WO2023246938A1 - 用于实体肿瘤的治疗性mRNA及其应用 - Google Patents

用于实体肿瘤的治疗性mRNA及其应用 Download PDF

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WO2023246938A1
WO2023246938A1 PCT/CN2023/102158 CN2023102158W WO2023246938A1 WO 2023246938 A1 WO2023246938 A1 WO 2023246938A1 CN 2023102158 W CN2023102158 W CN 2023102158W WO 2023246938 A1 WO2023246938 A1 WO 2023246938A1
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mrna encoding
seq
mrna
encoding
csf
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PCT/CN2023/102158
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French (fr)
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胡勇
汤赞
蔡冠星
周瑞国
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深圳瑞吉生物科技有限公司
武汉瑞佶生物科技有限公司
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Publication of WO2023246938A1 publication Critical patent/WO2023246938A1/zh

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    • A—HUMAN NECESSITIES
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
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    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
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    • A61K38/19—Cytokines; Lymphokines; Interferons
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    • A—HUMAN NECESSITIES
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
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    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
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    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

  • the present invention relates to a therapeutic mRNA for solid tumors and related applications.
  • Solid tumors are difficult to treat due to their tumor microenvironment and heterogeneity.
  • Current treatments mainly include surgery, radiotherapy, chemotherapy and immunotherapy.
  • Surgery alone can treat locally distributed tumors, but large metastatic and malignant tumors are often difficult to treat surgically.
  • Other commonly used radiotherapy and chemotherapy have strong toxic side effects.
  • surgery and current treatments can kill large numbers of tumor cells, potential cancer stem cells may survive and form new tumors over time, causing tumor recurrence.
  • Cytokines are synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) after stimulation.
  • immune cells such as monocytes, macrophages, T cells, B cells, NK cells, etc.
  • non-immune cells endothelial cells, epidermal cells, fibroblasts, etc.
  • Cytokines generally regulate cell growth, differentiation and effects by binding to corresponding receptors, and regulate immune responses.
  • cytokines such as IL-2 have been approved by the FDA for the treatment of melanoma.
  • systemic administration of cytokines can cause systemic toxicity, the popularity of cytokines for treating tumors is not high, and existing cytokines for treating tumors have not achieved ideal efficacy.
  • One object of the present invention is to provide a new drug for preventing and treating solid tumors.
  • the present invention uses mRNA to encode at least two cytokines for treating tumors, which can change the microenvironment of the tumor, recruit and activate immune cells to kill tumor cells while reducing systemic toxicity. , can achieve the effect of reducing tumors and prolonging survival.
  • the invention provides an mRNA comprising an mRNA encoding at least two of the following cytokines:
  • IL-7 IL-12sc, IFN ⁇ , TNF ⁇ , IL-12A, IL-12B, GM-CSF.
  • the IFN ⁇ is preferably IFN ⁇ 4 and/or IFN ⁇ 2b.
  • one mRNA chain may encode only one cytokine or two or more cytokines.
  • one mRNA strand can encode both IL-12A and IL-12B proteins.
  • the mRNA of the present invention is a composition, which includes mRNA encoding IFN ⁇ , mRNA encoding IL-7, mRNA encoding TNF ⁇ , mRNA encoding IL-12A, and mRNA encoding IL-12B. , any of the mRNA encoding IL-12A and IL-12B, the mRNA encoding IL-12sc, and the mRNA encoding GM-CSF Two, three, four, five, six, seven or eight types of mRNA.
  • IFN ⁇ has the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.6 or SEQ ID NO.70;
  • IL-7 has the amino acid sequence shown in SEQ ID NO.11 or SEQ ID NO.65;
  • TNF ⁇ has the amino acid sequence shown in SEQ ID NO.18 or SEQ ID NO.75;
  • GM-CSF has the amino acid sequence shown in SEQ ID NO.35 or SEQ ID NO.80;
  • IL-12A has the amino acid sequence shown in SEQ ID NO.25 or SEQ ID NO.54;
  • IL-12B has the amino acid sequence shown in SEQ ID NO.30 or SEQ ID NO.57;
  • IL-12sc has the amino acid sequence shown in SEQ ID NO.43 or SEQ ID NO.49 or SEQ ID NO.60.
  • the mRNA encoding IFN ⁇ includes one or more of the following mRNAs: SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.10 , the mRNA of any sequence shown in SEQ ID NO.74, and any one of the sequences shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.74 mRNAs whose sequences have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% similarity.
  • the mRNA encoding IL-7 includes one or more of the following mRNAs: SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO .67, the mRNA of any sequence shown in SEQ ID NO.69, and any one of SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.67, SEQ ID NO.69
  • the sequences shown are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% similar to mRNA.
  • the mRNA encoding TNF ⁇ includes one or more of the following mRNAs: SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.77 , the mRNA of any sequence shown in SEQ ID NO.79, and any one of the sequences shown in SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.77, SEQ ID NO.79 mRNAs whose sequences have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% similarity.
  • the mRNA encoding IL-12A includes one or more of the following mRNAs: any one of SEQ ID NO.27, SEQ ID NO.29, and SEQ ID NO.56
  • the mRNA with the sequence shown has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, An mRNA that is at least 97%, at least 98%, at least 99% similar.
  • the mRNA encoding IL-12B includes one or more of the following mRNAs: any one of SEQ ID NO.32, SEQ ID NO.34, and SEQ ID NO.59
  • the mRNA with the sequence shown has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, An mRNA that is at least 97%, at least 98%, at least 99% similar.
  • the mRNA encoding IL-12A and IL-12B can be one mRNA encoding both IL-12A and IL-12B, wherein IL-12A and IL-12B can also be transmitted through a linker (such as SEQ ID NO.42 or SEQ ID NO.48, etc.) are connected to form IL-12sc (in the present invention, the protein formed by connecting the amino acid sequences of IL-12A and IL-12B through a linker is named IL-12sc, IL-12sc
  • the amino acid sequence from N-terminus to C-terminus can be IL-12A, IL-12B, or IL-12B, IL-12A).
  • the mRNA encoding IL-12sc includes one or more of the following mRNAs: SEQ ID NO.45, SEQ ID NO.47, SEQ ID NO.51, SEQ ID NO. 53.
  • the mRNA encoding GM-CSF includes one or more of the following mRNAs: any one of SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.41, SEQ ID NO.82, and SEQ ID NO.84
  • the mRNA with the sequence shown has at least 80%, at least 85%, and An mRNA that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% similar.
  • the mRNA of the present invention at least includes mRNA encoding IFN ⁇ , mRNA encoding IL-7 or mRNA encoding TNF ⁇ .
  • the mRNA of the present invention at least includes mRNA encoding IL-12A and IL-12B.
  • the mRNA of the present invention includes one or more groups of the following groups of mRNAs:
  • mRNA encoding IL-7 mRNA encoding TNF ⁇ , mRNA encoding IL-12A, mRNA encoding IL-12B, and mRNA encoding GM-CSF;
  • mRNA encoding IFN ⁇ mRNA encoding IL-7
  • mRNA encoding TNF ⁇ mRNA encoding IL-12sc
  • mRNA encoding GM-CSF mRNA encoding GM-CSF
  • the IFN ⁇ may specifically be IFN ⁇ 4 and/or IFN ⁇ 2b.
  • part or all of the mRNA of the present invention has one or more of the following characteristics:
  • the mRNA exists in the form of naked RNA, LNP package, LPX package or LPP package.
  • the chemically modified nucleoside is selected from the group consisting of 2-fluoro-2-deoxyadenosine, 2-fluoro-2-deoxyuridine, 2-fluoro-2-deoxycytidine, 2-fluoro-2-deoxyguanosine, 2-fluoro-2-deoxy-5-methylcytidine, 2-fluoro-2-deoxy-pseudouridine, 2-fluoro-2-deoxy-N1-methyl- Pseudouridine, 2-fluoro-2-deoxy-N7-methyl-guanosine, 2-fluoro-2-deoxy-5-methoxyuridine, 2-fluoro-2-deoxy-N4-acetylcytidine , 2-fluoro-2-deoxy-N6-methyladenosine, 5-methylcytidine, pseudouridine, N1-methyl-pseudouridine, N7-methyl-guanosine, 5-methoxyuridine
  • the 5' cap structure is selected from Cap0, Cap1, Cap2, ARCA, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza -Guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, 7-methyl-guanosine-5′-triphosphate-5′- Adenosine, guanosine-5′-triphosphate-5′-adenosine, 7-methyl-guanosine-5′-triphosphate-5′-guanosine, guanosine-5′-triphosphate-5′- One or more of guanosine and 7-methyl-guanosine-5′-triphosphate-5′-2-methoxyadenine-guanosine.
  • the poly-A tail contains at least 50, at least 80 or at least 100 nucleotides.
  • the integrity of each mRNA is greater than 70%.
  • the mRNA of the present invention is a composition including a composition encoding a single cytokine respectively.
  • the mRNA of the present invention is a composition, wherein the amount of the mRNA encoding each cytokine in the composition is 0.1 to 10 parts by weight, preferably 0.5 to 5 parts by weight, and more preferably 0.5 to 1 parts by weight.
  • the dosage ratio of the mRNA encoding each cytokine can also be optimized based on RT-PCR.
  • the amounts of the mRNA encoding each cytokine in the composition are substantially equal in mass, that is, the masses of the mRNA encoding each cytokine in the composition are substantially the same.
  • the amounts of each cytokine encoded by the mRNA of the present invention are substantially equal.
  • the present invention also provides DNA that can be transcribed into the above-mentioned mRNA.
  • the DNA is a composition corresponding to the above-mentioned mRNA combination.
  • the DNA includes nucleic acids encoding the mRNA of at least two of the aforementioned cytokines.
  • the present invention also provides a pharmaceutical composition, which pharmaceutical composition includes:
  • the mRNA of the present invention and pharmaceutically acceptable excipients.
  • the pharmaceutical composition of the present invention may be in a liquid dosage form, a solid dosage form or a combination form.
  • the present invention also provides the use of the mRNA or the pharmaceutical composition in preparing drugs for preventing and treating solid tumors.
  • the present invention also provides a method for preventing and/or treating solid tumors, which method includes administering to a subject an effective amount of the mRNA of the present invention or the pharmaceutical composition of the present invention.
  • prevention and treatment includes prevention and/or treatment (including adjuvant treatment).
  • the prevention and treatment of solid tumors includes inhibiting tumor growth, reducing tumor size, preventing tumor recurrence, and/or preventing tumor metastasis.
  • the drug for preventing and treating solid tumors is intratumoral injection or paratumoral injection.
  • the mRNA of the present invention can be used for intratumoral injection or paratumoral injection.
  • the drugs for preventing and treating solid tumors also include immune checkpoint inhibitors. That is, the mRNA of the present invention can be used in combination with immune checkpoint inhibitors.
  • the immune checkpoint inhibitors include PD-1 antibodies and/or CTLA-4 antibodies.
  • the mRNA of the present invention has a preventive and therapeutic effect on tumors, and can inhibit tumor growth, reduce tumor size, prevent tumor recurrence and/or prevent tumor metastasis.
  • Figure 1 shows the changes in tumor size in B16F10 tumor mice after injection of different mRNAs.
  • Figure 2 shows the survival curve of B16F10 tumor mice after injection of different mRNAs.
  • Figure 3 shows the changes in tumor size after another batch of experimental B16F10 tumor mice were injected with different mRNAs.
  • Figure 4 shows the changes in another batch of experimental CT26 tumor mice after intratumoral injection of mRNA.
  • CR Complete response.
  • Figure 5 shows the changes in tumor size after another batch of experimental CT26 tumor mice were injected with different mRNA combinations.
  • Figure 6 shows another batch of experimental CT26 tumor mice injected with 60 ⁇ g control mRNA or IFN ⁇ 4+IL-12sc+IL-7 cytokine mRNA mixture (20 ⁇ g mRNA/gene) on D9, D12, D15, D18, D21, and D25 respectively. Changes in tumor size.
  • each sequence is as follows:
  • SEQ ID NO.1 human IFNa4(amino acid)
  • SEQ ID NO.5 human IFNa4 optimized RNA encoding CDS
  • SEQ ID NO.15 human IL-7 optimized RNA encoding CDS 1
  • SEQ ID NO.17 human IL-7 optimized RNA encoding CDS 2
  • SEQ ID NO.18 human TNF- ⁇ (amino acid)
  • SEQ ID NO.20 human TNF ⁇ non optimized RNA encoding CDS
  • SEQ ID NO.21 human TNF ⁇ optimized CDS 1
  • SEQ ID NO.22 human TNF ⁇ optimized RNA encoding CDS 1
  • SEQ ID NO.24 human TNF ⁇ optimized RNA encoding CDS 2
  • SEQ ID NO.29 human IL-12A optimized RNA encoding CDS
  • SEQ ID NO.35 human GM-CSF amino acid
  • SEQ ID NO.36 human GM-CSF no optimized CDS
  • SEQ ID NO.38 human GM-CSF optimized CDS 1
  • SEQ ID NO.39 human GM-CSF optimized RNA encoding CDS 1
  • SEQ ID NO.40 human GM-CSF optimized CDS 2
  • SEQ ID NO.41 human GM-CSF optimized RNA encoding CDS 2
  • SEQ ID NO.45 human IL-12sc 1 non-optimized RNA encoding CDS
  • SEQ ID NO.62 murine IL-12sc non-optimized RNA encoding CDS
  • SEQ ID NO.80 murine GM-CSF amino acid
  • SEQ ID NO.82 murine GM-CSF non-optimized RNA encoding CDS
  • SEQ ID NO.90 luciferase RNA encoding CDS
  • mice 6-8 weeks old 18-20g female C57BL/6J (purchased from Zhuhai Beston) mice were kept in a mouse room with a temperature of 22 ⁇ 2°C and a relative humidity of 55% ⁇ 15%. The mice had free access to food and water. .
  • B16F10 cells purchased from ATCC
  • B16F10 cells were cultured with DMEM and 10% FBS at 37°C, 5% CO2.
  • B16F10 cells were cultured until the confluence was 80-90%, digested with 0.25% Trypin-EDTA and resuspended in DPBS. 1 ⁇ 106 cells/100 ⁇ l were subcutaneously injected into the right side of each mouse. On the 7th day after the injection of tumor cells, A B16F10 tumor mouse model capable of intratumoral injection of mRNA was obtained today.
  • Eligibility criteria A single band appears in electrophoresis detection and the size is correct.
  • NanoDrop to detect the concentration of the purified template and the ratios at 260/280 and 260/230. Samples were taken for DNA agarose gel electrophoresis detection (1.5% agarose, 5V/min, 40min).
  • Qualifying standards 260/280 between 1.8 and 2.1, 260/230 between 1.6 and 2.2.
  • Millipore 30Kd ultrafiltration tube concentrates the DNA template purified by FPLC, and elutes and dissolves it with RNase-free water. Use NanoDrop to detect the concentration of the template after ultrafiltration, as well as the ratios of A260/A280 and A260/A230. Finally dilute to 150ng/ml with RNase-free water.
  • Reaction volume 1600ml (placed in a 2ml RNase-free Tube, it is the reaction volume of a single tube, multiple tubes can be reacted at the same time): RNA-free water 440ml, 7.5mM ATP 160ml, 7.5mM UTP 160ml, 7.5mM CTP 160ml, 7.5mM GTP 160ml, 7.5mM M7G (2'OMeA) pG 160ml, 150ng/ml DNA template 40ml, 10 ⁇ Buffer 160ml and Enzyme Mix 160ml.
  • RNA The procedure for in vitro synthesis of RNA is 37°C, 10 h.
  • the recovered mRNA concentration detected by NanoDrop was 5 mg/ml, A260/A280 was 1.90, and A260/A230 was 2.0.
  • mice Inject control mRNA or cytokine mRNA mixture into the tumors of B16F10 model mice through intratumoral injection (8 to 9 mice in each group).
  • the mRNA injected into the mouse tumors is mixed with the same mass and diluted with physiological saline.
  • the mRNA used for intratumoral injection in mice was the mouse version of various cytokines (see Table 1 for experimental groupings).
  • the size of mouse tumors was measured with a vernier caliper every 3 to 4 days. When the tumor size exceeded 2000 mm 3 or the mouse body weight decreased by 20%, they were sacrificed.
  • the changes in tumor size of B16F10 tumor mice after injection of control mRNA, IFN ⁇ 4, IL-7, GM-CSF, IL-12A+IL-12B, TNF ⁇ , and cytokine mRNA mixture on D7, D10, D13, D17, and D20 are shown in the figure. 1.
  • 10 ⁇ g of single factor mRNA was given to each mouse
  • 60 ⁇ g of control mRNA was given to each mouse
  • the cytokine mRNA mixture was given to each mouse at 10 ⁇ g of mRNA/gene (i.e., a total of 20 ⁇ g of 2 cytokine mRNA mixtures were given to each mouse).
  • ⁇ g mRNA a mixture of 4 cytokines mRNA was given to each mouse, a total of 40 ⁇ g mRNA, a mixture of 6 cytokines mRNA was given to each mouse, a total of 60 ⁇ g mRNA), and tumor size was monitored until 31 days.
  • the survival curves of B16F10 tumor mice after injection of control mRNA and cytokine mRNA mixture are shown in Figure 2.
  • Mice were injected with 60 ⁇ g control mRNA and cytokine mRNA mixture (10 ⁇ g mRNA/gene) on D7, D10, D13, D17, and D20 and tumor size was monitored until 31 days.
  • mice The changes in tumor size of another batch of experimental B16F10 tumor mice after injection of control mRNA and different cytokine mRNA mixtures are shown in Figure 3.
  • Mice were injected with 60 ⁇ g control mRNA and cytokine mRNA mixture (10 ⁇ g mRNA/gene) on D7, D11, D15, D19, and D22 and tumor size was monitored until 25 days.
  • the mRNA composition of the present invention contains 2-6 components of the following mRNAs: mRNA encoding IFN ⁇ 4, mRNA encoding IL-7, mRNA encoding IL-12A, mRNA encoding IL-12B, and TNF ⁇ mRNA and mRNA encoding GM-CSF have therapeutic effects on tumors.
  • RNA preparation method is the same as in Example 1.
  • the mRNA of each cytokine was prepared. And grouped according to the scheme in Table 3.
  • Transplant 5x10 5 CT26 tumor cells subcutaneously in BALB/C mice When the tumors grow to 20-40mm 3 , control mRNA or cytokine mRNA mixture is injected into the tumors of CT26 model mice through intratumoral injection (8 mice per group). Only). The mRNA injected into the mouse tumor was mixed with the same mass and diluted to 50 ⁇ l with physiological saline. The mRNA used for intratumoral injection in the mouse was the mouse version of the five cytokines. The size of mouse tumors was measured with a vernier caliper every 3 to 4 days. When the tumor size exceeded 2000 mm 3 or the mouse body weight decreased by 20%, they were sacrificed.
  • CT26 tumor mice after injection of 100 ⁇ g control mRNA and IFN ⁇ 4+IL-7+GM-CSF+IL12sc+TNF ⁇ cytokine mRNA mixture (20 ⁇ g mRNA/gene) on D6, D8, D11, D13, and D15 are shown in the figure. 4. Monitor tumor size until day 39. It can be seen that the CT26 tumors injected with control mRNA have been growing, while the growth rate of CT26 tumors injected with the cytokine mRNA mixture is not as fast as control mRNA, and 7 of the 8 tumors disappeared completely.
  • Transplant 5x10 5 CT26 tumor cells subcutaneously in BALB/C mice When the tumors grow to 20-50mm 3 , control mRNA or cytokine mRNA mixture is injected into the tumors of CT26 model mice through intratumoral injection (9 mice per group). Only). The mRNA injected into the mouse tumor was mixed with the same mass and diluted to 50 ⁇ l with physiological saline. The mRNA used for intratumoral injection in the mouse was the mouse version of the five cytokines. The size of mouse tumors was measured with a vernier caliper every 3 to 4 days. When the tumor size exceeded 2000 mm 3 or the mouse body weight decreased by 20%, they were sacrificed.
  • CT26 tumor mice on D8, D10, D11, D14, D17, and D19 were injected with 60 ⁇ g control mRNA, IFN ⁇ 4+IL-7+GM-CSF, TNF ⁇ +IL-12sc+GM-CSF, IL-7+IL-12sc+GM-CSF, IFN ⁇ 4+TNF ⁇ +GM-CSF, IFN ⁇ 4+TNF ⁇ +IL-12sc, IFN ⁇ 4+GM-CSF+IL-7, IFN ⁇ 4+GM-CSF+IL-12sc, IFN ⁇ 4+IL-12sc+IL-7, TNF ⁇ +IL-12sc+IL -7.
  • 5x10 5 and 3x10 5 CT26 tumor cells were transplanted subcutaneously on the left and right sides of BALB/C mice respectively.
  • the left side was injected into the tumor by intratumoral injection.
  • Control mRNA or cytokine combination IFN ⁇ 4+IL-12sc+IL-7mRNA mixture (8 mice in each group) was injected into the side tumor, and isotype antibody or anti-PD-1 antibody was injected into the abdominal cavity respectively.
  • the mRNA injected into the mouse tumor was mixed with the same mass and diluted to 50 ⁇ l with physiological saline.
  • the mRNA used for intratumoral injection in the mouse was the mouse version of the five cytokines.
  • the size of mouse tumors was measured with a vernier caliper every 3 to 4 days. When the tumor size exceeded 2000 mm 3 or the mouse body weight decreased by 20%, they were sacrificed.
  • the changes in tumor size of CT26 tumor mice after injection of 60 ⁇ g control mRNA or IFN ⁇ 4+IL-12sc+IL-7 cytokine mRNA mixture (20 ⁇ g mRNA/gene) on D9, D12, D15, D18, D21, and D25 are shown in Figure 6. Tumor size was monitored until day 28.
  • CT26 tumors injected with control mRNA have been growing, while the growth rate of CT26 tumors injected with the cytokine mRNA mixture is not as fast as that of control mRNA.
  • both the treated side and the untreated side The tumor growth rate was significantly lower than that of other treatment groups.

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Abstract

一种用于实体肿瘤的治疗性mRNA及其应用。所述mRNA包括编码以下细胞因子中的至少两种细胞因子的mRNA：IFNα、IL-7、TNFα、IL-12A、IL-12B、GM-CSF、IL-12sc。所述mRNA对肿瘤有防治作用，可抑制肿瘤增长、减少肿瘤大小、防止肿瘤复发和/或者防止肿瘤转移。

Description

用于实体肿瘤的治疗性mRNA及其应用 技术领域
本发明是关于一种用于实体肿瘤的治疗性mRNA及其相关应用。
背景技术
实体肿瘤由于其肿瘤微环境、异质性等原因难以治疗。目前的治疗手段主要包括手术、放疗、化疗和免疫治疗。单独的手术治疗能够治疗局部分布的肿瘤，但是对于大的转移性和恶性肿瘤通常很难用手术进行治疗。其他常用的放疗和化疗都有很强的毒副反应。尽管手术和目前治疗方法能够杀伤大量的肿瘤细胞，但是潜在的肿瘤干细胞可能能够存活，这些细胞随着时间的进行又能够形成新的肿瘤而导致肿瘤的复发。
细胞因子是由免疫细胞(如单核、巨噬细胞、T细胞、B细胞、NK细胞等)和某些非免疫细胞(内皮细胞、表皮细胞、纤维母细胞等)经刺激而合成、分泌的一类具有广泛生物学活性的小分子蛋白质。细胞因子一般通过结合相应受体调节细胞生长、分化和效应，调控免疫应答。目前已有细胞因子如IL-2获得FDA批准用于治疗黑色素瘤。但是，由于细胞因子进行全身性给药会带来系统性的毒性，而导致细胞因子治疗肿瘤的普及率并不高，并且，现有细胞因子治疗肿瘤并没有取得理想的疗效。
发明内容
本发明的目的之一在于提供一种新的用于防治实体肿瘤的药物。
本发明为克服现有技术细胞因子治疗实体肿瘤的缺点，采用mRNA编码至少两种细胞因子用于治疗肿瘤，能够改变肿瘤的微环境，招募和激活免疫细胞对肿瘤细胞进行杀伤同时减少全身性毒性，可以达到减小肿瘤、延长生存期的效果。
一方面，本发明提供了一种mRNA，该mRNA包括编码以下细胞因子中的至少两种细胞因子的mRNA：
IL-7、IL-12sc、IFNα、TNFα、IL-12A、IL-12B、GM-CSF。
根据本发明的具体实施方案，所述IFNα优选为IFNα4和/或IFNα2b。
根据本发明的具体实施方案，本发明的mRNA中，一条mRNA链可以只编码一个细胞因子，也可以编码两个或更多个细胞因子。例如，一条mRNA链可以同时编码IL-12A和IL-12B蛋白。
根据本发明的具体实施方案，本发明的mRNA为组合物，该组合物包括编码IFNα的mRNA、编码IL-7的mRNA、编码TNFα的mRNA、编码IL-12A的mRNA、编码IL-12B的mRNA、编码IL-12A和IL-12B的mRNA、编码IL-12sc的mRNA、编码GM-CSF的mRNA中的任意 两种、三种、四种、五种、六种、七种或八种mRNA。
根据本发明的具体实施方案，本发明中：
IFNα具有如SEQ ID NO.1或SEQ ID NO.6或SEQ ID NO.70所示氨基酸序列；
IL-7具有如SEQ ID NO.11或SEQ ID NO.65所示氨基酸序列；
TNFα具有如SEQ ID NO.18或SEQ ID NO.75所示氨基酸序列；
GM-CSF具有如SEQ ID NO.35或SEQ ID NO.80所示氨基酸序列；
IL-12A具有如SEQ ID NO.25或SEQ ID NO.54所示氨基酸序列；
IL-12B具有如SEQ ID NO.30或SEQ ID NO.57所示氨基酸序列；
IL-12sc具有如SEQ ID NO.43或SEQ ID NO.49或SEQ ID NO.60所示氨基酸序列。
根据本发明的具体实施方案，本发明中，编码IFNα的mRNA包括以下mRNA中的一种或多种：SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.8、SEQ ID NO.10、SEQ ID NO.74中任一所示序列的mRNA，与SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.8、SEQ ID NO.10、SEQ ID NO.74中任一所示序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％相似性的mRNA。
根据本发明的具体实施方案，本发明中，编码IL-7的mRNA包括以下mRNA中的一种或多种：SEQ ID NO.13、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.67、SEQ ID NO.69中任一所示序列的mRNA，与SEQ ID NO.13、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.67、SEQ ID NO.69中任一所示序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％相似性的mRNA。
根据本发明的具体实施方案，本发明中，编码TNFα的mRNA包括以下mRNA中的一种或多种：SEQ ID NO.20、SEQ ID NO.22、SEQ ID NO.24、SEQ ID NO.77、SEQ ID NO.79中任一所示序列的mRNA，与SEQ ID NO.20、SEQ ID NO.22、SEQ ID NO.24、SEQ ID NO.77、SEQ ID NO.79中任一所示序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％相似性的mRNA。
根据本发明的具体实施方案，本发明中，编码IL-12A的mRNA包括以下mRNA中的一种或多种：SEQ ID NO.27、SEQ ID NO.29、SEQ ID NO.56中任一所示序列的mRNA，与SEQ ID NO.27、SEQ ID NO.29、SEQ ID NO.56中任一所示序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％相似性的mRNA。
根据本发明的具体实施方案，本发明中，编码IL-12B的mRNA包括以下mRNA中的一种或多种：SEQ ID NO.32、SEQ ID NO.34、SEQ ID NO.59中任一所示序列的mRNA，与SEQ ID NO.32、SEQ ID NO.34、SEQ ID NO.59中任一所示序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％相似性的mRNA。
根据本发明的具体实施方案，本发明中，编码IL-12A和IL-12B的mRNA可以是一条mRNA同时编码IL-12A和IL-12B，其中IL-12A和IL-12B也可以通过linker(例如SEQ ID NO.42或SEQ ID NO.48等)连接成IL-12sc(本发明中，将IL-12A与IL-12B的氨基酸序列通过linker连接而形成的蛋白命名为IL-12sc，IL-12sc的氨基酸序列从N端到C端可以依次是IL-12A、IL-12B，也可以依次是IL-12B、IL-12A)。即，本发明中，所述编码IL-12A和IL-12B的mRNA的一些具体方案为编码IL-12sc的mRNA。根据本发明的具体实施方案，优选地，编码IL-12sc的mRNA包括以下mRNA中的一种或多种：SEQ ID NO.45、SEQ ID NO.47、SEQ ID NO.51、SEQ ID NO.53、SEQ ID NO.62、SEQ ID NO.64中任一所示序列的mRNA，与SEQ ID NO.45、SEQ ID NO.47、SEQ ID NO.51、SEQ ID NO.53、SEQ ID NO.62、SEQ ID NO.64中任一所示序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％相似性的mRNA。
编码GM-CSF的mRNA包括以下mRNA中的一种或多种：SEQ ID NO.37、SEQ ID NO.39、SEQ ID NO.41、SEQ ID NO.82、SEQ ID NO.84中任一所示序列的mRNA，与SEQ ID NO.37、SEQ ID NO.39、SEQ ID NO.41、SEQ ID NO.82、SEQ ID NO.84中任一所示序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％相似性的mRNA。
根据本发明的具体实施方案，本发明的mRNA至少包括编码IFNα的mRNA、编码IL-7的mRNA或编码TNFα的mRNA。
根据本发明的具体实施方案，本发明的mRNA至少包括编码IL-12A以及IL-12B的mRNA。
根据本发明的具体实施方案，本发明的mRNA包括以下各组mRNA中的一组或多组：
(1)编码IL-12A的mRNA以及编码IL-12B的mRNA；
(2)编码IFNα的mRNA以及编码TNFα的mRNA；
(3)编码IL-7的mRNA以及编码TNFα的mRNA；
(4)编码IFNα的mRNA以及编码IL-7的mRNA；
(5)编码IFNα的mRNA以及编码GM-CSF的mRNA；
(6)编码IFNα的mRNA以及编码IL-12sc的mRNA；
(7)编码IL-7的mRNA以及编码IL-12sc的mRNA；
(8)编码IL-7的mRNA以及编码GM-CSF的mRNA；
(9)编码GM-CSF的mRNA以及编码IL-12sc的mRNA；
(10)编码GM-CSF的mRNA以及编码TNFα的mRNA；
(11)编码IL-12sc的mRNA以及编码TNFα的mRNA；
(12)编码IFNα的mRNA、编码IL-7的mRNA以及编码TNFα的mRNA；
(13)编码TNFα的mRNA、编码IL-12sc的mRNA以及编码GM-CSF的mRNA；
(14)编码GM-CSF的mRNA、编码IL-7的mRNA以及编码IL-12sc的mRNA；
(15)编码IFNα的mRNA、编码TNFα的mRNA以及编码GM-CSF的mRNA；
(16)编码IFNα的mRNA、编码IL-12sc的mRNA以及编码TNFα的mRNA；
(17)编码IFNα的mRNA、编码IL-7的mRNA以及编码GM-CSF的mRNA；
(18)编码IFNα的mRNA、编码IL-12sc的mRNA以及编码GM-CSF的mRNA；
(19)编码IFNα的mRNA、编码IL-12sc的mRNA以及编码IL-7的mRNA；
(20)编码TNFα的mRNA、编码Il-12sc的mRNA以及编码IL-7的mRNA；
(21)编码的TNFαmRNA、编码GM-CSF的mRNA以及编码IL-12sc的mRNA；
(22)编码IFNα的mRNA、编码IL-7的mRNA、编码TNFα的mRNA以及编码GM-CSF的mRNA；
(23)编码IFNα的mRNA、编码IL-7的mRNA、编码TNFα的mRNA、编码IL-12A的mRNA以及编码IL-12B的mRNA；
(24)编码IL-7的mRNA、编码TNFα的mRNA、编码IL-12A的mRNA、编码IL-12B的mRNA以及编码GM-CSF的mRNA；
(25)编码IFNα的mRNA、编码TNFα的mRNA、编码IL-12A的mRNA、编码IL-12B的mRNA以及编码GM-CSF的mRNA；
(26)编码IFNα的mRNA、编码IL-7的mRNA、编码IL-12A的mRNA、编码IL-12B的mRNA以及编码GM-CSF的mRNA；
(27)编码IFNα的mRNA、编码IL-7的mRNA、编码TNFα的mRNA、编码IL-12sc的mRNA；
(28)编码IL-7的mRNA、编码TNFα的mRNA、编码IL-12sc的mRNA以及编码GM-CSF的mRNA；
(29)编码IFNα的mRNA、编码TNFα的mRNA、编码IL-12sc的mRNA以及编码GM-CSF的mRNA；
(30)编码IFNα的mRNA、编码IL-7的mRNA、编码IL-12sc的mRNA以及编码GM-CSF的mRNA；
(31)编码IFNα的mRNA、编码IL-7的mRNA、编码TNFα的mRNA、编码IL-12A的mRNA、编码IL-12B的mRNA以及编码GM-CSF的mRNA；
(32)编码IFNα的mRNA、编码IL-7的mRNA、编码TNFα的mRNA、编码IL-12sc的mRNA以及编码GM-CSF的mRNA。
根据本发明的具体实施方案，本发明的上述mRNA的各组合方案中，所述IFNα具体可为IFNα4和/或IFNα2b。
根据本发明的具体实施方案，本发明的mRNA中，部分或全部mRNA具有以下一种或多种特征：
(1)包含尿嘧啶核苷、胞嘧啶核苷、腺嘌呤核苷、鸟嘌呤核苷或化学修饰核苷；
(2)包含5’帽子结构；
(3)包含5’UTR；
(4)包含3’UTR；
(5)包含poly-A尾；
(6)所述mRNA以naked RNA形式、LNP包裹的形式、LPX包裹的形式或LPP包裹的形式存在。
根据本发明的具体实施方案，本发明中，所述化学修饰核苷选自2-氟-2-脱氧腺苷、2-氟-2-脱氧尿苷、2-氟-2-脱氧胞苷、2-氟-2-脱氧鸟苷、2-氟-2-脱氧-5-甲基胞苷、2-氟-2-脱氧-假尿苷、2-氟-2-脱氧-N1-甲基-假尿苷、2-氟-2-脱氧-N7-甲基-鸟苷、2-氟-2-脱氧-5-甲氧基尿苷、2-氟-2-脱氧-N4-乙酰基胞苷、2-氟-2-脱氧-N6-甲基腺苷、5-甲基胞苷、假尿苷、N1-甲基-假尿苷、N7-甲基-鸟苷、5-甲氧基尿苷、N4-乙酰基胞苷和N6-甲基腺苷中的一种或几种。
根据本发明的具体实施方案，本发明中，所述5’帽子结构选自Cap0、Cap1、Cap2、ARCA、肌苷、N1-甲基-鸟苷、2′氟-鸟苷、7-脱氮-鸟苷、8-氧代-鸟苷、2-氨基-鸟苷、LNA-鸟苷、2-叠氮基-鸟苷、7-甲基-鸟苷-5′-三磷酸-5′-腺苷、鸟苷-5′-三磷酸-5′-腺苷、7-甲基-鸟苷-5′-三磷酸-5′-鸟苷、鸟苷-5′-三磷酸-5′-鸟苷和7-甲基-鸟苷-5′-三磷酸-5′-2-甲氧基腺嘌呤-鸟苷中的一种或几种。
根据本发明的具体实施方案，本发明中，所述poly-A尾含有至少50个、至少80个或至少100个核苷酸。
根据本发明的具体实施方案，本发明的mRNA中，各mRNA的完整性都大于70％。
根据本发明的具体实施方案，本发明的mRNA为包括分别编码单一细胞因子的组合物。
根据本发明的具体实施方案，本发明的mRNA为组合物，其中，编码各细胞因子的mRNA在组合物中的量分别为0.1～10重量份优选0.5～5重量份，更优选为0.5～1重量份。编码各细胞因子的mRNA的用量比例也可根据RT-PCR进行优化。优选地，编码各细胞因子的mRNA在组合物中的量为基本上等质量，即，组合物中编码各细胞因子的mRNA的质量基本相同。或者，本发明的mRNA所编码的各细胞因子基本上等物质的量。
另一方面，本发明还提供了可转录为上述mRNA的DNA。根据本发明的具体实施方案，所述DNA为对应于上述mRNA组合的组合物。在本发明的一些具体实施方案中，所述DNA包括编码前述至少两种细胞因子的mRNA的核酸。
另一方面，本发明还提供了一种药物组合物，该药物组合物包括：
本发明所述的mRNA，以及药学上可接受的辅料。
根据本发明的具体实施方案，本发明的药物组合物可以为液体剂型、固体剂型或者组合形式。
另一方面，本发明还提供了所述的mRNA或所述的药物组合物在制备用于防治实体瘤的药物中的应用。
另一方面，本发明还提供了一种预防和/或治疗实体瘤的方法，该方法包括给予受试者有效量的本发明所述的mRNA或本发明所述的药物组合物。
本发明中，所述“防治”包括预防和/或治疗(包括辅助治疗)。
根据本发明的具体实施方案，本发明中，所述防治实体瘤包括抑制肿瘤增长、减少肿瘤大小、防止肿瘤复发和/或者防止肿瘤转移。
根据本发明的具体实施方案，所述防治实体瘤的药物为瘤内注射或者瘤旁注射药物。本发明的mRNA可用于瘤内注射或者瘤旁注射。
根据本发明的具体实施方案，所述防治实体瘤的药物还包括免疫检查点抑制剂。即，本发明的mRNA可以和免疫检查点抑制剂联用。所述的免疫检查点抑制剂包括PD-1抗体和/或CTLA-4抗体。
本发明的mRNA，对肿瘤有防治作用，可抑制肿瘤增长、减少肿瘤大小、防止肿瘤复发和/或者防止肿瘤转移。
附图说明
图1显示B16F10肿瘤小鼠分别注射不同mRNA后肿瘤大小的变化。
图2显示B16F10肿瘤小鼠分别注射不同mRNA后的生存曲线。
图3显示另一批次实验B16F10肿瘤小鼠分别注射不同mRNA后肿瘤大小的变化。
图4显示另一批次实验CT26肿瘤小鼠瘤内注射mRNA后的变化。其中，图片A.小鼠注射control mRNA后每只小鼠肿瘤大小的变化(n＝8)；图片B.小鼠注射细胞因子混合物后每只小鼠肿瘤大小变化(n＝8)；CR:Complete response。
图5显示另一批次实验CT26肿瘤小鼠注射不同mRNA组合后肿瘤大小的变化。
图6显示另一批次实验CT26肿瘤小鼠在D9、D12、D15、D18、D21、D25分别注射60μg control mRNA或IFNα4+IL-12sc+IL-7细胞因子mRNA混合物(20μg mRNA/基因)后肿瘤大小的变化。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术方案进行以下详细说明，但不能理解为对本发明的可实施范围的限定。实施例中的实验方法，如无特殊说明，均为常规方法，或按照制造商所建议的条件和操作方法进行。下述实施例中所用的试验材料，如无特殊说明，均为自常规试剂商店购买得到的。
本发明中，各序列如下：
SEQ ID NO.1:human IFNa4(amino acid)
SEQ ID NO.2:human IFNa4 non-optimized CDS
SEQ ID NO.3:human IFNa4 non-optimized RNA encoding CDS
SEQ ID NO.4:human IFNa4 optimized CDS
SEQ ID NO.5:human IFNa4 optimized RNA encoding CDS
SEQ ID NO.6:human IFNα2b(amino acid)
SEQ ID NO.7:human IFNα2b non-optimized CDS
SEQ ID NO.8:human IFNα2b non-optimized RNA encoding CDS
SEQ ID NO.9:human IFNα2b optimized CDS
SEQ ID NO.10:human IFNα2b optimized RNA encoding CDS
SEQ ID NO.11:human IL-7(amino acid)
SEQ ID NO.12:human IL-7 non optimized CDS
SEQ ID NO.13:human IL-7 non optimized RNA encoding CDS
SEQ ID NO.14:human IL-7 optimized CDS 1
SEQ ID NO.15:human IL-7 optimized RNA encoding CDS 1
SEQ ID NO.16:human IL-7 optimized CDS 2
SEQ ID NO.17:human IL-7 optimized RNA encoding CDS 2
SEQ ID NO.18:human TNF-α(amino acid)
SEQ ID NO.19:human TNFαnon optimized CDS
SEQ ID NO.20:human TNFαnon optimized RNA encoding CDS
SEQ ID NO.21:human TNFαoptimized CDS 1
SEQ ID NO.22:human TNFαoptimized RNA encoding CDS 1
SEQ ID NO.23:human TNFαoptimized CDS 2
SEQ ID NO.24:human TNFαoptimized RNA encoding CDS 2
SEQ ID NO.25:human IL-12A amino acid
SEQ ID NO.26:human IL-12A non-optimized CDS
SEQ ID NO.27:IL-12A human non-optimized RNA encoding CDS
SEQ ID NO.28:human IL-12A optimized CDS
SEQ ID NO.29:human IL-12A optimized RNA encoding CDS
SEQ ID NO.30:human IL-12B amino acid
SEQ ID NO.31:human IL-12B non-optimized CDS
SEQ ID NO.32:human IL-12B non-optimized RNA encoding CDS
SEQ ID NO.33:human IL-12B optimized CDS
SEQ ID NO.34:human IL-12B optimized RNA encoding CDS
SEQ ID NO.35:human GM-CSF amino acid
SEQ ID NO.36:human GM-CSF no optimized CDS
SEQ ID NO.37:human GM-CSF no optimized RNA encoding CDS
SEQ ID NO.38:human GM-CSF optimized CDS 1
SEQ ID NO.39:human GM-CSF optimized RNA encoding CDS 1
SEQ ID NO.40:human GM-CSF optimized CDS 2
SEQ ID NO.41:human GM-CSF optimized RNA encoding CDS 2
SEQ ID NO.42:linker 1
SEQ ID NO.43:human IL-12sc 1 amino acid
SEQ ID NO.44:human IL-12sc 1 non-optimized CDS
SEQ ID NO.45:human IL-12sc 1 non-optimized RNA encoding CDS
SEQ ID NO.46:human IL-12sc 1 optimized CDS
SEQ ID NO.47:human IL-12sc 1 optimized RNA encoding CDS
SEQ ID NO.48:linker 2
SEQ ID NO.49:human IL-12sc 2 amino acid
SEQ ID NO.50:human IL-12sc 2 non-optimized CDS
SEQ ID NO.51:human IL-12sc 2 non-optimized RNA encoding CDS
SEQ ID NO.52:human IL-12sc 2 optimized CDS
SEQ ID NO.53:human IL-12sc 2 optimized RNA encoding CDS
SEQ ID NO.54:murine IL-12A amino acid
SEQ ID NO.55:murine IL-12A non-optimized CDS
SEQ ID NO.56:murine IL-12A non-optimized RNA encoding CDS
SEQ ID NO.57:murine IL-12B amino acid
SEQ ID NO.58:murine IL-12B non optimized CDS
SEQ ID NO.59:murine IL-12B non optimized RNA encoding CDS
SEQ ID NO.60:murine IL-12sc amino acid
SEQ ID NO.61:murine IL-12sc non-optimized CDS
SEQ ID NO.62:murine IL-12sc non-optimized RNA encoding CDS
SEQ ID NO.63:murine IL-12sc optimized CDS
SEQ ID NO.64:murine IL-12sc optimized RNA encoding CDS
SEQ ID NO.65:murine IL-7 amino acid
SEQ ID NO.66:murine IL-7 non optimized CDS
SEQ ID NO.67:murine IL-7 non optimized RNA encoding CDS
SEQ ID NO.68:murine IL-7 optimized CDS
SEQ ID NO.69:murine IL-7 optimized RNA encoding CDS
SEQ ID NO.70:murine IFNα4 amino acid
SEQ ID NO.71:murine IFNα4 non optimized CDS
SEQ ID NO.72:murine IFNα4 non optimized RNA encoding CDS
SEQ ID NO.73:murine IFNα4 optimized CDS
SEQ ID NO.74:murine IFNα4 optimized RNA encoding CDS
SEQ ID NO.75:murine TNFαamino acid
SEQ ID NO.76:murine TNFαnon optimized CDS
SEQ ID NO.77:murine TNFαnon optimized RNA encoding CDS
SEQ ID NO.78:murine TNFαoptimized CDS
SEQ ID NO.79:murine TNFαoptimized RNA encoding CDS
SEQ ID NO.80:murine GM-CSF amino acid
SEQ ID NO.81:murine GM-CSF non-optimized CDS
SEQ ID NO.82:murine GM-CSF non-optimized RNA encoding CDS
SEQ ID NO.83:murine GM-CSF optimized CDS
SEQ ID NO.84:murine GM-CSF optimized RNA encoding CDS
SEQ ID NO.85:5’UTR
SEQ ID NO.86:3’UTR
SEQ ID NO.87:poly-A
SEQ ID NO.88:luciferase amino acid
SEQ ID NO.89:luciferase CDS
SEQ ID NO.90:luciferase RNA encoding CDS
实施例1
1.B16F10肿瘤模型的建立
将6～8周龄18～20g雌性C57BL/6J(购自珠海百事通)小鼠饲养在温度为22±2℃、相对湿度为55％±15％鼠房中，小鼠能够自由获得食物和水。B16F10细胞(购自ATCC)用DMEM和10％FBS在37℃、5％CO2中培养。将B16F10细胞培养到融合度为80～90％时用0.25％Trypin-EDTA消化后用DPBS重悬，在每个小鼠的右侧皮下注射1×106细胞/100μl，在注射肿瘤细胞后第7天得到能够进行瘤内注射mRNA的B16F10肿瘤小鼠模型。
2.RNA制备
注射用各细胞因子的mRNA的具体合成方法如下：
1、将合成好的表达不同细胞因子的DNA克隆进含有5’-UTR(SEQ ID NO.85)、3’-UTR(SEQ ID NO.86)和poly-A尾(SEQ ID NO.87)质粒并按如下反应条件进行DNA模板的扩增：
预变性98℃3min；变性98℃10s，退火60℃5s，延伸72℃2min，34个循环；最后 延伸72℃，10min。
反应结束后，将反应液合并于1.5ml Tube管中。取10ml进行DNA琼脂糖凝胶电泳(1.5％琼脂糖，5V/min，40min)。根据电泳目的条带的大小对反应成功与否进行确认。
合格标准：电泳检测出现单一的条带，且大小正确。
2、DNA模板超滤
利用Millipore 30Kd超滤管浓缩上述获得的DNA模板。
3、DNA模板FPLC纯化
将上述超滤得到的DNA，加入等体积的苯酚/氯仿/异戊醇混合液(苯酚/氯仿/异戊醇＝25/24/1)，充分震荡后，12000g离心15min。
去掉沉淀，转移上清至新的离心管中，加入上清体积的1/10 3M NaAc(pH 5.2)，混匀，然后加入2倍体积的无水乙醇，混匀，至于-20℃静置30min。
4℃，12000g离心10min，弃上清。
用70％乙醇洗涤沉淀，12000g离心5min，取上清，于超净台晾干5min。
用适当的RNase-free水溶解纯化后的DNA模板。
用NanoDrop检测纯化后模板的浓度，以及260/280、260/230下的比值。取样进行DNA琼脂糖凝胶电泳检测(1.5％琼脂糖，5V/min，40min)。
合格标准：260/280介于1.8至2.1之间，260/230在1.6至2.2之间。
4、FPLC纯化后模板超滤
Millipore 30Kd超滤管浓缩FPLC纯化后的DNA模板，用RNase-free水洗脱溶解。用NanoDrop检测超滤后模板的浓度，以及A260/A280、A260/A230的比值。最终用RNase-free水稀释至150ng/ml。
5、mRNA的体外合成
在恒温反应器中，进行mRNA的体外合成。
按照如下合成体系进行(反应试剂按照从上至下添加)：
反应体积，1600ml(置于2ml RNase-free Tube管中，为单个管的反应体积，一次同时反应多管)：RNA-free水440ml、7.5mM的ATP 160ml、7.5mM的UTP 160ml、7.5mM的CTP160ml、7.5mM的GTP 160ml、7.5mM的M7G(2’OMeA)pG 160ml、150ng/ml的DNA模板40ml、10×Buffer 160ml和Enzyme Mix 160ml。
所述RNA体外合成的程序为37℃，10h。
6、DNase I消化去除DNA模板
向mRNA体外合成后的每个Tube管中各加入120ml DNase I。
上下颠倒10次混匀，1000rpm离心10s。
重新置于恒温反应器中，37℃，1h。
7、mRNA沉淀回收
向上一步骤中的每个50ml Tube管中，加入等体积的醋酸铵溶液。
上下颠倒10次混匀。
置于-20℃2h，沉淀。
17000g，4℃离心，30min。
去掉上清，用70％乙醇洗涤沉淀。
17000g，4℃离心，10min。
去掉70％乙醇，于超净台中蒸干，每管加入RNase-free水20ml。
静置10min后，用枪头轻吹混匀。
用NanoDrop检测回收后的mRNA浓度为5mg/ml，A260/A280为1.90、A260/A230为2.0。
取1ml，稀释10倍，进行RNA ScreenTape assay以及琼脂糖凝胶电泳检测其片段完整性。
8、LiCl沉淀纯化mRNA
将上一步骤中回收的mRNA按照其1.5倍体积加入Rnase-free水，混匀。
加入原mRNA 1.5倍体积-20预冷的LiCl溶液，混匀。
然后于-20℃静置2h。
16000g离心20min。
弃上清，用70％乙醇洗涤沉淀，16000g离心15min。
取上清，于超净台晾干5min。
用适当的RNase-free水溶解纯化后的mRNA。
本发明中，参照上述方法分别合成了如SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.8、SEQ ID NO.10、SEQ ID NO.72、SEQ ID NO.74、SEQ ID NO.13、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.67、SEQ ID NO.69、SEQ ID NO.20、SEQ ID NO.22、SEQ ID NO.24、SEQ ID NO.77、SEQ ID NO.79、SEQ ID NO.27、SEQ ID NO.29、SEQ ID NO.56、SEQ ID NO.32、SEQ ID NO.34、SEQ ID NO.59、SEQ ID NO.45、SEQ ID NO.47、SEQ ID NO.51、SEQ ID NO.53、SEQ ID NO.62、SEQ ID NO.64、SEQ ID NO.37、SEQ ID NO.39、SEQ ID NO.41、SEQ ID NO.82、SEQ ID NO.84所示序列的各mRNA。且各mRNA含有5’-UTR(SEQ ID NO.85)、3’-UTR(SEQ ID NO.86)和poly-A尾(SEQ ID NO.87)。
3.肿瘤治疗方法
通过肿瘤内注射的方式在B16F10模型小鼠肿瘤中注射control mRNA或者细胞因子mRNA混合物(每组小鼠8～9只)，小鼠肿瘤内注射的mRNA按相同质量进行混合后用生理盐水进行稀释至50μl，用于小鼠瘤内注射的mRNA是各种细胞因子的小鼠版本(实验分组参见表1)。 小鼠肿瘤大小每隔3～4天用游标卡尺测量一次，当肿瘤大小超过2000mm3或者小鼠体重下降20％时进行处死。
表1、实验分组
4.治疗结果
B16F10肿瘤小鼠在D7，D10，D13、D17，D20分别注射control mRNA、IFNα4、IL-7、GM-CSF、IL-12A+IL-12B、TNFα、细胞因子mRNA混合物后肿瘤大小的变化参见图1。其中单因子mRNA每只小鼠10μg，control mRNA每只小鼠60μg mRNA，细胞因子mRNA混合物按照10μg mRNA/基因给予每只小鼠(即，2种细胞因子mRNA混合物给予每只小鼠共20 μg mRNA，4种细胞因子mRNA混合物给予每只小鼠共40μg mRNA，6种细胞因子mRNA混合物给予每只小鼠共60μg mRNA)，并一直监测肿瘤大小直到31天。
B16F10肿瘤小鼠分别注射control mRNA和细胞因子mRNA混合物后小鼠的生存曲线参见图2。小鼠在D7，D10，D13、D17，D20注射60μg control mRNA和细胞因子mRNA混合物(10μg mRNA/基因)并一直监测肿瘤大小直到31天。
B16F10肿瘤小鼠在不同时间点的生存率参见表2。
表2、B16F10肿瘤小鼠在不同时间点的生存率
另一批实验的B16F10肿瘤小鼠分别注射control mRNA和不同细胞因子mRNA混合物后肿瘤大小的变化参见图3。小鼠在D7，D11，D15、D19，D22注射60μg control mRNA和细胞因子mRNA混合物(10μg mRNA/基因)并一直监测肿瘤大小直到25天。
本发明的实验表明，本发明的mRNA组合物，包含如下mRNA的2-6种组成：编码IFNα4的mRNA、编码IL-7的mRNA、编码IL-12A的mRNA、编码IL-12B的mRNA、编码TNFα的mRNA、编码GM-CSF的mRNA，对肿瘤有治疗作用。
实施例2
RNA制备方法同实施例1。本实施例中制备得到各细胞因子的mRNA。并按照表3方案分组。
表3、实验分组

在BALB/C小鼠皮下移植5x105CT26肿瘤细胞，待肿瘤长至20～40mm3，通过肿瘤内注射的方式在CT26模型小鼠肿瘤中注射control mRNA或者细胞因子mRNA混合物(每组小鼠8只)。小鼠肿瘤内注射的mRNA按相同质量进行混合后用生理盐水进行稀释至50μl，用于小鼠瘤内注射的mRNA是5种细胞因子的小鼠版本。小鼠肿瘤大小每隔3～4天用游标卡尺测量一次，当肿瘤大小超过2000mm3或者小鼠体重下降20％时进行处死。CT26肿瘤小鼠在D6、D8、D11、D13、D15分别注射100μg control mRNA、IFNα4+IL-7+GM-CSF+IL12sc+TNFα细胞因子mRNA混合物(20μg mRNA/基因)后肿瘤大小的变化参见图4，一直监测肿瘤大小直到39天。可以看到注射control mRNA后的CT26肿瘤一直在长大，而注射了细胞因子mRNA混合物的CT26肿瘤的增长速度不如control mRNA且8个肿瘤有7个肿瘤完全消失。
在BALB/C小鼠皮下移植5x105CT26肿瘤细胞，待肿瘤长至20～50mm3，通过肿瘤内注射的方式在CT26模型小鼠肿瘤中注射control mRNA或者细胞因子mRNA混合物(每组小鼠9只)。小鼠肿瘤内注射的mRNA按相同质量进行混合后用生理盐水进行稀释至50μl，用于小鼠瘤内注射的mRNA是5种细胞因子的小鼠版本。小鼠肿瘤大小每隔3～4天用游标卡尺测量一次，当肿瘤大小超过2000mm3或者小鼠体重下降20％时进行处死。CT26肿瘤小鼠在D8、 D10、D11、D14、D17、D19分别注射60μg control mRNA、IFNα4+IL-7+GM-CSF、TNFα+IL-12sc+GM-CSF、IL-7+IL-12sc+GM-CSF、IFNα4+TNFα+GM-CSF、IFNα4+TNFα+IL-12sc、IFNα4+GM-CSF+IL-7、IFNα4+GM-CSF+IL-12sc、IFNα4+IL-12sc+IL-7、TNFα+IL-12sc+IL-7、TNFα+GM-CSF+IL-7细胞因子mRNA混合物(；20μg mRNA/基因)后肿瘤大小的变化参见图5，一直监测肿瘤大小直到34天。可以看到注射control mRNA后的CT26肿瘤一直在长大，而注射了不同组合细胞因子mRNA混合物的CT26肿瘤的增长速度不如control mRNA且有许多组的肿瘤完全消失。
在BALB/C小鼠左右两侧腹皮下分别移植5x105和3x105CT26肿瘤细胞，待左侧肿瘤长至40～90mm3，右侧肿瘤大小40～70mm3，通过肿瘤内注射的方式在左侧肿瘤中注射control mRNA或者细胞因子组合IFNα4+IL-12sc+IL-7mRNA混合物(每组小鼠8只)，同时在腹腔分别注射isotype抗体或者抗PD-1抗体。小鼠肿瘤内注射的mRNA按相同质量进行混合后用生理盐水进行稀释至50μl用于小鼠瘤内注射的mRNA是5种细胞因子的小鼠版本。小鼠肿瘤大小每隔3～4天用游标卡尺测量一次，当肿瘤大小超过2000mm3或者小鼠体重下降20％时进行处死。CT26肿瘤小鼠在D9、D12、D15、D18、D21、D25分别注射60μg control mRNA或IFNα4+IL-12sc+IL-7细胞因子mRNA混合物(20μg mRNA/基因)后肿瘤大小的变化参见图6，一直监测肿瘤大小直到28天。可以看到注射control mRNA后的CT26肿瘤一直在长大，而注射了细胞因子mRNA混合物的CT26肿瘤的增长速度不如control mRNA且在联合使用抗PD-1抗体后不论是治疗侧或者未治疗侧的肿瘤增长速度都要显著低于其他处理组。

Claims (12)

  1. 一种mRNA，该mRNA包括编码以下细胞因子中的至少两种细胞因子的mRNA：
    IL-7、IL-12sc、IFNα、TNFα、IL-12A、IL-12B、GM-CSF。
  2. 根据权利要求1所述的mRNA，其为组合物，该组合物包括编码IFNα的mRNA、编码IL-7的mRNA、编码TNFα的mRNA、编码IL-12A的mRNA、编码IL-12B的mRNA、编码IL-12A和IL-12B的mRNA、编码IL-12sc的mRNA、编码GM-CSF的mRNA中的任意两种、三种、四种、五种、六种、七种或八种mRNA。
  3. 根据权利要求1或2所述的mRNA，其中：
    IFNα具有如SEQ ID NO.1或SEQ ID NO.6或SEQ ID NO.70所示氨基酸序列；
    IL-7具有如SEQ ID NO.11或SEQ ID NO.65所示氨基酸序列；
    TNFα具有如SEQ ID NO.18或SEQ ID NO.75所示氨基酸序列；
    IL-12A具有如SEQ ID NO.25或SEQ ID NO.54所示氨基酸序列；
    IL-12B具有如SEQ ID NO.30或SEQ ID NO.57所示氨基酸序列；
    IL-12sc具有如SEQ ID NO.43或SEQ ID NO.49或SEQ ID NO.60所示氨基酸序列；
    GM-CSF具有如SEQ ID NO.35或SEQ ID NO.80所示氨基酸序列。
  4. 根据权利要求1-3任一项所述的mRNA，其中：
    编码IFNα的mRNA包括以下mRNA中的一种或多种：SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.8、SEQ ID NO.10、SEQ ID NO.72、SEQ ID NO.74中任一所示序列的mRNA；
    编码IL-7的mRNA包括以下mRNA中的一种或多种：SEQ ID NO.13、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.67、SEQ ID NO.69中任一所示序列的mRNA；
    编码TNFα的mRNA包括以下mRNA中的一种或多种：SEQ ID NO.20、SEQ ID NO.22、SEQ ID NO.24、SEQ ID NO.77、SEQ ID NO.79中任一所示序列的mRNA；
    编码IL-12A的mRNA包括以下mRNA中的一种或多种：SEQ ID NO.27、SEQ ID NO.29、SEQ ID NO.56中任一所示序列的mRNA；
    编码IL-12B的mRNA包括以下mRNA中的一种或多种：SEQ ID NO.32、SEQ ID NO.34、SEQ ID NO.59中任一所示序列的mRNA；
    编码IL-12sc的mRNA包括以下mRNA中的一种或多种：SEQ ID NO.45、SEQ ID NO.47、SEQ ID NO.51、SEQ ID NO.53、SEQ ID NO.62、SEQ ID NO.64中任一所示序列的mRNA；
    编码GM-CSF的mRNA包括以下mRNA中的一种或多种：SEQ ID NO.37、SEQ ID NO.39、SEQ ID NO.41、SEQ ID NO.82、SEQ ID NO.84中任一所示序列的mRNA。
  5. 根据权利要求1-4任一项所述的mRNA，该mRNA至少包括编码IFNα的mRNA、 编码IL-7的mRNA或编码TNFα的mRNA；或者
    该mRNA至少包括编码IL-12A以及IL-12B或者IL-12sc的mRNA。
  6. 根据权利要求1-5任一项所述的mRNA，该mRNA包括以下各组mRNA中的一组或多组：
    (1)编码IL-12A的mRNA以及编码IL-12B的mRNA；
    (2)编码IFNα的mRNA以及编码TNFα的mRNA；
    (3)编码IL-7的mRNA以及编码TNFα的mRNA；
    (4)编码IFNα的mRNA以及编码IL-7的mRNA；
    (5)编码IFNα的mRNA以及编码GM-CSF的mRNA；
    (6)编码IFNα的mRNA以及编码IL-12sc的mRNA；
    (7)编码IL-7的mRNA以及编码IL-12sc的mRNA；
    (8)编码IL-7的mRNA以及编码GM-CSF的mRNA；
    (9)编码GM-CSF的mRNA以及编码IL-12sc的mRNA；
    (10)编码GM-CSF的mRNA以及编码TNFα的mRNA；
    (11)编码IL-12sc的mRNA以及编码TNFα的mRNA；
    (12)编码IFNα的mRNA、编码IL-7的mRNA以及编码TNFα的mRNA；
    (13)编码TNFα的mRNA、编码IL-12sc的mRNA以及编码GM-CSF的mRNA；
    (14)编码GM-CSF的mRNA、编码IL-7的mRNA以及编码IL-12sc的mRNA；
    (15)编码IFNα的mRNA、编码TNFα的mRNA以及编码GM-CSF的mRNA；
    (16)编码IFNα的mRNA、编码IL-12sc的mRNA以及编码TNFα的mRNA；
    (17)编码IFNα的mRNA、编码IL-7的mRNA以及编码GM-CSF的mRNA；
    (18)编码IFNα的mRNA、编码IL-12sc的mRNA以及编码GM-CSF的mRNA；
    (19)编码IFNα的mRNA、编码IL-12sc的mRNA以及编码IL-7的mRNA；
    (20)编码TNFα的mRNA、编码Il-12sc的mRNA以及编码IL-7的mRNA；
    (21)编码的TNFαmRNA、编码GM-CSF的mRNA以及编码IL-7的mRNA；
    (22)编码IFNα的mRNA、编码IL-7的mRNA、编码TNFα的mRNA以及编码GM-CSF的mRNA；
    (23)编码IFNα的mRNA、编码IL-7的mRNA、编码TNFα的mRNA、编码IL-12A的mRNA以及编码IL-12B的mRNA；
    (24)编码IL-7的mRNA、编码TNFα的mRNA、编码IL-12A的mRNA、编码IL-12B的mRNA以及编码GM-CSF的mRNA；
    (25)编码IFNα的mRNA、编码TNFα的mRNA、编码IL-12A的mRNA、编码IL-12B的mRNA以及编码GM-CSF的mRNA；
    (26)编码IFNα的mRNA、编码IL-7的mRNA、编码IL-12A的mRNA、编码IL-12B的mRNA以及编码GM-CSF的mRNA；
    (27)编码IFNα的mRNA、编码IL-7的mRNA、编码TNFα的mRNA、编码IL-12sc的mRNA；
    (28)编码IL-7的mRNA、编码TNFα的mRNA、编码IL-12sc的mRNA以及编码GM-CSF的mRNA；
    (29)编码IFNα的mRNA、编码TNFα的mRNA、编码IL-12sc的mRNA以及编码GM-CSF的mRNA；
    (30)编码IFNα的mRNA、编码IL-7的mRNA、编码IL-12sc的mRNA以及编码GM-CSF的mRNA；
    (31)编码IFNα的mRNA、编码IL-7的mRNA、编码TNFα的mRNA、编码IL-12A的mRNA、编码IL-12B的mRNA以及编码GM-CSF的mRNA；
    (32)编码IFNα的mRNA、编码IL-7的mRNA、编码TNFα的mRNA、编码IL-12sc的mRNA以及编码GM-CSF的mRNA。
  7. 根据权利要求1-6任一项所述的mRNA，其中，部分或全部mRNA具有以下一种或多种特征：
    (1)包含尿嘧啶核苷、胞嘧啶核苷、腺嘌呤核苷、鸟嘌呤核苷或化学修饰核苷；
    (2)包含5’帽子结构；
    (3)包含5’UTR；
    (4)包含3’UTR；
    (5)包含poly-A尾；
    (6)所述mRNA以naked RNA形式、RNA盐溶液的形式、LNP包裹的形式、LPX包裹的形式或LPP包裹的形式存在。
  8. 根据权利要求1-7任一项所述的mRNA，其为包括分别编码单一细胞因子的组合物，其中编码各细胞因子的mRNA在组合物中的量分别为0.1～10重量份优选0.5～5重量份更优选0.5～1重量份；优选地，编码各细胞因子的mRNA在组合物中的量为基本上等质量。
  9. 一种DNA，其包括编码权利要求1-8任一项所述的至少两种细胞因子的mRNA的核酸。
  10. 一种药物组合物，该药物组合物包括：
    权利要求1-8任一项所述的mRNA，以及
    药学上可接受的辅料；
    优选地，所述药物组合物为液体剂型、固体剂型或者组合形式。
  11. 权利要求1-8任一项所述的mRNA或权利要求9所述的DNA或权利要求10所述的药物组合物在制备用于防治实体瘤的药物中的应用；
    优选地，所述防治实体瘤包括抑制肿瘤增长、减少肿瘤大小、防止肿瘤复发和/或者防止肿瘤转移；
    优选地，所述防治实体瘤的药物为瘤内注射或者瘤旁注射药物；
    优选地，所述防治实体瘤的药物还包括免疫检查点抑制剂。
  12. 一种预防和/或治疗实体瘤的方法，该方法包括给予受试者有效量的权利要求1-8任一项所述的mRNA或权利要求10所述的药物组合物；
    优选地，所述防治实体瘤包括抑制肿瘤增长、减少肿瘤大小、防止肿瘤复发和/或者防止肿瘤转移；
    优选地，所述防治实体瘤的药物为瘤内注射或者瘤旁注射药物；
    优选地，所述防治实体瘤的药物还包括免疫检查点抑制剂。
PCT/CN2023/102158 2022-06-24 2023-06-25 用于实体肿瘤的治疗性mRNA及其应用 WO2023246938A1 (zh)

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