TW202026423A - Therapeutic rna for solid tumor cancers - Google Patents

Abstract

This disclosure relates to the field of therapeutic RNAs for treatment of solid tumor cancers.

Description

Therapeutic RNA for solid tumor cancer

This application claims the priority rights of U.S. Provisional Application No. 62/722,742 filed on August 24, 2018 and European Patent Application No. 19305092.9 filed on January 24, 2019; the contents of both are incorporated by reference in their entirety .

This disclosure relates to the field of therapeutic RNA for the treatment of solid tumors. The National Cancer Institute defines a solid tumor as an abnormal mass of tissue that does not usually contain cysts or fluid areas. Solid tumors include benign and malignant (cancerous) sarcomas, carcinomas, and lymphomas, and can be physically located in any tissue or organ including brain, ovary, breast, colon, and other tissues. Cancers are generally divided into two main types: solid tumor cancers and hematological (blood) cancers. It is estimated that more than 1.5 million cancer cases are diagnosed in the United States each year, and in the United States, more than 500,000 people will die of cancer each year.

Solid tumor cancers are particularly difficult to treat. Current treatments include surgery, radiation therapy, immunotherapy and chemotherapy. Surgery alone may be a suitable treatment for small local tumors, but large invasive tumors and most metastatic malignancies cannot usually be removed by surgery. Other common treatments (such as radiation therapy and chemotherapy) can be associated with undesirable side effects and damage to healthy cells.

Although surgery and current therapies can sometimes kill most solid tumors, additional cells (potentially including cancer stem cells) may survive the therapy. Over time, these cells may form A new tumor that causes the cancer to recur. Despite the multi-modal conventional therapy, for many types of solid tumors, the disease-free survival rate is less than 25%. Solid tumors that tolerate multimodal therapy or have recurred after therapy are even more difficult to treat, and have a long-term survival rate of less than 10%.

This paper discloses compositions, uses and methods that can overcome the current shortcomings in the treatment of solid tumors. The administration of therapeutic RNA disclosed herein can reduce tumor size, prolong survival time, and/or prevent tumor metastasis and/or recurrence.

Specific Example 1 is a composition or pharmaceutical preparation comprising RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, and RNA encoding IFNα protein.

Specific embodiment 2 is the pharmaceutical preparation or composition according to specific embodiment 1, which further comprises RNA encoding FLT3-L protein.

Specific example 3 is the composition according to specific example 1 or 2.

Specific embodiment 4 is the pharmaceutical preparation according to specific embodiment 1 or 2.

Specific embodiment 5 is the pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein the RNA has a ratio of 1:1:1 or 1:1:1:1, and optionally wherein The ratio was verified by quantitative RT-PCR.

Specific embodiment 6 is the pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein the RNA integrity is greater than or equal to 70%.

Specific embodiment 7 is the pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein the pharmaceutical preparation or composition comprises less than 250ng DNA/total mg of nucleic acid present.

Specific embodiment 8 is the pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein the IFNα protein is IFNα2b protein.

Specific embodiment 9 is a pharmaceutical preparation or composition according to any one of the foregoing specific embodiments, which in

The RNA encoding IL-12sc protein comprises the nucleotide sequence of SEQ ID NO: 17 or 18 or has at least 99%, 98%, 97%, 96%, and the nucleotide sequence of SEQ ID NO: 17 or 18. 95%, 90%, 85% or 80% identical nucleotide sequences; and/or

The IL-12sc protein comprises the amino acid sequence of SEQ ID NO: 14 or has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% of the amino acid sequence of SEQ ID NO: 14. % Or 80% identical amino acid sequence; and/or

The RNA encoding IL-12sc protein contains at least 99%, 98%, 97%, 96%, 95%, and the p40 portion of IL-12sc (nucleotides 1-984 of SEQ ID NO: 17 or 18). 90%, 85% or 80% identity and at least 99%, 98%, 97%, 96%, 95% with the p30 portion of IL-12sc (nucleotides 1027-1623 of SEQ ID NO: 17 or 18) , 90%, 85%, or 80% identical nucleotide sequence, and further comprise the nucleotide between the p40 part and the p35 part encoding the linker polypeptide.

Specific embodiment 10 is a pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein

The RNA encoding IL-15 sushi protein comprises the nucleotide sequence of SEQ ID NO: 26 or at least 99%, 98%, 97%, 96%, 95%, and the nucleotide sequence of SEQ ID NO: 26. A nucleotide sequence of 90%, 85% or 80% identity; and/or

The IL-15 sushi protein comprises the amino acid sequence of SEQ ID NO: 24 or has at least 99%, 98%, 97%, 96%, 95%, 90%, and the amino acid sequence of SEQ ID NO: 24. 85% or 80% identical amino acid sequence; and/or

The RNA encoding IL-15 sushi protein contains at least 99%, 98%, 97%, 96%, and the sushi domain of IL-15 receptor alpha (nucleotides 1-321 of SEQ ID NO: 26). 95%, 90%, 85%, or 80% identity and with mature IL-15 (nucleotides 382-729 of SEQ ID NO: 26) at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical nucleotide sequence, and optionally also Contains nucleotides between the sushi domain of IL-15 and the mature IL-15 encoding the linker polypeptide.

Specific embodiment 11 is a pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein

The RNA encoding IFNα protein comprises the nucleotide sequence of SEQ ID NO: 22 or 23 or has at least 99%, 98%, 97%, 96%, 95% of the nucleotide sequence of SEQ ID NO: 22 or 23. , 90%, 85% or 80% identical nucleotide sequence; and/or

The IFNα protein comprises the amino acid sequence of SEQ ID NO: 19 or has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or the amino acid sequence of SEQ ID NO: 19 80% identical amino acid sequence.

Specific embodiment 12 is the pharmaceutical preparation or composition according to any one of specific embodiments 2-11, wherein

The RNA encoding the FLT3-L protein comprises the nucleotide sequence of SEQ ID NO: 32 or is at least 99%, 98%, 97%, 96%, 95%, 90% to the nucleotide sequence of SEQ ID NO: 32. %, 85% or 80% identical nucleotide sequence; and/or

The FLT3-L protein comprises the amino acid sequence of SEQ ID NO: 30 or has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% of the amino acid sequence of SEQ ID NO: 30. % Or 80% identical amino acid sequence.

Specific embodiment 13 is the pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein at least one RNA comprises a modified nucleoside instead of at least one uridine.

Specific embodiment 14 is the pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein at least one RNA comprises a modified nucleoside instead of each uridine.

Specific embodiment 15 is the pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein each RNA comprises a modified nucleoside replacing at least one uridine.

Specific embodiment 16 is a pharmaceutical preparation or composition according to any one of the preceding specific embodiments, Each RNA contains modified nucleosides that replace each uridine.

Specific embodiment 17 is the pharmaceutical preparation or composition according to any one of specific embodiments 13-16, wherein the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U).

Specific embodiment 18 is a pharmaceutical preparation or composition according to any one of specific embodiments 13-17, wherein at least one RNA comprises more than one type of modified nucleosides, wherein the modified nucleosides are independently selected from pseudo Uridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U).

Specific embodiment 19 is the pharmaceutical preparation or composition according to specific embodiment 18, wherein the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

Specific embodiment 20 is the pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein at least one RNA comprises ^5'cap m ₂ ^7,3'-O Gppp(m ₁ ^2'-O ) ApG or 3'-O-Me-m ⁷ G(5')ppp(5')G.

Specific embodiment 21 is a pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein each RNA comprises ^5'cap m ₂ ^7,3'-O Gppp(m ₁ ^2'-O ) ApG or 3'-O-Me-m ⁷ G(5')ppp(5')G.

Specific embodiment 22 is a pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein at least one RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and 6 or a combination thereof The nucleotide sequence from the group consisting of SEQ ID NO: 4 and 6 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical 5'UTR.

Specific embodiment 23 is a pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein each RNA contains a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and 6 or a combination thereof The nucleotide sequence from the group consisting of SEQ ID NO: 4 and 6 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical 5'UTR.

Specific embodiment 24 is a pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein at least one RNA comprises a nucleotide sequence containing SEQ ID NO: 8 or a nucleus with SEQ ID NO: 8 The nucleotide sequence has a 3'UTR of a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical.

Specific embodiment 25 is a pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein each RNA comprises the nucleotide sequence of SEQ ID NO: 8 or the nucleotide sequence of SEQ ID NO: 8 The sequence has a 3'UTR of a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical.

Specific embodiment 26 is the pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein at least one RNA comprises a polyA tail.

Specific embodiment 27 is the pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein each RNA comprises a poly A tail.

Specific embodiment 28 is the pharmaceutical preparation or composition according to specific embodiment 26 or 27, wherein the poly A tail contains at least 100 nucleotides.

Specific embodiment 29 is the pharmaceutical formulation or composition according to any one of specific embodiments 26-28, wherein the poly A tail comprises the poly A tail shown in SEQ ID NO:66.

Specific embodiment 30 is the pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein one or more RNAs comprise:

^5'cap containing m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG or 3'-O-Me-m ⁷ G(5')ppp(5')G;

Containing (i) a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and 6 or (ii) having at least 99% of a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and 6, 5'UTR of a nucleotide sequence that is 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical;

Comprising (i) the nucleotide sequence of SEQ ID NO: 8 or (ii) the nucleotide sequence of SEQ ID NO: 8 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% The 3'UTR of a nucleotide sequence of% or 80% identity; and

A poly A tail containing at least 100 nucleotides.

Specific embodiment 31 is the pharmaceutical preparation or composition according to specific embodiment 30, wherein the poly A tail comprises SEQ ID NO:66.

Specific embodiment 32 is the pharmaceutical preparation or composition according to any one of the foregoing specific embodiments, which is a pharmaceutical composition comprising the RNA.

Specific embodiment 33 is the pharmaceutical preparation or composition according to specific embodiment 32, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.

Specific embodiment 34 is the pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein the RNA is formulated as a liquid, formulated as a solid, or a combination thereof.

Specific embodiment 35 is a pharmaceutical preparation or composition according to any one of the foregoing specific embodiments for pharmaceutical use.

Specific embodiment 36 is the pharmaceutical preparation or composition according to specific embodiment 35, wherein the pharmaceutical use includes the therapeutic treatment or preventive treatment of diseases or disorders.

Specific embodiment 37 is the pharmaceutical preparation or composition according to specific embodiment 36, wherein the therapeutic treatment or prophylactic treatment of the disease or disorder includes the treatment or prevention of solid tumors.

Specific embodiment 38 is the pharmaceutical preparation or composition according to specific embodiment 37, wherein the solid tumor is a sarcoma, carcinoma or lymphoma.

Specific embodiment 39 is the pharmaceutical preparation or composition according to any one of specific embodiments 37 or 38, wherein the solid tumor is in the lung, colon, ovary, cervix, uterus, peritoneum, testis, penis, tongue , Lymph nodes, pancreas, bone, breast, prostate, soft tissue, connective tissue, kidney, liver, brain, thyroid or skin.

Specific embodiment 40 is the pharmaceutical preparation or composition according to any one of specific embodiments 37-39, wherein the solid tumor is an epithelial tumor, Hodgkin’s lymphoma (HL), non-Hodgkin’s Lymphoma, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tumor, liver tumor, colorectal tumor, vascular tumor, mesothelioma tumor, pancreas tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor , Brain tumor, melanoma tumor, small cell lung tumor, neuroblastoma tumor, testicular tumor, cancer tumor, adenocarcinoma tumor, glioma tumor, seminoma tumor, retinoblastoma or osteosarcoma tumor.

Specific embodiment 41 is the pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein the RNA is used for intratumor or peripheral administration.

Specific embodiment 42 is the pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein the RNA is formulated for injection.

Specific embodiment 43 is a pharmaceutical preparation or composition according to any one of the foregoing specific embodiments, which is used for administration to humans.

Specific embodiment 44 is the pharmaceutical preparation or composition according to any one of specific embodiments 37-43, wherein treating or preventing the solid tumor includes reducing the size of a tumor, preventing the recurrence of cancer in remission, or preventing an individual’s Cancer metastasis.

Specific embodiment 45 is the pharmaceutical preparation or composition according to any one of specific embodiments 36-44, wherein the therapeutic treatment or prophylactic treatment of the disease or disorder further comprises the administration of another therapy.

Specific embodiment 46 is the pharmaceutical preparation or composition according to specific embodiment 45, wherein the additional therapy includes one or more selected from the group consisting of: (i) surgery to remove, excise or reduce tumors, (ii) immunity Therapies, (iii) radiotherapy and (iv) chemotherapy.

Specific embodiment 47 is the pharmaceutical formulation or composition according to any one of specific embodiments 45-46, wherein the additional therapy comprises the administration of another therapeutic agent.

Specific embodiment 48 is the pharmaceutical preparation or composition according to specific embodiment 47, wherein the additional therapeutic agent is an anti-cancer therapeutic agent.

Specific embodiment 49 is the pharmaceutical preparation or composition according to specific embodiment 47 or 48, wherein the additional therapeutic agent is a checkpoint modifier.

Specific embodiment 50 is the pharmaceutical preparation or composition according to specific embodiment 49, wherein the checkpoint modulator is an anti-PD1 antibody, an anti-CTLA-4 antibody or the group of an anti-PD1 antibody and an anti-CTLA-4 antibody Together.

Specific embodiment 51 is a method for treating solid tumors or reducing the possibility of solid tumors, which comprises administering a first RNA to an individual in need, wherein the first RNA encodes IL-12sc protein, IL-15 sushi protein , FLT3-L protein or IFNα protein, and the individual is further treated with additional RNA, wherein:

If the first RNA encodes IL-12sc protein, the additional RNA encodes IL-15 sushi protein, IFNα protein, and FLT3-L protein; or

If the first RNA encodes IL-15 sushi protein, the additional RNA encodes IL-12sc protein, IFNα protein, and FLT3-L protein; or

If the first RNA encodes IFNα protein, the additional RNA encodes IL-15 sushi protein, IL-12sc protein, and FLT3-L protein; or

If the first RNA encodes FLT3-L protein, the additional RNA encodes IL-15 sushi protein, IFNα protein, and IL-12sc protein; or

If the first RNA encodes IL-12sc protein, the additional RNA encodes IL-15 sushi protein and IFNα protein; or

If the first RNA encodes IL-15 sushi protein, the additional RNA encodes IL-12sc protein and IFNα protein; or

If the first RNA encodes IFNα protein, the additional RNA encodes IL-15 sushi protein and IL-12sc protein.

Embodiment 52 is a kit comprising the composition according to any one of Embodiments 1-50.

Specific embodiment 53 is the pharmaceutical preparation according to any one of specific embodiments 1-2 or 4-50, wherein the pharmaceutical preparation is a kit.

Specific embodiment 54 is the pharmaceutical preparation according to specific embodiment 53, or the kit according to specific embodiment 52, wherein the RNA is in a separate vial.

Specific embodiment 55 is the kit according to any one of specific embodiments 52-54, which further includes instructions for using the composition to treat or prevent solid tumors.

Specific Example 56 is an RNA for use in a method for treating or preventing solid tumors in an individual, wherein the method comprises administering RNA encoding IL-12sc protein, IL-15 sushi protein encoding RNA and RNA encoding IFNα protein.

Specific Example 57 is the use of RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, and RNA encoding IFNα protein for the treatment of solid tumors.

Specific embodiment 58 is the RNA or use according to any one of specific embodiments 56-57, which further includes RNA encoding FLT3-L protein.

Specific embodiment 59 is the RNA or use according to any one of specific embodiments 56-58, wherein the RNA has a ratio of 1:1:1 or 1:1:1:1, and optionally wherein The ratio was verified by quantitative RT-PCR.

Specific embodiment 60 is the RNA or use according to any one of specific embodiments 56-59, wherein the RNA integrity is greater than or equal to 70%.

Specific embodiment 61 is the RNA or use according to any one of specific embodiments 56-60, wherein the pharmaceutical preparation or composition comprises less than 250ng DNA/total mg of nucleic acid present.

Specific embodiment 62 is the RNA or use according to any one of specific embodiments 56-61, wherein the IFNα protein is an IFNα2b protein.

Specific embodiment 63 is the RNA or use according to any one of specific embodiments 56-62, wherein

The RNA encoding IL-12sc protein comprises the nucleotide sequence of SEQ ID NO: 17 or 18 or has at least 99%, 98%, 97%, 96%, and the nucleotide sequence of SEQ ID NO: 17 or 18. 95%, 90%, 85% or 80% identical nucleotide sequences; and/or

The IL-12sc protein comprises the amino acid sequence of SEQ ID NO: 14 or has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% of the amino acid sequence of SEQ ID NO: 14. % Or 80% identical Amino acid sequence; and/or

The RNA encoding IL-12sc protein contains at least 99%, 98%, 97%, 96%, 95%, and the p40 portion of IL-12sc (nucleotides 1-984 of SEQ ID NO: 17 or 18). 90%, 85% or 80% identity and at least 99%, 98%, 97%, 96%, 95% with the p30 portion of IL-12sc (nucleotides 1027-1623 of SEQ ID NO: 17 or 18) , 90%, 85%, or 80% identical nucleotide sequence, and further comprise the nucleotide between the p40 part and the p35 part encoding the linker polypeptide.

Specific embodiment 64 is the RNA or use according to any one of specific embodiments 56-63, wherein

The RNA encoding IL-15 sushi protein comprises the nucleotide sequence of SEQ ID NO: 26 or at least 99%, 98%, 97%, 96%, 95%, and the nucleotide sequence of SEQ ID NO: 26. A nucleotide sequence of 90%, 85% or 80% identity; and/or

The IL-15 sushi protein comprises the amino acid sequence of SEQ ID NO: 24 or has at least 99%, 98%, 97%, 96%, 95%, 90%, and the amino acid sequence of SEQ ID NO: 24. 85% or 80% identical amino acid sequence; and/or

The RNA encoding IL-15 sushi protein contains at least 99%, 98%, 97%, 96%, and the sushi domain of IL-15 receptor alpha (nucleotides 1-321 of SEQ ID NO: 26). 95%, 90%, 85%, or 80% identity and with mature IL-15 (nucleotides 382-729 of SEQ ID NO: 26) at least 99%, 98%, 97%, 96%, 95%, A nucleotide sequence of 90%, 85%, or 80% identity, and optionally further comprises a nucleotide encoding a linker polypeptide between the sushi domain of IL-15 and the mature IL-15.

Specific embodiment 65 is the RNA or use according to any one of specific embodiments 58-64, wherein

The RNA encoding the FLT3-L protein comprises the nucleotide sequence of SEQ ID NO: 32 or is at least 99%, 98%, 97%, 96%, 95%, 90% to the nucleotide sequence of SEQ ID NO: 32. %, 85% or 80% identical nucleotide sequence; and/or

The FLT3-L protein comprises the amino acid sequence of SEQ ID NO: 30 or has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% of the amino acid sequence of SEQ ID NO: 30. % Or 80% identical Amino acid sequence.

Specific embodiment 66 is the RNA or use according to any one of specific embodiments 56-65, wherein (i) the RNA encoding IFNα protein comprises the nucleotide sequence of SEQ ID NO: 22 or 23 or is the same as SEQ ID NO: 22 or 23. ID NO: the nucleotide sequence of 22 or 23 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical; and/or (ii) The IFNα protein comprises the amino acid sequence of SEQ ID NO: 19 or has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or the amino acid sequence of SEQ ID NO: 19 80% identical amino acid sequence.

Specific embodiment 67 is the RNA or use according to any one of specific embodiments 56-66, wherein at least one RNA comprises a modified nucleoside replacing at least one uridine.

Specific embodiment 68 is the RNA or use according to any one of specific embodiments 56-67, wherein at least one RNA comprises a modified nucleoside in place of each uridine.

Specific embodiment 69 is the RNA or use according to any one of specific embodiments 56-68, wherein each RNA comprises a modified nucleoside that replaces at least one uridine.

Specific embodiment 70 is the RNA or use according to any one of specific embodiments 56-69, wherein each RNA contains a modified nucleoside in place of each uridine.

Specific embodiment 71 is the RNA or use according to any one of specific embodiments 67-70, wherein the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) ) And 5-methyl-uridine (m5U).

Specific embodiment 72 is the RNA according to any one of specific embodiments 67-71, wherein at least one RNA comprises more than one type of modified nucleosides, wherein the modified nucleosides are independently selected from pseudouridine (ψ ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U).

Specific embodiment 73 is the RNA or use according to specific embodiment 72, wherein the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

Specific embodiment 74 is the RNA or use according to any one of specific embodiments 56-73, wherein to One less RNA contains 5'cap m27, 3'-OGppp(m12'-O)ApG or 3'-O-Me-m7G(5')ppp(5')G.

Specific embodiment 75 is the RNA or use according to any one of specific embodiments 56-75, wherein each RNA comprises 5'cap m27, 3'-OGppp(m12'-O)ApG or 3'-O- Me-m7G(5')ppp(5')G.

Specific embodiment 76 is the RNA or use according to any one of specific embodiments 56-75, wherein at least one RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and 6 or is selected The nucleotide sequence from the group consisting of SEQ ID NO: 4 and 6 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical 5'UTR.

Specific embodiment 77 is the RNA or use according to any one of specific embodiments 56-76, wherein each RNA contains a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and 6 or a combination thereof The nucleotide sequence from the group consisting of SEQ ID NO: 4 and 6 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical 5'UTR.

Specific embodiment 78 is the RNA or use according to any one of specific embodiments 56-77, wherein at least one RNA comprises the nucleotide sequence of SEQ ID NO: 8 or the nucleotide sequence of SEQ ID NO: 8 The sequence has a 3'UTR of a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical.

Specific embodiment 79 is the RNA or use according to any one of specific embodiments 56-78, wherein each RNA comprises the nucleotide sequence of SEQ ID NO: 8 or the nucleotide sequence of SEQ ID NO: 8 The sequence has a 3'UTR of a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical.

Specific embodiment 80 is the RNA or use according to any one of specific embodiments 56-79, wherein at least one RNA comprises a poly A tail.

Specific embodiment 81 is the RNA or use according to any one of specific embodiments 56-80, wherein each RNA comprises a poly A tail.

Specific embodiment 82 is the RNA or use according to specific embodiment 80 or 81, wherein the poly A The tail contains at least 100 nucleotides.

Specific embodiment 83 is the RNA or use according to any one of specific embodiments 80-82, wherein the poly A tail comprises the poly A tail shown in SEQ ID NO:66.

Specific embodiment 84 is the RNA according to any one of specific embodiments 56-83, wherein one or more RNAs comprise:

^5'cap containing m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG or 3'-O-Me-m ⁷ G(5')ppp(5')G;

Containing (i) a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and 6 or (ii) having at least 99% of a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and 6, 5'UTR of a nucleotide sequence that is 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical;

Comprising (i) the nucleotide sequence of SEQ ID NO: 8 or (ii) the nucleotide sequence of SEQ ID NO: 8 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% The 3'UTR of a nucleotide sequence of% or 80% identity; and

A poly A tail containing at least 100 nucleotides.

Specific embodiment 85 is the RNA or use according to specific embodiment 84, wherein the poly A tail comprises SEQ ID NO:66.

Specific embodiment 86 is the RNA or use according to any one of specific embodiments 56-85, wherein the method further comprises administering another therapy.

Specific embodiment 87 is the RNA or use according to specific embodiment 86, wherein the additional therapy includes one or more selected from the group consisting of: (i) surgery to remove, excise or reduce tumors, (ii) immunotherapy, (iii) Radiotherapy and (iv) Chemotherapy.

Specific embodiment 88 is the RNA or use according to specific embodiment 86 or 87, wherein the additional therapy comprises administration of another therapeutic agent.

Specific embodiment 89 is the RNA or use according to specific embodiment 88, wherein the additional therapeutic agent is an anti-cancer therapeutic agent.

Specific embodiment 90 is the RNA or use according to specific embodiment 86 or 87, wherein the additional therapeutic agent is a checkpoint modulator.

Specific embodiment 91 is the RNA or use according to specific embodiment 90, wherein the checkpoint modulator is an anti-PD1 antibody, an anti-CTLA-4 antibody or a combination of an anti-PD1 antibody and an anti-CTLA-4 antibody.

Specific embodiment 92 is the RNA or use according to any one of specific embodiments 56-91, wherein the solid tumor is a sarcoma, carcinoma or lymphoma.

Specific embodiment 93 is the RNA or use according to any one of specific embodiments 56-91, wherein the solid tumor is in the lung, colon, ovary, cervix, uterus, peritoneum, testis, penis, tongue, lymph node , Pancreas, bone, breast, prostate, soft tissue, connective tissue, kidney, liver, brain, thyroid or skin.

Specific embodiment 94 is the RNA or use according to any one of specific embodiments 56-93, wherein the solid tumor is an epithelial tumor, Hodgkin’s lymphoma (HL), non-Hodgkin’s lymphoma , Prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tumor, liver tumor, colorectal tumor, vascular tumor, mesothelioma tumor, pancreas tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, brain Tumor, melanoma tumor, small cell lung tumor, neuroblastoma tumor, testicular tumor, carcinoma tumor, adenocarcinoma tumor, glioma tumor, seminoma tumor, retinoblastoma or osteosarcoma tumor.

Specific embodiment 95 is the RNA or use according to any one of specific embodiments 56-94, wherein the RNA is administered in or around the tumor.

Specific embodiment 96 is the RNA or use according to any one of specific embodiments 56-95, wherein the RNA is formulated for injection.

Embodiment 97 is the RNA or use of any one of embodiments 86-96, wherein the additional therapeutic agent is administered systemically.

Specific embodiment 98 is the RNA or use according to any one of specific embodiments 56-97, wherein the individual is a human.

Specific embodiment 99 is the RNA or use according to any one of specific embodiments 56-98, wherein the RNA is administered simultaneously.

Specific embodiment 100 is the RNA or use according to any one of specific embodiments 56-99, wherein the RNA is administered by injection, wherein prior to injection, the RNA is mixed in a liquid solution together.

Specific embodiment 101 is the RNA or use according to any one of specific embodiments 56-100, wherein the RNA is administered by administering a composition comprising a combination of the RNA.

Specific embodiment 102 is the RNA or use according to any one of specific embodiments 56-100, wherein treating or preventing solid tumors includes reducing tumor size, preventing recurrence of cancer in remission, or preventing cancer metastasis in an individual.

Specific Example 103 is a composition or a pharmaceutical preparation comprising RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, RNA encoding IFNα protein, and RNA encoding FLT3-L protein.

Specific embodiment 104 is the composition according to specific embodiment 103.

Specific embodiment 105 is the pharmaceutical preparation according to specific embodiment 103.

Specific embodiment 106 is the pharmaceutical preparation or composition according to any one of specific embodiments 103-105, wherein the RNA has a ratio of 1:1:1 or 1:1:1:1, and optionally The ratio is verified by quantitative RT-PCR.

Specific embodiment 107 is the pharmaceutical preparation or composition according to any one of specific embodiments 103-106, wherein the RNA integrity is greater than or equal to 70%.

Specific embodiment 108 is the pharmaceutical preparation or composition according to any one of specific embodiments 103-107, wherein the pharmaceutical preparation or composition comprises less than 250ng DNA/total mg of nucleic acid present.

Specific embodiment 109 is the pharmaceutical preparation or composition according to any one of specific embodiments 103-108, wherein the IFNα protein is IFNα2b protein.

Specific embodiment 110 is the pharmaceutical preparation or composition according to specific embodiments 103-109, wherein

The RNA encoding IL-12sc protein comprises the nucleotide sequence of SEQ ID NO: 17 or 18 or has at least 99%, 98%, 97%, 96%, and the nucleotide sequence of SEQ ID NO: 17 or 18. 95%, 90%, 85% or 80% identical nucleotide sequences; and/or

The IL-12sc protein comprises the amino acid sequence of SEQ ID NO: 14 or has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% of the amino acid sequence of SEQ ID NO: 14. % Or 80% identical amino acid sequence; and/or

The RNA encoding IL-12sc protein contains at least 99%, 98%, 97%, 96%, 95%, and the p40 portion of IL-12sc (nucleotides 1-984 of SEQ ID NO: 17 or 18). 90%, 85% or 80% identity and at least 99%, 98%, 97%, 96%, 95% with the p30 portion of IL-12sc (nucleotides 1027-1623 of SEQ ID NO: 17 or 18) , 90%, 85%, or 80% identical nucleotide sequence, and further comprise the nucleotide between the p40 part and the p35 part encoding the linker polypeptide.

Specific embodiment 111 is the pharmaceutical preparation or composition according to specific embodiments 103-110, wherein

The RNA encoding IL-15 sushi protein comprises the nucleotide sequence of SEQ ID NO: 26 or at least 99%, 98%, 97%, 96%, 95%, and the nucleotide sequence of SEQ ID NO: 26. A nucleotide sequence of 90%, 85% or 80% identity; and/or

The IL-15 sushi protein comprises the amino acid sequence of SEQ ID NO: 24 or has at least 99%, 98%, 97%, 96%, 95%, 90%, and the amino acid sequence of SEQ ID NO: 24. 85% or 80% identical amino acid sequence; and/or

The RNA encoding IL-15 sushi protein contains at least 99%, 98%, 97%, 96%, and the sushi domain of IL-15 receptor alpha (nucleotides 1-321 of SEQ ID NO: 26). 95%, 90%, 85%, or 80% identity and with mature IL-15 (nucleotides 382-729 of SEQ ID NO: 26) at least 99%, 98%, 97%, 96%, 95%, A nucleotide sequence of 90%, 85%, or 80% identity, and optionally further comprises a nucleotide encoding a linker polypeptide between the sushi domain of IL-15 and the mature IL-15.

Specific embodiment 112 is the pharmaceutical preparation or composition according to specific embodiments 103-111, wherein

The RNA encoding IFNα protein comprises the nucleotide sequence of SEQ ID NO: 22 or 23 or has at least 99%, 98%, 97%, 96%, 95% of the nucleotide sequence of SEQ ID NO: 22 or 23. , 90%, 85% or 80% identical nucleotide sequence; and/or

The IFNα protein comprises the amino acid sequence of SEQ ID NO: 19 or has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or the amino acid sequence of SEQ ID NO: 19 80% identical amino acid sequence.

Specific embodiment 113 is the pharmaceutical preparation or composition according to specific embodiments 103-112, wherein

The RNA encoding the FLT3-L protein comprises the nucleotide sequence of SEQ ID NO: 32 or is at least 99%, 98%, 97%, 96%, 95%, 90% to the nucleotide sequence of SEQ ID NO: 32. %, 85% or 80% identical nucleotide sequence; and/or

The FLT3-L protein comprises the amino acid sequence of SEQ ID NO: 30 or has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% of the amino acid sequence of SEQ ID NO: 30. % Or 80% identical amino acid sequence.

Specific embodiment 114 is the pharmaceutical preparation or composition according to specific embodiments 103-113, which is a pharmaceutical composition containing the RNA.

Specific embodiment 115 is the pharmaceutical preparation or composition according to specific embodiment 114, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.

Specific embodiment 116 is the pharmaceutical preparation or composition according to any one of specific embodiments 103-115, wherein the RNA is formulated as a liquid, formulated as a solid, or a combination thereof.

Specific embodiment 117 is the pharmaceutical preparation or composition according to specific embodiments 103-116, which is used for medicinal purposes.

Specific embodiment 118 is the pharmaceutical preparation or composition according to specific embodiments 103-117, wherein the pharmaceutical use includes the therapeutic treatment or prophylactic treatment of diseases or disorders.

Specific embodiment 119 is a pharmaceutical preparation or composition according to specific embodiments 103-118, wherein The therapeutic treatment or prophylactic treatment of the disease or disorder includes the treatment or prevention of solid tumors.

Specific embodiment 120 is the pharmaceutical preparation or composition according to specific embodiment 119, wherein the solid tumor is a sarcoma, carcinoma or lymphoma.

Embodiment 121 is the pharmaceutical preparation or composition according to specific embodiments 119-120, wherein the solid tumor is in the lung, colon, ovary, cervix, uterus, peritoneum, testis, penis, tongue, lymph node, pancreas , Bone, breast, prostate, soft tissue, connective tissue, kidney, liver, brain, thyroid or skin.

Specific embodiment 122 is the pharmaceutical preparation or composition according to specific embodiments 103-121, wherein the solid tumor is an epithelial tumor, Hodgkin’s lymphoma (HL), non-Hodgkin’s lymphoma, Guardian tumors, ovarian tumors, renal cell tumors, gastrointestinal tumors, liver tumors, colorectal tumors, vascular tumors, mesothelioma tumors, pancreatic tumors, breast tumors, sarcoma tumors, lung tumors, colon tumors, brain tumors, Melanoma tumor, small cell lung tumor, neuroblastoma tumor, testicular tumor, cancer tumor, adenocarcinoma tumor, glioma tumor, seminoma tumor, retinoblastoma or osteosarcoma tumor.

Specific Example 123 is an RNA for use in a method for treating or preventing solid tumors in an individual, wherein the method comprises administering RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, RNA encoding IFNα protein and RNA encoding FLT3-L protein.

Specific example 124 is the use of RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, RNA encoding IFNα protein, and RNA encoding FLT3-L for the treatment of solid tumors.

Specific Example 125 is a composition or pharmaceutical preparation comprising RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, and RNA encoding IFNα protein (such as described herein), wherein

a. The RNA encoding the IL-12sc protein is not an RNA encoding the IL-12sc protein having at least 95% identity with the amino acid sequence of SEQ ID NO: 14, and/or does not contain the same as SEQ ID NO: 17 or 18 nucleotides are nucleotides with at least 95% identity;

b. The RNA encoding the IFNα2b protein does not have the same amino acid sequence as SEQ ID NO: 19 RNA encoding IFNα2b protein with less than 95% identity, and/or does not contain nucleotides with at least 95% identity with the nucleotides of SEQ ID NO: 22 or 23; and

c. The RNA encoding the IL-15 sushi protein is not the RNA encoding the IL-15 sushi protein that has at least 95% identity with the amino acid sequence of SEQ ID NO: 24, and/or does not contain the same as SEQ ID NO: The 26 nucleotides are nucleotides that are at least 95% identical.

Sequence description

Figures 1A-1H show the results of experiments in which RNA (10μg RNA/target or 40μg control RNA) was injected into the tumor of CT26 tumor-bearing mice on days 12, 15, 19 and 22, and the individual The tumor grows and is plotted to day 35. Figure 1A shows the results of treatment with a mixture of RNA encoding IL-15 sushi, IL-12sc, GM-CSF and IFNα. Figure 1B shows the treatment with an RNA mixture of IL-15 sushi, IL-12sc and IFNα (without GM-CSF). Figure 1C shows the treatment with an RNA mixture of IL-15 sushi, GM-CSF and IFNα (without IL-12sc). Figure ID shows the treatment with an RNA mixture of IL-12sc, GM-CSF and IFNα (no IL-15 sushi). Figure 1E shows the treatment with an RNA mixture of IL-15 sushi, IL-12sc and GM-CSF (without IFNα). Figure 1F shows treatment with control RNA encoding luciferase. Figure 1G shows the average tumor volume up to day 33 for each treatment group. Figure 1H shows tumor growth inhibition by day 19 for each treatment group.

Figures 2A-2C show the results of the experiment in which RNA (2μg RNA/target or 8μg control RNA) was injected into the tumor of B16F10 tumor-bearing mice on the 11th day, and individual tumor growth was monitored and plotted to The 62nd day. Figure 2A shows the treatment with the RNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFNα; Figure 2C shows the treatment with the RNA mixture of IL-15 sushi, IL-12sc and IFNα (without GM-CSF); And Figure 2C shows a luciferase control.

Figures 3A-3E show the results of experiments in which CT26 tumor-bearing mice were injected intratumorally on the 13, 16, 20 and 24 days (AB) or on the 11, 14, 17 and 21 days (CD) RNA (5 μg RNA/target or 20 μg control RNA), and individual tumor growth was monitored and plotted to day 50. Figure 3A and Figure 3C shows the treatment with the RNA mixture of IL-15 sushi, IL-12sc, IFNα and FLT3-L. Figure 3B and Figure 3D show the luciferase control. Figure 3E shows the treatment with the RNA mixture of IL-15 sushi, IL-12sc, IFNα and GM-CSF.

Figures 4A-4B show the results of the experiment in which RNA (2μg RNA/target or 8μg control RNA) was injected into the tumor of B16F10 tumor-bearing mice on the 11th day, and individual tumor growth was monitored and plotted to The 57th day. Figure 4A shows the treatment with the RNA mixture of IL-15 sushi, IL-12sc, IFNα and GM-CSF; Figure 4B shows the treatment with the RNA mixture of IL-15 sushi, IL-12sc, IFNα and FLT3-L; and 4C shows the luciferase control.

Figures 5A-5B show the results of experiments in which RNA (2μg RNA/target or 8μg control RNA) was injected into the tumor of B16F10 tumor-bearing mice, and the tumor was taken out and dissociated 7 days after the injection And stained with a set of antibodies to assess the immune population within the tumor. The numbers of CD8+ T cells and NK cells used in different treatment groups are listed.

Figures 6A-6C show the results of the experiment in which RNA (5μg RNA/target or 20μg control RNA) was injected into the tumor of CT26 tumor-bearing mice, and gp70 in the blood was evaluated on days 19 and 31 after treatment Percentage of specific CD8+ T cells. Figure 6A shows day 19 and Figure 6B shows day 31, from the same experiment. In a similarly designed independent study, gp70-specific CD8+ T cells in the blood were evaluated on day 24 (Figure 6C).

1. Definition

In some embodiments, the RNA comprises a modified nucleobase that replaces at least one (e.g., each) uridine. In some specific embodiments, the RNA includes a Cap1 structure at the 5'end of the RNA. In some embodiments, the RNA includes a modified nucleobase replacing at least one (e.g., each) uridine, and a Cap1 structure at the 5'end of the RNA. In some specific embodiments, the 5'UTR comprises SEQ ID NO: 4 or 6. In some embodiments, the RNA has been processed to reduce double-strength, for example, by purification on cellulose (as described in the examples and as known in the art) or by high-performance liquid chromatography (HPLC). Strand RNA (dsRNA). The "Cap1" structure can be generated by enzymatic capping after in vitro translation or during in vitro translation (co-transcription capping).

In some specific embodiments, the structural unit caps used to modify RNA are as follows, and the structural unit caps are used when co-transcriptional capping is below:

m ₂ ^7,3'-O Gppp(m ₁ ^2'-O ) ApG (sometimes also called m ₂ ^7,3'O G(5')ppp(5')m ^2'-O ApG), which Has the following structure:

The following is an exemplary Cap1 RNA after co-transcription and capping, which contains RNA and m ₂ ^7,3'O G(5')ppp(5')m ^2'-O ApG:

The following is another exemplary Cap1 RNA after enzymatic capping (no cap analog):

In some specific embodiments, the RNA is modified by the "Cap0" structure, which is used in a specific embodiment of the cap analogue anti-reverse cap (ARCA cap (m ₂ ^7,3'O G(5')ppp(5')G)) produced during in vitro translation (co-transcription capping), the cap analog anti-reverse cap has the following structure:

The following is an exemplary Cap0 RNA containing RNA and m ₂ ^7,3'O G(5')ppp(5')G:

In some specific embodiments, the "Cap0" structure is used for in vitro translation (co-transcription) using the cap analog β-S-ARCA (m ₂ ^7,2'O G(5')ppSp(5')G) The cap analog β-S-ARCA has the following structure:

The following is an exemplary Cap0 RNA containing β-S-ARCA (m ₂ ^7,2'O G(5')ppSp(5')G) and RNA.

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

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

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

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

"Pseudouridine" is an example of a modified nucleoside that is an isomer of uridine, in which uracil is attached to the pentose ring via a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond. For example, pseudouridine is described in Charette and Gray, Life ; 49:341-351 (2000).

Another exemplary modified nucleoside is N1-methylpseudouridine (m1Ψ), which has the following structure:

N1-methyl pseudo-UTP has the following structure:

As used herein, the term "poly A tail" or "poly A sequence" 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 can follow the 3'UTR in the RNA described herein. The uninterrupted poly A tail is characterized by continuous adenylate residues. In fact, uninterrupted poly A tail is typical. The RNA disclosed herein may have a poly A tail attached to the free 3'end of the RNA by a template-independent RNA polymerase after transcription, or a poly A tail that is encoded by DNA and transcribed by a template-dependent RNA polymerase. tail.

It has been shown that the poly A tail of about 120 A nucleotides has a beneficial effect on the level of RNA in transfected eukaryotic cells and 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. 4009-4017).

The poly A tail can have any length. In a specific embodiment, the poly A tail contains 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 nucleosides The acid and in particular about 120 A nucleotides consist essentially of or consist of it. In this context, "consisting essentially of" means most of the nucleotides in the poly A tail, typically at least 75%, at least 80%, at least 85% by number in the poly A tail , At least 90%, At least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides are A nucleotides, but the remaining nucleotides are allowed to be nucleotides other than A nucleotides, such as U nucleotide (uridine acid), G nucleotide (guanylic acid) or C nucleotide (cytidine acid). In this context, "consisting of" means that all nucleotides in the poly A tail, that is, 100% by number of nucleotides in the poly A tail are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

In some embodiments, during RNA transcription based on a DNA template, for example, during preparation of in vitro transcribed RNA, a poly A tail is attached, and the DNA template contains repeated dT nucleosides in a strand complementary to the coding strand. Acid (deoxythymidylate). The DNA sequence (coding strand) encoding the poly A tail is called the poly (A) box.

In some specific embodiments, the poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides, but is interrupted by a random sequence of four nucleotides (dA, dC, dG and dT) . The length of such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides. Such boxes are disclosed in WO 2016/005324 A1, which is hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 A1 can be used in the present invention. Poly (A) consisting essentially of dA nucleotides but interrupted by a random sequence having four nucleotides (dA, dC, dG, and dT) evenly distributed and having a length of, for example, 5 to 50 nucleotides The cassette shows that plasmid DNA that is constant at the DNA level reproduces in E. coli and is still associated with beneficial properties related to supporting RNA stability at the RNA level, and encompasses translation efficiency. Therefore, in some specific embodiments, the poly A tail contained in the RNA molecule described herein consists essentially of A nucleotides, but is composed of four nucleotides (A, C, G, U) randomly The sequence is interrupted. The length of such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides.

In some specific embodiments, no nucleotides other than the A nucleotide flanks the poly A tail at its 3'end, that is, the poly A tail is not covered or is not connected to the 3'end. Other nucleotides.

In some embodiments, the poly A tail contains the sequence:

(SEQ ID NO：66)，其也在表1中示出。

(SEQ ID NO: 66), which is also shown in Table 1.

"RNA" and "mRNA" are used interchangeably herein.

In this context, "IFNα" is used in the upper sense to describe any type I interferon α cytokines, including IFNα2b and IFNα4. Any IFNα can be incorporated into the composition and used in the methods described herein.

As used herein, the term "treatment" encompasses any administration or administration of a disease treatment agent to an individual, and includes inhibiting the disease, preventing its development, alleviating one or more symptoms of the disease, curing the disease, or preventing the recurrence of the disease. For example, treatment of solid tumors may include alleviating the symptoms of solid tumors, reducing the size of solid tumors, eliminating solid tumors, reducing further tumor growth, or reducing or eliminating the recurrence of solid tumors after treatment. Treatment can also be measured as a change in effectiveness biomarkers or imaging or radiographic measurements.

As used herein, the term "prevention" means to inhibit or prevent the development of cancer, including solid tumors, in individuals who are considered cancer-free.

"Metastasis" means a process in which cancer spreads from where it first appeared as the primary tumor to other locations in the body.

As used herein, the term "intratumor" means entering a tumor. For example, intratumor injection means to inject a therapeutic agent at any location in contact with the tumor.

As used herein, the term "peri-tumorally or peri-tumoral" is the area about 2-mm wide and adjacent to the invasive front around the tumor. The area surrounding the tumor contains host tissue. See, for example:

"Administration" means to provide a medicament or composition to an individual, and includes but not limited to administration by a medical professional and self-administration.

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

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

The terms "% identical", "% identity" or similar terms are intended to specifically refer to the percentage of identical nucleotides or amino acids in the optimal alignment between the sequences to be compared. The percentages are purely statistical, and the differences between the two sequences can but are not necessarily distributed randomly over the entire length of the sequence to be compared. The comparison of two sequences is usually done in the following way: after the best alignment, the sequences are compared with respect to fragments or "comparison windows" to identify local regions of the corresponding sequences. The best comparison of comparison can be done manually or with the local homology algorithm of Smith and Waterman, 1981, Ads App.Math.2,482, or with the local homology algorithm of Neddleman and Wunsch, 1970, J.Mol.Biol.48,443 , With the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of a computer program that uses the algorithm (GAP, BESTFIT, FASTA, BLAST P, BLAST P, BLAST N and TFASTA, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin).

Percent identity is obtained by determining the number of identical positions corresponding to the sequence to be compared, dividing this number by the number of positions compared (for example, the number of positions in the reference sequence), and then multiplying the result by 100.

In some specific 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% of the entire length of the reference sequence. % Or about 100%. For example, if the reference nucleic acid sequence consists of 200 nucleotides, it is for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides (in some specific In the examples, consecutive nucleotides) give the degree of identity. In some embodiments, the degree of identity is given for the entire length of the reference sequence.

A nucleic acid sequence or amino acid sequence having a specific degree of identity with a given nucleic acid sequence or amino acid sequence, respectively, may have at least one functional characteristic of the given sequence, for example, and in some cases, functionally equivalent to all State the given sequence. An important characteristic includes the ability to act as a cytokine, especially when administered to an individual. In some specific embodiments, a nucleic acid sequence and an amino acid sequence that have a certain degree of identity with a given nucleic acid sequence or amino acid sequence are functionally equivalent to the given sequence.

2. Compositions and pharmaceutical preparations

In some specific embodiments, a composition or pharmaceutical preparation is provided, which comprises RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, and RNA encoding IFNα protein. In some cases, the pharmaceutical preparation or composition further includes RNA encoding FLT3-L protein.

In some specific embodiments, a composition is provided, the composition comprising RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, and RNA encoding IFNα protein.

In some specific embodiments, a pharmaceutical preparation is provided, which comprises RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, and RNA encoding IFNα protein.

In some specific embodiments, a composition is provided, which comprises RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, RNA encoding IFNα protein, and RNA encoding FLT3-L protein.

In some specific embodiments, a pharmaceutical preparation is provided, which comprises RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, RNA encoding IFNα protein, and RNA encoding FLT3-L.

In some specific embodiments, the pharmaceutical preparation or composition comprises RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, RNA encoding IFNα protein, and optionally RNA encoding FLT3-L, wherein The RNA has a mass ratio of 1:1:1 or 1:1:1:1 (when FLT3-L is present), optionally wherein the ratio is verified by quantitative RT-PCR or similar methods. In some specific embodiments, the "RNA ratio" is determined by quantitative RT-PCR or similar methods. In some embodiments, the RNA ratio is determined by The determination is as follows: 1) reverse transcription of the RNA mixture; and 2) quantification of each cDNA corresponding to each RNA in the mixture by droplet digital PCR (for example, by the Bio-Rad method) using Taqman probe. Based on the absolute count of positive events, the ratio of individual sequences is determined, and thus the ratio of RNA in the mixture.

In some specific embodiments, the pharmaceutical preparation or composition comprises RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, RNA encoding IFNα protein, and optionally RNA encoding FLT3-L, wherein " RNA integrity" (ie, the percentage of intact RNA) is greater than or equal to 70%. In some specific embodiments, RNA integrity is the percentage of the mass ratio of full-length RNA (ie, undegraded RNA) relative to total RNA (ie, full-length RNA and degraded RNA). In some embodiments, the Experion fully automated electrophoresis system (Bio-Rad) or similar technology is used to determine RNA integrity. In some specific embodiments, RNA integrity is greater than or equal to 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% % Or 99%.

In some specific embodiments, each RNA in the composition or pharmaceutical preparation is present in a certain length so that the RNA produces the protein it encodes. In some specific embodiments, each RNA in the composition or pharmaceutical preparation has certain RNA integrity, and the RNA integrity maintains the production of the protein encoded by the RNA.

In some specific embodiments, the pharmaceutical preparation or composition comprises RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, RNA encoding IFNα protein, and optionally RNA encoding FLT3-L, wherein The pharmaceutical preparation or composition contains less than 250ng DNA/total mg of nucleic acid present. In some embodiments, the effective removal of the DNA template is verified by qPCR or similar techniques targeting the region within the DNA template. Then calculate the residual amount of DNA template based on the reference standard curve. In some specific embodiments, the pharmaceutical preparation or composition contains less than 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90 , 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 ng DNA/total mg of nucleic acid present.

In some specific embodiments, at least 95% of the composition or pharmaceutical preparation comprises therapeutic RNA (e.g., RNA encoding therapeutic cytokines, checkpoint inhibitors, and/or other RNA intended for treatment).

In some embodiments, the amount of each RNA in the composition and pharmaceutical preparation is sufficient to produce the protein it encodes.

In some specific embodiments, the amount of each RNA in the composition and pharmaceutical preparation is the amount that contributes to the overall effect of treating or preventing solid tumor cancer when administered within or around the tumor. That is, if the individual RNA in the composition or the pharmaceutical preparation is omitted from the composition or the pharmaceutical preparation, the treatment or prevention of solid tumor cancer will be compared with the composition or the pharmaceutical preparation containing the omitted RNA. Will decrease.

In some specific embodiments, the amount of each RNA in the composition and pharmaceutical preparation is sufficient to produce substantially the following in vitro biological effects:

The RNA is transfected into a suitable cell line, and the protein encoded by the RNA is expressed. The protein can be separated and the biological activity measured basically as follows:

a) The activity of human IL-15 sushi can be determined by measuring the proliferation of the CTLL-2 cell line (Paxton RJ.2001. Measurement of interleukin 15. Curr Protoc Immunol Chapter 6: Unit 6 22), the CTLL-2 The cell line is a murine T-cell line that depends on cytokines for growth, such as IL-2, IL-15 or other common receptor γ-chain cytokinin family members.

b) The activity of human IL-12sc can be determined by measuring the alkaline phosphatase activity of the IL-12-specific HEK-Blue cell line HEK-Blue ^TM IL-12, which expresses IL-12-dependent alkali Phosphatase (Breivik L, Oftedal BE, Boe Wolff AS, Bratland E, Orlova EM, Husebye ES. 2014. A novel cell-based assay for measuring neutralizing autoantibodies against type I interferons in patients with autoimmune polyendocrine syndrome type 1.Clin Immunol 153: 220-227). The detection of IL-12 biological activity is based on the activation of the STAT4 pathway via IL-12, because HEK-Blue ^™ IL-12 cells contain the STAT4-inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene system.

c) The activity of human IFNα2b can be determined by measuring alkaline phosphatase activity (Breivik et al.) in the IFNα-specific HEK-Blue cell line HEK-Blue ^TM IFN-α/β. The expression of the cell line depends on the base of IFNα.性phosphatase. The detection of IFNα2b activity is based on the activation of the JAK-STAT pathway via type I interferon, because HEK-Blue ^™ IFN-α/β cells contain the JAK-STAT inducible SEAP reporter gene system.

d) The activity of FLT3-L can be determined by measuring the induction of IL-6 production in mouse myeloid leukemia M1 cells using an IL-6 quantification-based ELISA in the presence of mouse LIF recombinant protein.

For each specific example, the RNA may be as described below and in Table 1.

A. Interleukin-12 single chain (IL-12sc)

In some specific embodiments, a composition or pharmaceutical preparation is provided, which comprises RNA encoding interleukin-12 single-stranded (IL-12sc). In some specific embodiments, the interleukin-12 single-stranded (IL-12sc) RNA is encoded by a DNA sequence encoding interleukin-12 single-stranded (IL-12sc) (e.g., SEQ ID NO: 14), The RNA includes IL-12 p40 (sometimes called IL-12B; encoded by nucleotides 1-984 of SEQ ID NO: 15), linkers (such as GS linkers), and IL-12 p35 (sometimes called IL-12B) IL-12A; encoded by nucleotides 1027-1623 of SEQ ID NO: 15). In some specific embodiments, the IL-12p40, linker and IL-12p35 are continuous without intervening nucleotides. An exemplary DNA sequence encoding IL-12sc is provided in SEQ ID NO: 15. In some specific embodiments, the interleukin-12 single-stranded (IL-12sc) RNA is provided on SEQ ID NO: 17 or 18 (both of which encode the protein of SEQ ID NO: 14). The RNA sequence of IL-12 p40 is shown in nucleotides 1-984 of SEQ ID NO: 17 or 18, and the RNA sequence of IL-12 p35 is shown in nucleotides 1027-1623 of SEQ ID NO: 17 or 18.

The alignment of codon-optimized IL-12 p40 and native IL-12 p40 is shown below, where "S" is native IL-12 p40 (NM_002187.2; nucleotides 1-984 of SEQ ID NO: 15) And "Q" is the codon-optimized IL-12 p40 (nucleotides 1-984 of SEQ ID NO: 16). The percent identity is 77%.

The alignment of codon-optimized IL-12 p35 and native IL-12 p35 is shown below, where "S" is native IL-12 p35 (NM_00882.3; nucleotides 1027-1623 of SEQ ID NO: 15) And "Q" is codon-optimized IL-12 p35 (nucleotides 1027-1623 of SEQ ID NO: 16). The percent identity is 80%.

In some embodiments, the IL-12sc RNA is encoded by a codon-optimized DNA sequence encoding IL-12sc. In some specific embodiments, the IL-12sc RNA encodes IL-12 p40 by the secret Code optimized DNA sequence coding. In some embodiments, the IL-12 sc RNA is encoded by a codon-optimized DNA sequence encoding IL-12 p35. In some specific embodiments, the codon-optimized DNA sequence comprises or consists of SEQ ID NO: 16. In some specific embodiments, the DNA sequence comprises 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 16. The codon optimized DNA sequence. In some embodiments, the codon-optimized DNA sequence encoding IL-12 p40 comprises nucleotides encoding IL-12sc-p40 (nucleotides 1-984 of SEQ ID NO: 16). In some embodiments, the codon-optimized DNA sequence encoding IL-12 p35 comprises nucleotides encoding IL-12sc-p35 (nucleotides 1027-1623 of SEQ ID NO: 16). In some embodiments, the codon-optimized DNA sequence encoding IL-12sc comprises SEQ ID NO: 16 encoding IL-12sc-p40 (nucleotides 1-984 of SEQ ID NO: 16) and -p35 (SEQ ID NO: 16). ID NO: 16 nucleotides 1027-1623) part of the nucleotides, and also includes nucleotides between the p40 part and the p35 part (for example, SEQ ID NO : 16 nucleotides 985-1026). Any linker known to those skilled in the art can be used. The p40 part can be 5'or 3'to p35 part.

In some specific embodiments, the IL-12sc RNA comprises an RNA sequence, which is transcribed, for example, from a DNA sequence encoding IL-12sc. RNA can also be produced recombinantly. In some specific embodiments, the RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NO: 15 or 16. In some specific embodiments, the RNA sequence comprises or consists of SEQ ID NO: 17 or 18. In some specific embodiments, the RNA sequence comprises 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO: 17 or 18. The same RNA sequence or consists of it. In some specific embodiments, the RNA sequence comprises SEQ ID NO: 17 or 18 encoding IL-12sc-p40 (nucleotides 1-984 of SEQ ID NO: 17 or 18) and -p35 (SEQ ID NO: 17 or 18 nucleotides 1027-1623) part of the nucleotides. In some embodiments, the codon-optimized RNA sequence encoding IL-12sc comprises SEQ ID NO: 18 encoding IL-12sc-p40 (nucleotides 1-984 of SEQ ID NO: 18) and -p35 (SEQ ID NO: 18). ID NO: 18 nucleotides 1027-1623) part of the nucleotides, and contains the nucleotides between the p40 part and the p35 part that encodes the linker polypeptide that connects the p40 and p35 parts. Any linker known to those skilled in the art can be used.

In some embodiments, one or more uridines in IL-12 sc RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U). In some specific embodiments, the RNA contains modified nucleosides in place of each uridine. In some specific embodiments, the modified nucleoside is N1-methyl-pseudouridine (m ¹ ψ).

In some specific embodiments, the IL-12 sc RNA comprises an altered nucleotide at the 5'end. In some specific embodiments, the RNA includes a 5'cap. Any 5'cap known in the art can be used. In some specific embodiments, the 5'cap includes 5'to 5'triphosphate bonds. In some specific embodiments, the 5'cap comprises 5'to 5'triphosphate linkages including phosphorothioate modification. In some specific embodiments, the 5'cap comprises 2'- 0 or 3'- 0 -ribose-methylated nucleotides. In some specific embodiments, the 5'cap comprises modified guanosine nucleotides or modified adenosine nucleotides. In some embodiments, the 5'cap comprises 7-methylguanylic acid. In some specific embodiments, the 5'cap is Cap0 or Cap1. Exemplary cap structure comprises a m7G (5 ') ppp (5 ') G, m7,2` O -mG (5 ') pp S p (5') G, m7G (5 ') ppp (5') 2` O -mg and m7,3` O -mG (5 ') ppp (5') 2` O -mA.

In some specific embodiments, the IL-12 sc RNA comprises a 5' untranslated region (UTR). In some specific embodiments, the 5'UTR is upstream of the start codon. In some specific embodiments, the 5'UTR regulates the translation of RNA. In some specific embodiments, the 5'UTR is a stabilizing sequence. In some specific embodiments, the 5'UTR extends the half-life of RNA. Any 5'UTR known in the art can be used. In some specific embodiments, the 5'UTR RNA sequence is transcribed from SEQ ID NO: 3 or 5. In some specific embodiments, the 5'UTR RNA sequence comprises or consists of SEQ ID NO: 4 or 6. In some specific embodiments, the 5'UTR RNA sequence is at least 70% of SEQ ID NO: 4 or 6, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% are the same.

In some specific embodiments, the IL-12 sc RNA comprises 3'UTR. In some embodiments, the 3'UTR is after the translation of the stop codon. In some specific embodiments, the 3'UTR regulates polyadenylation, translation efficiency, localization or stability of RNA. In some specific embodiments, the 3'UTR RNA sequence is transcribed from SEQ ID NO:7. In some specific embodiments, the 3'UTR RNA sequence comprises or consists of SEQ ID NO: 8. In some specific embodiments, the 3'UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO: 8 the same.

In some specific embodiments, the IL-12 sc RNA comprises both 5'UTR and 3'UTR. In some specific embodiments, the IL-12 sc RNA only contains 5'UTR. In some specific embodiments, the IL-12 sc RNA only contains 3'UTR.

In some specific embodiments, the IL-12 sc RNA comprises a poly A tail. In some specific embodiments, the RNA comprises a poly A tail of at least about 25, at least about 30, at least about 50 nucleotides, at least about 70 nucleotides, or at least about 100 nucleotides. In some specific embodiments, the poly A tail contains 200 or more nucleotides. In some specific embodiments, the poly A tail comprises or consists of SEQ ID NO: 60.

In some specific embodiments, the RNA includes a 5'cap, a 5'UTR, a nucleic acid encoding IL-12sc, a 3'UTR, and a poly A tail in order.

In some specific embodiments, the IL-12 sc RNA comprises a DNA sequence comprising a DNA sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96% identical to SEQ ID NO: 15 or 16. %, 97%, 98%, 99% or 100% identical and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% with SEQ ID NO: 3 or 5 , 99% or 100% identical nucleic acid sequence or composed of it.

In some specific embodiments, the IL-12sc RNA comprises an RNA sequence, the RNA sequence is, for example, transcribed from a DNA sequence, and the DNA sequence comprises at least 70%, 75%, 80% of SEQ ID NO: 15 or 16 %, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical and at least 70%, 75%, 80%, 85%, 90% with SEQ ID NO: 3 or 5 , 95%, 96%, 97%, 98%, 99% or 100% identical nucleic acid sequence or composed of it. RNA can also be produced recombinantly. In some embodiments, one or more uridines in IL-12 sc RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U). In some specific embodiments, the RNA contains modified nucleosides in place of each uridine. In some specific embodiments, the modified nucleoside is N1-methyl-pseudouridine (m ¹ ψ).

In some specific embodiments, the IL-12 sc RNA is encoded by a DNA sequence that contains at least 70%, 75%, 80%, 85%, 90%, 95% of SEQ ID NO: 15 or 16 %, 96%, 97%, 98%, 99%, or 100% and is the same as SEQ ID NO: 7 at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 %, 99% or 100% identical nucleic acid sequence or composed of it.

In some specific embodiments, the IL-12sc RNA comprises an RNA sequence, the RNA sequence is, for example, transcribed from a DNA sequence, and the DNA sequence comprises at least 70%, 75%, 80% of SEQ ID NO: 15 or 16 %, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% and are at least 70%, 75%, 80%, 85%, 90%, 95% identical to SEQ ID NO: 7 %, 96%, 97%, 98%, 99%, or 100% identical nucleic acid sequence or consist of it. RNA can also be produced recombinantly. In some embodiments, one or more uridines in IL-12 sc RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U). In some specific embodiments, the RNA contains modified nucleosides in place of each uridine. In some specific embodiments, the modified nucleoside is N1-methyl-pseudouridine (m ¹ ψ).

In some specific embodiments, the IL-12 sc RNA comprises a DNA sequence comprising a DNA sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96% identical to SEQ ID NO: 15 or 16. %, 97%, 98%, 99% or 100% identical; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% with SEQ ID NO: 3 or 5 , 99% or 100% identical; and at least 70%, 75%, 80%, and SEQ ID NO: 7 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical nucleic acid sequence or consist of the same.

In some specific embodiments, the IL-12sc RNA comprises an RNA sequence, the RNA sequence is, for example, transcribed from a DNA sequence, and the DNA sequence comprises at least 70%, 75%, 80% of SEQ ID NO: 15 or 16 %, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical; at least 70%, 75%, 80%, 85%, 90% with SEQ ID NO: 3 or 5 , 95%, 96%, 97%, 98%, 99% or 100%; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97 %, 98%, 99% or 100% identical nucleic acid sequence or consisting of it. RNA can also be produced recombinantly. In some embodiments, one or more uridines in IL-12 sc RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U). In some specific embodiments, the RNA contains modified nucleosides in place of each uridine. In some specific embodiments, the modified nucleoside is N1-methyl-pseudouridine (m ¹ ψ).

In some specific embodiments, the IL-12sc RNA comprises an RNA sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96% identical to SEQ ID NO: 17 or 18. %, 97%, 98%, 99% or 100% identical; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% with SEQ ID NO: 4 or 6 , 99% or 100% identical; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 8 Nucleic acid sequence or consists of it. In some embodiments, one or more uridines in IL-12 sc RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U).

B. Interferon α (IFNα)

In some specific embodiments, a composition or pharmaceutical preparation is provided, which comprises RNA encoding interferon alpha (IFN alpha). In some embodiments, the interferon alpha (IFN alpha) RNA is encoded by a DNA sequence encoding interferon alpha (IFN alpha) (for example, SEQ ID NO: 19). An exemplary DNA sequence encoding this IFNα is provided in SEQ ID NO: 20.

The alignment of codon-optimized IFNα and natural IFNα is shown below, where "S" is natural IFNα (NM_000605.3; SEQ ID NO: 20) and "Q" is codon-optimized IFNα (SEQ ID NO: 21 ). The percent identity is 79%.

In some embodiments, the IFNα RNA is encoded by codon-optimized DNA encoding IFNα. In some specific embodiments, the codon-optimized DNA sequence comprises or consists of the nucleotides of SEQ ID NO: 21. In some specific embodiments, the DNA sequence comprises 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 21 The codon-optimized DNA sequence or consists of it.

In some specific embodiments, the IFNα RNA comprises an RNA sequence, and the RNA sequence is, for example, Transcribed from the DNA sequence encoding IFNα. RNA can also be produced recombinantly. In some specific embodiments, the RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NO: 20 or 21. In some specific embodiments, the RNA sequence comprises or consists of SEQ ID NO: 22 or 23. In some specific embodiments, the RNA sequence contains 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO: 22 or 23. The same RNA sequence or consists of it.

In some embodiments, one or more uridines in IFNα RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U). In some embodiments, each uridine in the RNA is modified. In some specific embodiments, each uridine in the RNA is modified with N1-methyl-pseudouridine (m ¹ ψ).

In some specific embodiments, the IFNα RNA contains altered nucleotides at the 5'end. In some specific embodiments, the IFNα RNA comprises a 5'cap. Any 5'cap known in the art can be used. In some specific embodiments, the 5'cap includes 5'to 5'triphosphate bonds. In some specific embodiments, the 5'cap comprises 5'to 5'triphosphate linkages including phosphorothioate modification. In some specific embodiments, the 5'cap comprises 2'- 0 or 3'- 0 -ribose-methylated nucleotides. In some specific embodiments, the 5'cap comprises modified guanosine nucleotides or modified adenosine nucleotides. In some embodiments, the 5'cap comprises 7-methylguanylic acid. In some specific embodiments, the 5'cap is Cap0 or Cap1. Exemplary cap structure comprises a m7G (5 ') ppp (5 ') G, m7,2` O -mG (5 ') pp S p (5') G, m7G (5 ') ppp (5') 2` O -mg and m7,3` O -mG (5 ') ppp (5') 2` O -mA.

In some embodiments, the IFNα RNA comprises a 5' untranslated region (UTR). In some specific embodiments, the 5'UTR is upstream of the start codon. In some specific embodiments, the 5'UTR regulates the translation of RNA. In some specific embodiments, the 5'UTR is a stabilizing sequence. In some specific embodiments, the 5'UTR extends the half-life of RNA. Any 5'UTR known in the art can be used. In some specific embodiments, the 5'UTR RNA sequence is derived from SEQ ID NO: 3 or 5 The nucleotide sequence is transcribed. In some specific embodiments, the 5'UTR RNA sequence comprises or consists of SEQ ID NO: 4 or 6. In some specific embodiments, the 5'UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or the same as SEQ ID NO: 4 or 6. 99% same.

In some specific embodiments, the IFNα RNA comprises 3'UTR. In some embodiments, the 3'UTR is after the translation of the stop codon. In some specific embodiments, the 3'UTR regulates polyadenylation, translation efficiency, localization or stability of RNA. In some embodiments, the 3'UTR RNA sequence is transcribed from the nucleotide sequence comprising SEQ ID NO:7. In some specific embodiments, the 3'UTR RNA sequence comprises or consists of SEQ ID NO: 8. In some specific embodiments, the 3'UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO: 8 the same.

In some specific embodiments, the IFNα RNA comprises both 5'UTR and 3'UTR. In some embodiments, the composition contains only 5'UTR. In some embodiments, the composition contains only 3'UTR.

In some specific embodiments, the IFNα RNA comprises a poly A tail. In some specific embodiments, the IFNα RNA comprises a poly A tail of at least about 25, at least about 30, at least about 50 nucleotides, at least about 70 nucleotides, or at least about 100 nucleotides. In some specific embodiments, the poly A tail contains 200 or more nucleotides. In some specific embodiments, the poly A tail comprises or consists of SEQ ID NO: 66.

In some specific embodiments, the RNA includes a 5'cap, a 5'UTR, a nucleic acid encoding IFNα, a 3'UTR, and a poly A tail in order.

In some specific embodiments, the IFNα RNA is encoded by a DNA sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, SEQ ID NO: 20 or 21 96%, 97%, 98%, 99% or 100% identical and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 with SEQ ID NO: 3 or 5 %, 99% or 100% identical nucleic acid sequence or composed of it.

In some specific embodiments, the IFNα RNA comprises an RNA sequence, the RNA sequence is, for example, transcribed from a DNA sequence, the DNA sequence comprising SEQ ID NO: 20 or 21 at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical and at least 70%, 75%, 80%, 85%, 90%, 95% with SEQ ID NO: 3 or 5 %, 96%, 97%, 98%, 99%, or 100% identical nucleic acid sequence or consist of it. RNA can also be produced recombinantly. In some embodiments, one or more uridines in IFNα RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U). In some specific embodiments, the RNA contains modified nucleosides in place of each uridine. In some specific embodiments, the modified nucleoside is N1-methyl-pseudouridine (m ¹ ψ).

In some specific embodiments, the IFNα RNA is encoded by a DNA sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, SEQ ID NO: 20 or 21 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7 at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical nucleic acid sequences or consist of them.

In some specific embodiments, the IFNα RNA comprises an RNA sequence, the RNA sequence is, for example, transcribed from a DNA sequence, the DNA sequence comprising SEQ ID NO: 20 or 21 at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical and at least 70%, 75%, 80%, 85%, 90%, 95% with SEQ ID NO: 7 , 96%, 97%, 98%, 99% or 100% identical nucleic acid sequence or consist of it. In some embodiments, one or more uridines in IFNα RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U). In some specific embodiments, the RNA contains modified nucleosides in place of each uridine. In some specific embodiments, the modified nucleoside is N1-methyl-pseudouridine (m ¹ ψ).

In some specific embodiments, the IFNα RNA is encoded by a DNA sequence that contains at least 70%, 75%, 80%, 85%, 90%, 95%, 96% of SEQ ID NO: 20 or 21. , 97%, 98%, 99% or 100% identical; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID NO: 1, 3 or 5 Same; and is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to or consists of a nucleic acid sequence of SEQ ID NO: 7 .

In some specific embodiments, the IFNα RNA comprises an RNA sequence, the RNA sequence is, for example, transcribed from a DNA sequence, and the DNA sequence comprises at least 70%, 75%, 80%, 85% of SEQ ID NO: 20 or 21. %, 90%, 95%, 96%, 97%, 98%, 99% or 100%; same as SEQ ID NO: 3 or 5 at least 70%, 75%, 80%, 85%, 90%, 95% , 96%, 97%, 98%, 99% or 100%; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 %, 99% or 100% identical nucleic acid sequence or composed of it. RNA can also be produced recombinantly. In some embodiments, one or more uridines in IFNα RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U). In some specific embodiments, the RNA contains modified nucleosides in place of each uridine. In some specific embodiments, the modified nucleoside is N1-methyl-pseudouridine (m ¹ ψ). In some specific embodiments, the composition comprises an RNA sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, SEQ ID NO: 22 or 23. 97%, 98%, 99% or 100% identical; with SEQ ID NO: 4 or 6 at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 % Or 100% identical; and a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 8 Or consist of it. In some embodiments, one or more uridines in IFNα RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U).

C. Interleukin 15 (IL-15) sushi

In some specific embodiments, a composition or pharmaceutical preparation is provided, which comprises RNA encoding interleukin 15 (IL-15) sushi. As used herein, the term "IL-15 sushi" describes the soluble A construct of interleukin 15 (IL-15) receptor α sushi domain and mature interleukin α (IL-15) was used as a fusion protein. In some specific embodiments, the IL-15 sushi RNA is encoded by a DNA sequence encoding IL-15 sushi (SEQ ID NO: 24), and the DNA sequence includes a soluble IL-15 receptor alpha chain (sushi). It is the glycine-serine (GS) linker followed by the mature sequence of IL-15. The DNA sequence encoding this IL-15 sushi is provided in SEQ ID NO: 25.

In some specific embodiments, the IL-15 sushi RNA is, for example, an RNA sequence transcribed from a DNA sequence encoding IL-15 sushi. RNA can also be produced recombinantly. In some specific embodiments, the RNA sequence is transcribed from the nucleotide sequence comprising SEQ ID NO:25. In some embodiments, the nucleotide encoding the linker may be completely absent or partially or completely replaced by any nucleotide encoding a suitable linker. In some specific embodiments, the RNA sequence comprises or consists of SEQ ID NO: 26. In some specific embodiments, the RNA sequence comprises 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 26的RNA sequence. In some specific embodiments, the DNA or RNA sequence encoding IL-15 sushi comprises a sushi domain encoding IL-15 receptor alpha (for example, nucleotides 1-321 of SEQ ID NO: 25 or 26) and Nucleotides of mature IL-15 (eg, nucleotides 382-729 of SEQ ID NO: 25 or 26). In some specific embodiments, the DNA or RNA sequence encoding IL-15 sushi comprises a sushi domain encoding IL-15 receptor alpha (for example, nucleotides 1-321 of SEQ ID NO: 25 or 26) and The nucleotides of mature IL-15 (for example, nucleotides 382-729 of SEQ ID NO: 25 or 26), and also include nucleotides between these portions that encode the linker polypeptide that connects the portions. In some specific embodiments, the linker comprises nucleotides 322-381 of SEQ ID NO: 25 or 26. Any linker known to those skilled in the art can be used.

In some embodiments, one or more uridines in IL-15 sushi RNA are replaced with modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U). In some specific embodiments, the RNA contains modified nucleosides in place of each uridine. In some specific embodiments, the modified nucleoside is N1-methyl-pseudouridine (m ¹ ψ).

In some specific embodiments, the IL-15 sushi RNA includes an altered nucleotide at the 5'end. In some specific embodiments, the IL-15 sushi RNA includes a 5'cap. Any 5'cap known in the art can be used. In some specific embodiments, the 5'cap includes 5'to 5'triphosphate bonds. In some specific embodiments, the 5'cap comprises 5'to 5'triphosphate linkages including phosphorothioate modification. In some specific embodiments, the 5'cap comprises 2'- 0 or 3'- 0 -ribose-methylated nucleotides. In some specific embodiments, the 5'cap comprises modified guanosine nucleotides or modified adenosine nucleotides. In some embodiments, the 5'cap comprises 7-methylguanylic acid. In some specific embodiments, the 5'cap is Cap0 or Cap1. Exemplary cap structure comprises a m7G (5 ') ppp (5 ') G, m7,2` O -mG (5 ') pp S p (5') G, m7G (5 ') ppp (5') 2` O -mg and m7,3` O -mG (5 ') ppp (5') 2` O -mA.

In some specific embodiments, the IL-15 sushi RNA includes a 5'untranslated region (UTR). In some specific embodiments, the 5'UTR is upstream of the start codon. In some specific embodiments, the 5'UTR regulates the translation of RNA. In some specific embodiments, the 5'UTR is a stabilizing sequence. In some specific embodiments, the 5'UTR extends the half-life of RNA. Any 5'UTR known in the art can be used. In some specific embodiments, the 5'UTR RNA sequence is transcribed from SEQ ID NO: 3 or 5. In some specific embodiments, the 5'UTR RNA sequence comprises or consists of SEQ ID NO: 4 or 6. In some specific embodiments, the 5'UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or the same as SEQ ID NO: 4 or 6. 99% same.

In some specific embodiments, the IL-15 sushi RNA contains 3'UTR. In some embodiments, the 3'UTR is after the translation of the stop codon. In some specific embodiments, the 3'UTR regulates polyadenylation, translation efficiency, localization or stability of RNA. In some specific embodiments, the 3'UTR RNA sequence is transcribed from SEQ ID NO:7. In some specific embodiments, the 3'UTR RNA sequence comprises or consists of SEQ ID NO: 8. In some specific embodiments, the 3'UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% identical to SEQ ID NO: 8 Or 99% the same.

In some specific embodiments, the IL-15 sushi RNA includes both 5'UTR and 3'UTR. In some specific embodiments, the IL-15 sushi RNA only contains 5'UTR. In some specific embodiments, the IL-15 sushi RNA only contains 3'UTR.

In some specific embodiments, the IL-15 sushi RNA contains a poly A tail. In some specific embodiments, the RNA comprises a poly A tail of at least about 25, at least about 30, at least about 50 nucleotides, at least about 70 nucleotides, or at least about 100 nucleotides. In some specific embodiments, the poly A tail contains 200 or more nucleotides. In some specific embodiments, the poly A tail comprises or consists of SEQ ID NO: 66.

In some specific embodiments, the RNA includes a 5'cap, a 5'UTR, a nucleic acid encoding IL-15 sushi, a 3'UTR, and a poly A tail in order.

In some specific embodiments, the IL-15 sushi RNA is encoded by a DNA sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95% of SEQ ID NO: 25 , 96%, 97%, 98%, 99% or 100% and are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, and SEQ ID NO: 3 or 5 98%, 99% or 100% identical nucleic acid sequence or consist of it.

In some specific embodiments, the IL-15 sushi RNA comprises an RNA sequence, the RNA sequence is, for example, transcribed from a DNA sequence, and the DNA sequence comprises at least 70%, 75%, 80% of SEQ ID NO: 25 , 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% and are the same as SEQ ID NO: 3 or 5 at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical nucleic acid sequence or consisting of it. RNA can also be produced recombinantly. In some embodiments, one or more uridines in IFNα RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U). In some specific embodiments, the RNA contains modified nucleosides in place of each uridine. In some specific embodiments, the modified nucleoside is N1-methyl-pseudouridine (m ¹ ψ).

In some specific embodiments, the IL-15 sushi RNA comprises a DNA sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96% of SEQ ID NO: 25 , 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7 at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% Or 100% identical nucleic acid sequence or composed of it.

In some specific embodiments, the IL-15 sushi RNA comprises an RNA sequence, the RNA sequence is, for example, transcribed from a DNA sequence, and the DNA sequence comprises at least 70%, 75%, 80% of SEQ ID NO: 25 , 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% and are at least 70%, 75%, 80%, 85%, 90%, 95% identical to SEQ ID NO: 7 , 96%, 97%, 98%, 99% or 100% identical nucleic acid sequence or consist of it. RNA can also be produced recombinantly. In some embodiments, one or more uridines in IFNα RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U). In some specific embodiments, the RNA contains modified nucleosides in place of each uridine. In some specific embodiments, the modified nucleoside is N1-methyl-pseudouridine (m ¹ ψ).

In some specific embodiments, the IL-15 sushi RNA comprises a DNA sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96% of SEQ ID NO: 25 , 97%, 98%, 99%, or 100%; same as SEQ ID NO: 3 or 5; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical; and nucleic acid that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7 Sequence or consist of it.

In some specific embodiments, the IL-15 sushi RNA comprises an RNA sequence, the RNA sequence is, for example, transcribed from a DNA sequence, and the DNA sequence comprises at least 70%, 75%, 80% of SEQ ID NO: 25 , 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%; at least 70%, 75%, 80%, 85%, 90%, and SEQ ID NO: 3 or 5 95%, 96%, 97%, 98%, 99% or 100%; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% with SEQ ID NO: 7 , 98%, 99%, or 100% identical nucleic acid sequence or composed of it. In some embodiments, one or more uridines in the IFNα RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U). In some specific embodiments, the RNA contains modified nucleosides in place of each uridine. In some specific embodiments, the modified nucleoside is N1-methyl-pseudouridine (m ¹ ψ).

In some specific embodiments, the IL-15 sushi RNA comprises an RNA sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96% of SEQ ID NO: 26 , 97%, 98%, 99% or 100% identical; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and SEQ ID NO: 4 or 6 99% or 100% identical; and nucleic acid that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 8 Sequence or consist of it. In some embodiments, one or more uridines in IFNα RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U).

D. Fms-related tyrosine kinase 3 ligand (FLT3-L)

In some specific embodiments, a composition or pharmaceutical preparation comprising RNA encoding Fms-related tyrosine kinase 3 ligand (FLT3-L) is administered. In some embodiments, the FLT3-L RNA is encoded by a DNA sequence encoding FLT3-L (for example, SEQ ID NO: 31).

In some specific embodiments, the FLT3-L RNA comprises an RNA sequence, which is transcribed, for example, from a DNA sequence encoding FLT3-L. In some specific embodiments, the RNA sequence is transcribed from SEQ ID NO:31. RNA can also be produced recombinantly. In some specific embodiments, the RNA sequence comprises or consists of SEQ ID NO: 32. In some specific embodiments, the RNA sequence comprises 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 32的RNA sequence.

In some embodiments, one or more uridines in FLT3-L RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U). In some specific embodiments, the RNA contains modified nucleosides in place of each uridine. In some specific embodiments, the modified nucleoside is N1-methyl-pseudouridine (m ¹ ψ). In some specific embodiments, the FLT3-L RNA includes an altered nucleotide at the 5'end. In some specific embodiments, the RNA includes a 5'cap. Any 5'cap known in the art can be used. In some specific embodiments, the 5'cap includes 5'to 5'triphosphate bonds. In some specific embodiments, the 5'cap comprises 5'to 5'triphosphate linkages including phosphorothioate modification. In some specific embodiments, the 5'cap comprises 2'- 0 or 3'- 0 -ribose-methylated nucleotides. In some specific embodiments, the 5'cap comprises modified guanosine nucleotides or modified adenosine nucleotides. In some embodiments, the 5'cap comprises 7-methylguanylic acid. In some specific embodiments, the 5'cap is Cap0 or Cap1. Exemplary cap structure comprises a m7G (5 ') ppp (5 ') G, m7,2` O -mG (5 ') pp S p (5') G, m7G (5 ') ppp (5') 2` O -mg and m7,3` O -mG (5 ') ppp (5') 2` O -mA.

In some specific embodiments, the FLT3-L RNA comprises a 5' untranslated region (UTR). In some specific embodiments, the 5'UTR is upstream of the start codon. In some specific embodiments, the 5'UTR regulates the translation of RNA. In some specific embodiments, the 5'UTR is a stabilizing sequence. In some specific embodiments, the 5'UTR extends the half-life of RNA. Any 5'UTR known in the art can be used. In some specific embodiments, the 5'UTR RNA sequence is transcribed from SEQ ID NO: 3 or 5. In some specific embodiments, the 5'UTR RNA sequence comprises or consists of SEQ ID NO: 4 or 6. In some specific embodiments, the 5'UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or the same as SEQ ID NO: 4 or 6. 99% same.

In some specific embodiments, the FLT3-L RNA comprises 3'UTR. In some embodiments, the 3'UTR is after the translation of the stop codon. In some specific embodiments, the 3'UTR regulates polyadenylation, translation efficiency, localization or stability of RNA. In some specific embodiments, the 3'UTR RNA sequence is transcribed from SEQ ID NO:7. In some specific embodiments, the 3'UTR RNA sequence comprises or consists of SEQ ID NO: 8. In some embodiments, the 3'UTR RNA sequence is the same as SEQ ID NO: 8 is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% the same.

In some specific embodiments, the FLT3-L RNA comprises both 5'UTR and 3'UTR. In some embodiments, the RNA contains only 5'UTR. In some embodiments, the composition contains only 3'UTR.

In some specific embodiments, the FLT3-L RNA comprises a poly A tail. In some specific embodiments, the RNA comprises a poly A tail of at least about 25, at least about 30, at least about 50 nucleotides, at least about 70 nucleotides, or at least about 100 nucleotides. In some specific embodiments, the poly A tail contains 200 or more nucleotides. In some specific embodiments, the poly A tail comprises or consists of SEQ ID NO: 66.

In some specific embodiments, the FLT3-L RNA includes a 5'cap, a 5'UTR, a nucleic acid encoding FLT3-L, a 3'UTR, and a poly A tail in order.

In some specific embodiments, the FLT3-L RNA is encoded by a DNA sequence that contains at least 70%, 75%, 80%, 85%, 90%, 95%, SEQ ID NO: 31 96%, 97%, 98%, 99% or 100% identical and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 with SEQ ID NO: 3 or 5 %, 99% or 100% identical nucleic acid sequence or composed of it.

In some specific embodiments, the FLT3-L RNA comprises an RNA sequence, the RNA sequence is, for example, transcribed from a DNA sequence, and the DNA sequence comprises at least 70%, 75%, 80%, and SEQ ID NO: 31 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical and at least 70%, 75%, 80%, 85%, 90%, 95% with SEQ ID NO: 3 or 5 %, 96%, 97%, 98%, 99%, or 100% identical nucleic acid sequence or consist of it. RNA can also be produced recombinantly. In some embodiments, one or more uridines in FLT3-L RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U). In some specific embodiments, the RNA contains modified nucleosides in place of each uridine. In some specific embodiments, the modified nucleoside is N1-methyl-pseudouridine (m ¹ ψ).

In some specific embodiments, the FLT3-L RNA is encoded by a DNA sequence that contains at least 70%, 75%, 80%, 85%, 90%, 95%, SEQ ID NO: 31 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7 at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical nucleic acid sequences or consist of them.

In some specific embodiments, the FLT3-L RNA comprises an RNA sequence, the RNA sequence is, for example, transcribed from a DNA sequence, and the DNA sequence comprises at least 70%, 75%, 80%, and SEQ ID NO: 32 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% are the same as SEQ ID NO: 7 at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical nucleic acid sequence or consist of it. RNA can also be produced recombinantly. In some embodiments, one or more uridines in FLT3-L RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U). In some specific embodiments, the RNA contains modified nucleosides in place of each uridine. In some specific embodiments, the modified nucleoside is N1-methyl-pseudouridine (m ¹ ψ).

In some specific embodiments, the FLT3-L RNA comprises a DNA sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, SEQ ID NO: 31 97%, 98%, 99% or 100% identical; with SEQ ID NO: 3 or 5 at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 % Or 100% identical; and a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7 Or consist of it.

In some specific embodiments, the FLT3-L RNA comprises an RNA sequence, the RNA sequence is, for example, transcribed from a DNA sequence, and the DNA sequence comprises at least 70%, 75%, 80%, and SEQ ID NO: 32 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%; same as SEQ ID NO: 3 or 5 at least 70%, 75%, 80%, 85%, 90%, 95 %, 96%, 97%, 98%, 99% or 100%; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, and SEQ ID NO: 7 98%, 99% or 100% identical nucleic acid sequence or composed of it. RNA can also be produced recombinantly. In some embodiments, one or more uridines in FLT3-L RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U). In some specific embodiments, the RNA contains modified nucleosides in place of each uridine. In some specific embodiments, the modified nucleoside is N1-methyl-pseudouridine (m ¹ ψ).

In some specific embodiments, the FLT3-L RNA comprises an RNA sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, SEQ ID NO: 32 97%, 98%, 99% or 100% identical; with SEQ ID NO: 4 or 6 at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 % Or 100% identical; and a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 8 Or consist of it. In some embodiments, one or more uridines in FLT3-L RNA are replaced by modified nucleosides as described herein. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m ¹ ψ), or 5-methyl-uridine (m ⁵ U).

E. Modified RNA

Each RNA described herein can be modified in any manner known to those skilled in the art. In some specific embodiments, each RNA modification is as follows:

˙Replace each modified nucleobase of uridine;

˙Cap1 structure at the 5'end of RNA.

In some specific embodiments, the 5'UTR comprises SEQ ID NO: 4 or 6. In some embodiments, the RNA has been processed to reduce double-stranded RNA (dsRNA), as described above. The "Cap1" structure can be generated by enzymatic capping after in vitro translation or during in vitro translation (co-transcription capping).

In some specific embodiments, one or more uridines in the RNA are replaced by modified nucleosides. In some specific embodiments, the modified nucleoside is a modified uridine.

In some specific embodiments, the modified uridine that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine Glycoside (m1ψ) or 5-methyl-uridine (m5U).

In some embodiments, one or more cytosine, adenine or guanine in the RNA is replaced by one or more modified nucleobases. In a specific embodiment, the modified nucleobase replacing cytosine is 5-methylcytosine (m ⁵ C). In another specific embodiment, the modified nucleobase that replaces adenine is N ⁶ -methyl adenine (m ⁶ A). In another specific embodiment, any other modified nucleobases known in the art for reducing the immunogenicity of molecules can be used.

The modified nucleoside that replaces one or more uridine in RNA can be any one or more of the following: 3-methyl-uridine (m ³ U), 5-methoxy-uridine (mo ⁵ U) , 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s ² U), 4-thio-uridine (s ⁴ U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho ⁵ U), 5-aminoallyl-uridine, 5-halo Generation-uridine (for example, 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo ⁵ U), uridine 5-oxyacetic acid methyl ester (mcmo ⁵ U), 5-carboxymethyl-uridine (cm ⁵ U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm ⁵ U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm ⁵ U), 5-methoxycarbonylmethyl-uridine (mcm ⁵ U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm ⁵ s ² U), 5-amine Methyl-2-thio-uridine (nm ⁵ s ² U), 5-methylaminomethyl-uridine (mnm ⁵ U), 1-ethyl-pseudouridine, 5-methylamine Methyl-2-thio-uridine (mnm ⁵ s ² U), 5-methylaminomethyl-2-seleno-uridine (mnm ⁵ se ² U), 5-aminomethanyl Methyl-uridine (ncm ⁵ U), 5-carboxymethylaminomethyl-uridine (cmnm ⁵ U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm ⁵ s ² U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurylmethyl-uridine (τm ⁵ U), 1-taurylmethyl-pseudouridine , 5-taurylmethyl-2-thio-uridine (τm5s2U), 1-taurylmethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine ( m ⁵ s ² U), 1-methyl-4-thio-pseudouridine (m ¹ s ⁴ ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m ³ ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza -Pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m ⁵ D), 2-thio-dihydro Uridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methyl Oxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp ³ U), 1-methyl-3- (3-Amino-3-carboxypropyl)pseudouridine (acp ³ ψ), 5-(isopentenylaminomethyl)uridine (inm ⁵ U), 5-(isopentenylamino Methyl)-2-thio-uridine (inm ⁵ s ² U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m ⁵ Um), 2'-O -Methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s ² Um), 5-methoxycarbonylmethyl-2'-O-methyl-urine Glycoside (mcm ⁵ Um), 5-aminomethylaminomethyl-2'-O-methyl-uridine (ncm ⁵ Um), 5-carboxymethylaminomethyl-2'-O-methyl -Uridine (cmnm ⁵ Um), 3,2'-O-Dimethyl-uridine (m ³ Um), 5-(Isopentenylaminomethyl)-2'-O-methyl-urine Glycoside (inm ⁵ Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-( 2-Methoxycarbonylvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

3. Treatment methods

The compositions and pharmaceutical preparations provided herein can be used in methods, such as methods of treatment. In some embodiments, the composition, pharmaceutical preparation and RNA are administered to humans.

In some embodiments, the present invention includes the use of a composition or pharmaceutical preparation in a method of treatment, wherein the treatment method is used to treat solid tumors, and wherein the composition or pharmaceutical preparation comprises RNA, wherein the RNA is composed of It consists of RNA encoding IFNα, RNA encoding IL-15 sushi, RNA encoding IL-12sc, and RNA encoding FLT3-L.

In some embodiments, the present invention includes the use of a composition or pharmaceutical preparation in a method of treatment, wherein the treatment method is used to treat solid tumors, and wherein the composition or pharmaceutical preparation comprises RNA, wherein the RNA is composed of It consists of RNA encoding IFNα, RNA encoding IL-15 sushi, and RNA encoding IL-12sc.

In some specific embodiments, the composition or pharmaceutical preparation used in the treatment method includes RNA encoding IL-12sc protein, and further includes RNA selected from RNA encoding IFNα and RNA encoding IL-15 sushi protein.

In some specific embodiments, the composition or pharmaceutical preparation used in the treatment method includes RNA encoding IL-15 sushi, and further includes RNA encoding IFNα protein and RNA encoding IL-12sc protein.

In some specific embodiments, the composition or pharmaceutical preparation used in the treatment method includes RNA encoding IFNα protein, and further includes RNA encoding IL-12sc and RNA encoding IL-15 sushi.

In some specific embodiments, the composition or pharmaceutical preparation used in the treatment method comprises RNA encoding FLT3-L protein and one or more of the following: RNA encoding IL-12sc, RNA encoding IL-15 sushi, and IFNα RNA.

In some specific embodiments, the pharmaceutical preparations or compositions described herein are used for pharmaceutical use. In some specific embodiments, the pharmaceutical use includes therapeutic treatment or preventive treatment of diseases or disorders. In some cases, the therapeutic treatment or prophylactic treatment of the disease or disorder includes the treatment or prevention of solid tumors.

In some specific embodiments, the solid tumor is a sarcoma, carcinoma or lymphoma.

In some cases, the solid tumor is in the lung, colon, ovary, cervix, uterus, peritoneum, testis, penis, tongue, lymph node, pancreas, bone, breast, prostate, soft tissue, connective tissue, kidney, In the liver, brain, thyroid or skin.

In some specific embodiments, the solid tumor is an epithelial tumor, Hodgkin’s lymphoma (HL), non-Hodgkin’s lymphoma, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tumor, Liver tumors, colorectal tumors, vascular tumors, mesothelioma tumors, pancreatic tumors, breast tumors, sarcoma tumors, lung tumors, colon tumors, brain tumors, melanoma tumors, small cell lung tumors, neuroblastoma tumors, Testicular tumor, cancer tumor, adenocarcinoma tumor, glioma tumor, seminoma tumor, retinoblastoma or osteosarcoma tumor.

In some specific embodiments, based on equal RNA quality, the RNA is administered at a ratio of 1:1:1 or 1:1:1:1. For example, 20 μg of IL-15-sushi, 20 μg of IL-12sc, 20 μg of IFNα, and optionally 20 μg of FLT3-L.

In some specific embodiments, the pharmaceutical formulations or compositions described herein are for pharmaceutical use and are administered to an individual with another therapy. In some specific embodiments, the other therapy is an anti-PD1 antibody, an anti-CTLA4 antibody, or a combination of an anti-PD1 antibody and an anti-CTLA4 antibody.

In some specific embodiments, the anti-PD1 antibody is a chimeric, humanized or human antibody. In some specific embodiments, the anti-PD-1 antibody is isolated and/or recombinant. Examples of anti-PD-1 antibodies are nivolumab, pembrolizumab, cemiplimab, MEDI0608 (formerly known as AMP-514; see, for example, WO 2012/145493 and US Patent No. 9,205,148) , PDR001 (see, for example, WO 2015/112900), PF-06801591 (see, for example, WO 2016/092419), BGB-A317 (see, for example, WO 2015/035606).

In some embodiments, the anti-PD-1 antibodies are those disclosed in WO 2015/112800 (such as those referred to as H1M7789N, H1M7799N, H1M7800N, H2M7780N, H2M7788N, H2M7790N, H2M7791N, H2M7794N, H2M7795N, H2M7796N, H2M7798N, H4H9019P, H4xH9034P2, H4xH9035P2, H4xH9037P2, H4xH9045P2, H4xH9048P2, H4H9057P2, H4H9068P2, H4xH9119P2, H4xH9120P2, H4xH9128P2, H4xH9135P2, H4xH9145P2, H4xH8992P, H4xH8999P and those H4xH9008P, and in the PCT Publication One of those referred to as H4H7798N, H4H7795N2, H4H9008P and H4H9048P2 in Table 3). The disclosure of WO 2015/112800 is incorporated herein by reference in its entirety. For example, the antibodies and related antibodies disclosed in WO 2015/112800 (including antibodies and antigen-binding fragments having the CDR, VH and VL sequences or heavy chain and light chain sequences disclosed in the PCT publication) and binding to the Antibodies and antigen-binding fragments of the same PD-1 epitope as disclosed in the PCT publication can be used in conjunction with the RNA composition of the present invention to treat and/or prevent cancer.

In some specific embodiments, the anti-PD-1 antibody may comprise the heavy chain and light chain amino acid sequences shown in SEQ ID NOs: 79 and 80, respectively; in SEQ ID NOs: 87 and 88 VH and VL sequence Columns (shown in italics) or one or more (e.g., all six) CDRs (shown in bold boxes) in SEQ ID NOs: 79 and 80. In some specific embodiments, antibodies comprising the following CDRs are encompassed:

HCDR1=GFTFSNFG (SEQ ID NO: 81)

HCDR2=ISGGGRDT (SEQ ID NO: 82)

HCDR3=VKWGNIYFDY (SEQ ID NO: 83)

LCDR1=LSINTF(SEQ ID NO:84)

LCDR2=AAS (SEQ ID NO: 85)

LCDR3=QQSSNTPFT (SEQ ID NO: 86).

The antibody comprising the heavy chain containing the VH and VL sequences in SEQ ID NOs: 87 and 88 (shown in italics) is a fully human anti-PD-1 antibody, called REGN2810 (Sepritimab).

Anti-PD-1 Mab heavy chain

(SEQ ID NO：79)

(SEQ ID NO: 79)

HCDR1=GFTFSNFG (SEQ ID NO: 81)

HCDR2=ISGGGRDT (SEQ ID NO: 82)

HCDR3=VKWGNIYFDY (SEQ ID NO: 83)

Anti-PD-1 Mab light chain

(SEQ ID NO：80)

(SEQ ID NO: 80)

LCDR1=LSINTF(SEQ ID NO:84)

LCDR2=AAS (SEQ ID NO: 85)

LCDR3=QQ SSNTPFT (SEQ ID NO: 86)

In some embodiments, the RNA can be delivered via injection into a tumor (e.g., intratumor), near the tumor (peripheral tumor), or near the site of tumor removal, and the antibody can be delivered in the same manner or systemically (e.g., like Intestinal or parenteral, including via injection, infusion and implantation). The administration can be simultaneous or sequential. If it is sequential, it can be administered in any order and at any suitable time interval known to those skilled in the art.

In some embodiments, the therapeutic RNA composition is delivered with another therapy directly into the tumor or near the tumor or tumor removal site. In some embodiments, the therapeutic RNA composition is delivered directly into the tumor or near the tumor or tumor removal site, while the other agent is delivered systemically.

***

This description and exemplary specific embodiments should not be considered limiting. For the purpose of this specification and the scope of the appended invention application, unless otherwise indicated, in the specification and the invention application scope All numbers and other numerical values expressing quantities, percentages, or ratios used in should be understood to be modified by the term "about" to the extent that they cannot be modified in this way in all cases. "About" indicates the degree of change that does not substantially affect the characteristics of the subject, for example, within 10%, 5%, 2%, or 1%. Therefore, unless there are instructions to the contrary, the numerical parameters set forth in the following specification and the scope of the appended invention application are approximate values, and the approximate values may vary according to the desired characteristics that are attempted to be obtained. At least and not trying to limit the application of the principle of equivalence to the scope of patent applications for inventions, each numerical parameter should at least be interpreted as being based on the number of significant figures reported and by applying common rounding techniques.

Note that as used in this specification and the scope of the appended invention application, the singular forms "a, an" and "the" and any singular use of any words include plural indicators, unless explicitly And clearly limited to one indicator/type. As used herein, the term "including" and its grammatical variants are intended to be non-limiting, so that the elaboration of items in the list does not exclude other similar items that can be substituted or added to the listed items.

Instance

The following examples are provided to illustrate certain disclosed specific embodiments, and should not be construed as limiting the scope of this disclosure in any way.

Example 1-Materials and Methods

B16F10 tumor model : 6-8 weeks old female C57BL/6J mice (Jackson Laboratory; Bar Harbor, Maine) weighing between 17.0 g and 20.9 g were adapted to the environment for at least three days before study recruitment. The mice had free access to food (Harlan 2916 rodent diet, Massachusetts, USA) and sterile water, and were reared under a 12-hour light/dark cycle at 22°C ± 2°C and a relative humidity of 55% ± 15%. B16F10 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, Virginia, USA) (catalog number CRL-6475) and were supplemented with 10% heat in 5% CO2 at 37°C. Inactivated fetal bovine serum (HI FBS) (Life technologies, catalog number 10082-147) was cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Life technologies, catalog number 11995). The cells were harvested using 0.25% trypsin-EDTA (Life technologies, catalog number 25200-056) and resuspended in Dulbecco's phosphate-buffered saline (DPBS) (Life technologies, catalog number 14190-144), And 0.5 x 10^6 cells/200μl/mouse were subcutaneously (SC) implanted into the right abdomen of female C57BL/6J mice.

Tumor monitoring: B16F10 tumor was measured with calipers twice a week until the final sacrifice. When the tumor size reaches approximately 2000 mm ³ or there is an animal health problem (20% of the tumor is ulcerated), the animal is euthanized. Tumor regression is defined as: i) tumor volume at the end of the study <20mm3 or ii) T ^F /T ⁰ <1, where T ^F is equal to the final tumor volume, and T ⁰ is equal to the tumor volume on the day of the first intratumoral RNA injection.

CT26 tumor model: Regarding the study using the CT26 tumor model, female Balb/c Rj mice weighing between 17g and 24g (Janvier, Genest Island, France) were made 6-8 weeks old before recruiting. -St.-Îsle)) Adapt to the environment for at least six days. The mice have free access to food (ssniff MZ autoclavable Soest, Germany) and sterile water, and are reared under a 12-hour light/dark cycle at 22°C ± 2°C and a relative humidity of 55% ± 10%. CT26 cells were obtained from (ATCC® CRL-2638 ^™ ) and supplemented with 10% fetal bovine serum (FBS) (Biochrom, catalog number S 0115) at Roswell Park Memorial Institute ( Roswell Park Memorial Institute, RPMI) 1640 medium GlutaMAX ^™ (Life technologies, catalog number 61870-044). The cells were collected using StemPro® Accutase® cell dissociation reagent (Life technologies, catalog number A1110501), resuspended in DPBS (Life technologies, catalog number 14190-169), and 0.5 x 10^6 cells/100μl/small Mouse SC was implanted into the right shaved abdomen of female Balb/c Rj mice. Intratumor RNA injection was started 11 to 15 days after tumor inoculation. The tumor growth in the CT26 tumor model is evaluated by caliper measurement every 2-3 days and expressed as the product of the vertical diameter using the following formula: a2 * b/2, where b is the longer of the two diameters (a<b) .

Gp70 reactive CD8+ cells: In addition to tumor growth, gp70 reactive CD8+ T cells are measured in the indicated blood. Blood samples were collected using EDTA-coated tubes. Transfer 100 μL of blood to a FACS tube, and add an antibody mixture containing T-Select H-2Ld MuLV gp70 tetramer-SPSYVYHQF-APC (MBL (TS-M-521-2), 4 μL for 100 μL of blood ), anti-CD8a FITC (life technologies (MCD801), 1 μL for 100 μL blood) and anti-CD45 V500 (BD (561487), 1 μL for 100 μL blood). After incubating for 20 min at room temperature, blood lysis buffer (BD (349202), 300 μL/tube) was added, and the cells were further incubated for 6 min. Then the sample was washed twice with PBS-EDTA buffer. The FACS samples were analyzed on the FACS Canto II flow cytometer.

RNA modifications: Unless otherwise stated, all RNA modifications are as follows. The synthetic DNA fragment encoding the gene of interest is cloned into a commonly used starting vector containing 5'-UTR (in some cases, corresponding to the tobacco etch virus leader sequence TEV, SEQ ID NO: 3 ), a 3'UTR composed of two elements called F and I (in some cases, corresponding to SEQ ID NO: 7) and a poly (A) tail (A30-linked) of 110 nucleotides in total Sub-A70 structure; SEQ ID NO: 60). Linearization of plasmid DNA was performed downstream of poly(dA:dT) with class IIS restriction endonucleases to generate templates without additional nucleotides except poly(dA:dT) (see Holtkamp et al., Blood 108(13): 4009-172006 (2006)). In the presence of 7.5mM ATP, CTP, GTP and N1-methyl-pseudouridine triphosphate, linearized plasmid DNA was subjected to in vitro transcription with T7 RNA polymerase (Thermo Fisher) as described previously (see Grudzien -Nogalska E et al. Methods Mol Biol. 969: 55-72 (2013)). The RNA was then purified using magnetic particles ( Berensmeier 2006 ), and subsequently Cap1 was enzymatically introduced using a commercially available system based on vaccinia virus capping enzyme (NEB) and adding RNA cap 2'-O-methyltransferase (NEB) structure. Then, the RNA is subjected to further purification procedures by cellulose-based chromatography to remove double-stranded RNA impurities (see Day PR et al., Phytopathology 67:1393 (1977); Morris TJ et al., Phytopathology 69:854 -858 (1979); and Castillo A et al., Virol J. 8:38 (2011)). The RNA concentration and quality were evaluated by spectrophotometry and analyzed by capillary gel electrophoresis system. A dsRNA-specific J2 mAb (English & Scientific Consulting) was used to assess the presence of dsRNA in a Northwestern dot blot assay, as described in Karikó et al. Nucleic Acids Res. 39(21): e142 (2011).

Preparation of RNA for in vivo research : Prepare the corresponding RNA mixture for in vivo research by mixing equal amounts (micrograms) of RNA in water at 2 times the expected dose. The RNA injected into the mouse is the mouse version of RNA other than FLT3-L described in Table 1, wherein SEQ ID NO: 65 (human FLT3-L with murine optimized secretion sequence) is used. In some cases, the RNA mixture was frozen at -80°C until the day of intratumoral injection. On the day of injection, the RNA was thawed and mixed with an equal volume of 2X sterile Ringer's solution. In other cases, the RNA mixture is prepared fresh and diluted in Ringer's solution to a final concentration of 1X RNA/Ringer's solution. In both cases, the resulting 1X RNA/Ringer's solution was used for intratumor injection.

Example 2-The combination of three RNAs reduces tumor volume in vivo

We previously tested the effects of cytokines RNA mixtures containing three different combinations of RNA in CT26 tumor-bearing mice, as described above. Tumors treated with four injections of RNA encoding IL-15 sushi, IL-12sc, GM-CSF and IFNα (10 μg RNA/target) induced regression in 10 of the 10 treated tumors (Figure 1A; Table 2 ( The table and figure represent the same data; the table provides the original data)). Use i) IL-15 sushi, IL-12sc and IFNα (no GM-CSF), ii) IL-15 sushi, GM-CSF and IFNα (no IL-12sc), iii) IL-12sc, GM-CSF and IFNα (IL-15 sushi without IL-15), and iv) IL-15 sushi, IL-12sc and GM-CSF (without IFNα) 3 combined RNA mixture treated tumors resulted in 8 out of 10 regressions (IL-15 sushi, IL-15 -12sc and IFNα (no GM-CSF; Figure 1B; Table 3), 6 out of 10 regressions (IL-15 sushi, GM-CSF and IFNα (no IL-12sc; Figure 1C; Table 4), 10 8 out of 10 regressions (IL-12sc, GM-CSF and IFNα (without IL-15 sushi; Figure 1D; Table 5), 7 out of 10 regressions (IL-15 sushi, IL-12sc And GM-CSF (no IFNα; Figure 1E; Table 6). Tumors treated with control RNA (40 μg RNA injected per treatment) did not show tumor regression (Figure 1F; Table 7). To analyze the tumor growth kinetics, for each treatment group, the average tumor volume up to day 33 was calculated (Figure 1G). For each treatment group, tumor growth inhibition T/C (tumor/control based on average tumor volume) was plotted to day 19 (Figure 1H).

In a separate experiment using the B16F10 tumor model, the anti-tumor activity of the 4 combination RNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFNα and the 3 combination mixture of IL-15 sushi, IL-12sc and IFNα Compare. On day 11, the mice received a single injection of RNA within the tumor. Treatment with a 4-combined cytokine RNA mixture encoding IL-15 sushi, IL-12sc, GM-CSF and IFNα resulted in tumor regression in 6 out of 10 mice (Figure 2A). Tumors treated with the 3-combined RNA mixture of IL-15 sushi, IL-12sc and IFNα (without GM-CSF) had tumor regression in 8 out of 10 mice (Figure 2B), while the control encoding luciferase The RNA-treated mice did not notice tumor regression (Figure 2C).

Example 3-The combination of four RNAs reduces tumor volume and promotes immune response in vivo

On days 13, 16, 20, and 24, a 4-cell mixture of RNA encoding IL-15 sushi, IL-12sc, IFNα, and FLT3-L was injected into CT26 tumor-bearing mice, and tumor growth was monitored to 50 Day (5μg RNA/target and 20μg control RNA). As shown in Figure 3, intratumoral injection of a combination of four RNAs encoding IL-15 sushi, IL-12sc, IFNα and FIT3-L induced tumor regression in 4 out of 10 mice (Figure 3A). And the mice treated with the control RNA encoding luciferase showed tumor regression in 1 out of 10 animals (Figure 3B). These data were confirmed in repeated studies of similar design. In repeated experiments, a mixture of cytokines RNA encoding IL-15 sushi, IL-12sc, IFNα and FLT3-L (injection of 5μg RNA/target and 20μg control RNA on days 11, 14, 17 and 21) resulted in 9 out of 11 mice showed tumor regression (Figure 3C), while treatment with control RNA encoding luciferase showed tumor regression in 1 out of 11 mice (Figure 3D). Mice treated with a mixture of cytokine RNA encoding IL-15 sushi, IL-12sc, IFNα and GM-CSF showed tumor regression in 2 out of 10 animals (Figure 3E).

In a separate experiment using the B16F10 tumor model, on day 11, a mixture of RNAs encoding IL-15 sushi, IL-12sc, IFNα and GM-CSF (Figure 4A); encoding IL-15 sushi, IL-12sc A mixture of RNA of IFNα and FLT3-L (Figure 4B); or control RNA encoding luciferase (Figure 4C) (2μg RNA/target and 8μg control RNA) were injected into B16F10 tumor-bearing mice at a time, and Monitor tumor growth to day 57. As shown in Figure 4, intratumor injection of a combination of four RNAs encoding IL-15 sushi, IL-12sc, IFNα and GM-CSF induced tumor regression in 7 out of 10 mice (Figure 4A). Intratumoral injection The combination of the four RNAs encoding IL-15 sushi, IL-12sc, IFNα, and FLT3-L induced tumor regression in 6 out of 10 mice (Figure 4B), while those treated with control RNA encoding luciferase 0 out of 10 mice showed tumor regression (Figure 4C).

B16F10 tumor-bearing mice received a single injection of RNA (2 μg RNA/target or 8 μg control RNA), and 7 days after intratumor injection, the tumor was removed, dissociated, and stained with a set of antibodies to assess the intratumoral immune population. The numbers of CD8+ T cells and NK cells used in different treatment groups are listed. The results are shown in Figure 5A and Figure 5B.

On days 13, 16, 20, and 24, mice bearing a single CT26 tumor received four intratumoral injections 1) IL-15 sushi, GM-CSF, IFNα and IL-12sc cytokine RNA mixture; 2) IL -15 A mixture of cytokines RNA of sushi, FLT3L, IFNα and IL-12sc; or 3) RNA encoding luciferase (5 μg RNA/target/injection and 20 μg RNA/control injection). After the first intratumoral RNA administration Blood was collected on day 19 and day 31, and gp70 tumor antigen-specific T cells were quantified by flow cytometry. Compared with mice receiving control RNA, the frequency of gp70 tumor antigen-specific T cells in the blood was greatly increased in mice after intratumoral injection of RNA cytokine (Figure 6A (day 19) and Figure 6B ( Day 31)). On day 31, the frequency of gp70-specific T cells was higher for the mixture containing FLT3L compared to the mixture containing GM-CSF (Figure 6B). In a separate experiment, on day 24, compared with control RNA, IL-15 sushi, IL-12sc, IFNα, and FLT3-L cytokine RNA mixture resulted in a greatly increased frequency of gp70-specific T cells (Figure 6C ).

<110> SANOFI BIONTECH RNA PHARMACEUTICALS GMBH WAGENAAR, Timothy R., et al.

<120> Therapeutic RNA for solid tumor cancer

<130> 01183-0028-00PCT

<150> US 62/722,742

<151> 2018-08-24

<150> EP 19305092.9

<151> 2019-01-24

<160> 88

<170> PatentIn version 3.5

<210> 1

<400> 1

000

<210> 2

<400> 2

000

<210> 3

<211> 145

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis: Exemplary 5 UTR (DNA)

<400> 3

<210> 4

<211> 145

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesis: Exemplary 5 UTR(RNA)

<400> 4

<210> 5

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis: Exemplary alternative 5 UTR (DNA)

<400> 5

<210> 6

<211> 52

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesis: Exemplary alternative 5 UTR(RNA)

<400> 6

<210> 7

<211> 317

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis: Exemplary 3 UTR (DNA)

<400> 7

<210> 8

<211> 317

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesis: Exemplary 3 UTR(RNA)

<400> 8

<210> 9

<400> 9

000

<210> 10

<400> 10

000

<210> 11

<400> 11

000

<210> 12

<400> 12

000

<210> 13

<400> 13

000

<210> 14

<211> 539

<212> PRT

<213> Homo sapiens

<220>

<221> Features not yet classified

<222> (1)..(539)

<223> Human IL-12sc (amino acid)

<400> 14

<210> 15

<211> 1623

<212> DNA

<213> Homo sapiens

<220>

<221> Features not yet classified

<222> (1)..(1623)

<223> Human non-optimized IL-12sc (CDS DNA)

<400> 15

<210> 16

<211> 1623

<212> DNA

<213> Homo sapiens

<220>

<221> Features not yet classified

<222> (1)..(1623)

<223> Human optimized IL-12sc (CDS DNA)

<400> 16

<210> 17

<211> 1623

<212> RNA

<213> Homo sapiens

<220>

<221> Features not yet classified

<222> (1)..(1623)

<223> Human non-optimized IL-12sc (RNA encoding CDS)

<400> 17

<210> 18

<211> 1623

<212> RNA

<213> Homo sapiens

<220>

<221> Features not yet classified

<222> (1)..(1623)

<223> Human optimized IL-12sc (RNA encoding CDS)

<400> 18

<210> 19

<211> 188

<212> PRT

<213> Homo sapiens

<220>

<221> Features not yet classified

<222> (1)..(188)

<223> Human IFN-alpha-2b (amino acid)

<400> 19

<210> 20

<211> 570

<212> DNA

<213> Homo sapiens

<220>

<221> Features not yet classified

<222> (1)..(570)

<223> Human non-optimized IFN- α -2b (CDS DNA)

<400> 20

<210> 21

<211> 570

<212> DNA

<213> Homo sapiens

<220>

<221> Features not yet classified

<222> (1)..(570)

<223> Human optimized IFN- α -2b (CDS DNA)

<400> 21

<210> 22

<211> 570

<212> RNA

<213> Homo sapiens

<220>

<221> Features not yet classified

<222> (1)..(570)

<223> Human non-optimized IFN- α -2b (RNA encoding CDS)

<400> 22

<210> 23

<211> 570

<212> RNA

<213> Homo sapiens

<220>

<221> Features not yet classified

<222> (1)..(570)

<223> Human optimized IFN- α -2b (RNA encoding CDS)

<400> 23

<210> 24

<211> 241

<212> PRT

<213> Homo sapiens

<220>

<221> Features not yet classified

<222> (1)..(241)

<223> Human IL-15 sushi (amino acid)

<400> 24

<210> 25

<211> 729

<212> DNA

<213> Homo sapiens

<220>

<221> Features not yet classified

<222> (1)..(729)

<223> Human IL-15 sushi (CDS DNA)

<400> 25

<210> 26

<211> 729

<212> RNA

<213> Homo sapiens

<220>

<221> Features not yet classified

<222> (1)..(729)

<223> Human IL-15 sushi (RNA coded CDS)

<400> 26

<210> 27

<400> 27

000

<210> 28

<400> 28

000

<210> 29

<400> 29

000

<210> 30

<211> 185

<212> PRT

<213> Homo sapiens

<220>

<221> Features not yet classified

<222> (1)..(185)

<223> Human FLT3-L (amino acid)

<400> 30

<210> 31

<211> 555

<212> DNA

<213> Homo sapiens

<220>

<221> Features not yet classified

<222> (1)..(555)

<223> Human FLT3L (CDS DNA)

<400> 31

<210> 32

<211> 555

<212> RNA

<213> Homo sapiens

<220>

<221> Features not yet classified

<222> (1)..(555)

<223> Human FLT3L (RNA encoding CDS)

<400> 32

<210> 33

<400> 33

000

<210> 34

<400> 34

000

<210> 35

<400> 35

000

<210> 36

<400> 36

000

<210> 37

<400> 37

000

<210> 38

<400> 38

000

<210> 39

<211> 544

<212> PRT

<213> House mouse

<220>

<221> Features not yet classified

<222> (1)..(544)

<223> Mouse IL-12sc (amino acid)

<400> 39

<210> 40

<211> 2081

<212> DNA

<213> House mouse

<220>

<221> Features not yet classified

<222> (1)..(2081)

<223> Mouse IL-12sc (DNA: 5UTR-CDS-3UTR)

<400> 40

<210> 41

<211> 2081

<212> RNA

<213> House mouse

<220>

<221> Features not yet classified

<222> (1)..(2081)

<223> Mouse IL-12sc (RNA)

<400> 41

<210> 42

<400> 42

000

<210> 43

<400> 43

000

<210> 44

<400> 44

000

<210> 45

<211> 190

<212> PRT

<213> House mouse

<220>

<221> Features not yet classified

<222> (1)..(190)

<223> Mouse IFN- α -4 (amino acid)

<400> 45

<210> 46

<211> 1019

<212> DNA

<213> House mouse

<220>

<221> Features not yet classified

<222> (1)..(1019)

<223> Mouse IFN- α -4

<400> 46

<210> 47

<211> 1019

<212> RNA

<213> House mouse

<220>

<221> Features not yet classified

<222> (1)..(1019)

<223> Mouse IFN- α -4 (RNA)

<400> 47

<210> 48

<400> 48

000

<210> 49

<400> 49

000

<210> 50

<400> 50

000

<210> 51

<211> 232

<212> PRT

<213> House mouse

<220>

<221> Features not yet classified

<222> (1)..(232)

<223> Mouse IL-15 sushi (amino acid)

<400> 51

<210> 52

<211> 1145

<212> DNA

<213> House mouse

<220>

<221> Features not yet classified

<222> (1)..(1145)

<223> Mouse IL-15 sushi (DNA: 5UTR-CDS-3UTR)

<400> 52

<210> 53

<211> 1145

<212> RNA

<213> House mouse

<220>

<221> Features not yet classified

<222> (1)..(1145)

<223> Mouse IL-15 sushi (RNA: 5UTR-CDS-3UTR))

<400> 53

<210> 54

<400> 54

000

<210> 55

<400> 55

000

<210> 56

<400> 56

000

<210> 57

<211> 141

<212> PRT

<213> House mouse

<220>

<221> Features not yet classified

<222> (1)..(141)

<223> Mouse GM-CSF (amino acid)

<400> 57

<210> 58

<211> 891

<212> DNA

<213> House mouse

<220>

<221> Features not yet classified

<222> (1)..(891)

<223> Mouse GM-CSF

<400> 58

<210> 59

<211> 891

<212> RNA

<213> House mouse

<220>

<221> Features not yet classified

<222> (1)..(891)

<223> Mouse GM-CSF (RNA)

<400> 59

<210> 60

<400> 60

000

<210> 61

<400> 61

000

<210> 62

<400> 62

000

<210> 63

<211> 187

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic: FLT3-L (amino acid; human FLT3L in combination with a mouse optimized secretion sequence)

<400> 63

<210> 64

<211> 1010

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis: FLT3-L (DNA; 5UTR-human FLT3L-3 UTR combined with mouse optimized secretion sequence)

<400> 64

<210> 65

<211> 1010

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesis: FLT3L (RNA; 5UTR-human FLT3L-3 UTR combined with mouse optimized secretion sequence)

<400> 65

<210> 66

<211> 110

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesis: Exemplary Poly-A (Poly-A)

<400> 66

<210> 67

<400> 67

000

<210> 68

<400> 68

000

<210> 69

<400> 69

000

<210> 70

<400> 70

000

<210> 71

<400> 71

000

<210> 72

<400> 72

000

<210> 73

<400> 73

000

<210> 74

<400> 74

000

<210> 75

<400> 75

000

<210> 76

<400> 76

000

<210> 77

<400> 77

000

<210> 78

<400> 78

000

<210> 79

<211> 444

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis: anti-PD1 Mab heavy chain

<400> 79

<210> 80

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis: anti-PD1 Mab light chain

<400> 80

<210> 81

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis: HCDR1

<400> 81

<210> 82

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis: HCDR2

<400> 82

<210> 83

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis: HCDR3

<400> 83

<210> 84

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis: LCDR1

<400> 84

<210> 85

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis: LCDR2

<400> 85

<210> 86

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis: LCDR3

<400> 86

<210> 87

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis: Anti-PD1 Mab VH

<400> 87

<210> 88

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis: anti-PD1 Mab VL

<400> 88

Claims (124)

A composition or pharmaceutical preparation, which comprises RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, and RNA encoding IFNα protein. The pharmaceutical preparation or composition according to claim 1, which further comprises RNA encoding FLT3-L protein. The composition according to claim 1 or claim 2. The pharmaceutical preparation according to claim 1 or claim 2. The pharmaceutical preparation or composition according to any one of claims 1-4, wherein the RNA has a ratio of 1:1:1 or 1:1:1:1, and wherein the ratio is determined by quantitative RT- PCR verification. The pharmaceutical preparation or composition according to any one of claims 1-5, wherein the RNA integrity is greater than or equal to 70%. The pharmaceutical preparation or composition according to any one of claims 1-6, wherein the pharmaceutical preparation or composition contains less than 250ng DNA/total mg of nucleic acid present. The pharmaceutical preparation or composition according to any one of claims 1-7, wherein the IFNα protein is IFNα2b protein. The pharmaceutical preparation or composition according to any one of claims 1-8, wherein (i) The RNA encoding IL-12sc protein comprises the nucleotide sequence of SEQ ID NO: 17 or 18 or has at least 99%, 98%, 97%, and the nucleotide sequence of SEQ ID NO: 17 or 18; 96%, 95%, 90%, 85% or 80% identical nucleotide sequence; and/or (ii) The IL-12sc protein comprises the amino acid sequence of SEQ ID NO: 14 or has at least 99%, 98%, 97%, 96%, 95%, 90%, and the amino acid sequence of SEQ ID NO: 14 %, 85% or 80% identical amino acid sequence; and/or (iii) The RNA encoding IL-12sc protein contains at least 99%, 98%, 97%, 96%, and the p40 portion of IL-12sc (nucleotides 1-984 of SEQ ID NO: 17 or 18) 95%, 90%, 85% or 80% identity and at least 99%, 98%, 97%, 96% with the p30 portion of IL-12sc (nucleotides 1027-1623 of SEQ ID NO: 17 or 18) , 95%, 90%, 85%, or 80% identical nucleotide sequence, and further comprises nucleotides between the p40 part and the p35 part encoding the linker polypeptide. The pharmaceutical preparation or composition according to any one of claims 1-9, wherein (i) The RNA encoding IL-15 sushi protein comprises the nucleotide sequence of SEQ ID NO: 26 or has at least 99%, 98%, 97%, 96%, and the nucleotide sequence of SEQ ID NO: 26. 95%, 90%, 85% or 80% identical nucleotide sequences; and/or (ii) The IL-15 sushi protein comprises the amino acid sequence of SEQ ID NO: 24 or has at least 99%, 98%, 97%, 96%, 95%, and the amino acid sequence of SEQ ID NO: 24. 90%, 85% or 80% identical amino acid sequence; and/or (iii) The RNA encoding IL-15 sushi protein contains at least 99%, 98%, 97%, and the sushi domain of IL-15 receptor α (nucleotides 1-321 of SEQ ID NO: 26). 96%, 95%, 90%, 85%, or 80% identity and with mature IL-15 (nucleotides 382-729 of SEQ ID NO: 26) at least 99%, 98%, 97%, 96%, A nucleotide sequence of 95%, 90%, 85% or 80% identity, and optionally also contains a nucleoside encoding a linker polypeptide between the sushi domain of IL-15 and the mature IL-15 acid. The pharmaceutical preparation or composition according to any one of claims 1-10, wherein (i) The RNA encoding IFNα protein comprises the nucleotide sequence of SEQ ID NO: 22 or 23 or has at least 99%, 98%, 97%, 96% of the nucleotide sequence of SEQ ID NO: 22 or 23 , 95%, 90%, 85% or 80% identical nucleotide sequences; and/or (ii) The IFNα protein comprises the amino acid sequence of SEQ ID NO: 19 or has at least 99%, 98%, 97%, 96%, 95%, 90%, and the amino acid sequence of SEQ ID NO: 19; 85% or 80% identical amino acid sequence. The pharmaceutical preparation or composition according to any one of claims 2-11, wherein (i) The RNA encoding FLT3-L protein comprises the nucleotide sequence of SEQ ID NO: 32 or at least 99%, 98%, 97%, 96%, 95% of the nucleotide sequence of SEQ ID NO: 32 %, 90%, 85%, or 80% identical nucleotide sequence; and/or (ii) The FLT3-L protein comprises the amino acid sequence of SEQ ID NO: 30 or has at least 99%, 98%, 97%, 96%, 95%, 90%, and the amino acid sequence of SEQ ID NO: 30. %, 85%, or 80% identical amino acid sequence. The pharmaceutical preparation or composition according to any one of claims 1-12, wherein at least one RNA comprises a modified nucleoside replacing at least one uridine. The pharmaceutical preparation or composition according to any one of claims 1-13, wherein at least one RNA comprises a modified nucleoside instead of each uridine. The pharmaceutical preparation or composition according to any one of claims 1-14, wherein each RNA comprises a modified nucleoside replacing at least one uridine. The pharmaceutical preparation or composition according to any one of claims 1-15, wherein each RNA comprises a modified nucleoside instead of each uridine. The pharmaceutical preparation or composition according to any one of claims 13-16, wherein the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5- Methyl-uridine (m5U). The pharmaceutical preparation or composition according to any one of claims 13-17, wherein at least one RNA comprises more than one type of modified nucleosides, wherein the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U). The pharmaceutical preparation or composition according to claim 18, wherein the modified nucleoside is N1- Methyl-pseudouridine (m1ψ). The pharmaceutical preparation or composition according to claim 1-19, wherein at least one RNA comprises ^5'cap m ₂ ^{7, 3'-O} Gppp(m ₁ ^2'-O )ApG or 3'-O-Me-m ⁷ G(5')ppp(5')G. The pharmaceutical preparation or composition according to claim 1-20, wherein each RNA comprises ^5'cap m ₂ ^{7, 3'-O} Gppp(m ₁ ^2'-O )ApG or 3'-O-Me-m ⁷ G(5')ppp(5')G. The pharmaceutical preparation or composition according to claim 1-21, wherein at least one RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and 6 or a combination selected from SEQ ID NO: 4 and 6. The nucleotide sequence of the constituent group has a 5'UTR of a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical. The pharmaceutical preparation or composition according to any one of claims 1-22, wherein each RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and 6 or a combination selected from SEQ ID NO : The nucleotide sequence of the group consisting of 4 and 6 has a 5'UTR of a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical. The pharmaceutical preparation or composition according to any one of claims 1 to 23, wherein at least one RNA comprises the nucleotide sequence of SEQ ID NO: 8 or has at least 99% of the nucleotide sequence of SEQ ID NO: 8 The 3'UTR of a nucleotide sequence with %, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity. The pharmaceutical preparation or composition according to any one of claims 1-24, wherein each RNA comprises a nucleotide sequence containing SEQ ID NO: 8 or having at least 99% of the nucleotide sequence of SEQ ID NO: 8 The 3'UTR of a nucleotide sequence with %, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity. The pharmaceutical preparation or composition according to any one of the preceding claims, wherein at least one RNA comprises a poly-A (poly-A) tail. The pharmaceutical preparation or composition according to any one of claims 1-25, wherein each RNA comprises a poly A tail. The pharmaceutical preparation or composition according to claim 26 or 27, wherein the poly A tail contains at least 100 nucleotides. The pharmaceutical formulation or composition according to any one of claims 26-28, wherein the poly A tail comprises the poly A tail shown in SEQ ID NO:66. The pharmaceutical preparation or composition according to any one of claims 1-29, wherein the one or more RNAs comprise: a. ^5'cap containing m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG or 3'-O-Me-m ⁷ G(5')ppp(5')G; b. comprising (i) a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and 6 or (ii) a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and 6 having at least 99 %, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical nucleotide sequence 5'UTR; c. Comprising (i) the nucleotide sequence of SEQ ID NO: 8 or (ii) the nucleotide sequence of SEQ ID NO: 8 having at least 99%, 98%, 97%, 96%, 95%, 90% , 3'UTR of a nucleotide sequence with 85% or 80% identity; and d. A poly A tail containing at least 100 nucleotides. The pharmaceutical preparation or composition according to claim 30, wherein the poly A tail comprises SEQ ID NO:66. The pharmaceutical preparation or composition according to any one of claims 1-31, which is a pharmaceutical composition containing the RNA. The pharmaceutical preparation or composition according to claim 32, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients. The pharmaceutical preparation or composition according to any one of claims 1-33, wherein the RNA is formulated as a liquid, as a solid, or a combination thereof. The pharmaceutical preparation or composition according to any one of claims 1-34, which is used for pharmaceutical use. The pharmaceutical preparation or composition according to claim 35, wherein the pharmaceutical use includes therapeutic treatment or preventive treatment of diseases or disorders. The pharmaceutical preparation or composition according to claim 36, wherein the therapeutic treatment or preventive treatment of the disease or disorder includes treatment or prevention of solid tumors. The pharmaceutical preparation or composition according to claim 37, wherein the solid tumor is a sarcoma, carcinoma or lymphoma. The pharmaceutical preparation or composition according to any one of claim 37 or 38, wherein the solid tumor is in the lung, colon, ovary, cervix, uterus, peritoneum, testis, penis, tongue, lymph node, pancreas, Bone, breast, prostate, soft tissue, connective tissue, kidney, liver, brain, thyroid or skin. The pharmaceutical preparation or composition according to any one of claims 37-39, wherein the solid tumor is an epithelial tumor, Hodgkin’s lymphoma (HL), non-Hodgkin’s lymphoma, prostate Tumors, ovarian tumors, renal cell tumors, gastrointestinal tumors, liver tumors, colorectal tumors, vascular tumors, mesothelioma tumors, pancreatic tumors, breast tumors, sarcoma tumors, lung tumors, colon tumors, brain tumors, melanomas Tumor, small cell lung tumor, neuroblastoma tumor, testicular tumor, carcinoma tumor, adenocarcinoma tumor, glioma tumor, seminoma tumor, retinoblastoma or osteosarcoma tumor. The pharmaceutical preparation or composition according to any one of claims 1-40, wherein the RNA is for intratumor or peripheral administration. The pharmaceutical preparation or composition according to any one of claims 1-41, wherein the RNA is formulated for injection. The pharmaceutical preparation or composition according to any one of claims 1-42, wherein the RNA is for administration to humans. The pharmaceutical preparation or composition according to any one of claims 37-43, wherein treating or preventing the solid tumor includes reducing the size of a tumor, preventing the recurrence of cancer in remission, or preventing cancer in an individual Disease transfer. The pharmaceutical preparation or composition according to any one of claims 36 to 44, wherein the therapeutic treatment or prophylactic treatment of the disease or disorder further includes administration of another therapy. The pharmaceutical preparation or composition according to claim 45, wherein the additional therapy comprises one or more selected from the group consisting of (i) surgery to remove, excise or reduce tumors, (ii) immunotherapy, (iii) radiation Therapy and (iv) chemotherapy. The pharmaceutical preparation or composition according to any one of claims 45-46, wherein the additional therapy comprises administration of another therapeutic agent. The pharmaceutical preparation or composition according to claim 47, wherein the additional therapeutic agent is an anticancer therapeutic agent. The pharmaceutical preparation or composition according to claim 47 or 48, wherein the additional therapeutic agent is a checkpoint modifier. The pharmaceutical preparation or composition according to claim 49, wherein the checkpoint modulator is an anti-PD1 antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD1 antibody and an anti-CTLA-4 antibody. A method for treating solid tumors or reducing the possibility of solid tumors, which comprises administering a first RNA to an individual in need, wherein the first RNA encodes IL-12sc protein, IL-15 sushi protein, FLT3-L protein Or IFNα protein, and the individual is further treated with additional RNA, wherein: a. If the first RNA encodes IL-12sc protein, the additional RNA includes RNA encoding IL-15 sushi protein, RNA encoding IFNα protein, and RNA encoding FLT3-L protein; or b. If the first RNA encodes IL-15 sushi protein, the additional RNA includes RNA encoding IL-12sc protein, RNA encoding IFNα protein, and RNA encoding FLT3-L protein; or c. If the first RNA encodes IFNα protein, the additional RNA includes RNA encoding IL-15 sushi protein, RNA encoding IL-12sc protein, and RNA encoding FLT3-L protein; or d. If the first RNA encodes FLT3-L protein, then the additional RNA includes RNA encoding IL-15 sushi protein, RNA encoding IFNα protein, and RNA encoding IL-12sc protein; or e. If the first RNA encodes IL-12sc protein, the additional RNA includes RNA encoding IL-15 sushi protein and RNA encoding IFNα protein; or f. If the first RNA encodes IL-15 sushi protein, the additional RNA includes RNA encoding IL-12sc protein and RNA encoding IFNα protein; or g. If the first RNA encodes IFNα protein, the additional RNA includes RNA encoding IL-15 sushi protein and RNA encoding IL-12sc protein. A kit comprising the composition according to any one of claims 1-50. The pharmaceutical preparation according to any one of claims 1-2 or 4-50, wherein the pharmaceutical preparation is a kit. The pharmaceutical preparation according to claim 53 or the kit according to claim 52, wherein the RNA is in a separate vial. The kit according to any one of claims 52-54, which further comprises instructions for using the composition to treat or prevent solid tumors. An RNA for use in a method for treating or preventing solid tumors in an individual, wherein the method comprises administering RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, and RNA encoding IFNα protein . The use of RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein and RNA encoding IFNα protein for treating solid tumors. The RNA or use according to any one of claims 56-57, which further includes RNA encoding FLT3-L protein. The RNA or use according to any one of claims 56-58, wherein the RNA has a ratio of 1:1:1 or 1:1:1:1, and wherein the ratio is verified by quantitative RT-PCR . The RNA or use according to any one of claims 56-59, wherein the RNA integrity is greater than or equal to 70%. The RNA or use according to any one of claims 56-60, wherein the pharmaceutical preparation or composition contains less than 250ng DNA/total mg of nucleic acid present. The RNA or use according to any one of claims 56-61, wherein the IFNα protein is IFNα2b protein. The RNA or use according to any one of claims 56-62, wherein (i) The RNA encoding IL-12sc protein comprises the nucleotide sequence of SEQ ID NO: 17 or 18 or has at least 99%, 98%, 97%, and the nucleotide sequence of SEQ ID NO: 17 or 18; 96%, 95%, 90%, 85% or 80% identical nucleotide sequence; and/or (ii) The IL-12sc protein comprises the amino acid sequence of SEQ ID NO: 14 or has at least 99%, 98%, 97%, 96%, 95%, 90%, and the amino acid sequence of SEQ ID NO: 14 %, 85% or 80% identical amino acid sequence; and/or (iii) The RNA encoding IL-12sc protein contains at least 99%, 98%, 97%, 96%, and the p40 portion of IL-12sc (nucleotides 1-984 of SEQ ID NO: 17 or 18) 95%, 90%, 85% or 80% identity and at least 99%, 98%, 97%, 96% with the p30 portion of IL-12sc (nucleotides 1027-1623 of SEQ ID NO: 17 or 18) , 95%, 90%, 85%, or 80% identical nucleotide sequence, and further comprises nucleotides between the p40 part and the p35 part encoding the linker polypeptide. The RNA or use according to any one of claims 56-63, wherein (i) The RNA encoding IL-15 sushi protein comprises the nucleotide sequence of SEQ ID NO: 26 or has at least 99%, 98%, 97%, 96%, and the nucleotide sequence of SEQ ID NO: 26. 95%, 90%, 85% or 80% identical nucleotide sequences; and/or (ii) The IL-15 sushi protein comprises the amino acid sequence of SEQ ID NO: 24 or has at least 99%, 98%, 97%, 96%, 95%, and the amino acid sequence of SEQ ID NO: 24. 90%, 85% or 80% identical amino acid sequence; and/or (iii) The RNA encoding IL-15 sushi protein contains at least 99%, 98%, 97%, and the sushi domain of IL-15 receptor α (nucleotides 1-321 of SEQ ID NO: 26). 96%, 95%, 90%, 85%, or 80% identity and with mature IL-15 (nucleotides 382-729 of SEQ ID NO: 26) at least 99%, 98%, 97%, 96%, A nucleotide sequence of 95%, 90%, 85% or 80% identity, and optionally also contains a nucleoside encoding a linker polypeptide between the sushi domain of IL-15 and the mature IL-15 acid. The RNA or use according to any one of claims 58-64, wherein (i) The RNA encoding FLT3-L protein comprises the nucleotide sequence of SEQ ID NO: 32 or at least 99%, 98%, 97%, 96%, 95% of the nucleotide sequence of SEQ ID NO: 32 %, 90%, 85%, or 80% identical nucleotide sequence; and/or (ii) The FLT3-L protein comprises the amino acid sequence of SEQ ID NO: 30 or has at least 99%, 98%, 97%, 96%, 95%, 90%, and the amino acid sequence of SEQ ID NO: 30. %, 85%, or 80% identical amino acid sequence. The RNA or use according to any one of claims 56-65, wherein (i) The RNA encoding IFNα protein comprises the nucleotide sequence of SEQ ID NO: 22 or 23 or has at least 99%, 98%, 97%, 96% of the nucleotide sequence of SEQ ID NO: 22 or 23 , 95%, 90%, 85% or 80% identical nucleotide sequences; and/or (ii) The IFNα protein comprises the amino acid sequence of SEQ ID NO: 19 or has at least 99%, 98%, 97%, 96%, 95%, 90%, and the amino acid sequence of SEQ ID NO: 19; 85% or 80% identical amino acid sequence. The RNA or use according to any one of claims 56 to 66, wherein at least one RNA comprises a modified nucleoside in place of at least one uridine. The RNA or use according to any one of claims 56 to 67, wherein at least one RNA comprises a modified nucleoside in place of each uridine. The RNA or use according to any one of claims 56 to 68, wherein each RNA comprises a modified nucleoside in place of at least one uridine. The RNA or use according to any one of claims 56-69, wherein each RNA comprises a modified nucleoside in place of each uridine. The RNA or use according to any one of claims 67-70, wherein the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl -Uridine (m5U). The RNA according to any one of claims 67-71, wherein at least one RNA comprises more than one type of modified nucleosides, wherein the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl -Pseudouridine (m1ψ) and 5-methyl-uridine (m5U). The RNA or use according to claim 72, wherein the modified nucleoside is N1-methyl-pseudouridine (m1ψ). The RNA or use according to any one of claims 56-73, wherein at least one RNA comprises 5'cap m27, 3'-OGppp(m12'-O)ApG or 3'-O-Me-m7G(5' )ppp(5')G. The RNA or use according to any one of claims 56-73, wherein each RNA comprises 5'cap m27, 3'-OGppp(m12'-O)ApG or 3'-O-Me-m7G(5' )ppp(5')G. The RNA or use according to any one of claims 56-75, wherein at least one RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and 6 or is selected from SEQ ID NO: 4 The nucleotide sequence of the group consisting of and 6 has a 5'UTR of a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical. The RNA or the use according to any one of claims 56-76, wherein each RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and 6, or a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 The nucleotide sequence of the group consisting of and 6 has a 5'UTR of a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical. The RNA or use according to any one of claims 56-77, wherein at least one RNA comprises the nucleotide sequence of SEQ ID NO: 8 or has at least 99% of the nucleotide sequence of SEQ ID NO: 8, The 3'UTR of a nucleotide sequence that is 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical. The RNA or use according to any one of claims 56-78, wherein each RNA comprises a nucleotide sequence containing SEQ ID NO: 8 or at least 99% of the nucleotide sequence of SEQ ID NO: 8, The 3'UTR of a nucleotide sequence that is 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical. The RNA or use according to any one of claims 56-79, wherein at least one RNA comprises a poly A tail. The RNA or use according to any one of claims 56 to 80, wherein each RNA comprises a poly A tail. The RNA or use according to claim 80 or 81, wherein the poly A tail contains at least 100 nucleotides. The RNA or use according to any one of claims 80-82, wherein the poly A tail comprises the poly A tail shown in SEQ ID NO:66. The RNA or use according to any one of claims 56-83, wherein one or more RNAs comprise: a. ^5'cap containing m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG or 3'-O-Me-m ⁷ G(5')ppp(5')G; b. comprising (i) a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and 6 or (ii) a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and 6 having at least 99 %, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical nucleotide sequence 5'UTR; c. Comprising (i) the nucleotide sequence of SEQ ID NO: 8 or (ii) the nucleotide sequence of SEQ ID NO: 8 having at least 99%, 98%, 97%, 96%, 95%, 90% , 3'UTR of a nucleotide sequence with 85% or 80% identity; and d. A poly A tail containing at least 100 nucleotides. The RNA or use according to claim 84, wherein the poly A tail comprises SEQ ID NO:66. The RNA or use according to any one of claims 56-85, wherein the method further comprises administering another therapy. The RNA or use according to claim 86, wherein the additional therapy includes one or more selected from the group consisting of (i) surgery to remove, excise or reduce tumors, (ii) immunotherapy, (iii) radiotherapy, and (iv) Chemotherapy. The RNA or use according to claim 86 or 87, wherein the additional therapy comprises administration of another therapeutic agent. The RNA or use according to claim 88, wherein the additional therapeutic agent is an anticancer therapeutic agent. The RNA or use according to claim 86 or 87, wherein the additional therapeutic agent is a checkpoint modulator. The RNA or use according to claim 90, wherein the checkpoint modulator is an anti-PD1 antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD1 antibody and an anti-CTLA-4 antibody. The RNA or use according to any one of claims 56-91, wherein the solid tumor is a sarcoma, carcinoma or lymphoma. The RNA or use according to any one of claims 56-91, wherein the solid tumor is in the lung, colon, ovary, cervix, uterus, peritoneum, testis, penis, tongue, lymph node, pancreas, bone, In the breast, prostate, soft tissue, connective tissue, kidney, liver, brain, thyroid or skin. The RNA or use according to any one of claims 56-93, wherein the solid tumor is an epithelial tumor, Hodgkin’s lymphoma (HL), non-Hodgkin’s lymphoma, prostate tumor, Ovarian tumor, renal cell tumor, gastrointestinal tumor, liver tumor, colorectal tumor, vascular tumor, mesothelioma tumor, pancreas tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, brain tumor, melanoma tumor, Small cell lung tumor, neuroblastoma tumor, testicular tumor, cancer tumor, adenocarcinoma tumor, glioma tumor, seminoma tumor, retinoblastoma or osteosarcoma tumor. The RNA or use according to any one of claims 56-94, wherein the RNA is administered in or around a tumor. The RNA or use according to any one of claims 56-95, wherein the RNA is formulated for injection. The RNA or use according to any one of claims 86-96, wherein the additional therapeutic agent is administered systemically. The RNA or use according to any one of claims 56-97, wherein the individual is human. The RNA or use according to any one of claims 56-98, wherein the RNA is administered simultaneously. The RNA or use according to any one of claims 56 to 99, wherein the RNA is administered by injection, wherein the RNA is mixed together in a liquid solution before injection. The RNA or use according to any one of claims 56-100, wherein the RNA is administered by administering a composition comprising a combination of the RNA. The RNA or use according to any one of claims 56-100, wherein the treatment or prevention Solid tumors include reducing the size of tumors, preventing the recurrence of cancer in remission, or preventing cancer metastasis in an individual. A composition or pharmaceutical preparation comprising RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, RNA encoding IFNα protein, and RNA encoding FLT3-L protein. The composition according to claim 103. The pharmaceutical preparation described in claim 103. The pharmaceutical preparation or composition according to claim 103-105, wherein the RNA has a ratio of 1:1:1 or 1:1:1:1, and wherein the ratio is verified by quantitative RT-PCR. The pharmaceutical preparation or composition according to claim 103-106, wherein the RNA integrity is greater than or equal to 70%. The pharmaceutical preparation or composition according to claim 103-107, wherein the pharmaceutical preparation or composition contains less than 250ng DNA/total mg of nucleic acid present. The pharmaceutical preparation or composition according to claim 103-108, wherein the IFNα protein is IFNα2b protein. The pharmaceutical preparation or composition according to claim 103-109, wherein (i) The RNA encoding IL-12sc protein comprises the nucleotide sequence of SEQ ID NO: 17 or 18 or has at least 99%, 98%, 97%, and the nucleotide sequence of SEQ ID NO: 17 or 18; 96%, 95%, 90%, 85% or 80% identical nucleotide sequence; and/or (ii) The IL-12sc protein comprises the amino acid sequence of SEQ ID NO: 14 or has at least 99%, 98%, 97%, 96%, 95%, 90%, and the amino acid sequence of SEQ ID NO: 14 %, 85% or 80% identical amino acid sequence; and/or (iii) The RNA encoding IL-12sc protein contains at least 99%, 98%, 97%, 96%, and the p40 portion of IL-12sc (nucleotides 1-984 of SEQ ID NO: 17 or 18) 95%, 90%, 85% or 80% identity and at least 99%, 98%, 97%, 96% with the p30 portion of IL-12sc (nucleotides 1027-1623 of SEQ ID NO: 17 or 18) , 95%, 90%, 85% or 80% The nucleotide sequence of identity, and also includes the nucleotide between the p40 part and the p35 part that encodes the linker polypeptide. The pharmaceutical preparation or composition according to claim 103-110, wherein (i) The RNA encoding IL-15 sushi protein comprises the nucleotide sequence of SEQ ID NO: 26 or has at least 99%, 98%, 97%, 96%, and the nucleotide sequence of SEQ ID NO: 26. 95%, 90%, 85% or 80% identical nucleotide sequences; and/or (ii) The IL-15 sushi protein comprises the amino acid sequence of SEQ ID NO: 24 or has at least 99%, 98%, 97%, 96%, 95%, and the amino acid sequence of SEQ ID NO: 24. 90%, 85% or 80% identical amino acid sequence; and/or (iii) The RNA encoding IL-15 sushi protein contains at least 99%, 98%, 97%, and the sushi domain of IL-15 receptor α (nucleotides 1-321 of SEQ ID NO: 26). 96%, 95%, 90%, 85%, or 80% identity and with mature IL-15 (nucleotides 382-729 of SEQ ID NO: 26) at least 99%, 98%, 97%, 96%, A nucleotide sequence of 95%, 90%, 85% or 80% identity, and optionally also contains a nucleoside encoding a linker polypeptide between the sushi domain of IL-15 and the mature IL-15 acid. The pharmaceutical preparation or composition according to claim 103-111, wherein (i) The RNA encoding IFNα protein comprises the nucleotide sequence of SEQ ID NO: 22 or 23 or has at least 99%, 98%, 97%, 96% of the nucleotide sequence of SEQ ID NO: 22 or 23 , 95%, 90%, 85% or 80% identical nucleotide sequences; and/or (ii) The IFNα protein comprises the amino acid sequence of SEQ ID NO: 19 or has at least 99%, 98%, 97%, 96%, 95%, 90%, and the amino acid sequence of SEQ ID NO: 19; 85% or 80% identical amino acid sequence. The pharmaceutical preparation or composition according to claim 103-112, wherein (i) The RNA encoding FLT3-L protein comprises the nucleotide sequence of SEQ ID NO: 32 or at least 99%, 98%, 97%, 96%, 95% of the nucleotide sequence of SEQ ID NO: 32 %, 90%, 85%, or 80% identical nucleotide sequence; and/or (ii) The FLT3-L protein comprises the amino acid sequence of SEQ ID NO: 30 or has at least 99%, 98%, 97%, 96%, 95%, 90%, and the amino acid sequence of SEQ ID NO: 30. %, 85%, or 80% identical amino acid sequence. The pharmaceutical preparation or composition according to claim 103-113, which is a pharmaceutical composition containing the RNA. The pharmaceutical preparation or composition according to claim 114, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients. The pharmaceutical preparation or composition according to claims 103-115, wherein the RNA is formulated as a liquid, as a solid, or a combination thereof. The pharmaceutical preparation or composition according to claim 103-116, which is used for pharmaceutical use. The pharmaceutical preparation or composition according to claims 103-117, wherein the pharmaceutical use includes the therapeutic treatment or preventive treatment of diseases or disorders. The pharmaceutical preparation or composition according to claim 103-118, wherein the therapeutic treatment or prophylactic treatment of the disease or disorder includes the treatment or prevention of solid tumors. The pharmaceutical preparation or composition according to claim 119, wherein the solid tumor is sarcoma, carcinoma or lymphoma. The pharmaceutical preparation or composition according to claim 119-120, wherein the solid tumor is in the lung, colon, ovary, cervix, uterus, peritoneum, testis, penis, tongue, lymph node, pancreas, bone, breast, In the prostate, soft tissue, connective tissue, kidney, liver, brain, thyroid or skin. The pharmaceutical preparation or composition according to claim 103-121, wherein the solid tumor is epithelial tumor, Hodgkin’s lymphoma (HL), non-Hodgkin’s lymphoma, prostate tumor, ovarian Tumor, renal cell tumor, gastrointestinal tumor, liver tumor, colorectal tumor, vascular tumor, mesothelioma tumor, pancreas tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, brain tumor, melanoma tumor, small Cell lung tumor, neuroblastoma tumor, testicular tumor, carcinoma tumor, adenocarcinoma tumor, glioma tumor, seminoma tumor, retinoblastoma or osteosarcoma tumor. An RNA for use in a method for treating or preventing solid tumors in an individual, wherein the method comprises administering RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, RNA encoding IFNα protein And RNA encoding FLT3-L protein. An RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, RNA encoding IFNα protein and RNA encoding FLT3-L are used for treating solid tumors.

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