CN117327709A - Therapeutic mRNA for solid tumors and uses thereof - Google Patents

Therapeutic mRNA for solid tumors and uses thereof Download PDF

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Publication number
CN117327709A
CN117327709A CN202310327875.1A CN202310327875A CN117327709A CN 117327709 A CN117327709 A CN 117327709A CN 202310327875 A CN202310327875 A CN 202310327875A CN 117327709 A CN117327709 A CN 117327709A
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mrna encoding
seq
mrna
encoding
csf
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胡勇
汤赞
蔡冠星
周瑞国
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Wuhan Ruiji Biotechnology Co ltd
Shenzhen Ruiji Biotechnology Co ltd
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Wuhan Ruiji Biotechnology Co ltd
Shenzhen Ruiji Biotechnology Co ltd
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Priority to PCT/CN2023/102158 priority Critical patent/WO2023246938A1/en
Publication of CN117327709A publication Critical patent/CN117327709A/en
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Abstract

The invention provides a therapeutic mRNA for solid tumors and application thereof. The mRNA of the present invention includes mRNA encoding at least two cytokines of the following cytokines: IFNα, IL-7, TNF α, IL-12A, IL-12B, GM-CSF, IL-12sc. The mRNA of the invention has the function of preventing and treating tumor, and can inhibit tumor growth, reduce tumor size, prevent tumor recurrence and/or prevent tumor metastasis.

Description

Therapeutic mRNA for solid tumors and uses thereof
Technical Field
The present invention relates to a therapeutic mRNA for solid tumors and related applications.
Background
Solid tumors are difficult to treat due to their tumor microenvironment, heterogeneity, etc. Current treatments mainly include surgery, radiation therapy, chemotherapy and immunotherapy. Surgical treatment alone can treat locally distributed tumors, but is often difficult to treat surgically for large metastatic and malignant tumors. Other common radiotherapy and chemotherapy have strong toxic and side effects. Although surgery and current treatment methods can kill a large number of tumor cells, potential tumor stem cells may be able to survive, which over time can in turn form new tumors that lead to recurrence of the tumor.
Cytokines are small molecule proteins with broad biological activity that are synthesized and secreted by immune cells (e.g., monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) through stimulation. Cytokines generally regulate immune responses by binding to corresponding receptors to regulate cell growth, differentiation, and effects. Cytokines such as IL-2 are currently available with FDA approval for the treatment of melanoma. However, systemic administration of cytokines causes systemic toxicity, which results in a low prevalence of cytokines for tumor treatment and the existing cytokines do not achieve optimal therapeutic effect.
Disclosure of Invention
The invention aims at providing a novel medicine for preventing and treating solid tumors.
The invention aims to overcome the defects of the prior art that the cytokines treat solid tumors, adopts mRNA to code at least two cytokines for treating the tumors, can change the microenvironment of the tumors, recruit and activate immune cells to kill tumor cells and reduce systemic toxicity, and can achieve the effects of reducing the tumors and prolonging the life.
In one aspect, the invention provides an mRNA comprising mRNA encoding at least two of the following cytokines:
IFNα、IL-7、TNFα、IL-12A、IL-12B、IL-12sc、GM-CSF。
according to the invention of specific embodiments, the IFN alpha preferably IFN alpha 4 and/or IFN alpha 2b.
According to a specific embodiment of the invention, one mRNA strand may encode only one cytokine or may encode two or more cytokines in the mRNA of the invention. For example, one mRNA strand may encode both IL-12A and IL-12B proteins.
According to a specific embodiment of the invention, the mRNA of the invention is a composition comprising any two, three, four, five, six, seven or eight mRNAs of mRNA encoding IFN alpha, mRNA encoding IL-7, mRNA encoding TNF alpha, mRNA encoding IL-12A, mRNA encoding IL-12B, mRNA encoding IL-12A and IL-12B, mRNA encoding IL-12sc, mRNA encoding GM-CSF.
According to a specific embodiment of the invention, in the invention:
IFN alpha has an amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO.6 or SEQ ID NO. 70;
IL-7 has an amino acid sequence shown as SEQ ID NO.11 or SEQ ID NO. 65;
TNFα has an amino acid sequence as shown in SEQ ID NO.18 or SEQ ID NO. 75;
GM-CSF has an amino acid sequence as shown in SEQ ID NO.35 or SEQ ID NO. 80;
IL-12A has an amino acid sequence as shown in SEQ ID NO.25 or SEQ ID NO. 54;
IL-12B has an amino acid sequence as shown in SEQ ID NO.30 or SEQ ID NO. 57;
IL-12sc has an amino acid sequence as shown in SEQ ID NO.43 or SEQ ID NO.49 or SEQ ID NO. 60.
According to the invention of specific embodiments, the invention, encoding IFN alpha mRNA including one or more of the following mRNA: mRNA of any one of SEQ ID No.3, SEQ ID No.5, SEQ ID No.8, SEQ ID No.10, SEQ ID No.74 having 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 to mRNA of any one of SEQ ID No.3, SEQ ID No.5, SEQ ID No.8, SEQ ID No.10, SEQ ID No. 74.
According to a specific embodiment of the invention, the mRNA encoding IL-7 comprises one or more of the following mRNAs: mRNA of any one of SEQ ID No.13, SEQ ID No.15, SEQ ID No.17, SEQ ID No.67, SEQ ID No.69 has 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 to mRNA of any one of SEQ ID No.13, SEQ ID No.15, SEQ ID No.17, SEQ ID No.67, SEQ ID No. 69.
According to a specific embodiment of the invention, the mRNA encoding tnfα in the present invention comprises one or more of the following mrnas: mRNA of any one of SEQ ID No.20, SEQ ID No.22, SEQ ID No.24, SEQ ID No.77, SEQ ID No.79 having 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 to any one of SEQ ID No.20, SEQ ID No.22, SEQ ID No.24, SEQ ID No.77, SEQ ID No. 79.
According to a specific embodiment of the invention, the mRNA encoding IL-12A comprises one or more of the following mRNAs: mRNA having 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 to the sequence shown in any one of SEQ ID No.27, SEQ ID No.29, SEQ ID No. 56.
According to a specific embodiment of the invention, the mRNA encoding IL-12B comprises one or more of the following mRNAs: mRNA having 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 to the sequence shown in any one of SEQ ID No.32, SEQ ID No.34, SEQ ID No. 59.
According to the specific embodiment of the invention, the mRNA encoding IL-12A and IL-12B may be one mRNA encoding both IL-12A and IL-12B, wherein IL-12A and IL-12B may be linked to form IL-12sc by linker (for example, SEQ ID NO.42 or SEQ ID NO.48, etc.), in the present invention, the protein formed by linking the amino acid sequences of IL-12A and IL-12B by linker is named IL-12sc, and the amino acid sequence of IL-12sc may be IL-12A, IL-12B in order from N-terminus to C-terminus, or IL-12B, IL-12A in order. That is, in the present invention, the encoding IL-12A and IL-12B mRNA specific scheme is encoding IL-12sc mRNA. According to a specific embodiment of the invention, preferably, the mRNA encoding IL-12sc comprises one or more of the following mRNAs: mRNA of any one of SEQ ID No.45, SEQ ID No.47, SEQ ID No.51, SEQ ID No.53, SEQ ID No.62, SEQ ID No.64 having 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 to mRNA of any one of SEQ ID No.45, SEQ ID No.47, SEQ ID No.51, SEQ ID No.53, SEQ ID No.62, SEQ ID No. 64.
The mRNA encoding GM-CSF includes one or more of the following mRNAs: mRNA of any one of SEQ ID No.37, SEQ ID No.39, SEQ ID No.41, SEQ ID No.82, SEQ ID No.84 having 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 to any one of SEQ ID No.37, SEQ ID No.39, SEQ ID No.41, SEQ ID No.82, SEQ ID No. 84.
According to a specific embodiment of the invention, the mRNA of the invention at least includes encoding IFN alpha mRNA, encoding IL-7mRNA or encoding TNF alpha mRNA.
According to the invention of the specific embodiments, the mRNA at least includes encoding IL-12A and IL-12B mRNA.
According to a specific embodiment of the invention, the mRNA of the invention comprises one or more of the following sets of mrnas:
(1) mRNA encoding IL-12A and mRNA encoding IL-12B;
(2) mRNA encoding IFN alpha and mRNA encoding TNF alpha;
(3) mRNA encoding IL-7 and mRNA encoding TNF alpha;
(4) mRNA encoding IFN alpha and mRNA encoding IL-7;
(5) mRNA encoding IFN alpha and mRNA encoding GM-CSF;
(6) mRNA encoding IFN alpha and mRNA encoding IL-12 sc;
(7) mRNA encoding IL-7 and mRNA encoding IL-12 sc;
(8) mRNA encoding IL-7 and mRNA encoding GM-CSF;
(9) mRNA encoding GM-CSF and mRNA encoding IL-12 sc;
(10) mRNA encoding GM-CSF and mRNA encoding TNF α;
(11) mRNA encoding IL-12sc and mRNA encoding TNF alpha;
(12) mRNA encoding IFN alpha, mRNA encoding IL-7 and mRNA encoding TNF alpha;
(13) mRNA encoding TNF alpha, mRNA encoding IL-12sc, and mRNA encoding GM-CSF;
(14) mRNA encoding GM-CSF, mRNA encoding IL-7, and mRNA encoding IL-12 sc;
(15) mRNA encoding IFN alpha, mRNA encoding TNF alpha, and mRNA encoding GM-CSF;
(16) mRNA encoding IFN alpha, mRNA encoding IL-12sc and mRNA encoding TNF alpha;
(17) mRNA encoding IFN alpha, mRNA encoding IL-7 and mRNA encoding GM-CSF;
(18) mRNA encoding IFN alpha, mRNA encoding IL-12sc, and mRNA encoding GM-CSF;
(19) mRNA encoding IFN alpha, mRNA encoding IL-12sc and mRNA encoding IL-7;
(20) mRNA encoding TNF alpha, mRNA encoding IL-12sc, and mRNA encoding IL-7;
(21) An mRNA encoding TNF alpha, an mRNA encoding GM-CSF, and an mRNA encoding IL-12 sc;
(22) mRNA encoding IFN alpha, mRNA encoding IL-7, mRNA encoding TNF alpha, and mRNA encoding GM-CSF;
(23) mRNA encoding IFN alpha, mRNA encoding IL-7, mRNA encoding TNF alpha, mRNA encoding IL-12A, and mRNA encoding IL-12B;
(24) mRNA encoding IL-7, mRNA encoding TNF alpha, mRNA encoding IL-12A, mRNA encoding IL-12B, and mRNA encoding GM-CSF;
(25) mRNA encoding IFN alpha, mRNA encoding TNF alpha, mRNA encoding IL-12A, mRNA encoding IL-12B, and mRNA encoding GM-CSF;
(26) mRNA encoding IFN alpha, mRNA encoding IL-7, mRNA encoding IL-12A, mRNA encoding IL-12B, and mRNA encoding GM-CSF;
(27) mRNA encoding IFN alpha, mRNA encoding IL-7, mRNA encoding TNF alpha, mRNA encoding IL-12 sc;
(28) mRNA encoding IL-7, mRNA encoding TNF alpha, mRNA encoding IL-12sc, and mRNA encoding GM-CSF;
(29) mRNA encoding IFN alpha, mRNA encoding TNF alpha, mRNA encoding IL-12sc, and mRNA encoding GM-CSF;
(30) mRNA encoding IFN alpha, mRNA encoding IL-7, mRNA encoding IL-12sc, and mRNA encoding GM-CSF;
(31) mRNA encoding IFN alpha, mRNA encoding IL-7, mRNA encoding TNF alpha, mRNA encoding IL-12A, mRNA encoding IL-12B, and mRNA encoding GM-CSF;
(32) mRNA encoding IFN alpha, mRNA encoding IL-7, mRNA encoding TNF alpha, mRNA encoding IL-12sc, and mRNA encoding GM-CSF.
According to the specific embodiment of the present invention, in each combination scheme of the above mRNA of the present invention, the IFN alpha may be specifically IFN alpha 4 and/or IFN alpha 2b.
According to a specific embodiment of the invention, part or all of the mRNA of the invention has one or more of the following features:
(1) Comprising uridine, cytidine, adenine nucleoside, guanosine or a chemically modified nucleoside;
(2) Comprising a 5' cap structure;
(3) Comprising a 5' UTR;
(4) Comprising a 3' UTR;
(5) Comprising a poly-A tail;
(6) The mRNA is in the form of a naked RNA, LNP-coated, LPX-coated or LPP-coated form.
According to a specific embodiment of the present invention, the chemically modified nucleoside is selected from one or more 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, N4-acetylcytidine and N6-methyladenosine.
According to a specific embodiment of the present invention, the 5 'Cap structure is selected from one or more of 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, 7-methyl-guanosine-5 '-triphosphate-5' -guanosine, guanosine-5 '-triphosphate-5' -guanosine and 7-methyl-guanosine-5 '-triphosphate-5' -2-methoxyadenine-guanosine.
According to a specific embodiment of the invention, the poly-A tail comprises at least 50, at least 80 or at least 100 nucleotides.
According to a specific embodiment of the invention, each mRNA has an integrity of greater than 70% in the mRNA of the invention.
According to a specific embodiment of the invention, the mRNA of the invention is a composition comprising a single cytokine encoded by each.
According to a specific embodiment of the present invention, the mRNA of the present invention is a composition, wherein the amount of mRNA encoding each cytokine in the composition is 0.1 to 10 parts by weight, preferably 0.5 to 5 parts by weight, more preferably 0.5 to 1 part by weight, respectively. The proportion of mRNA encoding each cytokine may also be optimised for RT-PCR. Preferably, the amount of mRNA encoding each cytokine in the composition is substantially equal in mass, i.e., the mass of mRNA encoding each cytokine in the composition is substantially the same. Alternatively, the mRNA of the present invention encodes a cytokine in an amount substantially equal to the amount of the substance.
In another aspect, the invention also provides DNA transcribable into the mRNA described above. According to a specific embodiment of the invention, the DNA is a composition corresponding to the above mRNA combination. In some embodiments of the invention, the DNA comprises a nucleic acid encoding mRNA of the aforementioned at least two cytokines.
In another aspect, the present invention also provides a pharmaceutical composition comprising:
the mRNA and pharmaceutically acceptable auxiliary materials are provided.
According to particular embodiments of the present invention, the pharmaceutical composition of the present invention may be in liquid dosage form, solid dosage form or combination.
On the other hand, the invention also provides the application of the mRNA or the pharmaceutical composition in preparing medicines for preventing and treating solid tumors.
In the present invention, the term "control" includes prophylaxis and/or treatment (including adjuvant treatment).
According to a specific embodiment of the invention, the prevention and treatment of solid tumors comprises inhibition of tumor growth, reduction of tumor size, prevention of tumor recurrence and/or prevention of tumor metastasis.
According to a specific embodiment of the invention, the drug for preventing and treating solid tumors is an intratumoral injection or a peritumoral injection drug. The mRNA of the present invention can be used for intratumoral injection or paraneoplastic injection.
According to a specific embodiment of the invention, the medicament for controlling solid tumors further comprises an immune checkpoint inhibitor. That is, the mRNA of the present invention may be used in combination with an immune checkpoint inhibitor. The immune checkpoint inhibitor comprises PD-1 antibody and/or CTLA-4 antibody.
The mRNA of the invention has the function of preventing and treating tumor, and can inhibit tumor growth, reduce tumor size, prevent tumor recurrence and/or prevent tumor metastasis.
Drawings
FIG. 1 shows the change in tumor size of B16F10 tumor mice after injection of different mRNAs, respectively.
FIG. 2 shows survival curves of B16F10 tumor mice after injection of different mRNAs, respectively.
FIG. 3 shows the change in tumor size after injection of different mRNA in another batch of experimental B16F10 tumor mice.
Fig. 4 shows the change after intratumoral injection of mRNA from another experimental CT26 tumor mice. Panel a. Change in tumor size of each mouse after injection of control mRNA (n=8); panel b. tumor size change (n=8) in each mouse after injection of cytokine mixture; CR is Complete response.
Fig. 5 shows the change in tumor size after injection of different mRNA combinations in another experimental CT26 tumor mice.
FIG. 6 shows the change in tumor size of another experimental CT26 tumor mice after injection of 60. Mu.g of control mRNA or IFNα4+IL-12sc+IL-7 cytokine mRNA mixture (20. Mu.g mRNA/gene) respectively in D9, D12, D15, D18, D21, D25.
Detailed Description
The technical solution of the present invention will be described in detail below for a clearer understanding of technical features, objects and advantageous effects of the present invention, but should not be construed as limiting the scope of the present invention. The experimental methods in the examples, unless otherwise specified, were either conventional or performed according to the conditions and operating methods recommended by the manufacturer. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional reagent stores.
In the present invention, the sequences are as follows:
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 TNFα optimized CDS 1
SEQ ID NO.22:human TNFα optimized 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-12Aamino 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 RNAencoding 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 RNAencoding CDS
SEQ ID NO.63:murine IL-12sc optimized CDS
SEQ ID NO.64:murine IL-12sc optimized RNAencoding 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 TNFα amino acid
SEQ ID NO.76:murine TNFα non optimized CDS
SEQ ID NO.77:murine TNFα non optimized RNA encoding CDS
SEQ ID NO.78:murine TNFα optimized CDS
SEQ ID NO.79:murine TNFα optimized 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
example 1
Construction of B16F10 tumor model
Female C57BL/6J (purchased from Zhuhai Baitong) mice of 18-20 g 6-8 weeks old were kept in a room at 22+ -2deg.C and a relative humidity of 55% + -15% and were free to obtain food and water. B16F10 cells (purchased from ATCC) were cultured in DMEM and 10% FBS at 37 ℃ in 5% CO 2. B16F10 cells were cultured to a confluency of 80 to 90%, digested with 0.25% Trypin-EDTA, resuspended in DPBS, and injected subcutaneously 1×106 cells/100 μl on the right side of each mouse, and a B16F10 tumor mouse model capable of intratumorally injecting mRNA was obtained on day 7 after tumor cell injection.
RNA preparation
The specific synthesis method of mRNA of each cytokine for injection is as follows:
1. the synthesized DNAs expressing different cytokines were cloned into plasmids containing 5'-UTR (SEQ ID NO. 85), 3' -UTR (SEQ ID NO. 86) and poly-A tail (SEQ ID NO. 87) and amplified as DNA templates according to the following reaction conditions:
pre-denaturation at 98℃for 3min; denaturation at 98℃for 10s, annealing at 60℃for 5s, extension at 72℃for 2min,34 cycles; finally, the temperature is 72 ℃ and the time is 10min.
After completion of the reaction, the reaction mixture was combined with a 1.5ml Tube. 10ml of the sample was subjected to DNA agarose gel electrophoresis (1.5% agarose, 5V/min,40 min). And confirming whether the reaction is successful or not according to the size of the electrophoresis target band.
And (5) qualification standard: the electrophoresis detection shows a single band and is correct in size.
2. DNA template ultrafiltration
The DNA template obtained above was concentrated using a Millipore 30Kd ultrafiltration tube.
3. DNA template FPLC purification
The DNA obtained by the above ultrafiltration was added to an equal volume of a phenol/chloroform/isoamyl alcohol mixture (phenol/chloroform/isoamyl alcohol=25/24/1), and after sufficient shaking, 12000g was centrifuged for 15min.
Removing precipitate, transferring supernatant to a new centrifuge tube, adding 1/10 of the volume of NaAc (pH 5.2) of the supernatant, mixing, adding 2 times of absolute ethanol, mixing, and standing at-20deg.C for 30min.
Centrifuge at 12000g for 10min at 4℃and discard supernatant.
Washing the precipitate with 70% ethanol, centrifuging 12000g for 5min, collecting supernatant, and air drying on a super clean bench for 5min.
The purified DNA template was dissolved in appropriate RNase-free water.
The concentration of the purified template was measured using NanoDrop, and the ratios at 260/280, 260/230. Samples were taken for detection by DNA agarose gel electrophoresis (1.5% agarose, 5V/min,40 min).
And (5) qualification standard: 260/280 is between 1.8 and 2.1 and 260/230 is between 1.6 and 2.2.
4. Template ultrafiltration after FPLC purification
The DNA template purified by FPLC was concentrated in a Millipore 30Kd ultrafiltration tube, and eluted with RNase-free water. The concentration of template after ultrafiltration, and the ratio of A260/A280, A260/A230 were measured using NanoDrop. Finally, the mixture was diluted to 150ng/ml with RNase-free water.
5. In vitro synthesis of mRNA
In a isothermal reactor, in vitro synthesis of mRNA was performed.
The following synthesis system was carried out (reagents were added from top to bottom):
1600ml of reaction volume (in a 2ml RNase-free Tube, single Tube reaction volume, one simultaneous reaction multitube): 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 XBuffer 160ml and Enzyme Mix 160ml.
The in vitro synthesis procedure of RNA was 37℃for 10h.
6. DNase I digestion to remove DNA template
120ml DNase I was added to each Tube after in vitro mRNA synthesis.
Mix up and down 10 times and centrifuge at 1000rpm for 10s.
The mixture was placed in a constant temperature reactor again at 37℃for 1h.
7. mRNA precipitation recovery
To each 50ml Tube in the previous step, an equal volume of ammonium acetate solution was added.
Mix up and down for 10 times.
Placing at-20deg.C for 2 hr, and precipitating.
17000g, centrifuged at 4℃for 30min.
The supernatant was removed and the precipitate was washed with 70% ethanol.
17000g, centrifuged at 4℃for 10min.
70% ethanol was removed, evaporated to dryness in an ultra clean bench, and 20ml of RNase-free water was added to each tube.
Standing for 10min, and lightly blowing with gun head.
The mRNA concentration after the recovery was 5mg/ml by the NanoDrop assay, A260/A280 was 1.90 and A260/A230 was 2.0.
1ml was diluted 10-fold and subjected to RNA ScreenTape assay and agarose gel electrophoresis to examine the fragment integrity.
8. LiCl precipitation purification of mRNA
mRNA recovered in the previous step was added to RNase-free water in an amount of 1.5 times the volume of the mRNA, and the mixture was homogenized.
Adding 1.5 times of pre-cooled LiCl solution of the original mRNA, and uniformly mixing.
Then, the mixture was allowed to stand at-20℃for 2 hours.
Centrifuge at 16000g for 20min.
The supernatant was discarded, and the pellet was washed with 70% ethanol and centrifuged at 16000g for 15min.
Taking the supernatant, and airing the supernatant on an ultra-clean bench for 5min.
The purified mRNA was dissolved in appropriate RNase-free water.
In the present invention, mRNA of the sequences shown as 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 were synthesized by referring to the above methods. And each mRNA contained 5'-UTR (SEQ ID NO. 85), 3' -UTR (SEQ ID NO. 86) and poly-A tail (SEQ ID NO. 87).
3, methods of tumor treatment
Control mRNA or cytokine mRNA mixtures (8-9 per group of mice) were injected by intratumoral injection into tumors of B16F10 model mice, and the intratumoral injection mRNA was diluted to 50. Mu.l with physiological saline after mixing the same mass for the mice, and the mRNA for intratumoral injection was a mouse version of various cytokines (see Table 1 for experimental group). The tumor size of the mice is measured by vernier caliper every 3-4 days, when the tumor size exceeds 2000mm 3 Or mice were sacrificed when body weight was reduced by 20%.
TABLE 1 grouping of experiments
4 treatment results
The change in tumor size of B16F10 tumor mice after injection of the mixture of control mRNA, IFNα4, IL-7, GM-CSF, IL-12A+IL-12B, TNF α, cytokine mRNA, respectively, at D7, D10, D13, D17, and D20 is shown in FIG. 1. Where single factor mRNA was 10. Mu.g per mouse, control mRNA was 60. Mu.g per mouse, cytokine mRNA mixture was administered to each mouse at 10. Mu.g mRNA/gene (i.e., 2 cytokine mRNA mixtures were administered to each mouse with 20. Mu.g mRNA,4 cytokine mRNA mixtures were administered to each mouse with 40. Mu.g mRNA,6 cytokine mRNA mixtures were administered to each mouse with 60. Mu.g mRNA), and tumor size was monitored for up to 31 days.
The survival curves of B16F10 tumor mice after injection of the control mRNA and cytokine mRNA mixtures, respectively, are shown in FIG. 2. Mice were injected with 60 μg control mRNA and cytokine mRNA mixture (10 μg mRNA/gene) at D7, D10, D13, D17, D20 and tumor size was monitored for up to 31 days.
Survival of B16F10 tumor mice at various time points is shown in table 2.
Table 2, survival of B16F10 tumor mice at different time points
The change in tumor size after injection of control mRNA and different cytokine mRNA mixtures, respectively, in B16F10 tumor mice from another experiment is shown in FIG. 3. Mice were injected with 60 μg control mRNA and cytokine mRNA mixture (10 μg mRNA/gene) at D7, D11, D15, D19, D22 and tumor size was monitored until 25 days.
Experiments of the invention show that the mRNA composition of the invention comprises 2-6 compositions of the following mRNA: mRNA encoding IFN alpha 4, mRNA encoding IL-7, mRNA encoding IL-12A, mRNA encoding IL-12B, mRNA encoding TNF alpha, mRNA encoding GM-CSF, has therapeutic effect on tumors.
Example 2
RNA was prepared in the same manner as in example 1. mRNA of each cytokine was prepared in this example. And grouped according to the scheme of table 3.
TABLE 3 grouping of experiments
Subcutaneous transplantation 5X10 in BALB/C mice 5 CT26 tumor cells, when the tumor grows to 20-40 mm 3 Control mRNA or cytokine mRNA mixtures (8 mice per group) were injected by intratumoral injection into tumors in CT26 model mice. The mRNA injected intratumorally in mice was diluted to 50. Mu.l with physiological saline after mixing the same mass, and the mRNA injected intratumorally in mice was a mouse version of 5 cytokines. The tumor size of the mice is measured by vernier caliper every 3-4 days, when the tumor size exceeds 2000mm 3 Or mice were sacrificed when body weight was reduced by 20%. CT26 tumor mice were injected with 100. Mu.g control mRNA, IFNα4+IL-7+at D6, D8, D11, D13, D15, respectivelyTumor size changes after GM-CSF+IL12sc+TNF α cytokine mRNA mixture (20 μg mRNA/gene) see FIG. 4, tumor size was monitored for up to 39 days. It can be seen that the CT26 tumor was growing after injection of control mRNA, whereas CT26 tumor injected with cytokine mRNA mixture was not growing at the same rate as control mRNA and 7 tumors were completely disappeared from 8 tumors.
Subcutaneous transplantation 5X10 in BALB/C mice 5 CT26 tumor cells, when the tumor grows to 20-50 mm 3 Control mRNA or cytokine mRNA mixtures (9 mice per group) were injected by intratumoral injection into tumors in CT26 model mice. The mRNA injected intratumorally in mice was diluted to 50. Mu.l with physiological saline after mixing the same mass, and the mRNA injected intratumorally in mice was a mouse version of 5 cytokines. The tumor size of the mice is measured by vernier caliper every 3-4 days, when the tumor size exceeds 2000mm 3 Or mice were sacrificed when body weight was reduced by 20%. CT26 tumor mice were monitored for tumor size changes after injection of 60 μg of control mRNA, IFNα4+IL-7+GM-CSF, TNFα+IL-12sc+GM-CSF, IL-7+IL-12sc+GM-CSF, IFNα04+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, and a mixture of TNFα+GM-CSF+IL-7 cytokine mRNA (; 20 μg mRNA/gene), see FIG. 5, for up to 34 days. It can be seen that the CT26 tumors after injection of control mRNA were growing, whereas CT26 tumors injected with a mixture of cytokine mRNAs of different combinations did not grow as fast as control mRNA and many groups of tumors completely disappeared.
Transplanting 5x10 subcutaneously on the left and right flanks of BALB/C mice 5 And 3x10 5 CT26 tumor cells, when the left tumor grows to 40-90 mm 3 The size of the tumor on the right side is 40-70 mm 3 Control mRNA or cytokine combination IFNα4+IL-12sc+IL-7mRNA mixture (8 mice per group) was injected by intratumoral injection into the left tumor, while the isotype antibody or anti-PD-1 antibody was injected intraperitoneally, respectively. mRNA injected into tumor of mice was mixed in the same mass and diluted to 50. Mu.l with physiological salinemRNA for intratumoral injection in mice was a mouse version of 5 cytokines. The tumor size of the mice is measured by vernier caliper every 3-4 days, when the tumor size exceeds 2000mm 3 Or mice were sacrificed when body weight was reduced by 20%. CT26 tumor mice were monitored for tumor size up to 28 days after injection of 60 μg of control mRNA or IFNα4+IL-12sc+IL-7 cytokine mRNA mixture (20 μg mRNA/gene) respectively in D9, D12, D15, D18, D21, D25, see FIG. 6. It can be seen that CT26 tumors grew after injection of control mRNA, whereas CT26 tumors injected with cytokine mRNA mixtures grew less rapidly than control mRNA and significantly slower on either the treated or untreated side than the other treatment groups after combined use of anti-PD-1 antibodies.

Claims (11)

1. An mRNA comprising mRNA encoding at least two of the following cytokines:
IFNα、IL-7、TNFα、IL-12A、IL-12B、IL-12sc、GM-CSF。
2. the mRNA of claim 1, which is a composition comprising any two, three, four, five, six, seven, or eight mrnas of an mRNA encoding ifnα, an mRNA encoding IL-7, an mRNA encoding tnfα, an mRNA encoding IL-12A, an mRNA encoding IL-12B, an mRNA encoding IL-12A and IL-12B, an mRNA encoding IL-12sc, an mRNA encoding GM-CSF.
3. The mRNA according to claim 1 or 2, wherein:
IFN alpha has an amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO.6 or SEQ ID NO. 70;
IL-7 has an amino acid sequence shown as SEQ ID NO.11 or SEQ ID NO. 65;
TNFα has an amino acid sequence as shown in SEQ ID NO.18 or SEQ ID NO. 75;
IL-12A has an amino acid sequence as shown in SEQ ID NO.25 or SEQ ID NO. 54;
IL-12B has an amino acid sequence as shown in SEQ ID NO.30 or SEQ ID NO. 57;
IL-12sc has an amino acid sequence as shown in SEQ ID NO.43 or SEQ ID NO.49 or SEQ ID NO. 60;
GM-CSF has the amino acid sequence shown as SEQ ID NO.35 or SEQ ID NO. 80.
4. The mRNA of any one of claims 1-3, wherein:
the mRNA encoding IFN alpha includes one or more of the following mRNAs: mRNA of any one of SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.72 and SEQ ID NO. 74;
the mRNA encoding IL-7 includes one or more of the following mRNAs: mRNA of any one of SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.67 and SEQ ID NO. 69;
the mRNA encoding tnfα includes one or more of the following: mRNA of any one of SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.77 and SEQ ID NO. 79;
the mRNA encoding IL-12A includes one or more of the following mRNAs: mRNA of any one of SEQ ID NO.27, SEQ ID NO.29 and SEQ ID NO. 56;
the mRNA encoding IL-12B includes one or more of the following mRNAs: mRNA of any one of SEQ ID NO.32, SEQ ID NO.34 and SEQ ID NO. 59;
the mRNA encoding IL-12sc includes one or more of the following mRNAs: mRNA of any one of SEQ ID NO.45, SEQ ID NO.47, SEQ ID NO.51, SEQ ID NO.53, SEQ ID NO.62 and SEQ ID NO. 64;
the mRNA encoding GM-CSF includes one or more of the following mRNAs: mRNA of any one of SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.41, SEQ ID NO.82 and SEQ ID NO. 84.
5. The mRNA according to any one of claims 1 to 4, which comprises at least mRNA encoding IFN alpha, mRNA encoding IL-7 or mRNA encoding TNF alpha; or alternatively
The mRNA includes at least mRNA encoding IL-12A, IL-12B or IL-12sc.
6. An mRNA according to any one of claims 1 to 5, comprising one or more of the following sets of mrnas:
(1) mRNA encoding IL-12A and mRNA encoding IL-12B;
(2) mRNA encoding IFN alpha and mRNA encoding TNF alpha;
(3) mRNA encoding IL-7 and mRNA encoding TNF alpha;
(4) mRNA encoding IFN alpha and mRNA encoding IL-7;
(5) mRNA encoding IFN alpha and mRNA encoding GM-CSF;
(6) mRNA encoding IFN alpha and mRNA encoding IL-12 sc;
(7) mRNA encoding IL-7 and mRNA encoding IL-12 sc;
(8) mRNA encoding IL-7 and mRNA encoding GM-CSF;
(9) mRNA encoding GM-CSF and mRNA encoding IL-12 sc;
(10) mRNA encoding GM-CSF and mRNA encoding TNF α;
(11) mRNA encoding IL-12sc and mRNA encoding TNF alpha;
(12) mRNA encoding IFN alpha, mRNA encoding IL-7 and mRNA encoding TNF alpha;
(13) mRNA encoding TNF alpha, mRNA encoding IL-12sc, and mRNA encoding GM-CSF;
(14) mRNA encoding GM-CSF, mRNA encoding IL-7, and mRNA encoding IL-12 sc;
(15) mRNA encoding IFN alpha, mRNA encoding TNF alpha, and mRNA encoding GM-CSF;
(16) mRNA encoding IFN alpha, mRNA encoding IL-12sc and mRNA encoding TNF alpha;
(17) mRNA encoding IFN alpha, mRNA encoding IL-7 and mRNA encoding GM-CSF;
(18) mRNA encoding IFN alpha, mRNA encoding IL-12sc, and mRNA encoding GM-CSF;
(19) mRNA encoding IFN alpha, mRNA encoding IL-12sc and mRNA encoding IL-7;
(20) mRNA encoding TNF alpha, mRNA encoding IL-12sc, and mRNA encoding IL-7;
(21) An mRNA encoding TNF alpha, an mRNA encoding GM-CSF, and an mRNA encoding IL-7;
(22) mRNA encoding IFN alpha, mRNA encoding IL-7, mRNA encoding TNF alpha, and mRNA encoding GM-CSF;
(23) mRNA encoding IFN alpha, mRNA encoding IL-7, mRNA encoding TNF alpha, mRNA encoding IL-12A, and mRNA encoding IL-12B;
(24) mRNA encoding IL-7, mRNA encoding TNF alpha, mRNA encoding IL-12A, mRNA encoding IL-12B, and mRNA encoding GM-CSF;
(25) mRNA encoding IFN alpha, mRNA encoding TNF alpha, mRNA encoding IL-12A, mRNA encoding IL-12B, and mRNA encoding GM-CSF;
(26) mRNA encoding IFN alpha, mRNA encoding IL-7, mRNA encoding IL-12A, mRNA encoding IL-12B, and mRNA encoding GM-CSF;
(27) mRNA encoding IFN alpha, mRNA encoding IL-7, mRNA encoding TNF alpha, mRNA encoding IL-12 sc;
(28) mRNA encoding IL-7, mRNA encoding TNF alpha, mRNA encoding IL-12sc, and mRNA encoding GM-CSF;
(29) mRNA encoding IFN alpha, mRNA encoding TNF alpha, mRNA encoding IL-12sc, and mRNA encoding GM-CSF;
(30) mRNA encoding IFN alpha, mRNA encoding IL-7, mRNA encoding IL-12sc, and mRNA encoding GM-CSF;
(31) mRNA encoding IFN alpha, mRNA encoding IL-7, mRNA encoding TNF alpha, mRNA encoding IL-12A, mRNA encoding IL-12B, and mRNA encoding GM-CSF;
(32) mRNA encoding IFN alpha, mRNA encoding IL-7, mRNA encoding TNF alpha, mRNA encoding IL-12sc, and mRNA encoding GM-CSF.
7. The mRNA of any one of claims 1-6, wherein a portion or all of the mRNA has one or more of the following characteristics:
(1) Comprising uridine, cytidine, adenine nucleoside, guanosine or a chemically modified nucleoside;
(2) Comprising a 5' cap structure;
(3) Comprising a 5' UTR;
(4) Comprising a 3' UTR;
(5) Comprising a poly-A tail;
(6) The mRNA is in the form of a naked RNA, a RNA salt solution, an LNP-coated form, an LPX-coated form, or an LPP-coated form.
8. The mRNA according to any one of claims 1 to 7, which is a composition comprising the individual cytokines encoded, wherein the amount of mRNA encoding each cytokine in the composition is 0.1 to 10 parts by weight, preferably 0.5 to 5 parts by weight, more preferably 0.5 to 1 part by weight, respectively; preferably, the amount of mRNA encoding each cytokine in the composition is substantially equal in quality.
9. A DNA comprising a nucleic acid encoding mRNA of at least two cytokines of any one of claims 1-8.
10. A pharmaceutical composition, the pharmaceutical composition comprising:
the mRNA of any one of claims 1-8, and
pharmaceutically acceptable auxiliary materials;
preferably, the pharmaceutical composition is in a liquid dosage form, a solid dosage form or a combination.
11. Use of an mRNA according to any one of claims 1 to 8 or a DNA according to claim 9 or a pharmaceutical composition according to claim 10 for the preparation of a medicament for the prevention and treatment of solid tumors;
preferably, the prevention and treatment of solid tumors comprises inhibition of tumor growth, reduction of tumor size, prevention of tumor recurrence and/or prevention of tumor metastasis;
preferably, the medicine for preventing and treating the solid tumor is an intratumoral injection or a peritumoral injection medicine;
preferably, the medicament for preventing and treating solid tumors further comprises an immune checkpoint inhibitor.
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