TW202026423A

TW202026423A - Therapeutic rna for solid tumor cancers

Info

Publication number: TW202026423A
Application number: TW108130212A
Authority: TW
Inventors: 提摩西瓦赫納爾; 弗里德里克吉澤克; 烏格沙辛; 德米特里威德斯奇; 克里斯汀霍爾茨
Original assignee: 法商賽諾菲公司; 德商拜恩迪克Ｒｎａ製藥有限公司
Priority date: 2018-08-24
Filing date: 2019-08-23
Publication date: 2020-07-16
Also published as: WO2020041655A1

Abstract

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

Description

用於實體瘤癌症的治療性RNA Therapeutic RNA for solid tumor cancer

本申請要求2018年8月24日提交的美國臨時申請號62/722,742和2019年1月24日提交的歐洲專利申請號19305092.9的優先權權益；兩者的內容係藉由引用以其整體併入。 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 .

本揭示文本涉及治療實體瘤的治療性RNA的領域。美國國家癌症研究所將實體瘤定義為通常不含有囊腫或液體區域的異常組織塊。實體瘤包括良性和惡性(癌性)肉瘤、癌和淋巴瘤，並且可以物理地位於包括腦、卵巢、***、結腸和其他組織的任何組織或器官中。癌症通常分為兩種主要類型：實體瘤癌症和血液學(血液)癌症。據估計，在美國每年診斷出超過150萬例癌症病例，並且在美國，每年有超過500,000人將死於癌症。 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.

實體瘤癌症尤其難以治療。目前的治療包括手術、放射療法、免疫療法和化學療法。單獨手術可能是針對小局部腫瘤的合適治療，但是大的浸潤性腫瘤和大多數轉移性惡性腫瘤通常不能藉由手術切除。其他常見的治療(諸如放射療法和化學療法)會關聯不希望的副作用和對健康細胞的損害。 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.

儘管手術和當前療法有時能夠殺死大部分實體瘤，但是另外的細胞(潛在地包括癌症幹細胞)可能在療法中存活。隨著時間的流逝，這些細胞可能形成導致癌症復發的新腫瘤。儘管有多模式的常規療法，但是對於許多類型的實體瘤，無病生存率小於25%。耐受多模式的療法的或在療法後已復發的實體瘤甚至更難以治療，並且長期生存率小於10%。 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%.

本文揭示可以克服目前在實體瘤的治療中的缺點的組合物、用途和方法。本文揭示的治療性RNA的投予可以減小腫瘤尺寸、延長生存時間和/或防止腫瘤的轉移和/或復發。 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.

具體實施例1是一種組合物或醫藥製劑，其包含編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA和編碼IFNα蛋白的RNA。 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.

具體實施例2是根據具體實施例1所述的醫藥製劑或組合物，其還包含編碼FLT3-L蛋白的RNA。 Specific embodiment 2 is the pharmaceutical preparation or composition according to specific embodiment 1, which further comprises RNA encoding FLT3-L protein.

具體實施例3是根據具體實施例1或2所述的組合物。 Specific example 3 is the composition according to specific example 1 or 2.

具體實施例4是根據具體實施例1或2所述的醫藥製劑。 Specific embodiment 4 is the pharmaceutical preparation according to specific embodiment 1 or 2.

具體實施例5是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中所述RNA具有1：1：1或1：1：1：1的比率，並且任選地其中所述比率藉由定量RT-PCR驗證。 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.

具體實施例6是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中所述RNA完整性大於或等於70%。 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%.

具體實施例7是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中所述醫藥製劑或組合物包含少於250ng DNA/存在的核酸的總mg。 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.

具體實施例8是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中所述IFNα蛋白是IFNα2b蛋白。 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.

具體實施例9是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中 Specific embodiment 9 is a pharmaceutical preparation or composition according to any one of the foregoing specific embodiments, which in

所述編碼IL-12sc蛋白的RNA包含SEQ ID NO：17或18的核苷酸序列或與SEQ ID NO：17或18的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列；和/或 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

所述IL-12sc蛋白包含SEQ ID NO：14的胺基酸序列或與SEQ ID NO：14的胺基酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的胺基酸序列；和/或 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

所述編碼IL-12sc蛋白的RNA包含與IL-12sc的p40部分(SEQ ID NO：17或18的核苷酸1-984)具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性並且與IL-12sc的p30部分(SEQ ID NO：17或18的核苷酸1027-1623)具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列，並且還包含編碼連接子多肽的在所述p40部分與p35部分之間的核苷酸。 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.

具體實施例10是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中 Specific embodiment 10 is a pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein

所述編碼IL-15 sushi蛋白的RNA包含SEQ ID NO：26的核苷酸序列或與SEQ ID NO：26的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列；和/或 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

所述IL-15 sushi蛋白包含SEQ ID NO：24的胺基酸序列或與SEQ ID NO：24的胺基酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的胺基酸序列；和/或 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

所述編碼IL-15 sushi蛋白的RNA包含與IL-15受體α的sushi結構域(SEQ ID NO：26的核苷酸1-321)具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性並且與成熟IL-15(SEQ ID NO：26的核苷酸382-729)具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列，並且任選地還包含編碼連接子多肽的在IL-15的sushi結構域與所述成熟IL-15之間的核苷酸。 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.

具體實施例11是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中 Specific embodiment 11 is a pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein

所述編碼IFNα蛋白的RNA包含SEQ ID NO：22或23的核苷酸序列或與SEQ ID NO：22或23的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列；和/或 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

所述IFNα蛋白包含SEQ ID NO：19的胺基酸序列或與SEQ ID NO：19的胺基酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的胺基酸序列。 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.

具體實施例12是根據具體實施例2-11中任一項所述的醫藥製劑或組合物，其中 Specific embodiment 12 is the pharmaceutical preparation or composition according to any one of specific embodiments 2-11, wherein

所述編碼FLT3-L蛋白的RNA包含SEQ ID NO：32的核苷酸序列或與SEQ ID NO：32的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列；和/或 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

所述FLT3-L蛋白包含SEQ ID NO：30的胺基酸序列或與SEQ ID NO：30的胺基酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的胺基酸序列。 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.

具體實施例13是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中至少一種RNA包含替代至少一個尿苷的修飾核苷。 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.

具體實施例14是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中至少一種RNA包含替代每個尿苷的修飾核苷。 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.

具體實施例15是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中每種RNA包含替代至少一個尿苷的修飾核苷。 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.

具體實施例16是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中每種RNA包含替代每個尿苷的修飾核苷。 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.

具體實施例17是根據具體實施例13-16中任一項所述的醫藥製劑或組合物，其中所述修飾核苷獨立地選自假尿苷(ψ)、N1-甲基-假尿苷(m1ψ)和5-甲基-尿苷(m5U)。 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).

具體實施例18是根據具體實施例13-17中任一項所述的醫藥製劑或組合物，其中至少一種RNA包含多於一種類型的修飾核苷，其中所述修飾核苷獨立地選自假尿苷(ψ)、N1-甲基-假尿苷(m1ψ)和5-甲基-尿苷(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).

具體實施例19是根據具體實施例18所述的醫藥製劑或組合物，其中所述修飾核苷是N1-甲基-假尿苷(m1ψ)。 Specific embodiment 19 is the pharmaceutical preparation or composition according to specific embodiment 18, wherein the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

具體實施例20是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中至少一種RNA包含5’帽m₂ ^7,3’-OGppp(m₁ ^2’-O)ApG或3’-O-Me-m⁷G(5’)ppp(5’)G。 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.

具體實施例21是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中每種RNA包含5’帽m₂ ^7,3’-OGppp(m₁ ^2’-O)ApG或3’-O-Me-m⁷G(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.

具體實施例22是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中至少一種RNA包含含有選自SEQ ID NO：4和6組成之群組的核苷酸序列或與選自SEQ ID NO：4和6組成之群組的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的5’ UTR。 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.

具體實施例23是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中每種RNA包含含有選自SEQ ID NO：4和6組成之群組的核苷酸序列或與選自SEQ ID NO：4和6組成之群組的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的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.

具體實施例24是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中至少一種RNA包含含有SEQ ID NO：8的核苷酸序列或與SEQ ID NO：8的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的3’ 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.

具體實施例25是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中每種RNA包含含有SEQ ID NO：8的核苷酸序列或與SEQ ID NO：8的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的3’ UTR。 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.

具體實施例26是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中至少一種RNA包含多聚A尾(polyA tail)。 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.

具體實施例27是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中每種RNA包含多聚A尾。 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.

具體實施例28是根據具體實施例26或27所述的醫藥製劑或組合物，其中所述多聚A尾包含至少100個核苷酸。 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.

具體實施例29是根據具體實施例26-28中任一項所述的醫藥製劑或組合物，其中所述多聚A尾包含示於SEQ ID NO：66的多聚A尾。 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.

具體實施例30是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中一種或多種RNA包含： Specific embodiment 30 is the pharmaceutical preparation or composition according to any one of the preceding specific embodiments, wherein one or more RNAs comprise:

包含m₂ ^7,3’-OGppp(m₁ ^2’-O)ApG或3’-O-Me-m⁷G(5’)ppp(5’)G的5’帽； ^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;

包含(i)選自SEQ ID NO：4和6組成之群組的核苷酸序列或(ii)與選自SEQ ID NO：4和6組成之群組的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的5’ UTR； 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;

包含(i)SEQ ID NO：8的核苷酸序列或(ii)與SEQ ID NO：8的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的3’ UTR；和 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

包含至少100個核苷酸的多聚A尾。 A poly A tail containing at least 100 nucleotides.

具體實施例31是根據具體實施例30所述的醫藥製劑或組合物，其中所述多聚A尾包含SEQ ID NO：66。 Specific embodiment 31 is the pharmaceutical preparation or composition according to specific embodiment 30, wherein the poly A tail comprises SEQ ID NO:66.

具體實施例32是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其是包含所述RNA的藥物組合物。 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.

具體實施例33是根據具體實施例32所述的醫藥製劑或組合物，其中所述藥物組合物還包含一種或多種藥學上可接受的載體、稀釋劑和/或賦形劑。 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.

具體實施例34是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中所述RNA被配製為液體、配製為固體或其組合。 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.

具體實施例35是根據前述具體實施例中任一項所述的醫藥製劑或組合物，用於藥物用途。 Specific embodiment 35 is a pharmaceutical preparation or composition according to any one of the foregoing specific embodiments for pharmaceutical use.

具體實施例36是根據具體實施例35所述的醫藥製劑或組合物，其中所述藥物用途包括疾病或障礙的治療性治療或預防性治療。 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.

具體實施例37是根據具體實施例36所述的醫藥製劑或組合物，其中所述疾病或障礙的治療性治療或預防性治療包括治療或預防實體瘤。 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.

具體實施例38是根據具體實施例37所述的醫藥製劑或組合物，其中所述實體瘤是肉瘤、癌或淋巴瘤。 Specific embodiment 38 is the pharmaceutical preparation or composition according to specific embodiment 37, wherein the solid tumor is a sarcoma, carcinoma or lymphoma.

具體實施例39是根據具體實施例37或38中任一項所述的醫藥製劑或組合物，其中所述實體瘤是在肺、結腸、卵巢、子宮頸、子宮、腹膜、睾丸、陰莖、舌、淋巴結、胰臟、骨、***、攝護腺、軟組織、結締組織、腎臟、肝臟、腦、甲狀腺或皮膚中。 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.

具體實施例40是根據具體實施例37-39中任一項所述的醫藥製劑或組合物，其中所述實體瘤是上皮性腫瘤、霍奇金氏淋巴瘤(HL)、非霍奇金氏淋巴瘤、攝護腺腫瘤、卵巢腫瘤、腎細胞腫瘤、胃腸道腫瘤、肝腫瘤、結直腸腫瘤、脈管腫瘤、間皮瘤腫瘤、胰臟腫瘤、***腫瘤、肉瘤腫瘤、肺腫瘤、結腸腫瘤、腦腫瘤、黑色素瘤腫瘤、小細胞肺腫瘤、神經母細胞瘤腫瘤、睾丸腫瘤、癌腫瘤、腺癌腫瘤、膠質瘤腫瘤、精原細胞瘤腫瘤、視網膜母細胞瘤或骨肉瘤腫瘤。 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.

具體實施例41是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中所述RNA用於腫瘤內或腫瘤周邊投予。 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.

具體實施例42是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其中所述RNA被配製用於注射。 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.

具體實施例43是根據前述具體實施例中任一項所述的醫藥製劑或組合物，其用於向人投予。 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.

具體實施例44是根據具體實施例37-43中任一項所述的醫藥製劑或組合物，其中治療或預防所述實體瘤包括減小腫瘤的尺寸、預防緩解期癌症的復發或預防個體的癌症轉移。 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.

具體實施例45是根據具體實施例36-44中任一項所述的醫藥製劑或組合物，其中所述疾病或障礙的治療性治療或預防性治療還包括投予另外的療法。 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.

具體實施例46是根據具體實施例45所述的醫藥製劑或組合物，其中所述另外的療法包括選自以下的一種或多種：(i)手術以切除、割除或消減腫瘤，(ii)免疫療法，(iii)放射療法和(iv)化學療法。 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.

具體實施例47是根據具體實施例45-46中任一項所述的醫藥製劑或組合物，其中所述另外的療法包括投予另外的治療劑。 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.

具體實施例48是根據具體實施例47所述的醫藥製劑或組合物，其中所述另外的治療劑是抗癌治療劑。 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.

具體實施例49是根據具體實施例47或48所述的醫藥製劑或組合物，其中所述另外的治療劑是檢查點調節劑。 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.

具體實施例50是根據具體實施例49所述的醫藥製劑或組合物，其中所述檢查點調節劑是抗PD1抗體、抗CTLA-4抗體或抗PD1抗體和抗CTLA-4抗體的組合。 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.

具體實施例51是一種用於治療實體瘤或減少實體瘤可能性的方法，其包括向有需要的個體投予第一RNA，其中所述第一RNA編碼IL-12sc蛋白、IL-15 sushi蛋白、FLT3-L蛋白或IFNα蛋白，並且所述個體進一步用另外的RNA治療，其中： 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:

如果所述第一RNA編碼IL-12sc蛋白，則所述另外的RNA編碼IL-15 sushi蛋白、IFNα蛋白和FLT3-L蛋白；或 If the first RNA encodes IL-12sc protein, the additional RNA encodes IL-15 sushi protein, IFNα protein, and FLT3-L protein; or

如果所述第一RNA編碼IL-15 sushi蛋白，則所述另外的RNA編碼IL-12sc蛋白、IFNα蛋白和FLT3-L蛋白；或 If the first RNA encodes IL-15 sushi protein, the additional RNA encodes IL-12sc protein, IFNα protein, and FLT3-L protein; or

如果所述第一RNA編碼IFNα蛋白，則所述另外的RNA編碼IL-15 sushi蛋白、IL-12sc蛋白和FLT3-L蛋白；或 If the first RNA encodes IFNα protein, the additional RNA encodes IL-15 sushi protein, IL-12sc protein, and FLT3-L protein; or

如果所述第一RNA編碼FLT3-L蛋白，則所述另外的RNA編碼IL-15 sushi蛋白、IFNα蛋白和IL-12sc蛋白；或 If the first RNA encodes FLT3-L protein, the additional RNA encodes IL-15 sushi protein, IFNα protein, and IL-12sc protein; or

如果所述第一RNA編碼IL-12sc蛋白，則所述另外的RNA編碼IL-15 sushi蛋白和IFNα蛋白；或 If the first RNA encodes IL-12sc protein, the additional RNA encodes IL-15 sushi protein and IFNα protein; or

如果所述第一RNA編碼IL-15 sushi蛋白，則所述另外的RNA編碼IL-12sc蛋白和IFNα蛋白；或 If the first RNA encodes IL-15 sushi protein, the additional RNA encodes IL-12sc protein and IFNα protein; or

如果所述第一RNA編碼IFNα蛋白，則所述另外的RNA編碼IL-15 sushi蛋白和IL-12sc蛋白。 If the first RNA encodes IFNα protein, the additional RNA encodes IL-15 sushi protein and IL-12sc protein.

具體實施例52是一種套組，其包括根據具體實施例1-50中任一項所述的組合物。 Embodiment 52 is a kit comprising the composition according to any one of Embodiments 1-50.

具體實施例53是根據具體實施例1-2或4-50中任一項所述的醫藥製劑，其中所述醫藥製劑是套組。 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.

具體實施例54是根據具體實施例53所述的醫藥製劑或根據具體實施例52所述的套組，其中所述RNA在單獨的小瓶中。 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.

具體實施例55是根據具體實施例52-54中任一項所述的套組，其還包含用於使用所述組合物治療或預防實體瘤的說明。 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.

具體實施例56是一種RNA，所述RNA用於在用於治療或預防個體的實體瘤的方法中使用，其中所述方法包括投予編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA和編碼IFNα蛋白的RNA。 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.

具體實施例57是編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA和編碼IFNα蛋白的RNA用於治療實體瘤的用途。 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.

具體實施例58是根據具體實施例56-57中任一項所述的RNA或用途，其還包括編碼FLT3-L蛋白的RNA。 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.

具體實施例59是根據具體實施例56-58中任一項所述的RNA或用途，其中所述RNA具有1：1：1或1：1：1：1的比率，並且任選地其中所述比率藉由定量RT-PCR驗證。 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.

具體實施例60是根據具體實施例56-59中任一項所述的RNA或用途，其中所述RNA完整性大於或等於70%。 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%.

具體實施例61是根據具體實施例56-60中任一項所述的RNA或用途，其中所述醫藥製劑或組合物包含少於250ng DNA/存在的核酸的總mg。 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.

具體實施例62是根據具體實施例56-61中任一項所述的RNA或用途，其中所述IFNα蛋白是IFNα2b蛋白。 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.

具體實施例63是根據具體實施例56-62中任一項所述的RNA或用途，其中 Specific embodiment 63 is the RNA or use according to any one of specific embodiments 56-62, wherein

具體實施例64是根據具體實施例56-63中任一項所述的RNA或用途，其中 Specific embodiment 64 is the RNA or use according to any one of specific embodiments 56-63, wherein

所述編碼IL-15 sushi蛋白的RNA包含與IL-15受體α的sushi結構域(SEQ ID NO：26的核苷酸1-321)具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性並且與成熟IL-15(SEQ ID NO：26的核苷酸382-729)具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列，並且任選地還包含編碼連接子多肽的在IL-15的sushi結構域與所述成熟IL-15之間的核苷酸。 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.

具體實施例65是根據具體實施例58-64中任一項所述的RNA或用途，其中 Specific embodiment 65 is the RNA or use according to any one of specific embodiments 58-64, wherein

具體實施例66是根據具體實施例56-65中任一項所述的RNA或用途，其中(i)所述編碼IFNα蛋白的RNA包含SEQ ID NO：22或23的核苷酸序列或與SEQ ID NO：22或23的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列；和/或(ii)所述IFNα蛋白包含SEQ ID NO：19的胺基酸序列或與SEQ ID NO：19的胺基酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的胺基酸序列。 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.

具體實施例67是根據具體實施例56-66中任一項所述的RNA或用途，其中至少一種RNA包含替代至少一個尿苷的修飾核苷。 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.

具體實施例68是根據具體實施例56-67中任一項所述的RNA或用途，其中至少一種RNA包含替代每個尿苷的修飾核苷。 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.

具體實施例69是根據具體實施例56-68中任一項所述的RNA或用途，其中每種RNA包含替代至少一個尿苷的修飾核苷。 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.

具體實施例70是根據具體實施例56-69中任一項所述的RNA或用途，其中每種RNA包含替代每個尿苷的修飾核苷。 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.

具體實施例71是根據具體實施例67-70中任一項所述的RNA或用途，其中所述修飾核苷獨立地選自假尿苷(ψ)、N1-甲基-假尿苷(m1ψ)和5-甲基-尿苷(m5U)。 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).

具體實施例72是根據具體實施例67-71中任一項所述的RNA，其中至少一種RNA包含多於一種類型的修飾核苷，其中所述修飾核苷獨立地選自假尿苷(ψ)、N1-甲基-假尿苷(m1ψ)和5-甲基-尿苷(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).

具體實施例73是根據具體實施例72所述的RNA或用途，其中所述修飾核苷是N1-甲基-假尿苷(m1ψ)。 Specific embodiment 73 is the RNA or use according to specific embodiment 72, wherein the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

具體實施例74是根據具體實施例56-73中任一項所述的RNA或用途，其中至少一種RNA包含5’帽m27,3’-OGppp(m12’-O)ApG或3’-O-Me-m7G(5’)ppp(5’)G。 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.

具體實施例75是根據具體實施例56-75中任一項所述的RNA或用途，其中每種RNA包含5’帽m27,3’-OGppp(m12’-O)ApG或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.

具體實施例76是根據具體實施例56-75中任一項所述的RNA或用途，其中至少一種RNA包含含有選自SEQ ID NO：4和6組成之群組的核苷酸序列或與選自SEQ ID NO：4和6組成之群組的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的5’ UTR。 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.

具體實施例77是根據具體實施例56-76中任一項所述的RNA或用途，其中每種RNA包含含有選自SEQ ID NO：4和6組成之群組的核苷酸序列或與選自SEQ ID NO：4和6組成之群組的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的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.

具體實施例78是根據具體實施例56-77中任一項所述的RNA或用途，其中至少一種RNA包含含有SEQ ID NO：8的核苷酸序列或與SEQ ID NO：8的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的3’ 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.

具體實施例79是根據具體實施例56-78中任一項所述的RNA或用途，其中每種RNA包含含有SEQ ID NO：8的核苷酸序列或與SEQ ID NO：8的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的3’ UTR。 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.

具體實施例80是根據具體實施例56-79中任一項所述的RNA或用途，其中至少一種RNA包含多聚A尾。 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.

具體實施例81是根據具體實施例56-80中任一項所述的RNA或用途，其中每種RNA包含多聚A尾。 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.

具體實施例82是根據具體實施例80或81所述的RNA或用途，其中所述多聚A 尾包含至少100個核苷酸。 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.

具體實施例83是根據具體實施例80-82中任一項所述的RNA或用途，其中所述多聚A尾包含示於SEQ ID NO：66的多聚A尾。 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.

具體實施例84是根據具體實施例56-83中任一項所述的RNA或，其中一種或多種RNA包含： Specific embodiment 84 is the RNA according to any one of specific embodiments 56-83, wherein one or more RNAs comprise:

具體實施例85是根據具體實施例84所述的RNA或用途，其中所述多聚A尾包含SEQ ID NO：66。 Specific embodiment 85 is the RNA or use according to specific embodiment 84, wherein the poly A tail comprises SEQ ID NO:66.

具體實施例86是根據具體實施例56-85中任一項所述的RNA或用途，其中所述方法還包括投予另外的療法。 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.

具體實施例87是根據具體實施例86所述的RNA或用途，其中所述另外的療法包括選自以下的一種或多種：(i)手術以切除、割除或消減腫瘤，(ii)免疫療法，(iii)放射療法和(iv)化學療法。 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.

具體實施例88是根據具體實施例86或87所述的RNA或用途，其中所述另外的療法包括投予另外的治療劑。 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.

具體實施例89是根據具體實施例88所述的RNA或用途，其中所述另外的治療劑是抗癌治療劑。 Specific embodiment 89 is the RNA or use according to specific embodiment 88, wherein the additional therapeutic agent is an anti-cancer therapeutic agent.

具體實施例90是根具體據實施例86或87所述的RNA或用途，其中所述另外的治療劑是檢查點調節劑。 Specific embodiment 90 is the RNA or use according to specific embodiment 86 or 87, wherein the additional therapeutic agent is a checkpoint modulator.

具體實施例91是根據具體實施例90所述的RNA或用途，其中所述檢查點調節劑是抗PD1抗體、抗CTLA-4抗體或抗PD1抗體和抗CTLA-4抗體的組合。 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.

具體實施例92是根據具體實施例56-91中任一項所述的RNA或用途，其中所述實體瘤是肉瘤、癌或淋巴瘤。 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.

具體實施例93是根據具體實施例56-91中任一項所述的RNA或用途，其中所述實體瘤是在肺、結腸、卵巢、子宮頸、子宮、腹膜、睾丸、陰莖、舌、淋巴結、胰臟、骨、***、攝護腺、軟組織、結締組織、腎臟、肝臟、腦、甲狀腺或皮膚中。 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.

具體實施例94是根據具體實施例56-93中任一項所述的RNA或用途，其中所述實體瘤是上皮性腫瘤、霍奇金氏淋巴瘤(HL)、非霍奇金氏淋巴瘤、攝護腺腫瘤、卵巢腫瘤、腎細胞腫瘤、胃腸道腫瘤、肝腫瘤、結直腸腫瘤、脈管腫瘤、間皮瘤腫瘤、胰臟腫瘤、***腫瘤、肉瘤腫瘤、肺腫瘤、結腸腫瘤、腦腫瘤、黑色素瘤腫瘤、小細胞肺腫瘤、神經母細胞瘤腫瘤、睾丸腫瘤、癌腫瘤、腺癌腫瘤、膠質瘤腫瘤、精原細胞瘤腫瘤、視網膜母細胞瘤或骨肉瘤腫瘤。 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.

具體實施例95是根據具體實施例56-94中任一項所述的RNA或用途，其中所述RNA是腫瘤內或腫瘤周邊投予的。 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.

具體實施例96是根據具體實施例56-95中任一項所述的RNA或用途，其中所述RNA被配製用於注射。 Specific embodiment 96 is the RNA or use according to any one of specific embodiments 56-95, wherein the RNA is formulated for injection.

實施例97是根據實施例86-96中任一項所述的RNA或用途，其中所述另外的治療劑是全身投予的。 Embodiment 97 is the RNA or use of any one of embodiments 86-96, wherein the additional therapeutic agent is administered systemically.

具體實施例98是根據具體實施例56-97中任一項所述的RNA或用途，其中所述個體是人。 Specific embodiment 98 is the RNA or use according to any one of specific embodiments 56-97, wherein the individual is a human.

具體實施例99是根據具體實施例56-98中任一項所述的RNA或用途，其中所述RNA是同時投予的。 Specific embodiment 99 is the RNA or use according to any one of specific embodiments 56-98, wherein the RNA is administered simultaneously.

具體實施例100是根據具體實施例56-99中任一項所述的RNA或用途，其中所述RNA是藉由注射投予的，其中在注射之前，將所述RNA在液體溶液中混合在一起。 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.

具體實施例101是根據具體實施例56-100中任一項所述的RNA或用途，其中所述RNA是藉由投予包含所述RNA的組合的組合物投予的。 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.

具體實施例102是根據具體實施例56-100中任一項所述的RNA或用途，其中治療或預防實體瘤包括減小腫瘤的尺寸、預防緩解期癌症的復發或預防個體的癌症轉移。 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.

具體實施例103是一種組合物或醫藥製劑，其包含編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA、編碼IFNα蛋白的RNA和編碼FLT3-L蛋白的RNA。 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.

具體實施例104是根據具體實施例103所述的組合物。 Specific embodiment 104 is the composition according to specific embodiment 103.

具體實施例105是根據具體實施例103所述的醫藥製劑。 Specific embodiment 105 is the pharmaceutical preparation according to specific embodiment 103.

具體實施例106是根據具體實施例103-105中任一項所述的醫藥製劑或組合物，其中所述RNA具有1：1：1或1：1：1：1的比率，並且任選地其中所述比率藉由定量RT-PCR驗證。 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.

具體實施例107是根據具體實施例103-106中任一項所述的醫藥製劑或組合物，其中所述RNA完整性大於或等於70%。 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%.

具體實施例108是根據具體實施例103-107中任一項所述的醫藥製劑或組合物，其中所述醫藥製劑或組合物包含少於250ng DNA/存在的核酸的總mg。 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.

具體實施例109是根據具體實施例103-108中任一項所述的醫藥製劑或組合物，其中所述IFNα蛋白是IFNα2b蛋白。 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.

具體實施例110是根據具體實施例103-109所述的醫藥製劑或組合物，其中 Specific embodiment 110 is the pharmaceutical preparation or composition according to specific embodiments 103-109, wherein

具體實施例111是根據具體實施例103-110所述的醫藥製劑或組合物，其中 Specific embodiment 111 is the pharmaceutical preparation or composition according to specific embodiments 103-110, wherein

具體實施例112是根據具體實施例103-111所述的醫藥製劑或組合物，其中 Specific embodiment 112 is the pharmaceutical preparation or composition according to specific embodiments 103-111, wherein

具體實施例113是根據具體實施例103-112所述的醫藥製劑或組合物，其中 Specific embodiment 113 is the pharmaceutical preparation or composition according to specific embodiments 103-112, wherein

具體實施例114是根據具體實施例103-113所述的醫藥製劑或組合物，其是包含所述RNA的藥物組合物。 Specific embodiment 114 is the pharmaceutical preparation or composition according to specific embodiments 103-113, which is a pharmaceutical composition containing the RNA.

具體實施例115是根據具體實施例114所述的醫藥製劑或組合物，其中所述藥物組合物還包含一種或多種藥學上可接受的載體、稀釋劑和/或賦形劑。 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.

具體實施例116是根據具體實施例103-115中任一項所述的醫藥製劑或組合物，其中所述RNA被配製為液體、配製為固體或其組合。 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.

具體實施例117是根據具體實施例103-116所述的醫藥製劑或組合物，用於藥物用途。 Specific embodiment 117 is the pharmaceutical preparation or composition according to specific embodiments 103-116, which is used for medicinal purposes.

具體實施例118是根據具體實施例103-117所述的醫藥製劑或組合物，其中所述藥物用途包括疾病或障礙的治療性治療或預防性治療。 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.

具體實施例119是根據具體實施例103-118所述的醫藥製劑或組合物，其中所述疾病或障礙的治療性治療或預防性治療包括治療或預防實體瘤。 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.

具體實施例120是根據具體實施例119所述的醫藥製劑或組合物，其中所述實體瘤是肉瘤、癌或淋巴瘤。 Specific embodiment 120 is the pharmaceutical preparation or composition according to specific embodiment 119, wherein the solid tumor is a sarcoma, carcinoma or lymphoma.

實施例121是根據具體實施例119-120所述的醫藥製劑或組合物，其中所述實體瘤是在肺、結腸、卵巢、子宮頸、子宮、腹膜、睾丸、陰莖、舌、淋巴結、胰臟、骨、***、攝護腺、軟組織、結締組織、腎臟、肝臟、腦、甲狀腺或皮膚中。 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.

具體實施例122是根據具體實施例103-121所述的醫藥製劑或組合物，其中所述實體瘤是上皮性腫瘤、霍奇金氏淋巴瘤(HL)、非霍奇金氏淋巴瘤、攝護腺腫瘤、卵巢腫瘤、腎細胞腫瘤、胃腸道腫瘤、肝腫瘤、結直腸腫瘤、脈管腫瘤、間皮瘤腫瘤、胰臟腫瘤、***腫瘤、肉瘤腫瘤、肺腫瘤、結腸腫瘤、腦腫瘤、黑色素瘤腫瘤、小細胞肺腫瘤、神經母細胞瘤腫瘤、睾丸腫瘤、癌腫瘤、腺癌腫瘤、膠質瘤腫瘤、精原細胞瘤腫瘤、視網膜母細胞瘤或骨肉瘤腫瘤。 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.

具體實施例123是一種RNA，其用於在用於治療或預防個體的實體瘤的方法中使用，其中所述方法包括投予編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA、編碼IFNα蛋白的RNA和編碼FLT3-L蛋白的RNA。 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.

具體實施例124是編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA、編碼IFNα蛋白的RNA和編碼FLT3-L的RNA用於治療實體瘤的用途。 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.

具體實施例125是一種組合物或醫藥製劑，其包含編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA和編碼IFNα蛋白的RNA(諸如本文所述的)，其中 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.所述編碼IL-12sc蛋白的RNA不是編碼與SEQ ID NO：14的胺基酸序列具有至少95%同一性的IL-12sc蛋白的RNA，和/或不包含與SEQ ID NO：17或18的核苷酸具有至少95%同一性的核苷酸； 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.所述編碼IFNα2b蛋白的RNA不是與SEQ ID NO：19的胺基酸序列具有至少95%同一性的編碼IFNα2b蛋白的RNA，和/或不包含與SEQ ID NO：22或23的核苷酸具有至少95%同一性的核苷酸；並且 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.所述編碼IL-15 sushi蛋白的RNA不是與SEQ ID NO：24的胺基酸序列具有至少95%同一性的編碼IL-15 sushi蛋白的RNA，和/或不包含與SEQ ID NO：26的核苷酸具有至少95%同一性的核苷酸。 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.

序列說明Sequence description

圖1A-圖1H顯示實驗結果，在所述實驗中在第12、15、19和22天向CT26荷瘤小鼠腫瘤內注射RNA(10μg RNA/標靶或40μg對照RNA)，並且監測單獨的腫瘤生長並且標繪至第35天。圖1A顯示用編碼IL-15 sushi、IL-12sc、GM-CSF和IFNα的RNA的混合物處理的結果。圖1B顯示用IL-15 sushi、IL-12sc和IFNα(無GM-CSF)的RNA混合物的處理。圖1C顯示用IL-15 sushi、GM-CSF和IFNα(無IL-12sc)的RNA混合物的處理。圖1D顯示用IL-12sc、GM-CSF和IFNα(無IL-15 sushi)的RNA混合物的處理。圖1E顯示用IL-15 sushi、IL-12sc和GM-CSF(無IFNα)的RNA混合物的處理。圖1F顯示用編碼螢光素酶的對照RNA的處理。圖1G顯示對於每個處理組直到第33天的平均腫瘤體積。圖1H顯示對於每個處理組到第19天的腫瘤生長抑制。 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.

圖2A-圖2C顯示實驗結果，在所述實驗中在第11天向B16F10荷瘤小鼠腫瘤內注射RNA(2μg RNA/標靶或8μg對照RNA)，並且監測單獨的腫瘤生長並且標繪至第62天。圖2A顯示用IL-15 sushi、IL-12sc、GM-CSF和IFNα的RNA混合物的處理；圖2C顯示用IL-15 sushi、IL-12sc和IFNα(無GM-CSF)的RNA混合物的處理；並且圖2C顯示螢光素酶對照。 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.

圖3A-圖3E顯示實驗結果，在所述實驗中在第13、16、20和24天(A-B)或在第11、14、17和21天(C-D)向CT26荷瘤小鼠腫瘤內注射RNA(5μg RNA/標靶或20μg對照RNA)，並且監測單獨的腫瘤生長並且標繪至第50天。圖3A和圖3C顯示用IL-15 sushi、IL-12sc、IFNα和FLT3-L的RNA混合物的處理。圖3B和圖3D顯示螢光素酶對照。圖3E顯示用IL-15 sushi、IL-12sc、IFNα和GM-CSF的RNA混合物的處理。 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.

圖4A-圖4B顯示實驗結果，在所述實驗中在第11天向B16F10荷瘤小鼠腫瘤內注射RNA(2μg RNA/標靶或8μg對照RNA)，並且監測單獨的腫瘤生長並且標繪至第57天。圖4A顯示用IL-15 sushi、IL-12sc、IFNα和GM-CSF的RNA混合物的處理；圖4B顯示用IL-15 sushi、IL-12sc、IFNα和FLT3-L的RNA混合物的處理；並且圖4C顯示螢光素酶對照。 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.

圖5A-圖5B顯示實驗結果，在所述實驗中向B16F10荷瘤小鼠腫瘤內注射RNA(2μg RNA/標靶或8μg對照RNA)，並且在腫瘤內注射後7天，將腫瘤取出、解離並且用一組抗體染色以評估腫瘤內免疫群。列舉了用於不同處理組的CD8+ T細胞和NK細胞的數目。 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.

圖6A-圖6C顯示實驗結果，在所述實驗中向CT26荷瘤小鼠腫瘤內注射RNA(5μg RNA/標靶或20μg對照RNA)，並且在處理後第19和31天，評估血液中gp70特異性CD8+ T細胞的百分比。圖6A顯示第19天並且圖6B顯示第31天，來自同一實驗。在相似設計的獨立研究中，在第24天評估血液中gp70特異性CD8+ T細胞(圖6C)。 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.定義1. Definition

在一些具體實施例中，所述RNA包含替代至少一個(例如，每個)尿苷的修飾核鹼基。在一些具體實施例中，所述RNA包含在RNA的5’末端的Cap1結構。在一些具體實施例中，所述RNA包含替代至少一個(例如，每個)尿苷的修飾核鹼基，和在RNA的5’末端的Cap1結構。在一些具體實施例中，所述5’ UTR包含SEQ ID NO：4或6。在一些具體實施例中，已例如像藉由在纖維素上純化(如實例中所述的和如本領域已知的)或藉由高效液相色譜法(HPLC)對RNA進行處理以減少雙鏈RNA(dsRNA)。“Cap1”結構可以在體外轉譯後藉由酶促加帽或在體外轉譯(共轉錄加帽)期間生成。 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).

在一些具體實施例中，用於修飾RNA的結構單元帽如下，所述結構單元帽在共轉錄加帽以下時使用： 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:

m₂ ^7,3’-OGppp(m₁ ^2’-O)ApG(有時也稱為m₂ ^7,3`OG(5’)ppp(5’)m^2’-OApG)，其具有以下結構： 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:

以下是在共轉錄加帽後的示例性Cap1 RNA，其包含RNA和m₂ ^7,3`OG(5’)ppp(5’)m^2’-OApG： 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:

以下是在酶促加帽(無帽類似物)後的另一個示例性Cap1 RNA： The following is another exemplary Cap1 RNA after enzymatic capping (no cap analog):

在一些具體實施例中，所述RNA被“Cap0”結構修飾，所述結構是用在一個具體實施例中帽類似物抗反向帽(ARCA帽(m₂ ^7,3`OG(5’)ppp(5’)G))進行體外轉譯(共轉錄加帽)期間產生的，所述帽類似物抗反向帽具有以下結構： 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:

以下是包含RNA和m₂ ^7,3`OG(5’)ppp(5’)G的示例性Cap0 RNA： The following is an exemplary Cap0 RNA containing RNA and m ₂ ^7,3'O G(5')ppp(5')G:

在一些具體實施例中，所述“Cap0”結構是在使用帽類似物β-S-ARCA(m₂ ^7,2`OG(5’)ppSp(5’)G)進行體外轉譯(共轉錄加帽)時產生的，所述帽類似物β-S-ARCA具有以下結構： 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:

以下是包含β-S-ARCA(m₂ ^7,2`OG(5’)ppSp(5’)G)和RNA的示例性Cap0 RNA。 The following is an exemplary Cap0 RNA containing β-S-ARCA (m ₂ ^7,2'O G(5')ppSp(5')G) and RNA.

如本文所用，術語“尿嘧啶”描述可以在RNA的核酸中出現的核鹼基中的一種。尿嘧啶的結構是： 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:

如本文所用，術語“尿苷”描述可以在RNA中出現的核苷中的一種。尿苷的結構是： As used herein, the term "uridine" describes one of the nucleosides that can occur in RNA. The structure of uridine is:

UTP(尿苷5’-三磷酸酯)具有以下結構： UTP (uridine 5'-triphosphate) has the following structure:

假-UTP(假尿苷5’-三磷酸酯)具有以下結構： Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure:

“假尿苷”是作為尿苷異構體的修飾核苷的一個例子，其中尿嘧啶藉由碳-碳鍵而不是氮-碳糖苷鍵附接到戊糖環上。例如，假尿苷描述於Charette和Gray,Life；49：341-351(2000)中。 "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).

另一個示例性修飾核苷是N1-甲基假尿苷(m1Ψ)，其具有以下結構： Another exemplary modified nucleoside is N1-methylpseudouridine (m1Ψ), which has the following structure:

N1-甲基假-UTP具有以下結構： N1-methyl pseudo-UTP has the following structure:

如本文所用，術語“多聚A尾”或“多聚A序列”是指典型地位於RNA分子的3’端的腺苷酸殘基的不中斷或中斷序列。多聚A尾或多聚A序列是本領域技術人員已知的，並且可以在本文所述的RNA中的3’UTR之後。不中斷多聚A尾的特徵在於連續的腺苷酸殘基。實際上，不中斷多聚A尾是典型的。本文揭示的RNA可以具有在轉錄後藉由非模板依賴性RNA聚合酶附接到RNA的游離3’端的多聚A尾或藉由DNA編碼並且藉由模板依賴性RNA聚合酶轉錄的多聚A尾。 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.

已證明，約120個A核苷酸的多聚A尾對轉染的真核細胞中RNA的水平以及從存在於多聚A尾上游(5’)的開放閱讀框轉譯的蛋白質的水平具有有益的影響(Holtkamp等人，2006,Blood，第108卷，第4009-4017頁)。 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).

多聚A尾可以具有任何長度。在一個具體實施例中，多聚A尾包含至少20、至少30、至少40、至少80或至少100並且最多達500、最多達400、最多達300、最多達200或最多達150個A核苷酸並且特別是約120個A核苷酸，基本上由其組成或由其組成。在此上下文中，“基本上由...組成”意指多聚A尾中的大多數核苷酸，典型地多聚A尾中的按數目計至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%或至少99%的核苷酸是A核苷酸，但允許其餘核苷酸是除A核苷酸之外的其他核苷酸，諸如U核苷酸(尿苷酸)、G核苷酸(鳥苷酸)或C核苷酸(胞苷酸)。在此上下文中，“由...組成”意指多聚A尾中的所有核苷酸，即多聚A尾中的按數目計100%的核苷酸是A核苷酸。術語“A核苷酸”或“A”是指腺苷酸。 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.

在一些具體實施例中，在基於DNA模板進行RNA轉錄期間，例如在製備體外轉錄的RNA期間，附接多聚A尾，所述DNA模板在與編碼鏈互補的鏈中包含重複的dT核苷酸(脫氧胸苷酸)。編碼多聚A尾的DNA序列(編碼鏈)稱為多聚(A)盒。 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.

在一些具體實施例中，存在於DNA的編碼股中的多聚(A)盒基本上由dA核苷酸組成，但是被四種核苷酸(dA、dC、dG和dT)的隨機序列中斷。此類隨機序列的長度可以是5至50、10至30或10至20個核苷酸。此類盒公開於WO 2016/005324 A1中，將其特此藉由引用併入。WO 2016/005324 A1中揭示的任何多聚(A)盒可以用於本發明。基本上由dA核苷酸組成但是被具有平均分佈的四種核苷酸(dA、dC、dG和dT)並且具有例如5至50個核苷酸的長度的隨機序列中斷的多聚(A)盒顯示出在大腸桿菌中在DNA水平上恒定的質粒DNA繁殖並且在RNA水平上仍關聯與支持RNA穩定性有關的有益特性，並且涵蓋轉譯效率。因此，在一些具體實施例中，在本文所述的RNA分子中含有的多聚A尾基本上由A核苷酸組成，但是被四種核苷酸(A、C、G、U)的隨機序列中斷。此類隨機序列的長度可以是5至50、10至30或10至20個核苷酸。 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.

在一些具體實施例中，沒有除A核苷酸外的其他核苷酸將多聚A尾側接在其3’末端，即多聚A尾未被掩蓋或在其3’末端不接連除A外的其他核苷酸。 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.

在一些具體實施例中，多聚A尾包含序列： 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”和“mRNA”在本文中可互換使用。 "RNA" and "mRNA" are used interchangeably herein.

本文中上義性使用“IFNα”來描述任何I型干擾素α細胞介素，包括IFNα2b和IFNα4。可以將任何IFNα摻入組合物中並且用於本文所述的方法。 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.

如本文所用，術語“治療”涵蓋對於個體的疾病治療劑的任何投予或施用，並且包括抑制疾病、阻止其發展、緩解疾病的一種或多種症狀、治癒疾病、或預防疾病的復發。例如，實體瘤的治療可以包括減輕實體瘤的症狀、減小實體瘤的尺寸、消除實體瘤、降低腫瘤的進一步生長、或在治療後減少或消除實體瘤的復發。治療也可以作為有效性生物標誌物上的或成像或射線照相測量上的變化來測量。 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.

如本文所用，術語“預防”意指抑制被認為無癌症的個體的包括實體瘤在內的癌症或阻止其發展。 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.

“轉移”意指其中癌症從其最初作為原發腫瘤首先出現的位置擴散到身體其他位置的過程。 "Metastasis" means a process in which cancer spreads from where it first appeared as the primary tumor to other locations in the body.

如本文所用，術語“腫瘤內”意指進入腫瘤。例如，腫瘤內注射意指在接觸腫瘤的任何位置注射治療劑。 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.

如本文所用，術語“腫瘤周邊(peri-tumorally或peri-tumoral)”是約2-mm寬並且與腫瘤周圍的浸潤性前沿相鄰的區域。腫瘤周邊區域包含宿主組織。參見，例如： 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:

“投予”意指向個體提供藥劑或組合物，並且包括但不限於由醫學專業人員投予和自投予。 "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.

本揭示文本描述了分別與給定核酸序列或胺基酸序列(參考序列)具有一定同一性程度的核酸序列和胺基酸序列。 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.

兩個核酸序列之間的“序列同一性”指示序列之間相同的核苷酸的百分比。兩個胺基酸序列之間的“序列同一性”指示序列之間相同的胺基酸的百分比。 "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.

術語“%相同”、“%同一性”或類似術語旨在具體是指在待比較的序列之間的最佳比對中相同的核苷酸或胺基酸的百分比。所述百分比是純粹統計學的，並且兩個序列之間的差異可以但不一定隨機分佈在待比較的序列的整個長度上。兩個序列的比較通常藉由以下方式進行：在最佳比對之後，關於片段或“比較窗口”比較所述序列，以鑑定相應序列的局部區域。比較的最佳比對可以手動進行或者借助Smith和Waterman,1981,Ads App.Math.2,482的局部同源性算法、借助Neddleman和Wunsch,1970,J.Mol.Biol.48,443的局部同源性算法、借助Pearson和Lipman,1988,Proc.Natl Acad.Sci.USA 88,2444的相似度搜索算法、或借助使用所述算法的電腦程式(Wisconsin Genetics Software Package中的GAP、BESTFIT、FASTA、BLAST P、BLAST N和TFASTA,Genetics Computer Group,575 Science Drive，威斯康星州麥迪遜)來進行。 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).

百分比同一性藉由以下方式獲得：確定待比較的序列對應的相同位置的數目，用此數目除以比較的位置的數目(例如，參考序列中的位置的數目)，並且然後將此結果乘以100。 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.

在一些具體實施例中，同一性程度是針對區域給出的，所述區域是參考序列的整個長度的至少約50%、至少約60%、至少約70%、至少約80%、至少約90%或約100%。例如，如果參考核酸序列由200個核苷酸組成，則針對至少約100、至少約120、至少約140、至少約160、至少約180或約200個核苷酸(在一些具體實施例中為連續核苷酸)給出同一性程度。在一些具體實施例中，針對參考序列的整個長度給出同一性程度。 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.

分別與給定核酸序列或胺基酸序列具有特定同一性程度的核酸序列或胺基酸序列可具有所述給定序列的至少一種功能特性，例如並且在一些情況下，在功能上等同於所述給定序列。一個重要的特性包括充當細胞介素的能力，特別是當投予個體時。在一些具體實施例中，與給定核酸序列或胺基酸序列具有一定同一性程度的核酸序列和胺基酸序列在功能上等同於所述給定序列。 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.組合物和醫藥製劑2. Compositions and pharmaceutical preparations

在一些具體實施例中，提供了一種組合物或醫藥製劑，其包含編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA和編碼IFNα蛋白的RNA。在一些情況下，所述醫藥製劑或組合物還包含編碼FLT3-L蛋白的RNA。 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.

在一些具體實施例中，提供了一種組合物，所述組合物包含編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA和編碼IFNα蛋白的RNA。 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.

在一些具體實施例中，提供了一種醫藥製劑，其包含編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA和編碼IFNα蛋白的RNA。 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.

在一些具體實施例中，提供了一種組合物，其包含編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA、編碼IFNα蛋白的RNA和編碼FLT3-L蛋白的RNA。 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.

在一些具體實施例中，提供了一種醫藥製劑，其包含編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA、編碼IFNα蛋白的RNA和編碼FLT3-L的RNA。 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.

在一些具體實施例中，所述醫藥製劑或組合物包含編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA、編碼IFNα蛋白的RNA和任選地編碼FLT3-L的RNA，其中所述RNA具有1：1：1或1：1：1：1(當存在FLT3-L時)的質量比率，任選地其中所述比率藉由定量RT-PCR或相似方法驗證。在一些具體實施例中，“RNA比率”藉由定量RT-PCR或相似方法確定。在一些具體實施例中，RNA比率藉由以下方式確定：1)逆轉錄RNA混合物；並且2)使用Taqman探針藉由微滴式數字PCR(例如，藉由Bio-Rad方法)定量與混合物中每種RNA對應的每種cDNA。基於陽性事件的絕對計數，確定單獨的序列的比率，並且從而確定混合物中RNA的比率。 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.

在一些具體實施例中，所述醫藥製劑或組合物包含編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA、編碼IFNα蛋白的RNA和任選地編碼FLT3-L的RNA，其中“RNA完整性”(即，完整的RNA的百分比)大於或等於70%。在一些具體實施例中，RNA完整性是全長RNA(即，未降解的RNA)相對於總RNA(即，全長RNA和降解的RNA)的質量比率百分比。在一些具體實施例中，使用Experion全自動電泳系統(Bio-Rad)或相似技術確定RNA完整性。在一些具體實施例中，RNA完整性大於或等於70%、71%、72%、73%、74%、75%、80%、85%、90%、95%、96%、97%、98%或99%。 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%.

在一些具體實施例中，所述組合物或醫藥製劑中的每種RNA以一定長度存在，使得所述RNA產生其編碼的蛋白質。在一些具體實施例中，所述組合物或醫藥製劑中的每種RNA都具有一定RNA完整性，所述RNA完整性維持由所述RNA編碼的蛋白質的產生。 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.

在一些具體實施例中，所述醫藥製劑或組合物包含編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA、編碼IFNα蛋白的RNA和任選地編碼FLT3-L的RNA，其中所述醫藥製劑或組合物包含少於250ng DNA/存在的核酸的總mg。在一些具體實施例中，藉由針對DNA模板內的區域的qPCR或相似技術來驗證DNA模板的有效去除。然後基於參考標準曲線計算DNA模板的殘餘量。在一些具體實施例中，所述醫藥製劑或組合物包含少於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或1ng DNA/存在的核酸的總mg。 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.

在一些具體實施例中，所述組合物或醫藥製劑的至少95%包含治療性RNA(例如，編碼治療性細胞介素的RNA、檢查點抑制劑和/或旨在治療的其他RNA)。 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).

在一些具體實施例中，所述組合物和醫藥製劑中每種RNA的數量足以產生其編碼的蛋白質。 In some embodiments, the amount of each RNA in the composition and pharmaceutical preparation is sufficient to produce the protein it encodes.

在一些具體實施例中，所述組合物和醫藥製劑中每種RNA的數量是當被腫瘤內或腫瘤周邊投予時其有助於治療或預防實體瘤癌症的總體效果的量。也就是說，如果從組合物或醫藥製劑中省略所述組合物或醫藥製劑中的單獨的RNA，則如與包含被省略的RNA的組合物或醫藥製劑相比，實體瘤癌症的治療或預防將降低。 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.

在一些具體實施例中，組合物和醫藥製劑中每種RNA的數量足以產生基本上如下體外生物效應： 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:

將RNA轉染到合適的細胞株中，並且表達所述RNA編碼的蛋白質。可以分離所述蛋白質，並且基本上如下測量生物活性： 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)人IL-15 sushi的活性可以藉由測量CTLL-2細胞株的增殖(Paxton RJ.2001.Measurement of interleukin 15.Curr Protoc Immunol第6章：單元6 22)來確定，所述CTLL-2細胞株是生長依賴細胞介素(諸如IL-2、IL-15或其他常見受體γ-鏈細胞介素家族成員)的鼠T-細胞株。 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)人IL-12sc的活性可以藉由IL-12-特異性HEK-Blue細胞株HEK-Blue^TM IL-12測量鹼性磷酸酶活性來確定，所述細胞株表達依賴於IL-12的鹼性磷酸酶(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)。IL-12生物活性的檢測基於經由IL-12的STAT4途徑的激活，因為HEK-Blue^TM IL-12細胞含有STAT4誘導性SEAP(分泌的胚胎鹼性磷酸酶)報告基因系統。 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)人IFNα2b的活性可以在IFNα特異性HEK-Blue細胞株HEK-Blue^TM IFN-α/β中測量鹼性磷酸酶活性(Breivik等人)來確定，所述細胞株表達依賴於IFNα的鹼性磷酸酶。IFNα2b活性的檢測基於經由I型干擾素的JAK-STAT途徑的激活，因為HEK-Blue^TM IFN-α/β細胞含有JAK-STAT誘導性SEAP報告基因系統。 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)FLT3-L的活性可以藉由在小鼠LIF重組蛋白的存在下使用基於IL-6定量的ELISA測量小鼠髓性白血病M1細胞中IL-6產生的誘導來確定。 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.

對於每個具體實施例，所述RNA可以是如下和表1中所述的。 For each specific example, the RNA may be as described below and in Table 1.

A.白細胞介素-12單鏈(IL-12sc)A. Interleukin-12 single chain (IL-12sc)

在一些具體實施例中，提供了一種組合物或醫藥製劑，其包含編碼白細胞介素-12單鏈(IL-12sc)的RNA。在一些具體實施例中，所述白細胞介素-12單鏈(IL-12sc)RNA由編碼白細胞介素-12單鏈(IL-12sc)(例如，SEQ ID NO：14)的DNA序列編碼，所述RNA包含IL-12 p40(有時稱為IL-12B；由SEQ ID NO：15的核苷酸1-984編碼)、連接子(諸如GS連接子)和IL-12 p35(有時稱為IL-12A；由SEQ ID NO：15的核苷酸1027-1623編碼)。在一些具體實施例中，所述IL-12p40、連接子和IL-12p35是連續的，其中沒有間插核苷酸。SEQ ID NO：15中提供了編碼IL-12sc的示例性DNA序列。在一些具體實施例中，所述白細胞介素-12單鏈(IL-12sc)RNA提供在SEQ ID NO：17或18(兩者均編碼SEQ ID NO：14的蛋白質)上。IL-12 p40的RNA序列示於SEQ ID NO：17或18的核苷酸1-984，並且IL-12 p35的RNA序列示於SEQ ID NO：17或18的核苷酸1027-1623。 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.

密碼子優化的IL-12 p40與天然IL-12 p40的比對在以下示出，其中“S”是天然IL-12 p40(NM_002187.2；SEQ ID NO：15的核苷酸1-984)並且“Q”是密碼子優化的IL-12 p40(SEQ ID NO：16的核苷酸1-984)。百分比同一性是77%。 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%.

密碼子優化的IL-12 p35與天然IL-12 p35的比對在以下示出，其中“S”是天然IL-12 p35(NM_00882.3；SEQ ID NO：15的核苷酸1027-1623)並且“Q”是密碼子優化的IL-12 p35(SEQ ID NO：16的核苷酸1027-1623)。百分比同一性是80%。 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%.

在一些具體實施例中，所述IL-12sc RNA藉由編碼IL-12sc的密碼子優化的DNA序列編碼。在一些具體實施例中，所述IL-12sc RNA藉由編碼IL-12 p40的密碼子優化的DNA序列編碼。在一些具體實施例中，所述IL-12sc RNA藉由編碼IL-12 p35的密碼子優化的DNA序列編碼。在一些具體實施例中，所述密碼子優化的DNA序列包含SEQ ID NO：16或由其組成。在一些具體實施例中，所述DNA序列包含與SEQ ID NO：16具有70%、75%、80%、85%、90%、95%、96%、97%、98%或99%同一性的密碼子優化的DNA序列。在一些具體實施例中，編碼IL-12 p40的密碼子優化的DNA序列包含編碼IL-12sc-p40的核苷酸(SEQ ID NO：16的核苷酸1-984)。在一些具體實施例中，編碼IL-12 p35的密碼子優化的DNA序列包含編碼IL-12sc-p35的核苷酸(SEQ ID NO：16的核苷酸1027-1623)。在一些具體實施例中，編碼IL-12sc的密碼子優化的DNA序列包含SEQ ID NO：16的編碼IL-12sc-p40(SEQ ID NO：16的核苷酸1-984)和-p35(SEQ ID NO：16的核苷酸1027-1623)部分的核苷酸，並且還包含編碼連接p40和p35部分的連接子多肽的在p40部分與p35部分之間的核苷酸(例如，SEQ ID NO：16的核苷酸985-1026)。可以使用本領域技術人員已知的任何連接子。p40部分可以是5’或3’到p35部分的。 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.

在一些具體實施例中，所述IL-12sc RNA包含RNA序列，所述RNA序列是例如從編碼IL-12sc的DNA序列轉錄的。RNA也可以重組產生。在一些具體實施例中，所述RNA序列是從包含SEQ ID NO：15或16的核苷酸序列轉錄的。在一些具體實施例中，所述RNA序列包含SEQ ID NO：17或18或由其組成。在一些具體實施例中，所述RNA序列包含與SEQ ID NO：17或18具有70%、75%、80%、85%、90%、95%、96%、97%、98%或99%同一性的RNA序列或由其組成。在一些具體實施例中，所述RNA序列包含SEQ ID NO：17或18的編碼IL-12sc-p40(SEQ ID NO：17或18的核苷酸1-984)和-p35(SEQ ID NO：17或18的核苷酸1027-1623)部分的核苷酸。在一些具體實施例中，編碼IL-12sc的密碼子優化的RNA序列包含SEQ ID NO：18的編碼IL-12sc-p40(SEQ ID NO：18的核苷酸1-984)和-p35(SEQ ID NO：18的核苷酸1027-1623)部分的核苷酸，並且含編碼連接p40和p35部分的還包連接子多肽的在p40部分與p35部分之間的核苷酸。可以使用本領域技術人員已知的任何連接子。 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.

在一些具體實施例中，IL-12sc RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。在一些具體實施例中，所述RNA包含替代每個尿苷的修飾核苷。在一些具體實施例中，所述修飾核苷是N1-甲基-假尿苷(m¹ψ)。 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).

在一些具體實施例中，所述IL-12sc RNA包含在5’末端的改變的核苷酸。在一些具體實施例中，所述RNA包含5’帽。可以使用本領域已知的任何5’帽。在一些具體實施例中，所述5’帽包含5’至5’三磷酸鍵。在一些具體實施例中，所述5’帽包含包括硫代磷酸酯修飾的5’至5’三磷酸鍵。在一些具體實施例中，所述5’帽包含2’-O或3’-O-核糖-甲基化核苷酸。在一些具體實施例中，所述5’帽包含修飾鳥苷核苷酸或修飾腺苷核苷酸。在一些具體實施例中，所述5’帽包含7-甲基鳥苷酸。在一些具體實施例中，所述5’帽是Cap0或Cap1。示例性帽結構包括m7G(5’)ppp(5’)G、m7,2`O-mG(5’)pp_Sp(5’)G、m7G(5’)ppp(5’)2`O-mG和m7,3`O-mG(5’)ppp(5’)2`O-mA。 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.

在一些具體實施例中，所述IL-12sc RNA包含5’非轉譯區(UTR)。在一些具體實施例中，所述5’ UTR在起始密碼子的上游。在一些具體實施例中，所述5’ UTR調節RNA的轉譯。在一些具體實施例中，所述5’ UTR是穩定化序列。在一些具體實施例中，所述5’ UTR延長RNA的半衰期。可以使用本領域中已知的任何5’ UTR。在一些具體實施例中，所述5’ UTR RNA序列是從SEQ ID NO：3或5轉錄的。在一些具體實施例中，所述5’ UTR RNA序列包含SEQ ID NO：4或6或由其組成。在一些具體實施例中，所述5’ UTR RNA序列與SEQ ID NO：4或6至少70%、 75%、80%、85%、90%、95%、96%、97%、98%或99%相同。 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.

在一些具體實施例中，所述IL-12sc RNA包含3’ UTR。在一些具體實施例中，所述3’ UTR在轉譯終止密碼子之後。在一些具體實施例中，所述3’ UTR調節RNA的聚腺苷酸化、轉譯效率、定位或穩定性。在一些具體實施例中，所述3’ UTR RNA序列是從SEQ ID NO：7轉錄的。在一些具體實施例中，所述3’ UTR RNA序列包含SEQ ID NO：8或由其組成。在一些具體實施例中，所述3’ UTR RNA序列與SEQ ID NO：8至少70%、75%、80%、85%、90%、95%、96%、97%、98%或99%相同。 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.

在一些具體實施例中，所述IL-12sc RNA包含5’ UTR和3’ UTR兩者。在一些具體實施例中，所述IL-12sc RNA僅包含5’ UTR。在一些具體實施例中，所述IL-12sc RNA僅包含3’ UTR。 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.

在一些具體實施例中，所述IL-12sc RNA包含多聚A尾。在一些具體實施例中，所述RNA包含至少約25、至少約30、至少約50個核苷酸、至少約70個核苷酸或至少約個100核苷酸的多聚A尾。在一些具體實施例中，所述多聚A尾包含200個或更多個核苷酸。在一些具體實施例中，所述多聚A尾包含SEQ ID NO：60或由其組成。 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.

在一些具體實施例中，所述RNA按順序包含5’帽、5’ UTR、編碼IL-12sc的核酸、3’ UTR和多聚A尾。 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.

在一些具體實施例中，所述IL-12sc RNA包含DNA序列，所述DNA序列包含與SEQ ID NO：15或16至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同並且與SEQ ID NO：3或5至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。 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.

在一些具體實施例中，所述IL-12sc RNA包含RNA序列，所述RNA序列是例如從DNA序列轉錄的，所述DNA序列包含與SEQ ID NO：15或16至少70%、75%、 80%、85%、90%、95%、96%、97%、98%、99%或100%相同並且與SEQ ID NO：3或5至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。RNA也可以重組產生。在一些具體實施例中，IL-12sc RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。在一些具體實施例中，所述RNA包含替代每個尿苷的修飾核苷。在一些具體實施例中，所述修飾核苷是N1-甲基-假尿苷(m¹ψ)。 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).

在一些具體實施例中，所述IL-12sc RNA是由DNA序列編碼的，所述DNA序列包含與SEQ ID NO：15或16至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同並且與SEQ ID NO：7至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。 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.

在一些具體實施例中，所述IL-12sc RNA包含RNA序列，所述RNA序列是例如從DNA序列轉錄的，所述DNA序列包含與SEQ ID NO：15或16至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同並且與SEQ ID NO：7至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。RNA也可以重組產生。在一些具體實施例中，IL-12sc RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。在一些具體實施例中，所述RNA包含替代每個尿苷的修飾核苷。在一些具體實施例中，所述修飾核苷是N1-甲基-假尿苷(m¹ψ)。 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).

在一些具體實施例中，所述IL-12sc RNA包含DNA序列，所述DNA序列包含與SEQ ID NO：15或16至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；與SEQ ID NO：3或5至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；並且與SEQ ID NO：7至少70%、75%、80%、 85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。 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.

在一些具體實施例中，所述IL-12sc RNA包含RNA序列，所述RNA序列是例如從DNA序列轉錄的，所述DNA序列包含與SEQ ID NO：15或16至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；與SEQ ID NO：3或5至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；並且與SEQ ID NO：7至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。RNA也可以重組產生。在一些具體實施例中，IL-12sc RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。在一些具體實施例中，所述RNA包含替代每個尿苷的修飾核苷。在一些具體實施例中，所述修飾核苷是N1-甲基-假尿苷(m¹ψ)。 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).

在一些具體實施例中，所述IL-12sc RNA包含RNA序列，所述RNA序列包含與SEQ ID NO：17或18至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；與SEQ ID NO：4或6至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；並且與SEQ ID NO：8至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。在一些具體實施例中，IL-12sc RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。 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.干擾素α(IFNα)B. Interferon α (IFNα)

在一些具體實施例中，提供了一種組合物或醫藥製劑，其包含編碼干擾素α(IFNα)的RNA。在一些具體實施例中，所述干擾素α(IFNα)RNA藉由編碼干擾素α(IFNα)(例如，SEQ ID NO：19)的DNA序列編碼。SEQ ID NO：20中提供了編碼此IFNα的示例性DNA序列。 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.

密碼子優化的IFNα與天然IFNα的比對在以下示出，其中“S”是天然IFNα(NM_000605.3；SEQ ID NO：20)並且“Q”是密碼子優化的IFNα(SEQ ID NO：21)。百分比同一性是79%。 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%.

在一些具體實施例中，所述IFNα RNA藉由編碼IFNα的密碼子優化的DNA編碼。在一些具體實施例中，所述密碼子優化的DNA序列包含SEQ ID NO：21的核苷酸或由其組成。在一些具體實施例中，所述DNA序列包含與SEQ ID NO：21具有70%、75%、80%、85%、90%、95%、96%、97%、98%或99%同一性的密碼子優化的DNA序列或由其組成。 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.

在一些具體實施例中，所述IFNα RNA包含RNA序列，所述RNA序列是例如從編碼IFNα的DNA序列轉錄的。RNA也可以重組產生。在一些具體實施例中，所述RNA序列是從包含SEQ ID NO：20或21的核苷酸序列轉錄的。在一些具體實施例中，所述RNA序列包含SEQ ID NO：22或23或由其組成。在一些具體實施例中，所述RNA序列包含與SEQ ID NO：22或23具有70%、75%、80%、85%、90%、95%、96%、97%、98%或99%同一性的RNA序列或由其組成。 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.

在一些具體實施例中，IFNα RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。在一些具體實施例中，RNA中的每個尿苷都被修飾。在一些具體實施例中，RNA中的每個尿苷都用N1-甲基-假尿苷(m¹ψ)修飾。 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).

在一些具體實施例中，所述IFNα RNA包含在5’末端的改變的核苷酸。在一些具體實施例中，所述IFNα RNA包含5’帽。可以使用本領域已知的任何5’帽。在一些具體實施例中，所述5’帽包含5’至5’三磷酸鍵。在一些具體實施例中，所述5’帽包含包括硫代磷酸酯修飾的5’至5’三磷酸鍵。在一些具體實施例中，所述5’帽包含2’-O或3’-O-核糖-甲基化核苷酸。在一些具體實施例中，所述5’帽包含修飾鳥苷核苷酸或修飾腺苷核苷酸。在一些具體實施例中，所述5’帽包含7-甲基鳥苷酸。在一些具體實施例中，所述5’帽是Cap0或Cap1。示例性帽結構包括m7G(5’)ppp(5’)G、m7,2`O-mG(5’)pp_Sp(5’)G、m7G(5’)ppp(5’)2`O-mG和m7,3`O-mG(5’)ppp(5’)2`O-mA。 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.

在一些具體實施例中，所述IFNα RNA包含5’非轉譯區(UTR)。在一些具體實施例中，所述5’ UTR在起始密碼子的上游。在一些具體實施例中，所述5’ UTR調節RNA的轉譯。在一些具體實施例中，所述5’ UTR是穩定化序列。在一些具體實施例中，所述5’ UTR延長RNA的半衰期。可以使用本領域中已知的任何5’ UTR。在一些具體實施例中，所述5’ UTR RNA序列是從包含SEQ ID NO：3或5 的核苷酸序列轉錄的。在一些具體實施例中，所述5’ UTR RNA序列包含SEQ ID NO：4或6或由其組成。在一些具體實施例中，所述5’ UTR RNA序列與SEQ ID NO：4或6至少70%、75%、80%、85%、90%、95%、96%、97%、98%或99%相同。 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.

在一些具體實施例中，所述IFNα RNA包含3’ UTR。在一些具體實施例中，所述3’ UTR在轉譯終止密碼子之後。在一些具體實施例中，所述3’ UTR調節RNA的聚腺苷酸化、轉譯效率、定位或穩定性。在一些具體實施例中，所述3’ UTR RNA序列是從包含SEQ ID NO：7的核苷酸序列轉錄的。在一些具體實施例中，所述3’ UTR RNA序列包含SEQ ID NO：8或由其組成。在一些具體實施例中，所述3’ UTR RNA序列與SEQ ID NO：8至少70%、75%、80%、85%、90%、95%、96%、97%、98%或99%相同。 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.

在一些具體實施例中，所述IFNα RNA包含5’ UTR和3’ UTR兩者。在一些具體實施例中，所述組合物僅包含5’ UTR。在一些具體實施例中，所述組合物僅包含3’ UTR。 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.

在一些具體實施例中，所述IFNα RNA包含多聚A尾。在一些具體實施例中，所述IFNα RNA包含至少約25、至少約30、至少約50個核苷酸、至少約70個核苷酸或至少約100個核苷酸的多聚A尾。在一些具體實施例中，所述多聚A尾包含200個或更多個核苷酸。在一些具體實施例中，所述多聚A尾包含SEQ ID NO：66或由其組成。 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.

在一些具體實施例中，所述RNA按順序包含5’帽、5’ UTR、編碼IFNα的核酸、3’ UTR和多聚A尾。 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.

在一些具體實施例中，所述IFNα RNA是由DNA序列編碼的，所述DNA序列包含與SEQ ID NO：20或21至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同並且與SEQ ID NO：3或5至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。 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.

在一些具體實施例中，所述IFNα RNA包含RNA序列，所述RNA序列是例如從DNA序列轉錄的，所述DNA序列包含與SEQ ID NO：20或21至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同並且與SEQ ID NO：3或5至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。RNA也可以重組產生。在一些具體實施例中，IFNα RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。在一些具體實施例中，所述RNA包含替代每個尿苷的修飾核苷。在一些具體實施例中，所述修飾核苷是N1-甲基-假尿苷(m¹ψ)。 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).

在一些具體實施例中，所述IFNα RNA是由DNA序列編碼的，所述DNA序列包含與SEQ ID NO：20或21至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同並且與SEQ ID NO：7至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。 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.

在一些具體實施例中，所述IFNα RNA包含RNA序列，所述RNA序列是例如從DNA序列轉錄的，所述DNA序列包含與SEQ ID NO：20或21至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的並且與SEQ ID NO：7至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。在一些具體實施例中，IFNα RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。在一些具體實施例中，所述RNA包含替代每個尿苷的修飾核苷。在一些具體實施例中，所述修飾核苷是N1-甲基-假尿苷(m¹ψ)。 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).

在一些具體實施例中，所述IFNα RNA由DNA序列編碼，所述DNA序列包含與SEQ ID NO：20或21至少70%、75%、80%、85%、90%、95%、96%、97%、98%、 99%或100%相同；與SEQ ID NO：1、3或5至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；並且與SEQ ID NO：7至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。 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 .

在一些具體實施例中，所述IFNα RNA包含RNA序列，所述RNA序列是例如從DNA序列轉錄，所述DNA序列包含與SEQ ID NO：20或21至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；與SEQ ID NO：3或5至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；並且與SEQ ID NO：7至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。RNA也可以重組產生。在一些具體實施例中，IFNα RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。在一些具體實施例中，所述RNA包含替代每個尿苷的修飾核苷。在一些具體實施例中，所述修飾核苷是N1-甲基-假尿苷(m¹ψ)。在一些具體實施例中，所述組合物包含RNA序列，所述RNA序列包含與SEQ ID NO：22或23至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；與SEQ ID NO：4或6至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；並且與SEQ ID NO：8至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。在一些具體實施例中，IFNα RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。 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.白細胞介素15(IL-15)sushiC. Interleukin 15 (IL-15) sushi

在一些具體實施例中，提供了一種組合物或醫藥製劑，其包含編碼白細胞介素15(IL-15)sushi的RNA。如本文所用，術語“IL-15 sushi”描述了包含可溶性白細胞介素15(IL-15)受體α sushi結構域和成熟白細胞介素α(IL-15)的構建體作為融合蛋白。在一些具體實施例中，所述IL-15 sushi RNA由編碼IL-15 sushi(SEQ ID NO：24)的DNA序列編碼，所述DNA序列包含可溶性IL-15受體α鏈(sushi)其後是甘胺酸-絲胺酸(GS)連接子其後是IL-15的成熟序列。SEQ ID NO：25中提供了編碼此IL-15 sushi的DNA序列。 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.

在一些具體實施例中，所述IL-15 sushi RNA是例如從編碼IL-15 sushi的DNA序列轉錄的RNA序列。RNA也可以重組產生。在一些具體實施例中，所述RNA序列是從包含SEQ ID NO：25的核苷酸序列轉錄的。在一些具體實施例中，編碼所述連接子的核苷酸可以完全不存在或部分或全部被編碼合適連接子的任何核苷酸替代。在一些具體實施例中，所述RNA序列包含SEQ ID NO：26或由其組成。在一些具體實施例中，所述RNA序列包含與SEQ ID NO：26具有70%、75%、80%、85%、90%、95%、96%、97%、98%或99%同一性的RNA序列。在一些具體實施例中，所述編碼IL-15 sushi的DNA或RNA序列包含編碼IL-15受體α的sushi結構域(例如，SEQ ID NO：25或26的核苷酸1-321)和成熟IL-15(例如，SEQ ID NO：25或26的核苷酸382-729)的核苷酸。在一些具體實施例中，所述編碼IL-15 sushi的DNA或RNA序列包含編碼IL-15受體α的sushi結構域(例如，SEQ ID NO：25或26的核苷酸1-321)和成熟IL-15(例如，SEQ ID NO：25或26的核苷酸382-729)的核苷酸，並且還包含編碼連接所述部分的連接子多肽的在這些部分之間的核苷酸。在一些具體實施例中，所述連接子包含SEQ ID NO：25或26的核苷酸322-381。可以使用本領域技術人員已知的任何連接子。 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.

在一些具體實施例中，IL-15 sushi RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。在一些具體實施例中，所述RNA包含替代每個尿苷的修飾核苷。在一些具體實施例中，所述修飾核苷是N1-甲基 -假尿苷(m¹ψ)。 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).

在一些具體實施例中，所述IL-15 sushi RNA包含在5’末端的改變的核苷酸。在一些具體實施例中，所述IL-15 sushi RNA包含5’帽。可以使用本領域已知的任何5’帽。在一些具體實施例中，所述5’帽包含5’至5’三磷酸鍵。在一些具體實施例中，所述5’帽包含包括硫代磷酸酯修飾的5’至5’三磷酸鍵。在一些具體實施例中，所述5’帽包含2’-O或3’-O-核糖-甲基化核苷酸。在一些具體實施例中，所述5’帽包含修飾鳥苷核苷酸或修飾腺苷核苷酸。在一些具體實施例中，所述5’帽包含7-甲基鳥苷酸。在一些具體實施例中，所述5’帽是Cap0或Cap1。示例性帽結構包括m7G(5’)ppp(5’)G、m7,2`O-mG(5’)pp_Sp(5’)G、m7G(5’)ppp(5’)2`O-mG和m7,3`O-mG(5’)ppp(5’)2`O-mA。 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.

在一些具體實施例中，所述IL-15 sushi RNA包含5’非轉譯區(UTR)。在一些具體實施例中，所述5’ UTR在起始密碼子的上游。在一些具體實施例中，所述5’ UTR調節RNA的轉譯。在一些具體實施例中，所述5’ UTR是穩定化序列。在一些具體實施例中，所述5’ UTR延長RNA的半衰期。可以使用本領域中已知的任何5’ UTR。在一些具體實施例中，所述5’ UTR RNA序列是從SEQ ID NO：3或5轉錄的。在一些具體實施例中，所述5’ UTR RNA序列包含SEQ ID NO：4或6或由其組成。在一些具體實施例中，所述5’ UTR RNA序列與SEQ ID NO：4或6至少70%、75%、80%、85%、90%、95%、96%、97%、98%或99%相同。 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.

在一些具體實施例中，所述IL-15 sushi RNA包含3’ UTR。在一些具體實施例中，所述3’ UTR在轉譯終止密碼子之後。在一些具體實施例中，所述3’ UTR調節RNA的聚腺苷酸化、轉譯效率、定位或穩定性。在一些具體實施例中，所述3’ UTR RNA序列是從SEQ ID NO：7轉錄的。在一些具體實施例中，所述3’ UTR RNA序列包含SEQ ID NO：8或由其組成。在一些具體實施例中，所述3’ UTR RNA序列與SEQ ID NO：8至少70%、75%、80%、85%、90%、95%、96%、97%、98% 或99%相同。 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.

在一些具體實施例中，所述IL-15 sushi RNA包含5’ UTR和3’ UTR兩者。在一些具體實施例中，所述IL-15 sushi RNA僅包含5’ UTR。在一些具體實施例中，所述IL-15 sushi RNA僅包含3’ UTR。 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.

在一些具體實施例中，所述IL-15 sushi RNA包含多聚A尾。在一些具體實施例中，所述RNA包含至少約25、至少約30、至少約50個核苷酸、至少約70個核苷酸或至少約個100核苷酸的多聚A尾。在一些具體實施例中，所述多聚A尾包含200個或更多個核苷酸。在一些具體實施例中，所述多聚A尾包含SEQ ID NO：66或由其組成。 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.

在一些具體實施例中，所述RNA按順序包含5’帽、5’ UTR、編碼IL-15 sushi的核酸、3’ UTR和多聚A尾。 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.

在一些具體實施例中，所述IL-15 sushi RNA是由DNA序列編碼的，所述DNA序列包含與SEQ ID NO：25至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同並且與SEQ ID NO：3或5至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。 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.

在一些具體實施例中，所述IL-15 sushi RNA包含RNA序列，所述RNA序列是例如從DNA序列轉錄的，所述DNA序列包含與SEQ ID NO：25至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同並且與SEQ ID NO：3或5至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。RNA也可以重組產生。在一些具體實施例中，IFNα RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。在一些具體實施例中，所述RNA包含替代每個尿苷的修飾核苷。在一些具體實施例中，所述修飾核苷是N1-甲基-假尿苷(m¹ψ)。 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).

在一些具體實施例中，所述IL-15 sushi RNA包含DNA序列，所述DNA序列包含與SEQ ID NO：25至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同並且與SEQ ID NO：7至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。 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.

在一些具體實施例中，所述IL-15 sushi RNA包含RNA序列，所述RNA序列是例如從DNA序列轉錄的，所述DNA序列包含與SEQ ID NO：25至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同並且與SEQ ID NO：7至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。RNA也可以重組產生。在一些具體實施例中，IFNα RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。在一些具體實施例中，所述RNA包含替代每個尿苷的修飾核苷。在一些具體實施例中，所述修飾核苷是N1-甲基-假尿苷(m¹ψ)。 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).

在一些具體實施例中，所述IL-15 sushi RNA包含DNA序列，所述DNA序列包含與SEQ ID NO：25至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；與SEQ ID NO：3或5至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；並且與SEQ ID NO：7至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。 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.

在一些具體實施例中，所述IL-15 sushi RNA包含RNA序列，所述RNA序列是例如從DNA序列轉錄的，所述DNA序列包含與SEQ ID NO：25至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；與SEQ ID NO：3或5至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；並且與SEQ ID NO：7至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。在一些具體實施例中，IFNα RNA 中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。在一些具體實施例中，所述RNA包含替代每個尿苷的修飾核苷。在一些具體實施例中，所述修飾核苷是N1-甲基-假尿苷(m¹ψ)。 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).

在一些具體實施例中，所述IL-15 sushi RNA包含RNA序列，所述RNA序列包含與SEQ ID NO：26至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；與SEQ ID NO：4或6至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；並且與SEQ ID NO：8至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。在一些具體實施例中，IFNα RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。 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相關酪胺酸激酶3配體(FLT3-L)D. Fms-related tyrosine kinase 3 ligand (FLT3-L)

在一些具體實施例中，投予包含編碼Fms相關酪胺酸激酶3配體(FLT3-L)的RNA的組合物或醫藥製劑。在一些具體實施例中，所述FLT3-L RNA藉由編碼FLT3-L的DNA序列(例如，SEQ ID NO：31)編碼。 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).

在一些具體實施例中，所述FLT3-L RNA包含RNA序列，所述RNA序列是例如從編碼FLT3-L的DNA序列轉錄的。在一些具體實施例中，所述RNA序列是從SEQ ID NO：31轉錄的。RNA也可以重組產生。在一些具體實施例中，所述RNA序列包含SEQ ID NO：32或由其組成。在一些具體實施例中，所述RNA序列包含與SEQ ID NO：32具有70%、75%、80%、85%、90%、95%、96%、97%、98%或99%同一性的RNA序列。 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.

在一些具體實施例中，FLT3-L RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1- 甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。在一些具體實施例中，所述RNA包含替代每個尿苷的修飾核苷。在一些具體實施例中，所述修飾核苷是N1-甲基-假尿苷(m¹ψ)。在一些具體實施例中，所述FLT3-L RNA包含在5’末端的改變的核苷酸。在一些具體實施例中，所述RNA包含5’帽。可以使用本領域已知的任何5’帽。在一些具體實施例中，所述5’帽包含5’至5’三磷酸鍵。在一些具體實施例中，所述5’帽包含包括硫代磷酸酯修飾的5’至5’三磷酸鍵。在一些具體實施例中，所述5’帽包含2’-O或3’-O-核糖-甲基化核苷酸。在一些具體實施例中，所述5’帽包含修飾鳥苷核苷酸或修飾腺苷核苷酸。在一些具體實施例中，所述5’帽包含7-甲基鳥苷酸。在一些具體實施例中，所述5’帽是Cap0或Cap1。示例性帽結構包括m7G(5’)ppp(5’)G、m7,2`O-mG(5’)pp_Sp(5’)G、m7G(5’)ppp(5’)2`O-mG和m7,3`O-mG(5’)ppp(5’)2`O-mA。 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.

在一些具體實施例中，所述FLT3-L RNA包含5’非轉譯區(UTR)。在一些具體實施例中，所述5’ UTR在起始密碼子的上游。在一些具體實施例中，所述5’ UTR調節RNA的轉譯。在一些具體實施例中，所述5’ UTR是穩定化序列。在一些具體實施例中，所述5’ UTR延長RNA的半衰期。可以使用本領域中已知的任何5’ UTR。在一些具體實施例中，所述5’ UTR RNA序列是從SEQ ID NO：3或5轉錄的。在一些具體實施例中，所述5’ UTR RNA序列包含SEQ ID NO：4或6或由其組成。在一些具體實施例中，所述5’ UTR RNA序列與SEQ ID NO：4或6至少70%、75%、80%、85%、90%、95%、96%、97%、98%或99%相同。 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.

在一些具體實施例中，所述FLT3-L RNA包含3’ UTR。在一些具體實施例中，所述3’ UTR在轉譯終止密碼子之後。在一些具體實施例中，所述3’ UTR調節RNA的聚腺苷酸化、轉譯效率、定位或穩定性。在一些具體實施例中，所述3’ UTR RNA序列是從SEQ ID NO：7轉錄的。在一些具體實施例中，所述3’ UTR RNA序列包含SEQ ID NO：8或由其組成。在一些具體實施例中，所述3’ UTR RNA序列與SEQ ID NO：8至少70%、75%、80%、85%、90%、95%、96%、97%、98%或99%相同。 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.

在一些具體實施例中，所述FLT3-L RNA包含5’ UTR和3’ UTR兩者。在一些具體實施例中，所述RNA僅包含5’ UTR。在一些具體實施例中，所述組合物僅包含3’ UTR。 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.

在一些具體實施例中，所述FLT3-L RNA包含多聚A尾。在一些具體實施例中，所述RNA包含至少約25、至少約30、至少約50個核苷酸、至少約70個核苷酸或至少約個100核苷酸的多聚A尾。在一些具體實施例中，所述多聚A尾包含200個或更多個核苷酸。在一些具體實施例中，所述多聚A尾包含SEQ ID NO：66或由其組成。 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.

在一些具體實施例中，所述FLT3-L RNA按順序包含5’帽、5’ UTR、編碼FLT3-L的核酸、3’ UTR和多聚A尾。 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.

在一些具體實施例中，所述FLT3-L RNA是由DNA序列編碼的，所述DNA序列包含與SEQ ID NO：31至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同並且與SEQ ID NO：3或5至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。 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.

在一些具體實施例中，所述FLT3-L RNA包含RNA序列，所述RNA序列是例如從DNA序列轉錄的，所述DNA序列包含與SEQ ID NO：31至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同並且與SEQ ID NO：3或5至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。RNA也可以重組產生。在一些具體實施例中，FLT3-L RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。在一些具體實施例中，所述RNA包含替代每個尿苷的修飾核苷。在一些具體實施例中，所述修飾核苷是N1-甲基-假尿苷(m¹ψ)。 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).

在一些具體實施例中，所述FLT3-L RNA是由DNA序列編碼的，所述DNA序列包含與SEQ ID NO：31至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同並且與SEQ ID NO：7至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。 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.

在一些具體實施例中，所述FLT3-L RNA包含RNA序列，所述RNA序列是例如從DNA序列轉錄的，所述DNA序列包含與SEQ ID NO：32至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同並且與SEQ ID NO：7至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。RNA也可以重組產生。在一些具體實施例中，FLT3-L RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。在一些具體實施例中，所述RNA包含替代每個尿苷的修飾核苷。在一些具體實施例中，所述修飾核苷是N1-甲基-假尿苷(m¹ψ)。 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).

在一些具體實施例中，所述FLT3-L RNA包含DNA序列，所述DNA序列包含與SEQ ID NO：31至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；與SEQ ID NO：3或5至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；並且與SEQ ID NO：7至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。 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.

在一些具體實施例中，所述FLT3-L RNA包含RNA序列，所述RNA序列是例如從DNA序列轉錄的，所述DNA序列包含與SEQ ID NO：32至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；與SEQ ID NO：3或5至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；並且與SEQ ID NO：7至少70%、75%、80%、85%、90%、95%、96%、97%、98%、 99%或100%相同的核酸序列或由其組成。RNA也可以重組產生。在一些具體實施例中，FLT3-L RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。在一些具體實施例中，所述RNA包含替代每個尿苷的修飾核苷。在一些具體實施例中，所述修飾核苷是N1-甲基-假尿苷(m¹ψ)。 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).

在一些具體實施例中，所述FLT3-L RNA包含RNA序列，所述RNA序列包含與SEQ ID NO：32至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；與SEQ ID NO：4或6至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同；並且與SEQ ID NO：8至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%相同的核酸序列或由其組成。在一些具體實施例中，FLT3-L RNA中的一個或多個尿苷被如本文所述的修飾核苷替代。在一些具體實施例中，替代尿苷的修飾核苷是假尿苷(ψ)、N1-甲基-假尿苷(m¹ψ)或5-甲基-尿苷(m⁵U)。 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.修飾RNAE. Modified RNA

本文所述的每種RNA可以本領域技術人員已知的任何方式修飾。在一些具體實施例中，每種RNA修飾如下： 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:

˙替代每個尿苷的修飾核鹼基； ˙Replace each modified nucleobase of uridine;

˙在RNA的5’末端的Cap1結構。 ˙Cap1 structure at the 5'end of RNA.

在一些具體實施例中，所述5’ UTR包含SEQ ID NO：4或6。在一些具體實施例中，已經處理所述RNA以減少雙鏈RNA(dsRNA)，如上所述。“Cap1”結構可以在體外轉譯後藉由酶促加帽或在體外轉譯(共轉錄加帽)期間生成。 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).

在一些具體實施例中，所述RNA中的一個或多個尿苷被修飾核苷替代。在一些具體實施例中，所述修飾核苷是修飾尿苷。 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.

在一些具體實施例中，替代尿苷的修飾尿苷是假尿苷(ψ)、N1-甲基-假尿苷(m1ψ)或5-甲基-尿苷(m5U)。 In some specific embodiments, the modified uridine that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine Glycoside (m1ψ) or 5-methyl-uridine (m5U).

在一些具體實施例中，所述RNA中的一個或多個胞嘧啶、腺嘌呤或鳥嘌呤被一個或多個修飾核鹼基替代。在一個具體實施例中，替代胞嘧啶的修飾核鹼基是5-甲基胞嘧啶(m⁵C)。在另一個具體實施例中，替代腺嘌呤的修飾核鹼基是N⁶-甲基腺嘌呤(m⁶A)。在另一個具體實施例中，可以使用本領域已知用於降低分子的免疫原性的任何其他修飾核鹼基。 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.

替代RNA中的一個或多個尿苷的修飾核苷可以是以下中的任何一種或多種：3-甲基-尿苷(m³U)、5-甲氧基-尿苷(mo⁵U)、5-氮雜-尿苷、6-氮雜-尿苷、2-硫代-5-氮雜-尿苷、2-硫代-尿苷(s²U)、4-硫代-尿苷(s⁴U)、4-硫代-假尿苷、2-硫代-假尿苷、5-羥基-尿苷(ho⁵U)、5-胺基烯丙基-尿苷、5-鹵代-尿苷(例如，5-碘-尿苷或5-溴-尿苷)、尿苷5-氧基乙酸(cmo⁵U)、尿苷5-氧基乙酸甲酯(mcmo⁵U)、5-羧甲基-尿苷(cm⁵U)、1-羧甲基-假尿苷、5-羧基羥甲基-尿苷(chm⁵U)、5-羧基羥甲基-尿苷甲酯(mchm⁵U)、5-甲氧基羰基甲基-尿苷(mcm⁵U)、5-甲氧基羰基甲基-2-硫代-尿苷(mcm⁵s²U)、5-胺基甲基-2-硫代-尿苷(nm⁵s²U)、5-甲基胺基甲基-尿苷(mnm⁵U)、1-乙基-假尿苷、5-甲基胺基甲基-2-硫代-尿苷(mnm⁵s²U)、5-甲基胺基甲基-2-硒基-尿苷(mnm⁵se²U)、5-胺基甲醯基甲基-尿苷(ncm⁵U)、5-羧甲基胺基甲基-尿苷(cmnm⁵U)、5-羧甲基胺基甲基-2-硫代-尿苷(cmnm⁵s²U)、5-丙炔基-尿苷、1-丙炔基-假尿苷、5-牛磺醯甲基-尿苷(τm⁵U)、1-牛磺醯甲基-假尿苷、5-牛磺醯甲基-2-硫代-尿苷(τm5s2U)、1-牛磺醯甲基-4-硫代-假尿苷、5-甲基-2-硫代-尿苷(m⁵s²U)、1-甲基-4-硫代-假尿苷(m¹s⁴ψ)、4-硫代-1-甲基-假尿苷、3-甲基-假尿苷(m³ψ)、2-硫代-1-甲基-假尿苷、1-甲基-1-去氮雜-假尿苷、2-硫代-1-甲基-1-去氮雜-假尿苷、二氫尿苷(D)、二氫假尿苷、5,6-二氫尿苷、5-甲基-二氫尿苷(m⁵D)、2-硫代-二氫尿苷、2-硫代-二氫假尿苷、2-甲氧基-尿苷、2-甲氧基-4-硫代-尿苷、4-甲氧基-假尿苷、4- 甲氧基-2-硫代-假尿苷、N1-甲基-假尿苷、3-(3-胺基-3-羧丙基)尿苷(acp³U)、1-甲基-3-(3-胺基-3-羧丙基)假尿苷(acp³ψ)、5-(異戊烯基胺基甲基)尿苷(inm⁵U)、5-(異戊烯基胺基甲基)-2-硫代-尿苷(inm⁵s²U)、α-硫代-尿苷、2’-O-甲基-尿苷(Um)、5,2’-O-二甲基-尿苷(m⁵Um)、2’-O-甲基-假尿苷(ψm)、2-硫代-2’-O-甲基-尿苷(s²Um)、5-甲氧基羰基甲基-2’-O-甲基-尿苷(mcm⁵Um)、5-胺基甲醯基甲基-2’-O-甲基-尿苷(ncm⁵Um)、5-羧甲基胺基甲基-2’-O-甲基-尿苷(cmnm⁵Um)、3,2’-O-二甲基-尿苷(m³Um)、5-(異戊烯基胺基甲基)-2’-O-甲基-尿苷(inm⁵Um)、1-硫代-尿苷、脫氧胸苷、2’-F-ara-尿苷、2’-F-尿苷、2’-OH-ara-尿苷、5-(2-甲氧羰基乙烯基)尿苷、5-[3-(1-E-丙烯基胺基)尿苷或本領域已知的任何其他修飾尿苷。 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.治療方法3. Treatment methods

本文提供的組合物和醫藥製劑可以用於方法，例如治療方法。在一些具體實施例中，向人投予所述組合物、醫藥製劑和RNA。 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.

在一些具體實施例中，本發明包括組合物或醫藥製劑在治療方法中的用途，其中所述治療方法用於治療實體瘤，並且其中所述組合物或醫藥製劑包含RNA，其中所述RNA由編碼IFNα的RNA、編碼IL-15 sushi的RNA、編碼IL-12sc的RNA和編碼FLT3-L的RNA組成。 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.

在一些具體實施例中，本發明包括組合物或醫藥製劑在治療方法中的用途，其中所述治療方法用於治療實體瘤，並且其中所述組合物或醫藥製劑包含RNA，其中所述RNA由編碼IFNα的RNA、編碼IL-15 sushi的RNA和編碼IL-12sc的RNA組成。 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.

在一些具體實施例中，用於治療方法的組合物或醫藥製劑包含編碼IL-12sc蛋白的RNA，並且還包含選自編碼IFNα的RNA和編碼IL-15 sushi蛋白的RNA的RNA。 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.

在一些具體實施例中，用於治療方法的組合物或醫藥製劑包含編碼IL-15 sushi的RNA，並且還包含編碼IFNα蛋白的RNA和編碼IL-12sc蛋白的RNA。 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.

在一些具體實施例中，用於治療方法的組合物或醫藥製劑包含編碼IFNα蛋白的RNA，並且還包含編碼IL-12sc的RNA和編碼IL-15 sushi的RNA。 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.

在一些具體實施例中，用於治療方法的組合物或醫藥製劑包含編碼FLT3-L蛋白的RNA和以下中的一種或多種：編碼IL-12sc的RNA、編碼IL-15 sushi的RNA和編碼IFNα的RNA。 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.

在一些具體實施例中，本文所述的醫藥製劑或組合物用於藥物用途。在一些具體實施例中，所述藥物用途包括疾病或障礙的治療性治療或預防性治療。在一些情況下，所述疾病或障礙的治療性治療或預防性治療包括治療或預防實體瘤。 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.

在一些具體實施例中，所述實體瘤是肉瘤、癌或淋巴瘤。 In some specific embodiments, the solid tumor is a sarcoma, carcinoma or lymphoma.

在一些情況下，所述實體瘤是在肺、結腸、卵巢、子宮頸、子宮、腹膜、睾丸、陰莖、舌、淋巴結、胰臟、骨、***、攝護腺、軟組織、結締組織、腎臟、肝臟、腦、甲狀腺或皮膚中。 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.

在一些具體實施例中，所述實體瘤是上皮性腫瘤、霍奇金氏淋巴瘤(HL)、非霍奇金氏淋巴瘤、攝護腺腫瘤、卵巢腫瘤、腎細胞腫瘤、胃腸道腫瘤、肝腫瘤、結直腸腫瘤、脈管腫瘤、間皮瘤腫瘤、胰臟腫瘤、***腫瘤、肉瘤腫瘤、肺腫瘤、結腸腫瘤、腦腫瘤、黑色素瘤腫瘤、小細胞肺腫瘤、神經母細胞瘤腫瘤、睾丸腫瘤、癌腫瘤、腺癌腫瘤、膠質瘤腫瘤、精原細胞瘤腫瘤、視網膜母細胞瘤或骨肉瘤腫瘤。 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.

在一些具體實施例中，基於相等的RNA質量，所述RNA以1：1：1或1：1：1：1的比率投予。例如，20μg的IL-15-sushi、20μg的IL-12sc、20μg的IFNα和任選地20μg FLT3-L。 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.

在一些具體實施例中，本文所述的醫藥製劑或組合物用於藥物用途並且與另一種療法一起投予個體。在一些具體實施例中，所述其他療法是抗PD1抗體、抗CTLA4抗體、或抗PD1抗體和抗CTLA4抗體的組合。 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.

在一些具體實施例中，所述抗PD1抗體是嵌合的、人源化的或人抗體。在一些具體實施例中，所述抗PD-1抗體是分離和/或重組的。抗PD-1抗體的例子是納武單抗、派姆單抗、塞普利單抗(cemiplimab)、MEDI0608(曾用名AMP-514；參見，例如，WO 2012/145493和美國專利號9,205,148)、PDR001(參見，例如，WO 2015/112900)、PF-06801591(參見，例如，WO 2016/092419)、BGB-A317(參見，例如，WO 2015/035606)。 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).

在一些具體實施例中，所述抗PD-1抗體是在WO 2015/112800中揭示的那些(諸如在所述PCT揭示的表1中被稱為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和H4xH9008P的那些，和在所述PCT公開的表3中被稱為H4H7798N、H4H7795N2、H4H9008P和H4H9048P2的那些)之一。將WO 2015/112800的揭示內容藉由引用以其整體併入本文。例如，WO 2015/112800中揭示的抗體和相關抗體(包括具有在所述PCT公開案中揭示的CDR、VH和VL序列或重鏈和輕鏈序列的抗體和抗原結合片段)以及結合至與所述PCT公開案中揭示的抗體相同的PD-1表位的抗體和抗原結合片段可以與本發明的RNA組合物配合使用以治療和/或預防癌症。 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.

在一些具體實施例中，所述抗PD-1抗體可以包含分別以如下SEQ ID NO：79和80分別所示的重鏈和輕鏈胺基酸序列；在SEQ ID NO：87和88中的VH和VL序列(以斜體示出)或在SEQ ID NO：79和80中的一個或多個(例如，全部六個)CDR(以粗框示出)。在一些具體實施例中，涵蓋包含以下CDR的抗體： 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) HCDR1=GFTFSNFG (SEQ ID NO: 81)

HCDR2=ISGGGRDT(SEQ ID NO：82) HCDR2=ISGGGRDT (SEQ ID NO: 82)

HCDR3=VKWGNIYFDY(SEQ ID NO：83) HCDR3=VKWGNIYFDY (SEQ ID NO: 83)

LCDR1=LSINTF(SEQ ID NO：84) LCDR1=LSINTF(SEQ ID NO:84)

LCDR2=AAS(SEQ ID NO：85) LCDR2=AAS (SEQ ID NO: 85)

LCDR3=QQSSNTPFT(SEQ ID NO：86)。 LCDR3=QQSSNTPFT (SEQ ID NO: 86).

包含含有SEQ ID NO：87和88中的VH和VL序列(以斜體示出)的重鏈的抗體是完全人抗PD-1抗體，稱為REGN2810(塞普利單抗)。 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).

抗PD-1 Mab重鏈 Anti-PD-1 Mab heavy chain

(SEQ ID NO：79)

(SEQ ID NO: 79)

HCDR1=GFTFSNFG(SEQ ID NO：81) HCDR1=GFTFSNFG (SEQ ID NO: 81)

HCDR2=ISGGGRDT(SEQ ID NO：82) HCDR2=ISGGGRDT (SEQ ID NO: 82)

HCDR3=VKWGNIYFDY(SEQ ID NO：83) HCDR3=VKWGNIYFDY (SEQ ID NO: 83)

抗PD-1 Mab輕鏈 Anti-PD-1 Mab light chain

(SEQ ID NO：80)

(SEQ ID NO: 80)

LCDR1=LSINTF(SEQ ID NO：84) LCDR1=LSINTF(SEQ ID NO:84)

LCDR2=AAS(SEQ ID NO：85) LCDR2=AAS (SEQ ID NO: 85)

LCDR3=QQ SSNTPFT(SEQ ID NO：86) LCDR3=QQ SSNTPFT (SEQ ID NO: 86)

在一些具體實施例中，所述RNA可以經由注射到腫瘤中(例如，腫瘤內)、腫瘤(腫瘤周邊)附近或腫瘤去除部位附近來遞送，並且抗體可以以相同的方式或全身地(例如像腸內或腸胃外，包括經由注射、輸注和植入)來遞送。投予可以是同時或順序的。如果是順序的，則可以以任何順序並且以本領域技術人員已知的任何適當的時間間隔投予。 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.

在一些具體實施例中，將治療性RNA組合物與另一種療法一起直接遞送到腫瘤中或者腫瘤或腫瘤去除部位附近。在一些具體實施例中，將治療性RNA組合物直接遞送到腫瘤中或者腫瘤或腫瘤去除部位附近，而另一種藥劑全身遞送。 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.

*** ***

此描述和示例性具體實施例不應被視為是限制性的。為了本說明書和所附發明申請專利範圍的目的，除非另有指示，否則在說明書和發明申請專利範圍中使用的所有表達數量、百分比或比例的數字和其他數值應被理解為在所有情況下被術語“約”修飾至已無法將其如此修飾的程度。“約”指示實質上不影響所述主題的特性的變化程度，例如在10%、5%、2%或1%之內。因此，除非有相反的指示，否則以下說明書和所附發明申請專利範圍中闡述的數值參數是近似值，所述近似值可以根據試圖獲得的所需特性而變化。至少並且不試圖限制將等同原則應用於發明申請專利範圍，每個數字參數至少應被解釋為是根據所報告的有效數字的數量並且藉由應用普通修約技術的。 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.

注意，如本說明書和所附發明申請專利範圍所用，單數形式“一個/種(a、an)”和“所述(the)”以及任何詞語的任何單數使用包括複數個指示物，除非明確地並且清楚地限於一個/種指示物。如本文所用，術語“包括”及其語法變體旨在是非限制性的，使得列表中項目的詳述不排除可以取代或添加到所列項目的其他類似項目。 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.

實例Instance

提供以下實例以說明某些揭示的具體實施例，並且不應解釋為以任何方式限制本揭示文本的範圍。 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.

實例1-材料和方法Example 1-Materials and Methods

B16F10腫瘤模型：在研究招募前，使6-8周齡並且稱重在17.0g與20.9g之間的雌性C57BL/6J小鼠(Jackson Laboratory；緬因州巴爾港)適應環境至少三天。小鼠自由獲取食物(Harlan 2916齧齒動物飲食，美國馬薩諸塞州)和無菌水，並且在22℃±2℃且相對濕度為55%±15%的12小時光照/黑暗週期下飼養。B16F10細胞從美國典型培養物保藏中心(American Type Culture Collection，ATCC)(美國弗吉尼亞州馬納薩斯)(目錄號CRL-6475)獲得並且在37℃下在 5% CO2中在補充有10%熱滅活的胎牛血清(HI FBS)(Life technologies，目錄號10082-147)的杜氏改良伊格爾培養基(Dulbecco’s Modified Eagle’s Medium，DMEM)(Life technologies，目錄號11995)中培養。將細胞使用0.25%胰蛋白酶-EDTA(Life technologies，目錄號25200-056)收穫，重懸於杜氏磷酸鹽緩衝鹽水(Dulbecco’s phosphate-buffered saline，DPBS)(Life technologies，目錄號14190-144)中，並且以0.5 x 10^6個細胞/200μl/只小鼠經皮下(SC)植入雌性C57BL/6J小鼠的右側腹中。 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.

腫瘤監測：每週兩次用卡尺測量B16F10腫瘤直至最終處死。當腫瘤尺寸達到大約2000mm³或存在動物健康問題(腫瘤的20%面積潰瘍)時，對動物實施安樂死。腫瘤消退定義為：i)在研究結束時腫瘤體積<20mm3或ii)T^F/T⁰<1，其中T^F等於最終腫瘤體積，並且T⁰等於首次腫瘤內RNA注射當天的腫瘤體積。 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腫瘤模型：關於利用CT26腫瘤模型的研究，在研究招募前，使重量在17g與24g之間的6-8周齡雌性Balb/c Rj小鼠(Janvier，法國勒基奈斯聖島(Genest-St.-Îsle))適應環境至少六天。小鼠自由獲取食物(ssniff M-Z autoclavable Soest，德國)和無菌水，並且在22℃±2℃且相對濕度為55%±10%的12小時光照/黑暗週期下飼養。CT26細胞從(ATCC® CRL-2638^TM)獲得並且在37℃下在5% CO2中在補充有10%胎牛血清(FBS)(Biochrom，目錄號S 0115)的羅斯威爾公園紀念研究所(Roswell Park Memorial Institute，RPMI)1640培養基GlutaMAX^TM(Life technologies，目錄號61870-044)中培養。將細胞使用StemPro® Accutase®細胞解離試劑(Life technologies，目錄號A1110501)收集，重懸於DPBS(Life technologies，目錄號14190-169)中，並且以0.5 x 10^6個細胞/100μl/只小鼠SC植入雌性Balb/c Rj小鼠的右側剃毛腹中。接種腫瘤後11天-15天開始腫瘤內RNA注射。每2-3天藉由卡尺測量評估CT26腫瘤模型中的腫瘤生長，並且使用以下公式表示為垂直直徑的乘積：a2 * b/2，其中b是兩個直徑中的較長者(a<b)。 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反應性CD8+細胞：除腫瘤生長外，在指示的血液中測量gp70反應性CD8+ T細胞。使用EDTA塗覆的管採集血液樣品。將100μL血液轉移到FACS管中，並且添加抗體混合物，所述抗體混合物含有T-Select H-2Ld MuLV gp70四聚體-SPSYVYHQF-APC(MBL(TS-M-521-2)，對於100μL血液4μL)、抗CD8a FITC(life technologies(MCD801)，對於100μL血液1μL)和抗CD45 V500(BD(561487)，對於100μL血液1μL)。在室溫下培育20min後，添加血液裂解緩衝液(BD(349202)，300μL/管)，並且進一步培育6min。然後將樣品用PBS-EDTA緩衝液洗滌兩次。在FACS Canto II流式細胞儀上分析FACS樣品。 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修飾：除非另有說明，否則所有RNA修飾如下。將編碼感興趣的基因的合成DNA片段克隆到常用起始載體中，所述起始載體包含5’-UTR(在某些情況下，對應於煙草蝕紋病毒先導序列TEV，SEQ ID NO：3)、由稱為F和I的兩個元件組成的3’UTR(在某些情況下，對應於SEQ ID NO：7)和總共110個核苷酸的多聚(A)尾(A30-連接子-A70結構；SEQ ID NO：60)。用IIS類限制性內切酶在多聚(dA：dT)的下游進行質粒DNA的線性化，以生成除多聚(dA：dT)外沒有另外的核苷酸的模板(參見Holtkamp等人，Blood 108(13)：4009-172006(2006))。在7.5mM ATP、CTP、GTP和N1-甲基-假尿苷三磷酸酯的存在下，使線性化的質粒DNA經受如前所述用T7 RNA聚合酶(Thermo Fisher)的體外轉錄(參見Grudzien-Nogalska E等人，Methods Mol Biol.969：55-72(2013))。然後使用磁性顆粒(Berensmeier 2006)純化RNA，並且隨後使用可商購獲得的基於牛痘病毒加帽酶(NEB)並且添加RNA帽2’-O-甲基轉移酶(NEB)的系統酶促引入Cap1結構。然後，使所述RNA經受藉由基於纖維素的色譜法進行的進一步純化程序以除去雙鏈RNA雜質(參見Day PR等人，Phytopathology 67：1393(1977)；Morris TJ等人，Phytopathology 69：854-858(1979)；和Castillo A等人，Virol J.8：38(2011))。將RNA濃度和品質分別藉由分光光度法評估並且藉由毛細管凝膠電泳系統分析。使用dsRNA特異性J2 mAb(English & Scientific Consulting)在Northwestern點印跡測定中評估dsRNA的存在，如Karikó等人Nucleic Acids Res.39(21)：e142(2011)中描述的。 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).

用於體內研究的RNA的製備：藉由將等量(微克)的RNA以2倍的預期劑量在水中混合來製備用於體內研究的相應RNA混合物。注射到小鼠中的RNA是表1中描述的除了FLT3-L以外的小鼠版本的RNA，其中使用SEQ ID NO：65(具有鼠優化的分泌序列的人FLT3-L)。在一些情況下，將所述RNA混合物在-80℃下冷凍直至腫瘤內注射的那天。在注射當天，將RNA解凍並且與等體積的2X無菌林格氏溶液混合。在其他情況下，新鮮製備RNA混合物，並且在林格氏溶液中稀釋至1X RNA/林格氏溶液的最終濃度。在兩種情況下，將所得1X RNA/林格氏溶液用於腫瘤內注射。 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.

實例2-三種RNA的組合減小體內腫瘤體積Example 2-The combination of three RNAs reduces tumor volume in vivo

我們先前測試了包含三種RNA的不同組合的細胞介素RNA混合物在CT26荷瘤小鼠中的作用，如上所述。用四次注射編碼IL-15 sushi、IL-12sc、GM-CSF和IFNα的RNA(10μg RNA/標靶)處理的腫瘤在10個處理的腫瘤中有10個誘導消退(圖1A；表2(表和圖代表相同的數據；表提供原始數據))。用i)IL-15 sushi、IL-12sc和IFNα(無GM-CSF)，ii)IL-15 sushi、GM-CSF和IFNα(無IL-12sc)，iii)IL-12sc、GM-CSF和IFNα(無IL-15 sushi)，以及iv)IL-15 sushi、IL-12sc和GM-CSF(無IFNα)的3組合RNA混合物處理的腫瘤導致10個中有8個消退(IL-15 sushi、IL-12sc和IFNα(無GM-CSF；圖1B；表3)、10個中有6個消退(IL-15 sushi、GM-CSF和IFNα(無IL-12sc；圖1C；表4)、10個中有8個消退(IL-12sc、GM-CSF和IFNα(無IL-15 sushi；圖1D；表5)、10個中有7個消退(IL-15 sushi、IL-12sc 和GM-CSF(無IFNα；圖1E；表6)。用對照RNA處理的腫瘤(每次處理均注射40μg RNA)沒有顯示出腫瘤消退(圖1F；表7)。為了分析腫瘤生長動力學，對於每個處理組，計算直到第33天的平均腫瘤體積(圖1G)。對於每個處理組，將腫瘤生長抑制T/C(基於平均腫瘤體積的腫瘤/對照)繪製至第19天(圖1H)。 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).

在使用B16F10腫瘤模型的單獨的實驗中，將IL-15 sushi、IL-12sc、GM-CSF和IFNα的4組合RNA混合物的抗腫瘤活性與IL-15 sushi、IL-12sc和IFNα的3組合混合物進行比較。在第11天，小鼠接受腫瘤內單次RNA注射。用編碼IL-15 sushi、IL-12sc、GM-CSF和IFNα的4組合細胞介素RNA混合物的處理導致10只小鼠中有6只腫瘤消退(圖2A)。用IL-15 sushi、IL-12sc和IFNα(無GM-CSF)的3組合RNA混合物處理的腫瘤在10只小鼠中有8只腫瘤消退(圖2B)，而用編碼螢光素酶的對照RNA處理的小鼠沒有注意到腫瘤消退(圖2C)。 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).

實例3-四種RNA的組合減小腫瘤體積並且促進體內免疫反應Example 3-The combination of four RNAs reduces tumor volume and promotes immune response in vivo

在第13、16、20和24天將編碼IL-15 sushi、IL-12sc、IFNα和FLT3-L的RNA的4細胞介素混合物注射到CT26荷瘤小鼠中，並且監測腫瘤生長至第50天(5μg RNA/標靶和20μg對照RNA)。如圖3所示，腫瘤內注射編碼IL-15 sushi、IL-12sc、IFNα和FIT3-L的四種RNA的組合在10只小鼠中有4只誘導腫瘤消退(圖3A)，而用編碼螢光素酶的對照RNA處理的小鼠在10只動物中有1只顯示腫瘤消退(圖3B)。這些數據在相似設計的重複研究中被證實。在重複實驗中，編碼IL-15 sushi、IL-12sc、IFNα和FLT3-L的細胞介素RNA的混合物(在第11、14、17和21天注射5μg RNA/標靶和20μg對照RNA)導致在11只小鼠中有9只腫瘤消退(圖3C)，而用編碼螢光素酶的對照RNA的處理在11只小鼠中有1只顯示腫瘤消退(圖3D)。用編碼IL-15 sushi、IL-12sc、IFNα和GM-CSF的細胞介素RNA的混合物處理的小鼠在10只動物中有2只顯示腫瘤消退(圖3E)。 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).

在利用B16F10腫瘤模型的單獨的實驗中，在第11天，將編碼IL-15 sushi、IL-12sc、IFNα和GM-CSF的RNA的混合物(圖4A)；編碼IL-15 sushi、IL-12sc、IFNα和FLT3-L的RNA的混合物(圖4B)；或編碼螢光素酶的對照RNA(圖4C)(2μg RNA/標靶和8μg對照RNA)一次注射到B16F10荷瘤小鼠中，並且監測腫瘤生長至第57天。如圖4所示，腫瘤內注射編碼IL-15 sushi、IL-12sc、IFNα和GM-CSF的四種RNA的組合在10只小鼠中有7只誘導腫瘤消退(圖4A)，腫瘤內注射編碼IL-15 sushi、IL-12sc、IFNα和FLT3-L的四種RNA的組合在10只小鼠中有6只誘導腫瘤消退(圖4B)，而用編碼螢光素酶的對照RNA處理的小鼠在10只動物中有0只顯示腫瘤消退(圖4C)。 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荷瘤小鼠接受單次注射RNA(2μg RNA/標靶或8μg對照RNA)，並且在腫瘤內注射後7天，將腫瘤取出、解離並且用一組抗體染色以評估腫瘤內免疫群。列舉了用於不同處理組的CD8+ T細胞和NK細胞的數目。結果示於圖5A和圖5B中。 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.

在第13、16、20、24天，攜帶單個CT26腫瘤的小鼠接受四次腫瘤內注射1)IL-15 sushi、GM-CSF、IFNα和IL-12sc的細胞介素RNA混合物；2)IL-15 sushi、FLT3L、IFNα和IL-12sc的細胞介素RNA混合物；或3)編碼螢光素酶的RNA(5μg RNA/標靶/次次注射和20μg RNA/次對照注射)。在第一次腫瘤內RNA投予後第 19天和第31天收集血液，並且藉由流式細胞術對gp70腫瘤抗原特異性T細胞定量。與接受對照RNA的小鼠相比，在腫瘤內注射RNA細胞介素後的小鼠中，gp70腫瘤抗原特異性T細胞在血液中的頻率大大增加(圖6A(第19天)和圖6B(第31天))。在第31天，與含有GM-CSF的混合物相比，對於含有FLT3L的混合物，gp70特異性T細胞的頻率更高(圖6B)。在單獨的實驗中，在第24天，與對照RNA相比，IL-15 sushi、IL-12sc、IFNα和FLT3-L的細胞介素RNA混合物導致gp70特異性T細胞的頻率大大增加(圖6C)。 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 ).

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<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成：示例性3 UTR(DNA) <223> Synthesis: Exemplary 3 UTR (DNA)

<400> 7 <400> 7

<210> 8 <210> 8

<211> 317 <211> 317

<212> RNA <212> RNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成：示例性3 UTR(RNA) <223> Synthesis: Exemplary 3 UTR(RNA)

<400> 8 <400> 8

<210> 9 <210> 9

<400> 9 <400> 9

000 000

<210> 10 <210> 10

<400> 10 <400> 10

000 000

<210> 11 <210> 11

<400> 11 <400> 11

000 000

<210> 12 <210> 12

<400> 12 <400> 12

000 000

<210> 13 <210> 13

<400> 13 <400> 13

000 000

<210> 14 <210> 14

<211> 539 <211> 539

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(539) <222> (1)..(539)

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

<400> 14 <400> 14

<210> 15 <210> 15

<211> 1623 <211> 1623

<212> DNA <212> DNA

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(1623) <222> (1)..(1623)

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

<400> 15 <400> 15

<210> 16 <210> 16

<211> 1623 <211> 1623

<212> DNA <212> DNA

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(1623) <222> (1)..(1623)

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

<400> 16 <400> 16

<210> 17 <210> 17

<211> 1623 <211> 1623

<212> RNA <212> RNA

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(1623) <222> (1)..(1623)

<223> 人非優化IL-12sc(RNA編碼CDS) <223> Human non-optimized IL-12sc (RNA encoding CDS)

<400> 17 <400> 17

<210> 18 <210> 18

<211> 1623 <211> 1623

<212> RNA <212> RNA

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(1623) <222> (1)..(1623)

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

<400> 18 <400> 18

<210> 19 <210> 19

<211> 188 <211> 188

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(188) <222> (1)..(188)

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

<400> 19 <400> 19

<210> 20 <210> 20

<211> 570 <211> 570

<212> DNA <212> DNA

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(570) <222> (1)..(570)

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

<400> 20 <400> 20

<210> 21 <210> 21

<211> 570 <211> 570

<212> DNA <212> DNA

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(570) <222> (1)..(570)

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

<400> 21 <400> 21

<210> 22 <210> 22

<211> 570 <211> 570

<212> RNA <212> RNA

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(570) <222> (1)..(570)

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

<400> 22 <400> 22

<210> 23 <210> 23

<211> 570 <211> 570

<212> RNA <212> RNA

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(570) <222> (1)..(570)

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

<400> 23 <400> 23

<210> 24 <210> 24

<211> 241 <211> 241

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(241) <222> (1)..(241)

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

<400> 24 <400> 24

<210> 25 <210> 25

<211> 729 <211> 729

<212> DNA <212> DNA

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(729) <222> (1)..(729)

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

<400> 25 <400> 25

<210> 26 <210> 26

<211> 729 <211> 729

<212> RNA <212> RNA

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(729) <222> (1)..(729)

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

<400> 26 <400> 26

<210> 27 <210> 27

<400> 27 <400> 27

000 000

<210> 28 <210> 28

<400> 28 <400> 28

000 000

<210> 29 <210> 29

<400> 29 <400> 29

000 000

<210> 30 <210> 30

<211> 185 <211> 185

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(185) <222> (1)..(185)

<223> 人FLT3-L(胺基酸) <223> Human FLT3-L (amino acid)

<400> 30 <400> 30

<210> 31 <210> 31

<211> 555 <211> 555

<212> DNA <212> DNA

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(555) <222> (1)..(555)

<223> 人FLT3L(CDS DNA) <223> Human FLT3L (CDS DNA)

<400> 31 <400> 31

<210> 32 <210> 32

<211> 555 <211> 555

<212> RNA <212> RNA

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(555) <222> (1)..(555)

<223> 人FLT3L(RNA編碼CDS) <223> Human FLT3L (RNA encoding CDS)

<400> 32 <400> 32

<210> 33 <210> 33

<400> 33 <400> 33

000 000

<210> 34 <210> 34

<400> 34 <400> 34

000 000

<210> 35 <210> 35

<400> 35 <400> 35

000 000

<210> 36 <210> 36

<400> 36 <400> 36

000 000

<210> 37 <210> 37

<400> 37 <400> 37

000 000

<210> 38 <210> 38

<400> 38 <400> 38

000 000

<210> 39 <210> 39

<211> 544 <211> 544

<212> PRT <212> PRT

<213> 小家鼠 <213> House mouse

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(544) <222> (1)..(544)

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

<400> 39 <400> 39

<210> 40 <210> 40

<211> 2081 <211> 2081

<212> DNA <212> DNA

<213> 小家鼠 <213> House mouse

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(2081) <222> (1)..(2081)

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

<400> 40 <400> 40

<210> 41 <210> 41

<211> 2081 <211> 2081

<212> RNA <212> RNA

<213> 小家鼠 <213> House mouse

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(2081) <222> (1)..(2081)

<223> 鼠IL-12sc(RNA) <223> Mouse IL-12sc (RNA)

<400> 41 <400> 41

<210> 42 <210> 42

<400> 42 <400> 42

000 000

<210> 43 <210> 43

<400> 43 <400> 43

000 000

<210> 44 <210> 44

<400> 44 <400> 44

000 000

<210> 45 <210> 45

<211> 190 <211> 190

<212> PRT <212> PRT

<213> 小家鼠 <213> House mouse

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(190) <222> (1)..(190)

<223> 鼠IFN-α-4(胺基酸) <223> Mouse IFN- α -4 (amino acid)

<400> 45 <400> 45

<210> 46 <210> 46

<211> 1019 <211> 1019

<212> DNA <212> DNA

<213> 小家鼠 <213> House mouse

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(1019) <222> (1)..(1019)

<223> 鼠IFN-α-4 <223> Mouse IFN- α -4

<400> 46 <400> 46

<210> 47 <210> 47

<211> 1019 <211> 1019

<212> RNA <212> RNA

<213> 小家鼠 <213> House mouse

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(1019) <222> (1)..(1019)

<223> 鼠IFN-α-4(RNA) <223> Mouse IFN- α -4 (RNA)

<400> 47 <400> 47

<210> 48 <210> 48

<400> 48 <400> 48

000 000

<210> 49 <210> 49

<400> 49 <400> 49

000 000

<210> 50 <210> 50

<400> 50 <400> 50

000 000

<210> 51 <210> 51

<211> 232 <211> 232

<212> PRT <212> PRT

<213> 小家鼠 <213> House mouse

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(232) <222> (1)..(232)

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

<400> 51 <400> 51

<210> 52 <210> 52

<211> 1145 <211> 1145

<212> DNA <212> DNA

<213> 小家鼠 <213> House mouse

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(1145) <222> (1)..(1145)

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

<400> 52 <400> 52

<210> 53 <210> 53

<211> 1145 <211> 1145

<212> RNA <212> RNA

<213> 小家鼠 <213> House mouse

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(1145) <222> (1)..(1145)

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

<400> 53 <400> 53

<210> 54 <210> 54

<400> 54 <400> 54

000 000

<210> 55 <210> 55

<400> 55 <400> 55

000 000

<210> 56 <210> 56

<400> 56 <400> 56

000 000

<210> 57 <210> 57

<211> 141 <211> 141

<212> PRT <212> PRT

<213> 小家鼠 <213> House mouse

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(141) <222> (1)..(141)

<223> 鼠GM-CSF(胺基酸) <223> Mouse GM-CSF (amino acid)

<400> 57 <400> 57

<210> 58 <210> 58

<211> 891 <211> 891

<212> DNA <212> DNA

<213> 小家鼠 <213> House mouse

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(891) <222> (1)..(891)

<223> 鼠GM-CSF <223> Mouse GM-CSF

<400> 58 <400> 58

<210> 59 <210> 59

<211> 891 <211> 891

<212> RNA <212> RNA

<213> 小家鼠 <213> House mouse

<220> <220>

<221> 尚未歸類的特徵 <221> Features not yet classified

<222> (1)..(891) <222> (1)..(891)

<223> 鼠GM-CSF(RNA) <223> Mouse GM-CSF (RNA)

<400> 59 <400> 59

<210> 60 <210> 60

<400> 60 <400> 60

000 000

<210> 61 <210> 61

<400> 61 <400> 61

000 000

<210> 62 <210> 62

<400> 62 <400> 62

000 000

<210> 63 <210> 63

<211> 187 <211> 187

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

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

<400> 63 <400> 63

<210> 64 <210> 64

<211> 1010 <211> 1010

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成：FLT3-L(DNA；5UTR-與小鼠優化分泌序列組合的人FLT3L-3 UTR) <223> Synthesis: FLT3-L (DNA; 5UTR-human FLT3L-3 UTR combined with mouse optimized secretion sequence)

<400> 64 <400> 64

<210> 65 <210> 65

<211> 1010 <211> 1010

<212> RNA <212> RNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成：FLT3L(RNA；5UTR-與小鼠優化分泌序列組合的人FLT3L-3 UTR) <223> Synthesis: FLT3L (RNA; 5UTR-human FLT3L-3 UTR combined with mouse optimized secretion sequence)

<400> 65 <400> 65

<210> 66 <210> 66

<211> 110 <211> 110

<212> RNA <212> RNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成：示例性多聚A(Poly-A) <223> Synthesis: Exemplary Poly-A (Poly-A)

<400> 66 <400> 66

<210> 67 <210> 67

<400> 67 <400> 67

000 000

<210> 68 <210> 68

<400> 68 <400> 68

000 000

<210> 69 <210> 69

<400> 69 <400> 69

000 000

<210> 70 <210> 70

<400> 70 <400> 70

000 000

<210> 71 <210> 71

<400> 71 <400> 71

000 000

<210> 72 <210> 72

<400> 72 <400> 72

000 000

<210> 73 <210> 73

<400> 73 <400> 73

000 000

<210> 74 <210> 74

<400> 74 <400> 74

000 000

<210> 75 <210> 75

<400> 75 <400> 75

000 000

<210> 76 <210> 76

<400> 76 <400> 76

000 000

<210> 77 <210> 77

<400> 77 <400> 77

000 000

<210> 78 <210> 78

<400> 78 <400> 78

000 000

<210> 79 <210> 79

<211> 444 <211> 444

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成：抗PD1 Mab重鏈 <223> Synthesis: anti-PD1 Mab heavy chain

<400> 79 <400> 79

<210> 80 <210> 80

<211> 214 <211> 214

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成：抗PD1 Mab輕鏈 <223> Synthesis: anti-PD1 Mab light chain

<400> 80 <400> 80

<210> 81 <210> 81

<211> 8 <211> 8

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成：HCDR1 <223> Synthesis: HCDR1

<400> 81 <400> 81

<210> 82 <210> 82

<211> 8 <211> 8

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成：HCDR2 <223> Synthesis: HCDR2

<400> 82 <400> 82

<210> 83 <210> 83

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成：HCDR3 <223> Synthesis: HCDR3

<400> 83 <400> 83

<210> 84 <210> 84

<211> 6 <211> 6

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成：LCDR1 <223> Synthesis: LCDR1

<400> 84 <400> 84

<210> 85 <210> 85

<211> 3 <211> 3

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成：LCDR2 <223> Synthesis: LCDR2

<400> 85 <400> 85

<210> 86 <210> 86

<211> 9 <211> 9

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成：LCDR3 <223> Synthesis: LCDR3

<400> 86 <400> 86

<210> 87 <210> 87

<211> 117 <211> 117

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成：抗PD1 Mab VH <223> Synthesis: Anti-PD1 Mab VH

<400> 87 <400> 87

<210> 88 <210> 88

<211> 107 <211> 107

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成：抗PD1 Mab VL <223> Synthesis: anti-PD1 Mab VL

<400> 88 <400> 88

Claims

一種組合物或醫藥製劑，其包含編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA和編碼IFNα蛋白的RNA。 A composition or pharmaceutical preparation, which comprises RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein, and RNA encoding IFNα protein.

如請求項1所述的醫藥製劑或組合物，其還包含編碼FLT3-L蛋白的RNA。 The pharmaceutical preparation or composition according to claim 1, which further comprises RNA encoding FLT3-L protein.

如請求項1或如請求項2所述的組合物。 The composition according to claim 1 or claim 2.

如請求項1或如請求項2所述的醫藥製劑。 The pharmaceutical preparation according to claim 1 or claim 2.

如請求項1-4中任一項所述的醫藥製劑或組合物，其中所述RNA具有1：1：1或1：1：1：1的比率，並且其中所述比率藉由定量RT-PCR驗證。 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.

如請求項1-5中任一項所述的醫藥製劑或組合物，其中所述RNA完整性大於或等於70%。 The pharmaceutical preparation or composition according to any one of claims 1-5, wherein the RNA integrity is greater than or equal to 70%.

如請求項1-6中任一項所述的醫藥製劑或組合物，其中所述醫藥製劑或組合物包含少於250ng DNA/存在的核酸的總mg。 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.

如請求項1-7中任一項所述的醫藥製劑或組合物，其中所述IFNα蛋白是IFNα2b蛋白。 The pharmaceutical preparation or composition according to any one of claims 1-7, wherein the IFNα protein is IFNα2b protein.

如請求項1-8中任一項所述的醫藥製劑或組合物，其中 The pharmaceutical preparation or composition according to any one of claims 1-8, wherein

(i)所述編碼IL-12sc蛋白的RNA包含SEQ ID NO：17或18的核苷酸序列或與SEQ ID NO：17或18的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列；和/或 (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)所述IL-12sc蛋白包含SEQ ID NO：14的胺基酸序列或與SEQ ID NO：14的胺基酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的胺基酸序列；和/或 (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)所述編碼IL-12sc蛋白的RNA包含與IL-12sc的p40部分(SEQ ID NO：17或18的核苷酸1-984)具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性並且與IL-12sc的p30部分(SEQ ID NO：17或18的核苷酸1027-1623)具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列，並且還包含編碼連接子多肽的在所述p40部分與p35部分之間的核苷酸。 (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.

如請求項1-9中任一項所述的醫藥製劑或組合物，其中 The pharmaceutical preparation or composition according to any one of claims 1-9, wherein

(i)所述編碼IL-15 sushi蛋白的RNA包含SEQ ID NO：26的核苷酸序列或與SEQ ID NO：26的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列；和/或 (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)所述IL-15 sushi蛋白包含SEQ ID NO：24的胺基酸序列或與SEQ ID NO：24的胺基酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的胺基酸序列；和/或 (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)所述編碼IL-15 sushi蛋白的RNA包含與IL-15受體α的sushi結構域(SEQ ID NO：26的核苷酸1-321)具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性並且與成熟IL-15(SEQ ID NO：26的核苷酸382-729)具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列，並且任選地還包含編碼連接子多肽的在IL-15的sushi結構域與所述成熟IL-15之間的核苷酸。 (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.

如請求項1-10中任一項所述的醫藥製劑或組合物，其中 The pharmaceutical preparation or composition according to any one of claims 1-10, wherein

(i)所述編碼IFNα蛋白的RNA包含SEQ ID NO：22或23的核苷酸序列或與SEQ ID NO：22或23的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列；和/或 (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)所述IFNα蛋白包含SEQ ID NO：19的胺基酸序列或與SEQ ID NO：19的胺基酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的胺基酸序列。 (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.

如請求項2-11中任一項所述的醫藥製劑或組合物，其中 The pharmaceutical preparation or composition according to any one of claims 2-11, wherein

(i)所述編碼FLT3-L蛋白的RNA包含SEQ ID NO：32的核苷酸序列或與SEQ ID NO：32的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列；和/或 (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)所述FLT3-L蛋白包含SEQ ID NO：30的胺基酸序列或與SEQ ID NO：30的胺基酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的胺基酸序列。 (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.

如請求項1-12中任一項所述的醫藥製劑或組合物，其中至少一種RNA包含替代至少一個尿苷的修飾核苷。 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.

如請求項1-13中任一項所述的醫藥製劑或組合物，其中至少一種RNA包含替代每個尿苷的修飾核苷。 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.

如請求項1-14中任一項所述的醫藥製劑或組合物，其中每種RNA包含替代至少一個尿苷的修飾核苷。 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.

如請求項1-15中任一項所述的醫藥製劑或組合物，其中每種RNA包含替代每個尿苷的修飾核苷。 The pharmaceutical preparation or composition according to any one of claims 1-15, wherein each RNA comprises a modified nucleoside instead of each uridine.

如請求項13-16中任一項所述的醫藥製劑或組合物，其中所述修飾核苷獨立地選自假尿苷(ψ)、N1-甲基-假尿苷(m1ψ)和5-甲基-尿苷(m5U)。 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).

如請求項13-17中任一項所述的醫藥製劑或組合物，其中至少一種RNA包含多於一種類型的修飾核苷，其中所述修飾核苷獨立地選自假尿苷(ψ)、N1-甲基-假尿苷(m1ψ)和5-甲基-尿苷(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).

如請求項18所述的醫藥製劑或組合物，其中所述修飾核苷是N1- 甲基-假尿苷(m1ψ)。 The pharmaceutical preparation or composition according to claim 18, wherein the modified nucleoside is N1- Methyl-pseudouridine (m1ψ).

如請求項1-19所述的醫藥製劑或組合物，其中至少一種RNA包含5’帽m₂ ^7,3’-OGppp(m₁ ^2’-O)ApG或3’-O-Me-m⁷G(5’)ppp(5’)G。 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.

如請求項1-20所述的醫藥製劑或組合物，其中每種RNA包含5’帽m₂ ^7,3’-OGppp(m₁ ^2’-O)ApG或3’-O-Me-m⁷G(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.

如請求項1-21所述的醫藥製劑或組合物，其中至少一種RNA包含含有選自SEQ ID NO：4和6組成之群組的核苷酸序列或與選自SEQ ID NO：4和6組成之群組的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的5’ UTR。 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.

如請求項1-22中任一項所述的醫藥製劑或組合物，其中每種RNA包含含有選自SEQ ID NO：4和6組成之群組的核苷酸序列或與選自SEQ ID NO：4和6組成之群組的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的5’ UTR。 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.

如請求項1-23中任一項所述的醫藥製劑或組合物，其中至少一種RNA包含含有SEQ ID NO：8的核苷酸序列或與SEQ ID NO：8的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的3’ UTR。 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.

如請求項1-24中任一項所述的醫藥製劑或組合物，其中每種RNA包含含有SEQ ID NO：8的核苷酸序列或與SEQ ID NO：8的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的3’ UTR。 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.

如前述請求項中任一項所述的醫藥製劑或組合物，其中至少一種RNA包含多聚A(poly-A)尾。 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.

如請求項1-25中任一項所述的醫藥製劑或組合物，其中每種RNA包含多聚A尾。 The pharmaceutical preparation or composition according to any one of claims 1-25, wherein each RNA comprises a poly A tail.

如請求項26或27所述的醫藥製劑或組合物，其中所述多聚A尾包含至少100個核苷酸。 The pharmaceutical preparation or composition according to claim 26 or 27, wherein the poly A tail contains at least 100 nucleotides.

如請求項26-28中任一項所述的醫藥製劑或組合物，其中所述多聚A尾包含示於SEQ ID NO：66的多聚A尾。 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.

如請求項1-29中任一項所述的醫藥製劑或組合物，其中一種或多種RNA包含： The pharmaceutical preparation or composition according to any one of claims 1-29, wherein the one or more RNAs comprise:

a.包含m₂ ^7,3’-OGppp(m₁ ^2’-O)ApG或3’-O-Me-m⁷G(5’)ppp(5’)G的5’帽； 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.包含(i)選自SEQ ID NO：4和6組成之群組的核苷酸序列或(ii)與選自SEQ ID NO：4和6組成之群組的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的5’ UTR； 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.包含(i)SEQ ID NO：8的核苷酸序列或(ii)與SEQ ID NO：8的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的3’ UTR；和 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.包含至少100個核苷酸的多聚A尾。 d. A poly A tail containing at least 100 nucleotides.

如請求項30所述的醫藥製劑或組合物，其中所述多聚A尾包含SEQ ID NO：66。 The pharmaceutical preparation or composition according to claim 30, wherein the poly A tail comprises SEQ ID NO:66.

如請求項1-31中任一項所述的醫藥製劑或組合物，其是包含所述RNA的藥物組合物。 The pharmaceutical preparation or composition according to any one of claims 1-31, which is a pharmaceutical composition containing the RNA.

如請求項32所述的醫藥製劑或組合物，其中所述藥物組合物還包含一種或多種藥學上可接受的載體、稀釋劑和/或賦形劑。 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.

如請求項1-33中任一項所述的醫藥製劑或組合物，其中所述RNA被配製為液體、配製為固體或其組合。 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.

如請求項1-34中任一項所述的醫藥製劑或組合物，其用於藥物用途。 The pharmaceutical preparation or composition according to any one of claims 1-34, which is used for pharmaceutical use.

如請求項35所述的醫藥製劑或組合物，其中所述藥物用途包括疾病或障礙的治療性治療或預防性治療。 The pharmaceutical preparation or composition according to claim 35, wherein the pharmaceutical use includes therapeutic treatment or preventive treatment of diseases or disorders.

如請求項36所述的醫藥製劑或組合物，其中所述疾病或障礙的治療性治療或預防性治療包括治療或預防實體瘤。 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.

如請求項37所述的醫藥製劑或組合物，其中所述實體瘤是肉瘤、癌或淋巴瘤。 The pharmaceutical preparation or composition according to claim 37, wherein the solid tumor is a sarcoma, carcinoma or lymphoma.

如請求項37或38中任一項所述的醫藥製劑或組合物，其中所述實體瘤是在肺、結腸、卵巢、子宮頸、子宮、腹膜、睾丸、陰莖、舌、淋巴結、胰臟、骨、***、攝護腺、軟組織、結締組織、腎臟、肝臟、腦、甲狀腺或皮膚中。 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.

如請求項37-39中任一項所述的醫藥製劑或組合物，其中所述實體瘤是上皮性腫瘤、霍奇金氏淋巴瘤(HL)、非霍奇金氏淋巴瘤、攝護腺腫瘤、卵巢腫瘤、腎細胞腫瘤、胃腸道腫瘤、肝腫瘤、結直腸腫瘤、脈管腫瘤、間皮瘤腫瘤、胰臟腫瘤、***腫瘤、肉瘤腫瘤、肺腫瘤、結腸腫瘤、腦腫瘤、黑色素瘤腫瘤、小細胞肺腫瘤、神經母細胞瘤腫瘤、睾丸腫瘤、癌腫瘤、腺癌腫瘤、膠質瘤腫瘤、精原細胞瘤腫瘤、視網膜母細胞瘤或骨肉瘤腫瘤。 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.

如請求項1-40中任一項所述的醫藥製劑或組合物，其中所述RNA用於腫瘤內或腫瘤周邊投予。 The pharmaceutical preparation or composition according to any one of claims 1-40, wherein the RNA is for intratumor or peripheral administration.

如請求項1-41中任一項所述的醫藥製劑或組合物，其中所述RNA被配製用於注射。 The pharmaceutical preparation or composition according to any one of claims 1-41, wherein the RNA is formulated for injection.

如請求項1-42中任一項所述的醫藥製劑或組合物，其中所述RNA用於向人投予。 The pharmaceutical preparation or composition according to any one of claims 1-42, wherein the RNA is for administration to humans.

如請求項37-43中任一項所述的醫藥製劑或組合物，其中治療或預防所述實體瘤包括減小腫瘤的尺寸、預防緩解期癌症的復發或預防個體的癌症轉移。 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.

如請求項36-44中任一項所述的醫藥製劑或組合物，其中所述疾病或障礙的治療性治療或預防性治療還包括投予另外的療法。 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.

如請求項45所述的醫藥製劑或組合物，其中所述另外的療法包括選自以下的一種或多種：(i)手術以切除、割除或消減腫瘤，(ii)免疫療法，(iii)放射療法和(iv)化學療法。 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.

如請求項45-46中任一項所述的醫藥製劑或組合物，其中所述另外的療法包括投予另外的治療劑。 The pharmaceutical preparation or composition according to any one of claims 45-46, wherein the additional therapy comprises administration of another therapeutic agent.

如請求項47所述的醫藥製劑或組合物，其中所述另外的治療劑是抗癌治療劑。 The pharmaceutical preparation or composition according to claim 47, wherein the additional therapeutic agent is an anticancer therapeutic agent.

如請求項47或48所述的醫藥製劑或組合物，其中所述另外的治療劑是檢查點調節劑。 The pharmaceutical preparation or composition according to claim 47 or 48, wherein the additional therapeutic agent is a checkpoint modifier.

如請求項49所述的醫藥製劑或組合物，其中所述檢查點調節劑是抗PD1抗體、抗CTLA-4抗體或抗PD1抗體和抗CTLA-4抗體的組合。 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.

一種用於治療實體瘤或減少實體瘤可能性的方法，其包括向有需要的個體投予第一RNA，其中所述第一RNA編碼IL-12sc蛋白、IL-15 sushi蛋白、FLT3-L蛋白或IFNα蛋白，並且所述個體進一步用另外的RNA治療，其中： 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.如果所述第一RNA編碼IL-12sc蛋白，則所述另外的RNA包含編碼IL-15 sushi蛋白的RNA、編碼IFNα蛋白的RNA和編碼FLT3-L蛋白的RNA；或 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.如果所述第一RNA編碼IL-15 sushi蛋白，則所述另外的RNA包含編碼IL-12sc蛋白的RNA、編碼IFNα蛋白的RNA和編碼FLT3-L蛋白的RNA；或 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.如果所述第一RNA編碼IFNα蛋白，則所述另外的RNA包含編碼IL-15 sushi蛋白的RNA、編碼IL-12sc蛋白的RNA和編碼FLT3-L蛋白的RNA；或 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.如果所述第一RNA編碼FLT3-L蛋白，則所述另外的RNA包含編碼IL-15 sushi蛋白的RNA、編碼IFNα蛋白的RNA和編碼IL-12sc蛋白的RNA；或 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.如果所述第一RNA編碼IL-12sc蛋白，則所述另外的RNA包含編碼IL-15 sushi蛋白的RNA和編碼IFNα蛋白的RNA；或 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.如果所述第一RNA編碼IL-15 sushi蛋白，則所述另外的RNA包含編碼IL-12sc蛋白的RNA和編碼IFNα蛋白的RNA；或 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.如果所述第一RNA編碼IFNα蛋白，則所述另外的RNA包含編碼IL-15 sushi蛋白的RNA和編碼IL-12sc蛋白的RNA。 g. If the first RNA encodes IFNα protein, the additional RNA includes RNA encoding IL-15 sushi protein and RNA encoding IL-12sc protein.

一種套組，其包括如請求項1-50中任一項所述的組合物。 A kit comprising the composition according to any one of claims 1-50.

如請求項1-2或4-50中任一項所述的醫藥製劑，其中所述醫藥製劑是套組。 The pharmaceutical preparation according to any one of claims 1-2 or 4-50, wherein the pharmaceutical preparation is a kit.

如請求項53所述的醫藥製劑或如請求項52所述的套組，其中所述RNA在單獨的小瓶中。 The pharmaceutical preparation according to claim 53 or the kit according to claim 52, wherein the RNA is in a separate vial.

如請求項52-54中任一項所述的套組，其還包含用於使用所述組合物治療或預防實體瘤的說明。 The kit according to any one of claims 52-54, which further comprises instructions for using the composition to treat or prevent solid tumors.

一種RNA，其用於在用於治療或預防個體的實體瘤的方法中使用，其中所述方法包括投予編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA和編碼IFNα蛋白的RNA。 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 .

一種編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA和編碼IFNα蛋白的RNA用於治療實體瘤的用途。 The use of RNA encoding IL-12sc protein, RNA encoding IL-15 sushi protein and RNA encoding IFNα protein for treating solid tumors.

如請求項56-57中任一項所述的RNA或用途，其還包括編碼FLT3-L蛋白的RNA。 The RNA or use according to any one of claims 56-57, which further includes RNA encoding FLT3-L protein.

如請求項56-58中任一項所述的RNA或用途，其中所述RNA具有1：1：1或1：1：1：1的比率，並且其中所述比率藉由定量RT-PCR驗證。 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 .

如請求項56-59中任一項所述的RNA或用途，其中所述RNA完整性大於或等於70%。 The RNA or use according to any one of claims 56-59, wherein the RNA integrity is greater than or equal to 70%.

如請求項56-60中任一項所述的RNA或用途，其中所述醫藥製劑或組合物包含少於250ng DNA/存在的核酸的總mg。 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.

如請求項56-61中任一項所述的RNA或用途，其中所述IFNα蛋白是IFNα2b蛋白。 The RNA or use according to any one of claims 56-61, wherein the IFNα protein is IFNα2b protein.

如請求項56-62中任一項所述的RNA或用途，其中 The RNA or use according to any one of claims 56-62, wherein

如請求項56-63中任一項所述的RNA或用途，其中 The RNA or use according to any one of claims 56-63, wherein

如請求項58-64中任一項所述的RNA或用途，其中 The RNA or use according to any one of claims 58-64, wherein

如請求項56-65中任一項所述的RNA或用途，其中 The RNA or use according to any one of claims 56-65, wherein

如請求項56-66中任一項所述的RNA或用途，其中至少一種RNA包含替代至少一個尿苷的修飾核苷。 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.

如請求項56-67中任一項所述的RNA或用途，其中至少一種RNA包含替代每個尿苷的修飾核苷。 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.

如請求項56-68中任一項所述的RNA或用途，其中每種RNA包含替代至少一個尿苷的修飾核苷。 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.

如請求項56-69中任一項所述的RNA或用途，其中每種RNA包含替代每個尿苷的修飾核苷。 The RNA or use according to any one of claims 56-69, wherein each RNA comprises a modified nucleoside in place of each uridine.

如請求項67-70中任一項所述的RNA或用途，其中所述修飾核苷獨立地選自假尿苷(ψ)、N1-甲基-假尿苷(m1ψ)和5-甲基-尿苷(m5U)。 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).

如請求項67-71中任一項所述的RNA，其中至少一種RNA包含多於一種類型的修飾核苷，其中所述修飾核苷獨立地選自假尿苷(ψ)、N1-甲基-假尿苷(m1ψ)和5-甲基-尿苷(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).

如請求項72所述的RNA或用途，其中所述修飾核苷是N1-甲基-假尿苷(m1ψ)。 The RNA or use according to claim 72, wherein the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

如請求項56-73中任一項所述的RNA或用途，其中至少一種RNA包含5’帽m27,3’-OGppp(m12’-O)ApG或3’-O-Me-m7G(5’)ppp(5’)G。 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.

如請求項56-73中任一項所述的RNA或用途，其中每種RNA包含5’帽m27,3’-OGppp(m12’-O)ApG或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.

如請求項56-75中任一項所述的RNA或用途，其中至少一種RNA包含含有選自SEQ ID NO：4和6組成之群組的核苷酸序列或與選自SEQ ID NO：4 和6組成之群組的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的5’ UTR。 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.

如請求項56-76中任一項所述的RNA或用途，其中每種RNA包含含有選自SEQ ID NO：4和6組成之群組的核苷酸序列或與選自SEQ ID NO：4和6組成之群組的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的5’ UTR。 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.

如請求項56-77中任一項所述的RNA或用途，其中至少一種RNA包含含有SEQ ID NO：8的核苷酸序列或與SEQ ID NO：8的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的3’ UTR。 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.

如請求項56-78中任一項所述的RNA或用途，其中每種RNA包含含有SEQ ID NO：8的核苷酸序列或與SEQ ID NO：8的核苷酸序列具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性的核苷酸序列的3’ UTR。 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.

如請求項56-79中任一項所述的RNA或用途，其中至少一種RNA包含多聚A尾。 The RNA or use according to any one of claims 56-79, wherein at least one RNA comprises a poly A tail.

如請求項56-80中任一項所述的RNA或用途，其中每種RNA包含多聚A尾。 The RNA or use according to any one of claims 56 to 80, wherein each RNA comprises a poly A tail.

如請求項80或81所述的RNA或用途，其中所述多聚A尾包含至少100個核苷酸。 The RNA or use according to claim 80 or 81, wherein the poly A tail contains at least 100 nucleotides.

如請求項80-82中任一項所述的RNA或用途，其中所述多聚A尾包含示於SEQ ID NO：66的多聚A尾。 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.

如請求項56-83中任一項所述的RNA或用途，其中一種或多種RNA包含： The RNA or use according to any one of claims 56-83, wherein one or more RNAs comprise:

如請求項84所述的RNA或用途，其中所述多聚A尾包含SEQ ID NO：66。 The RNA or use according to claim 84, wherein the poly A tail comprises SEQ ID NO:66.

如請求項56-85中任一項所述的RNA或用途，其中所述方法還包括投予另外的療法。 The RNA or use according to any one of claims 56-85, wherein the method further comprises administering another therapy.

如請求項86所述的RNA或用途，其中所述另外的療法包括選自以下的一種或多種：(i)手術以切除、割除或消減腫瘤，(ii)免疫療法，(iii)放射療法和(iv)化學療法。 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.

如請求項86或87所述的RNA或用途，其中所述另外的療法包括投予另外的治療劑。 The RNA or use according to claim 86 or 87, wherein the additional therapy comprises administration of another therapeutic agent.

如請求項88所述的RNA或用途，其中所述另外的治療劑是抗癌治療劑。 The RNA or use according to claim 88, wherein the additional therapeutic agent is an anticancer therapeutic agent.

如請求項86或87所述的RNA或用途，其中所述另外的治療劑是檢查點調節劑。 The RNA or use according to claim 86 or 87, wherein the additional therapeutic agent is a checkpoint modulator.

如請求項90所述的RNA或用途，其中所述檢查點調節劑是抗PD1抗體、抗CTLA-4抗體或抗PD1抗體和抗CTLA-4抗體的組合。 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.

如請求項56-91中任一項所述的RNA或用途，其中所述實體瘤是肉瘤、癌或淋巴瘤。 The RNA or use according to any one of claims 56-91, wherein the solid tumor is a sarcoma, carcinoma or lymphoma.

如請求項56-91中任一項所述的RNA或用途，其中所述實體瘤是在肺、結腸、卵巢、子宮頸、子宮、腹膜、睾丸、陰莖、舌、淋巴結、胰臟、骨、***、攝護腺、軟組織、結締組織、腎臟、肝臟、腦、甲狀腺或皮膚中。 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.

如請求項56-93中任一項所述的RNA或用途，其中所述實體瘤是上皮性腫瘤、霍奇金氏淋巴瘤(HL)、非霍奇金氏淋巴瘤、攝護腺腫瘤、卵巢腫瘤、腎細胞腫瘤、胃腸道腫瘤、肝腫瘤、結直腸腫瘤、脈管腫瘤、間皮瘤腫瘤、胰臟腫瘤、***腫瘤、肉瘤腫瘤、肺腫瘤、結腸腫瘤、腦腫瘤、黑色素瘤腫瘤、小細胞肺腫瘤、神經母細胞瘤腫瘤、睾丸腫瘤、癌腫瘤、腺癌腫瘤、膠質瘤腫瘤、精原細胞瘤腫瘤、視網膜母細胞瘤或骨肉瘤腫瘤。 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.

如請求項56-94中任一項所述的RNA或用途，其中所述RNA是腫瘤內或腫瘤周邊投予的。 The RNA or use according to any one of claims 56-94, wherein the RNA is administered in or around a tumor.

如請求項56-95中任一項所述的RNA或用途，其中所述RNA被配製用於注射。 The RNA or use according to any one of claims 56-95, wherein the RNA is formulated for injection.

如請求項86-96中任一項所述的RNA或用途，其中所述另外的治療劑是全身投予的。 The RNA or use according to any one of claims 86-96, wherein the additional therapeutic agent is administered systemically.

如請求項56-97中任一項所述的RNA或用途，其中所述個體是人。 The RNA or use according to any one of claims 56-97, wherein the individual is human.

如請求項56-98中任一項所述的RNA或用途，其中所述RNA是同時投予的。 The RNA or use according to any one of claims 56-98, wherein the RNA is administered simultaneously.

如請求項56-99中任一項所述的RNA或用途，其中所述RNA是藉由注射投予的，其中在注射之前，將所述RNA在液體溶液中混合在一起。 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.

如請求項56-100中任一項所述的RNA或用途，其中所述RNA是藉由投予包含所述RNA的組合的組合物投予的。 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.

如請求項56-100中任一項所述的RNA或用途，其中治療或預防實體瘤包括減小腫瘤的尺寸、預防緩解期癌症的復發或預防個體的癌症轉移。 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.

一種組合物或醫藥製劑，其包含編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA、編碼IFNα蛋白的RNA和編碼FLT3-L蛋白的RNA。 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.

如請求項103所述的組合物。 The composition according to claim 103.

如請求項103所述的醫藥製劑。 The pharmaceutical preparation described in claim 103.

如請求項103-105所述的醫藥製劑或組合物，其中所述RNA具有1：1：1或1：1：1：1的比率，並且其中所述比率藉由定量RT-PCR驗證。 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.

如請求項103-106所述的醫藥製劑或組合物，其中所述RNA完整性大於或等於70%。 The pharmaceutical preparation or composition according to claim 103-106, wherein the RNA integrity is greater than or equal to 70%.

如請求項103-107所述的醫藥製劑或組合物，其中所述醫藥製劑或組合物包含少於250ng DNA/存在的核酸的總mg。 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.

如請求項103-108所述的醫藥製劑或組合物，其中所述IFNα蛋白是IFNα2b蛋白。 The pharmaceutical preparation or composition according to claim 103-108, wherein the IFNα protein is IFNα2b protein.

如請求項103-109所述的醫藥製劑或組合物，其中 The pharmaceutical preparation or composition according to claim 103-109, wherein

(iii)所述編碼IL-12sc蛋白的RNA包含與IL-12sc的p40部分(SEQ ID NO：17或18的核苷酸1-984)具有至少99%、98%、97%、96%、95%、90%、85%或80%同一性並且與IL-12sc的p30部分(SEQ ID NO：17或18的核苷酸1027-1623)具有至少99%、98%、97%、96%、95%、90%、85%或80% 同一性的核苷酸序列，並且還包含編碼連接子多肽的在所述p40部分與p35部分之間的核苷酸。 (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.

如請求項103-110所述的醫藥製劑或組合物，其中 The pharmaceutical preparation or composition according to claim 103-110, wherein

如請求項103-111所述的醫藥製劑或組合物，其中 The pharmaceutical preparation or composition according to claim 103-111, wherein

如請求項103-112所述的醫藥製劑或組合物，其中 The pharmaceutical preparation or composition according to claim 103-112, wherein

如請求項103-113所述的醫藥製劑或組合物，其是包含所述RNA的藥物組合物。 The pharmaceutical preparation or composition according to claim 103-113, which is a pharmaceutical composition containing the RNA.

如請求項114所述的醫藥製劑或組合物，其中所述藥物組合物還包含一種或多種藥學上可接受的載體、稀釋劑和/或賦形劑。 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.

如請求項103-115所述的醫藥製劑或組合物，其中所述RNA被配製為液體、配製為固體或其組合。 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.

如請求項103-116所述的醫藥製劑或組合物，其用於藥物用途。 The pharmaceutical preparation or composition according to claim 103-116, which is used for pharmaceutical use.

如請求項103-117所述的醫藥製劑或組合物，其中所述藥物用途包括疾病或障礙的治療性治療或預防性治療。 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.

如請求項103-118所述的醫藥製劑或組合物，其中所述疾病或障礙的治療性治療或預防性治療包括治療或預防實體瘤。 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.

如請求項119所述的醫藥製劑或組合物，其中所述實體瘤是肉瘤、癌或淋巴瘤。 The pharmaceutical preparation or composition according to claim 119, wherein the solid tumor is sarcoma, carcinoma or lymphoma.

如請求項119-120所述的醫藥製劑或組合物，其中所述實體瘤是在肺、結腸、卵巢、子宮頸、子宮、腹膜、睾丸、陰莖、舌、淋巴結、胰臟、骨、***、攝護腺、軟組織、結締組織、腎臟、肝臟、腦、甲狀腺或皮膚中。 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.

如請求項103-121所述的醫藥製劑或組合物，其中所述實體瘤是上皮性腫瘤、霍奇金氏淋巴瘤(HL)、非霍奇金氏淋巴瘤、攝護腺腫瘤、卵巢腫瘤、腎細胞腫瘤、胃腸道腫瘤、肝腫瘤、結直腸腫瘤、脈管腫瘤、間皮瘤腫瘤、胰臟腫瘤、***腫瘤、肉瘤腫瘤、肺腫瘤、結腸腫瘤、腦腫瘤、黑色素瘤腫瘤、小細胞肺腫瘤、神經母細胞瘤腫瘤、睾丸腫瘤、癌腫瘤、腺癌腫瘤、膠質瘤腫瘤、精原細胞瘤腫瘤、視網膜母細胞瘤或骨肉瘤腫瘤。 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.

一種RNA，其用於在用於治療或預防個體的實體瘤的方法中使用，其中所述方法包括投予編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA、編碼IFNα蛋白的RNA和編碼FLT3-L蛋白的RNA。 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.

一種編碼IL-12sc蛋白的RNA、編碼IL-15 sushi蛋白的RNA、編碼IFNα蛋白的RNA和編碼FLT3-L的RNA用於治療實體瘤的用途。 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.