database of human proteins was performed to determine whether the stitching points associated with the linker sequences and antigens had sequence homology to known human proteins.

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

For a peptide subsequence that is 100% homologous, no significant alignment to the human protein sequence could be found.

Section 4: safety pharmacology

ICH guidelines S7A describe a series of core studies including functional assessments of the respiratory system, Central Nervous System (CNS) and cardiovascular system that should be performed prior to human exposure to any drug product. Thus, the safety pharmacology of rna (lip) was tested as an integral part of the six GLP toxicology studies described below.

In a key repeated dose toxicity study, potential effects on the functioning of the CNS and respiratory system were evaluated and no relevant effects on any of the tested projects in animals were shown.

For the cardiovascular system, a risk analysis of the potential role of rna (lip) vaccines was performed. Systemically distributed RNA is degraded in the circulation and RNA formulated as RNA (lip) is cleared from the blood within minutes and distributed mainly to the spleen and liver as shown in the biodistribution studies (see below). The data obtained did not indicate that RNA (LIP) would accumulate in the cardiovascular system. The potential systemic side effects of rna (lip) vaccination are expected to be associated with a transient increase in IFN- α, which is not expected to lead to cardiovascular side effects, as documented in thousands of patients receiving IFN- α. Thus, no ICH S7A/B compliant GLP cardiovascular safety pharmacological study was performed. However, supportive ECG and blood pressure data from non-GLP pharmacological studies in cynomolgus monkeys treated with rna (lip) were available and addressed the assessment of cardiovascular function after treatment with rna (lip) vaccine.

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

Safety of respiration

Respiratory safety was included in repeated dose toxicity studies (LPT nos. 28864 and 30283, see below for study design) in mice using GLP compliant TAA-encoding RNA DP. For example, plethysmography was tested in a study using RNA (LIP) (LPT No.30283) using four animals/sex/group treated with control buffer, low dose and high dose (5 and 50 μ g of RNA formulated with 9 μ g and 90 μ g of liposomes, respectively). Positive controls for animals treated with 30mg carbamoyl- β -methylcholine chloride (bethanecol)/kg b.w. were also included. Plethysmography was performed one day after the 4 th to 7 th doses. The tests included evaluation of respiratory rate, tidal volume, minute volume, inspiration time, expiration time, peak expiration and inspiration flow, expiration time, and airway resistance index. None of the tested lung parameters showed any test item related changes in the treated animals compared to the control group. Only the animals of the positive control group showed the expected change.

CNS safety

CNS safety was included in repeated dose toxicity studies (LPT nos. 28864 and 30283, see below for study design) in mice using GLP compliant TAA-encoding RNA DP. For example, in a study using RNA (LIP) (LPT No.30283), the observational screening was tested in five animals/sex approximately 24 hours after the 5 th dose with control buffer, low dose and high dose (5. mu.g and 50. mu.g of RNA formulated with 9. mu.g and 90. mu.g of liposomes, respectively). The following tests were included in the observational screening: righting reflex (righting reflex), body temperature, salivation, startle response (startle response), respiration, mouth breathing, urination, convulsions, erections, diarrhea, pupil size, pupillary response (pupil response), lacrimation, impaired gait, sculpting, toe-in (toe-in), tail-in (tail-in), line-action (wire maneuver), hind leg-in (hind-leg), position-passive (position-activity), tremor, mediterranean, limb rotation, and auditory function. In addition, functional tests to evaluate grip strength and autonomic activity were included.

Neurological screening did not show any test item related effects on mice attributable to neurotoxicity. These findings were confirmed by the results of GLP compliance repeat dose toxicity study LPT No.28864, performed using different RNAs for the Lipo-MERIT study.

Cardiovascular safety

Since RNA degrades within seconds in circulation and there is no indication that RNA (lip) will accumulate in the cardiovascular system, no cardiovascular safety studies according to ICH S7 were performed.

However, supporting data from non-GLP pharmacological studies in cynomolgus monkeys with rna (lip) targeting melanoma-associated antigens used in Lipo-MERIT studies is available. In this study, 12 cynomolgus monkeys were treated in 6 groups (see table 11 for study design) and ECG and blood pressure measurements were taken at three time points after the 4 th dose, before the dose, 5 hours after the completion of the dose and 24 hours after the dose.

In cynomolgus monkeys, tolerance to treatment with rna (lip) was very good (no clinical observations were observed). None of the measured parameters (blood pressure, heart rate, QTc values, QT interval, P segment, PQ, QRS) showed any test item related effects. In addition, serum levels of CK-MB and troponin-I were measured to exclude the possibility of necrotic injury to myocardial tissue. All measured parameters were negative, supporting no toxic effect of rna (lip) on the cardiovascular system at the tested dose levels in the study.

Discussion and conclusions

The mode of action and major pharmacodynamics of rna (lip) in mice and in human in vitro test systems have been extensively studied. Preclinical studies have shown that following IV administration, rna (lip) vaccines target spleen and lymphoid tissues. Rna (lip) vaccines elicit a dual role, i.e. induction of antigen-specific T cell responses and cellular activation processes and immune regulation following TLR triggering.

The data generated confirm that all antigenic RNA leads applied in vivo induce antigen specific T cell responses, including tetanus toxoid helper epitopes.

The functional properties of RNA (lip) agents are (i) RNA protection in serum and (ii) efficient in vivo targeting of APCs capable of presenting antigenic peptides and becoming activated after TLR7 triggering. The immunomodulatory activity of RNA results in dose-dependent cytokine induction in human samples, mice and cynomolgus monkeys, which all show different degrees of induction of IFN-. alpha.IP-10 and IL-6, depending on the test species or test system applied. RNA (lip) -mediated cytokine induction in PBMC is expected because of good evidence from self RNA studies and literature. In addition to these expectations, moderate induction of IFN- α and induction of chemokine IP-10(CXCL10) are more likely to reflect the onset of the expected pharmacological effects than the undesirable immunotoxicological events.

Data generated in mice indicate that splenocytes are the major source of TLR 7-dependent IFN- α secretion, as IFN- α secretion is reduced in TLR 7-/-mice. We believe that the observed transient and fully reversible cytokine response serves as the intended pharmacodynamic effect contributing to the efficient induction of vaccine-induced anti-tumor T cell responses. The favourable immunological properties are combined with a good tolerance of the rna (lip) vaccine in mice and cynomolgus monkeys.

We also investigated the secondary effects of treatment with rna (lip) vaccines in several in vitro and in vivo studies using human, cynomolgus monkey and mouse test systems. Of particular interest are the immunomodulatory effects of RNA (lip) vaccines, as these are stronger than what we have observed for non-formulated RNA vaccines administered into lymph nodes, which only result in local cell activation and cytokine induction.

Experiments using whole blood samples and PBMCs from human and cynomolgus donors were performed, excluding non-specific or uncontrolled cellular activation of human immune cells by rna (lip) vaccines, but still showing modest induction of cytokines as expected. In these experiments, human cells and cynomolgus monkeys were treated at doses covering and above the highest expected clinical dose cohort.

Although differences were found in cytokine levels between donors, different in vitro test systems (cultured PBMC vs whole blood) or species, the cytokine patterns and transient nature of cytokine responses observed in all studies were similar, except for a few, for example no IP-10 induction was observed in cynomolgus monkeys. Human PBMC show IP-10 induction, and low response to IL-6, and even lower levels of IFN- α when examined in whole blood. Cynomolgus monkeys showed a very low IFN- α response, without showing any IP-10 induction and a more pronounced IL-6 response at the dose levels tested. Mice showed strong responses to IFN-. alpha.IP-10 and IL-6, however at doses approximately 10-fold higher (based on the dose per kg b.w.) than were tested in monkeys. In one aspect, differences in cytokine expression between mice and monkeys can be explained by testing different doses. On the other hand, mice have different activities of TLR7/8, which can also be a reasonable explanation for different cytokine expression patterns.

The cytokine response pattern observed in cynomolgus monkeys was better reflected by the whole blood test system compared to the PBMC test system, with a broader, higher cytokine response observed at lower dose levels in PBMCs. Nevertheless, the findings in the more sensitive PBMCs are integrated into a strategy that defines a safe starting dose for the patient. A side-by-side comparison of cytokine secretion in human and cynomolgus monkey whole blood reveals that the two species are highly comparable for the induction of pro-inflammatory cytokines after rna (lip) treatment, suggesting that cynomolgus monkeys are a suitable animal model to predict secondary pharmacodynamic effects that may arise following vaccination with rna (lip) in patients.

In addition to the cell activation process and cytokine induction following exposure to rna (lip), BioNTech also evaluated hematological changes in mouse and cynomolgus monkey studies. Here, transient lymphopenia was equally observed in mice and monkeys at all dose levels. In summary, monkeys treated with rna (lip) showed similar responses in cytokine profile and hematological parameters to those observed with monkeys treated with other TLR agonists. This is consistent with the hypothesis that the main activation process of cytokine expression by rna (lip) occurs through TLR stimulation. In wild type, TLR7^-/-And IFNAR^-/-Extensive pharmacodynamic studies in mice have shown that hematology investigations result in a secondary effect of rna (lip) -induced cytokines. RNA (LIP) and unformulated naked RNA have been shown to activate TLRs. TLR activation has been shown to induce lymphopenia and B cell accumulation in the spleen. Supportive, histopathological data generated in toxicology testing indicate that transient lymphoproliferation is found in the spleen, but not in any other organ or tissue. This is consistent with the lymphopenia observed in blood and underscores the expected targeting of rna (lip), and subsequently the expected attraction of effector cells to lymphoid organs.

Safety pharmacological studies performed indicate a safety profile for rna (lip). In any of the tests performed, the neurological screening did not show any test item related effects on mice. None of the lung parameters tested showed any change in mice treated with RNA (LIP). There is no indication of cardiovascular effects in cynomolgus monkeys. Overall, rna (lip) showed a very good overall safety profile in terms of safety pharmacological parameters.

Section 5: pharmacokinetics

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

The in vitro transcribed RNA consists of ribonucleotides and thus has the same structure as RNA synthesized by human cells, with the exception of the 5' -cap structure. Thus, RNA undergoes the same degradation process as native mRNA. Especially in the extracellular space and in serum, abundant ribonucleases lead to rapid degradation of RNA.

As shown below, the distribution/arrangement (displacement) and potential accumulation of RNA in the spleen, liver and lung was studied in pharmacokinetic studies. In addition, potential plasmid DNA impurities in gonads from mice treated with rna (lip) were quantified.

The biodistribution and persistence of the synthetic cationic lipid DOTMA was first studied in vivo studies. Synthetic DOPE cannot be distinguished from the body's own native phospholipid DOPE, should follow the natural metabolic pathway, and therefore biodistribution and accumulation have not been further investigated.

Biodistribution

RNA

During GLP repeat dose toxicity studies (LPT No.28864) conducted for clinical trial Lipo-MERIT, the biodistribution of rna (lip) was studied in detail in mice by organ sampling. The quantitative real-time reverse transcription PCR (RT-qPCR) method developed by IMGM laboratory GmbH, Martinsried, Germany was applied to analyze the sum of all IVT-RNAs of the organs under non-GLP conditions (study ID: RS 297). In summary, RNA is cleared very rapidly from the blood, with an estimated half-life of about 5 minutes. After 48 hours and after 7 days, only marginal level (marginal level) of RNA was detected in blood and organs, indicating that it was rapidly degraded and did not persist.

Residual plasmid impurities

The biodistribution of residual plasmid impurities from rna (lip) vaccination was investigated using samples from GLP repeat dose toxicity study (LPT No.28864). A method for the analysis of residual plasmid impurities in organ samples was developed according to GLP in BioNTech IMFS GmbH, Idar-Oberstein, Germany. All test samples were below or slightly above the lower limit of detection (LLOD), indicating that plasmid DNA did not accumulate or persist in the gonads (study ID: 36X 130313).

DOTMA

Biodistribution of the two synthetic lipids used in the rna (lip) formulation may provide insight into the physical distribution of the lipid complex carrier particles over time. The synthetic cationic lipid DOTMA was chosen for biodistribution studies because it is not a naturally occurring molecule and can therefore be easily detected in the context of biological matrices.

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

DOTMA can be clearly determined from all the organs studied and significant differences between the findings of different organs can be observed. The highest DOTMA survey was in the spleen, according to the proposed mode of action.

On the basis of these first results, a single administration of rna (lip) study was performed (report _ BN _14_ 004). The concentrations of DOTMA in selected organs were assessed over a period of up to 28 days (day 0, day 1, day 4, day 7, day 14, day 21 and day 28). In this experiment, 200 μ L of RNA (LIP) containing 20 μ g of RNA and 26 μ g of DOTMA (in the first study, 60 μ g per injection) was administered. The concentration of DOTMA in the applied product was 195 μ M. Three mice/time point were studied.

Clearly, DOTMA is mainly present in the spleen and liver with indications of slightly different accumulation kinetics. In all other organs/tissues studied (lung, heart, kidney, lymph nodes, fat pad, bone marrow, brain), the findings were lower by a factor of 10 to 50 than the previous one. From the data in the liver and spleen, the pharmacokinetics of DOTMA can be estimated: the maximum concentration was detected several days after administration. Within 20 days, the DOTMA concentration decreased to about 50% of the maximum. These findings support the hypothesis that: DOTMA is cleared from organs within an acceptable time scale and fails to indicate (make out) an indication of the risk of permanent accumulation in any organ.

In subsequent studies, DOTMA concentrations in selected organs were assessed before (control), during and after eight weekly RNA (lip) injections, each containing 20 μ g RNA (RBL005.2) and 26 μ g DOTMA (report _ RB _15_004_ V02). Organs were sampled from mice one hour after the first rna (lip) administration, and then every other week after the previous administration. After the completion of the eight application cycles, mice were sacrificed after additional 3, 6, 9, 12, and 15 weeks to study DOTMA clearance in the organs. Repeated administration of rna (lip) test items and organ sampling was performed internally, while extraction and quantification of DOTMA from the provided organ samples was performed by Charles River Laboratories Edinburgh ltd. (study No. 322915). The results are in full agreement with our previous studies: also, the highest concentrations of DOTMA were observed in the spleen, which is the primary target organ, followed by the liver. In all other organs, no more than about 5% of the concentration present in spleen samples was found (data not shown). DOTMA concentration increased with increasing number of rna (lip) injections and then continued to decay during the recovery period after the last application. The terminal half-life of DOTMA in plasma (7.07 weeks), spleen (6.76 weeks) and liver (6.57 weeks) was comparable to that obtained from animals in recovery phase after the 8 th dose.

In summary, DOTMA is delivered to the spleen (and other organs) rapidly (within less than one hour) as an indicator of lipid carrier following IV RNA (LIP) administration. Except for the spleen, DOTMA accumulated mainly in the liver, whereas no RNA translation was observed in the liver. In absolute numbers, the amount of DOTMA present in both organs was close to the total cumulative DOTMA amount injected absolutely, while the amount of DOTMA in all other organ samples was almost negligible.

According to the evaluation of animals from the convalescent group, accumulated DOTMA was cleared from the organs after repeated rna (lip) applications, the kinetics of which could reasonably be represented by a first order decay, with an approximate half-life of about 6 to 7 weeks. Such clearance kinetics are also consistent with the findings from repeated applications in which transient accumulation was observed.

Taken together with all results, these findings support the following assumptions: DOTMA is cleared from organs within an acceptable time frame and the potential risk of permanent lipid accumulation in plasma, liver, spleen, lung, heart, brain, kidney, uterus, lymph nodes and bone marrow is quite low.

Discussion and conclusions

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

Results of RNA (lip) biodistribution studies indicate high levels of RNA in the blood shortly after injection of RNA (lip). RNA is rapidly cleared from the blood and is subsequently found in the spleen and liver, albeit at much lower levels, while only marginal amounts are found in the lungs. Since RNA distribution to the liver may lead to transient immune activation through TLR triggering, liver enzymes will be closely monitored in patients after the first injection and throughout the study. After 48 hours and 7 days, only residual amounts of RNA were found in blood and organs, indicating that RNA did not accumulate or persist in any organ. Comparison of Cmax levels after the 1 st and 8 th injections also did not show any cumulative effect.

In the gonads, no plasmid DNA was detected or the sample was slightly higher than LLOD, indicating that there is only a small risk of integration of plasmid residues (e.g. kanamycin resistance gene) into the germ line cell genome.

It has been shown that the biodistribution of DOTMA is mainly present in spleen and liver, identifying spleen as the main target organ for rna (lip) vaccination, and significantly low exposure to plasma and other tissues after single and eight repeated rna (lip) administrations. DOTMA is cleared from plasma, spleen and liver with a comparable terminal half-life of about 6 to 7 weeks. A more systematic analysis of the biodistribution and accumulation of DOTMA will be performed before advanced clinical tests are performed.

Section 6: toxicology

Toxicology programs for the RNA vaccine platform include several pharmacological studies testing RNA (lip) vaccination in different dose ranges, and repeated dose toxicity studies including local tolerability and safety pharmacological parameters and immunotoxicity studies. The study was performed under GLP conditions using RNA and liposome batches comparable to clinical trial materials in terms of manufacturing process and analytical quality control, external CRO (LPT, Hamburg, Germany).

GLP compliance studies included 6-week repeat dose toxicity studies in which 8 different rnapdps encoding breast cancer antigens (LPT No.30283) were administered IV to C57BL/6 mice.

In addition, a supplementary GLP compliance 6-week repeat dose toxicity study (LPT No.28864) was performed using an RNA vaccine platform targeting multiple melanoma-specific antigens. Although different RNA sequences were tested, toxicity data was also associated with the application of RNA DP encoding TAA, and important information could be added as the same type of liposomes was used for RNA (lip) preparation. Due to the fact that possible side effects are related to the inherent molecular properties of liposome-formulated RNA independent of RNA sequence and length, the toxicity profiles of the formulated RNA in both studies should be identical or at least comparable.

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

Selection of related species

We believe mice are a relevant species to test for potential toxic direct effects of rna (lip) vaccines for the following main reasons:

mice as a model system provided all relevant features of innate and adaptive immunity associated with characterizing the direct toxic effects of RNA DP encoding TAA. Mice display all expected primary and secondary pharmacological effects, from induction of CD4+/CD8+ T cell responses to immunomodulation that enhances the immune response and leads to subsequent TLR triggering, cell activation and cytokine secretion.

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

The targeting effect of a vaccine (on-target effect) cannot be fully studied in animal species. Thus, the use of other animal species does not provide additional information and therefore the use of higher mammals should not be considered.

Single dose toxicology

Dose range finding studies are usually performed to adjust the dose for critical toxicity studies and to obtain preliminary information about target organs and signs of toxicity. We performed several pharmacological studies to test rna (lip) in different dose ranges using a protocol similar to the expected clinical protocol. During these studies, it was found that administration of rna (lip) induced a favorable pharmacodynamic effect and was well tolerated.

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

The following data were generated from a series of internal and literature studies:

tolerable doses in mice that would provide an adequate safety margin for the first dose used in humans can be inferred from the pharmacological studies performed.

A single dose administration will not be sufficient to induce a significant immune response. Maximal immune responses were observed after at least three administrations.

RNA vaccines and lipid complex formulations are generally well tolerated.

Based on these conclusions, we decided not to conduct a single dose toxicity study, but rather to conduct a repeat dose toxicity study directly.

Toxicology of repeated dose

The RNA vaccine platform RNA (lip) products were analyzed for safety and toxicology in several GLP compliance repeat dose toxicity studies involving IV injection of RNA (lip) products. Table 13 provides an overview of GLP repeat dose toxicity studies supporting clinical phase 1 and phase 2 testing using rna (lip) vaccines.

Table 13: design of GLP repeat dose toxicity study.

ATM formulations

The composition, formulation and specifications of AMTs are planned as close as possible to the intended pharmaceutical product for humans.

Minor modifications to the rna (lip) preparation process are necessary for the following reasons:

high doses are obtained that increase the likelihood of capturing potential dose-dependent toxicological effects and thereby meet the criteria for toxicity testing as outlined in the ICH S6 or M3(R2) guidelines.

To prevent administration in mice above the maximum volume feasible, i.e. a volume of 250 μ L in a slow bolus injection. Higher injection volumes are not ethically recommended and risk of losing mice during injection.

The differences from the clinical protocol are:

the patient will obtain different RNA (LIP) products in a sequential manner. This is not possible in mice because of volume limitations. RNA (lip) was prepared separately for mice, then mixed, and all four RNA lipid complexes were injected simultaneously in a total volume of 250 μ L.

For the formulation of RNA (LIP) for treatment of patients, 150mM NaCl will be used. To obtain higher doses in toxicity studies, higher concentrations of NaCl solutions must be used for rna (lip) formation.

For the preparation of RNA (LIP) for patient treatment, a RNA drug product will be used at a concentration of 0.25 mg/mL. To achieve high doses in toxicity studies, in the study of LPT No.28864, a more concentrated RNA (i.e., 1mg/mL) must be used to prepare the RNA (LIP) product.

In this experiment, half-concentrated acetic acid-stabilized liposomes (L4) relative to the equivalent amount of acetic acid-stabilized liposomes (L2) previously used, were used. Given the same characteristics as the L2 liposome, no further bridging study was planned.

Our standpoint is that the above-described modifications to the formulation scheme of the ATM have little or no effect on the results or performance of the study.

Design of research

See table 13 for study design. Data from standard toxicity studies were evaluated for signs of immunotoxicity potential according to ICH S6 and S8. The following studies were performed according to FDA, ICH and CHMP guidelines: mortality, histopathology (especially the spleen), gross pathology (gross pathology) and organ weight, clinical observations, ophthalmology, local tolerance, injection site reactions, body weight, food consumption, standard hematologic parameters, and clinical chemistry and cytokines (IL-1 β, IL-2, IL-6, IL-10, IL-12, TNF- α, INF- γ, and IP-10).

Safety pharmacology studies were included to test the respiratory and central nervous systems, as described in section 4.

Results

Toxicological assessments in 6-week repeat dose toxicity studies using the vaccine platform revealed only mild effects primarily attributable to the expected pharmacological mode of action of rna (lip) (table 14). The desired immune modulatory effects of rna (lip) are TLR activation and cytokine release. Induction of IFN- α in mice leads to secondary effects such as leukopenia, thrombocytopenia and increased liver parameters (e.g. ALAT), effects usually described for patients treated with IFN- α.

In line with this, the test item-related changes in the treated animals were mainly transient (table 14). In addition, transient activation of cytokines IP-10, IFN- α, IL-6 and IFN- γ was observed. All induction returned to normal levels after 24 hours (except for IP-10 levels, which were still slightly above normal).

Hematological findings in mice included mainly lymphopenia with low, reversibly reduced total leukocytes, neutrophils, reticulocytes, and thrombocytopenia in all treatment groups. These findings were completely reversible. The lymphoproliferation of the spleen observed in histopathology was completely restored and the expected effects and expected targeting of the spleen by the test substances and lymphocytes were summarized.

Slight changes in the liver parameters observed (e.g., GLDH, LDH, ALAT, and ASAT) primarily affected the high dose group, while not noted in the animals in the recovery group, indicating that the effect was completely recovered in at least three weeks or less. Histopathology did not detect hepatotoxicity.

Since there were no findings in the low dose group in study LPT No.30283, NOAEL was met at a dose of 5 μ g total RNA per animal (i.e., about 0.2mg/kg b.w. in mice).

Additional 4-week repeat dose toxicity studies were performed to account for changes in liposome buffer composition (LPT No. 30586). The data demonstrate that the novel liposomes are completely comparable in measured parameters to the liposomes used in the main study.

Table 14: summary of toxicology findings from repeated dose toxicity studies using RNA (LIP) (LPT Nos. 28864, 30283, and 30586).

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

Genetic toxicity

The components (lipids and RNA) of the RNA (lip) product are not suspected of having genotoxic potential. Neither the impurities nor the components of the delivery system were subjected to genotoxicity testing. Genotoxicity studies were not planned according to the recommendations given in the guideline for preclinical safety assessment of ICH biotech-derived drugs S6(R1) (6 months 2011).

Carcinogenicity

The RNA itself and the lipids used as a carrier have no carcinogenic or tumorigenic potential. According to ICH S1A, there is no need to conduct long-term carcinogenic studies without the cause of concerns from laboratory and toxicological studies, and without the deliberate desire to apply the drug for long periods.

Reproductive and developmental toxicity

Macroscopic and microscopic evaluation of male and female reproductive tissues was included in repeated dose toxicity studies in mice treated with rna (lip). The findings were not noted in any of these studies, and therefore no specific fertility and developmental toxicity studies were performed until the start of phase 1 studies with rna (lip) vaccines. Rna (lip) is not expected to produce direct cytotoxic effects on reproductive tissues, as supported by experience from other cancer vaccines, suggesting no effect on reproduction and development. As the effects on reproduction cannot be ruled out, women with fertility potential will have to use effective contraception during the treatment period. No further long-term or reproductive toxicity studies are currently planned.

Local tolerance

Tests for local tolerability were evaluated in GLP repeat dose toxicity studies against IV injections according to ICH recommendations. No signs of local intolerance were observed during the study.

Other toxicity studies

Antigenicity

Because IVT-RNA rapidly decomposes extracellularly in seconds to minutes, no anti-drug antibodies (ADA) are expected to form. Thus, no specific antigenicity test for antibody induction is planned.

Immunotoxicity

Since the immune system is intended to be activated by the rna (lip) product, particular attention is paid to the immunotoxicological parameters in order to exclude unintended activation or inhibition. The immunotoxicology examination was performed in both 6-week repeat dose toxicity studies (LPT nos. 28864 and 30283). In addition to monitoring cytokine levels in serum, immunotoxicity was evaluated taking into account the following relevant parameters: body weight, body temperature, lymphatic organ weight, macroscopic and histopathology of lymphatic organs, absolute and relative differential blood counts, total serum protein, albumin/immunoglobulin ratio, myeloid/erythroid ratio in bone marrow, coagulation parameters.

Hematology

In both studies, a decrease in lymphocytes, white blood cell count (mainly due to lymphocyte decrease) and platelets was observed in all treatment groups at test day 44, i.e. about 24 hours after the 8 th injection. After two weeks, all effects were fully restored. The results of the LPT No.28864 and 30283 studies are shown in table 15 and table 16, respectively.

Table 15: hematological data (LPT No. 28864).

Samples for hematological determinations were collected on test day 44 (about 24 hours after the 8 th injection).

Statistically significant, p is less than or equal to 0.05; statistically significant, p ≦ 0.01 (Dunnett's test).

Table 16: hematological data (LPT No. 30283).

Samples for hematological determinations were collected on test day 44 (about 24 hours after the 8 th injection).

Statistically significant, p is less than or equal to 0.05; statistically significant, p ≦ 0.01 (Dunnett's test).

Cytokine determination

The secretion of the following cytokines was analyzed in a repeated dose toxicity study: IL-1 β, IL-2, IL-6, IL-10, IL-12p70, TNF- α, IFN- γ, and IP-10, known as sensitive indicators of immune activation or TLR7 signaling. In toxicity studies, LPT No.28864 mice showed dose-dependent and subject-related increases in cytokine IP-10. IP-10 levels increased transiently and reached a maximum 6 hours after the 4 th injection. After 24 hours, the level was still significantly higher compared to the control level, but had returned to the normal level. IP-10 showed a maximum induction of 28-fold and 16-fold after 6 hours (in males and females, respectively) compared to the control group. Significant increases were also observed for TNF- α (female only, groups 2, 3 and 4), IL-10 (female only, groups 3 and 4), IL-6 (male, group 4) and IFN- γ (male, group 4). The maximum induction was 3-fold for TNF-alpha, 4-fold for IL-10, 7-fold and 8-fold for IL-6 and 6-fold and 2-fold for IFN-gamma. After 24 hours, all effects were completely reversible (except for TNF- α levels in group 4 females). The results are summarized in table 17.

Table 17: cytokine levels in plasma (LPT No. 28864).

Samples for cytokine determination were taken 6 hours and 24 hours after the 4 th injection.

Statistically significant, p is less than or equal to 0.05; statistically significant, p ≦ 0.01 (Dunnett's test).

For cytokine determination in study LPT No.30283, serum samples were taken 6 hours and 24 hours after the 5 th injection. Serum levels of IL-2, IL-6, IP-10, IFN- α and IFN- γ were found to be elevated (Table 18). For IL-6, a significant dose-dependent induction was noted. For IFN- γ, induction was noted after high dose treatment (statistically significant at p ≦ 0.01), more significant in male animals. For IFN- α, induction was observed after low dose and after high dose treatment (statistically significant at p ≦ 0.01 or p ≦ 0.05), and more significant for females in group 5 (RNA group 2, high dose). All the above cytokine induction resolved 24 hours after administration.

At 6 and 24 hours after low or high dose treatment, relatively low but dose-related induction of IL-2 was noted for male and female animals.

At 6 hours after high dose treatment, a significant dose-dependent effect of IP-10 was detected at all dose levels for male and female animals compared to the control group. IP-10 levels remained elevated at all dose levels compared to the control at 24 hours after administration. This induction of IP-10 reflects the expected pharmacological effect and is not considered an undesirable immunotoxicological event.

Table 18: cytokine levels in plasma (LPT No. 30283).

Samples for cytokine determination were taken 6 hours and 24 hours after the 5 th injection.

incr.: a significant improvement was noted compared to the control group, however, since the control group value was set to "0.0", the improvement could not be expressed as a multiple.

Statistically significant, p is less than or equal to 0.05; statistically significant, p ≦ 0.01 (Dunnett's test).

During the study comparing L1 and L2 liposomes, a cytokine determination was also performed (LPT No. 30586). Here, cytokines were analyzed 6 and 24 hours after the 4 th immunization. No attention was paid to the subject item-related changes between groups.

Discussion and conclusions

Treatment with RNA (LIP) was well tolerated in mice as shown by a number of antigen-encoding RNAs evaluated in three different repeated dose toxicity studies (LPT Nos. 28864, 30283 and 30586). Overall, treatments with up to 8 IV injections were well tolerated, as well as in the high dose group of animals. No premature death associated with the test item was observed in study Nos. 30283 and 30586. In study No.28864 one animal died prematurely after administration of the test item. Since there were no findings in the low dose group of study No.30283, NOAEL was achieved at a dose of 5 μ g total RNA per animal (i.e., about 0.2mg/kg b.w. in mice). In addition, vaccination with the rna (lip) product was also very well tolerated in non-GLP pharmacological studies in 12 cynomolgus monkeys (no clinical observations).

Toxicological assessments of rna (lip) in repeated dose toxicity studies in mice revealed effects attributable to the test items, including transient induction of cytokines, hematological changes, and liver enzyme elevation. The observed effects were mainly the induction of cytokines IP-10, IFN- α, IFN- γ and IL-6 in the in vivo studies reported here and in the in vitro studies described and discussed above.

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

Effects such as lymphopenia and liver enzyme elevation are also reported in mice and monkeys after treatment with plasmid lipid complexes and activation by TLRs, and are often observed as secondary effects driven by IFN- α secretion, which is often described for patients treated with recombinant IFN- α sold for many years for the treatment of a variety of neoplastic and non-neoplastic diseases.

The changes in hepatic parameters observed in the high dose group in mice indicate that the liver is likely to be a toxic target for higher doses of liposome-formulated RNA. These changes include increased liver weight, increased plasma levels of GLDH, LDH, ASAT and ALAT. These changes were considered mild and were not observed in the recovery group of animals, indicating that the effect was fully recovered in at least three weeks. In addition, histopathology did not reveal any hepatotoxicity. In cynomolgus monkeys, the biochemical parameters of both the liposome-treated group of animals and the animals subjected to the test item were considered to be within the limits of normal biological variability compared to the control animals. On test days 9, 16 or 23, the increase in CK noted for individual animals of groups 4, 5 or 6 compared to control animals was primarily attributable to the CK-MM fraction and was considered to be related to stress.

Mild elevation of hepatic parameters in mice may be a response by immunomodulation that may be triggered by phagocytosis of rna (lip) by hepatic target cells, such as Kupffer cells. In contrast to the effect observed in the mouse spleen (lymphoproliferation), this did not result in leukocyte recruitment to the liver, suggesting that the desired pharmacological effects, such as TLR activation and lymphocyte transport, were restricted to lymphoid organs.

Complement activation of liposome-formulated materials has been previously reported. For rna (lip) vaccination, a slight increase in C5a levels was observed in female mice, but this was considered to be a low bio-related event. In addition, mice are not considered a good model to extrapolate complement effects to humans.

Overall, the immune responses seen in all three rna (lip) repeated dose toxicity studies (LPT nos. 28864, 30283 and 30586) indicate a comprehensive picture of increased spleen weight, cytokine/chemokine activation and lymphocyte transport (comprehensive picture). This reflects the induction of the expected pharmacological event and underscores the relevance of mice as a correct test model for toxicity studies. In the bridging study of LPT No.30583, which evaluates the toxicity of the pH adjusted L2 liposomal formulation, no significant difference was observed between the two liposomal formulations. In this experiment, L4, half concentrated as L2 liposomes, was applied as pH adjusted liposomes. Given the same characteristics as the L2 liposome, no further bridging studies were planned.

Example 3: induction of antigen-specific T cells in the spleen by KLK2-, KLK3-, ACPP-, NKX 3-1-and HOXB 13-encoding RNAs

A2/DR1 mice were immunized with 200 μ L of 0.15mg/mL RNA (LIP) solution corresponding to 30 μ g RNA (LIP) on days 1, 8, 15, and 22.

Five days after the end of four rna (lip) injections, splenocytes were obtained from immunized a2/DR1 mice and the in vivo induction of antigen-specific T cells was determined by ELISPOT analysis. To test for immunogenicity, isolated splenocytes were restimulated with a peptide pool (15mer, 11 amino acid overlap) spanning the corresponding human protein, KLK2, KLK3(PSA), acpp (pap), NKX3-1, or HOXB 13. The results are shown in fig. 20. The IFN- γ + spot counts induced by KLK2-, ACPP-and HOXB 13-encoding rna (lip) were statistically higher (P ═ 0.0056; P > 0.0001; P ═ 0.0095) compared to restimulation with the control peptide CMV pp65, according to the unpaired t-test. Restimulation of splenocytes with the control P2/P16/P17 peptide also resulted in high spot counts in all immunized mice, indicating successful immunization. On all ELISPOT plates, negative control medium alone induced only the lowest spot count regardless of which animal the splenocytes were derived from.

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