Cytokine mRNA (messenger ribonucleic acid) combined medicine and application thereof

Abstract

The invention provides a cytokine mRNA (messenger Ribonucleic Acid) combination medicine and application thereof. The combined medicine comprises mRNA (messenger ribonucleic acid) for coding 4-1BBL protein, mRNA for coding CD40L protein and mRNA for coding IL-36 gamma protein. After the combined medicine is given to a tumor model mouse, the tumor volume of the mouse can be remarkably reduced. Compared with drugs of other protein types, the cytokine mRNA combined drug has the advantages of short production cycle, good safety, quick effect and low price.

Description

Technical Field

[0001] The present invention belongs to the field of medicines, and in particular relates to a cytokine mRNA combination medicine and application thereof. Background Art

[0002] At present, the treatment methods of tumors include surgical resection, radiotherapy, chemotherapy, immunotherapy, etc. Among them, although surgical treatment can remove the tumor, it may not be completely removed or cause tumor metastasis and recurrence during the operation, and may also cause organ damage and reduced body immunity; chemotherapy uses chemical drugs to treat malignant tumors, which is suitable for systemic tumors, such as blood tumors, but there are many side effects, such as the toxicity of anticancer drugs, the resistance of cancer cells to drugs, etc., which will greatly reduce the efficacy of drugs, and cause systemic side effects such as nausea and loss of appetite in patients; radiotherapy uses various rays of different energies (X-rays, γ-rays, electron beams) to irradiate tumors. Local treatment methods can assist surgical treatment and improve the effect, but like chemotherapy, there is a problem of "not distinguishing between friend and foe", that is, cancer cells and normal cells are killed together, and there are also shortcomings such as the effective dose and the toxic dose are very close, and there are relatively large toxic side effects; immunotherapy targets the body's low or hyperimmune state, artificially enhances or suppresses the body's immune function to achieve the purpose of treating the disease. Compared with traditional treatment methods, immunotherapy has significant advantages of fast response, small side effects, and long-lasting efficacy. Therefore, tumor immunotherapy has also become a hot spot in global tumor treatment research, and the market share of tumor immunotherapy continues to increase.

[0003] Since tumor cells are abnormal cells, they often have gene mutations and overexpression of oncogenes. Therefore, the immune system in the human body, which has the function of eliminating "foreigners", can theoretically eliminate these abnormal cells at any time. However, tumor cells have also evolved a set of countermeasures to confuse the immune system and escape. Tumor immunotherapy is to prevent, control and eliminate cancer by activating the body's own immune system and using the power of the body's own immune system. Immunotherapy can educate the immune system to recognize and attack specific tumor cells, enhance immune cells to help them eliminate cancer, and provide the body with additional components to enhance the immune response. There are many types of tumor immunotherapy, such as monoclonal antibodies, immune checkpoint inhibitors, cytokines, tumor vaccines, immune cell therapy, oncolytic viruses, etc.

[0004] Among all tumor immunotherapies, immune checkpoint inhibitors are one of the most successful therapies, which have changed the treatment prospects of many tumors such as lung cancer and melanoma. However, existing immune checkpoint drugs such as PD-1 / PD-L1 inhibitors have inevitable limitations. For example, PD-1 / PD-L1 inhibitors may trigger immune attacks on normal tissues and cause off-target damage. In addition, PD-1 / PD-L1 inhibitors have drug resistance problems and cannot benefit all patients continuously. At present, in the vast majority of unselected solid tumors, the efficacy of PD-1 / PD-L1 inhibitors alone is only about 10% to 30%, which also makes new treatment methods and methods a major demand and a direction for active exploration in the future.

[0005] Cytokines are molecular messengers secreted by various cells (immune cells and non-immune cells) for communication with other cells. They have multiple functions such as regulating innate immunity and adaptive immunity, hematopoiesis, cell growth, and repair of damaged tissues. Cytokines can be divided into interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokine family, growth factors, etc. Many cytokines exert their effects in vivo through paracrine, autocrine or endocrine modes, and have multiple physiological characteristics such as pleiotropy, overlap, antagonism, and synergy, forming a very complex cytokine regulatory network. Many cytokines, such as interleukins, have the function of regulating cancer immune responses. During immunotherapy, cytokines can directly stimulate immune effector cells and stromal cells at the tumor site and enhance cytotoxicity. Through studies on animal tumor models, it has been found that cytokines have a wide range of anti-tumor activities.

[0006] Single cytokine therapy has limitations such as strong toxicity and low response rate. Currently, many new strategies have emerged to design a new generation of cytokines. New strategies are shifting from using natural forms of cytokines to complex engineering methods, such as improving receptor affinity (cytokine mutants), increasing the half-life of cytokines in the body (polyethanolized cytokines), and designing tumor-targeted fusion structures (fused with antibodies) to improve efficacy and reduce systemic toxicity. In order to increase the application rate of cytokine therapy, cytokines are also combined with other immunotherapies to help drug-resistant patients benefit from them. Another way is to increase the concentration of cytokines in the tumor microenvironment by local injection, promote in situ anti-tumor activity and reduce systemic toxicity, and use cells in the tumor to produce cytokines to achieve the purpose of treating tumors.

[0007] For example, CN108524918B discloses that an anti-tumor composition composed of IL-36γ and soluble PD-1 (sPD-1) can be used to fight melanoma. However, due to the nature of its free protein, cytokine drugs are very easy to be quickly cleared and degraded by enzymes in the serum, resulting in a short half-life of the drug in the body. In addition, the protein purification process is complicated, and specific cytokine proteins often need to develop specific purification processes that are suitable for themselves. These cause an increase in treatment costs, and may also lead to the generation of side effects and even over-activation of the immune system.

[0008] CN111849905B discloses a mesenchymal stem cell that expresses an immunostimulatory factor, which includes a chemokine CXCL9 and a cytokine OX40L. It can be used to treat cancer or tumors. However, this technology not only relies on the therapeutic effect of cytokines, but must also rely on mesenchymal stem cells. Mesenchymal stem cells can specifically attract and activate immune cells that kill tumor tissues at the tumor site, and mesenchymal stem cells have a synergistic effect with chemokines and / or cytokines to have an efficient immune effect. Summary of the invention

[0009] In view of this, the purpose of the present invention is to provide a cytokine mRNA combination drug, which can be used to treat tumors. The cytokines in mRNA dosage form have significant advantages over traditional protein and antibody drugs in terms of efficacy, biosafety, synthesis process, cost, etc.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] In one aspect, the present invention provides a cytokine mRNA combination drug, comprising mRNA encoding 4-1BBL protein, mRNA encoding CD40L protein, and mRNA encoding IL-36γ protein.

[0012] According to a specific embodiment of the present invention, the concentrations of mRNA encoding 4-1BBL, CD40L and IL-36γ proteins are equal.

[0013] According to a specific embodiment of the present invention, the mRNA encoding the 4-1BBL protein includes the nucleotide sequence shown in SEQ ID NO.1 or a nucleotide sequence having at least 80% homology thereto.

[0014] According to a specific embodiment of the present invention, the mRNA encoding the CD40L protein comprises the nucleotide sequence shown in SEQ ID NO. 3 or a nucleotide sequence having at least 80% homology thereto.

[0015] According to a specific embodiment of the present invention, the mRNA encoding the IL-36γ protein includes the nucleotide sequence shown in SEQ ID NO.5 or a nucleotide sequence having at least 80% homology thereto.

[0016] Preferably, the above homology is 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 99.5% or more.

[0017] According to a specific embodiment of the present invention, each mRNA in the composition is encapsulated by lipid nanoparticles (LNPs).

[0018] According to a specific embodiment of the present invention, wherein the mRNA combination drug is a liquid preparation, more preferably, the mRNA is encapsulated in LNP and then dissolved in physiological saline.

[0019] According to a specific embodiment of the present invention, the administration method of the mRNA combination drug includes intratumoral injection, subcutaneous injection, intramuscular injection or intravenous injection.

[0020] In another aspect, the present invention also provides the use of the above cytokine mRNA combination drug in the preparation of a drug for treating or preventing a tumor, preferably, the tumor is melanoma, non-small cell lung cancer, gastric cancer, liver cancer, breast cancer, colorectal cancer. Preferably, the tumor is colon cancer.

[0021] In another aspect, the present invention provides a method for treating or preventing tumors, comprising administering an effective amount of the cytokine mRNA combination drug of the present invention to a subject in need thereof.

[0022] In one embodiment, the administration comprises intratumoral injection, subcutaneous injection, intramuscular injection or intravenous injection.

[0023] Beneficial Effects

[0024] After the cytokine in the mRNA dosage form of the present invention is administered to tumor model mice, the tumor volume of the mice can be significantly reduced. Compared with other protein-type drugs, the cytokine mRNA combination drug in the present invention has the advantages of short production cycle, good safety, rapid onset and low price. The mRNA production process is generally universal, does not require continuous redevelopment of new processes, and production does not require cell lines, and belongs to the chemical synthesis route. In addition, when the mRNA drug is administered, human cells become the site of antibody production, which is more conducive to the control of antibody post-translational modification, and can directly target intracellular targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The curves showing the changes in tumor volume in mice are shown.

[0026] Figure 2 The curve of changes in mouse body weight is shown. DETAILED DESCRIPTION

[0027] The present invention provides a cytokine mRNA combination medicine, comprising mRNA encoding 4-1BBL protein, mRNA encoding CD40L protein and mRNA encoding IL-36γ protein.

[0028] Wherein, the mRNA and amino acid sequence encoding the 4-1BBL protein preferably include the sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; in the present invention, the specific sequence of SEQ ID NO.1 is as follows:

[0029] atggatcagcatacactggatgtggaggatacagctgatgctagacacccagctggaacctcttgtcctagcgatgctgctctgctgagagatacaggactgctggctgatgctgccctgctgtccgatacagtgagaccaacaaatgctgctctgcctactgatgctgcctaccccgccgtgaacgtgagggaccgggaggccgcctggcccccagccctgaacttctgctccaggcaccctaagctgtacggcctggtggccctggtgctgctgctgctgatcgccgcctgcgtgcccatcttcaccaggaccgagcccaggcccgccctgaccatcaccacctcccccaacctgggcaccagggagaacaacgccgaccaggtgaccccagtgagccacatcggctgccctaataccacccagcagggcagccctgtgttcgccaagctgctggccaagaaccaggccagcctgtgtaacaccaccctgaactggcactcccaggacggcgccggctccagctacctgagccagggcctgagatacgaggaggacaagaaggagctggtggtggacagccctggcctgtactacgtgttcctggagctgaagctgagccctaccttcaccaacaccggccacaaggtgcagggctgggtgagcctggtgctgcaggccaagccacaggtggacgacttcgacaacctggccctgaccgtggagctgttcccttgcagcatggagaacaaactggtggacagaagctggagccagctgctgctgctgaaggccggccataggctgagcgtgggcctgagggcctacctgcacggagcccaggacgcctacagggactgggagctgtcctaccctaacaccaccagcttcggcctgtttctggtgaagcctgataatccttgggag

[0030] The specific sequence of SEQ ID NO.2 is as follows:

[0031] MDQHTLDVEDTADARHPAGTSCPSDAALLRDTGLLADAALLSDTVRPTNAA

[0032] LPTDAAYPAVNVRDREAAWPPALNFCSRHPKLYGLVALVLLLLIAACVPIFTRTEPRP

[0033] ALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLCNTTLN

[0034] WHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQG

[0035] WVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLKAGHRLSVGL

[0036] RAYLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWE

[0037] Wherein, the mRNA and amino acid sequence encoding CD40L protein preferably include the sequences shown in SEQ ID NO.3 and SEQ ID NO.4 respectively; in the present invention, the specific sequence of SEQ ID NO.3 is as follows:

[0038] atgatcgagacctacagccagccctctcccagatccgtggcaaccggcctgcccgctagcatgaagatcttcatgtacttgctgacagtgttcctgattactcagatgatcggctccgtgctgtttgctgtgtacctgcacaggaggctggacaaggtggaagaggaagtgaatctgcatgaggacttcgtgttcatcaagaagctgaagcggtgcaacaagggcgagggcagcctgagcctgctgaactgtgaggaaatgagacggcagtttgaggatctggtgaaagacattaccctgaacaaggaagagaaaaaggagaacagtttcgagatgcagcggggagatgaggacccccagatcgctgcccacgtggtgtctgaggccaactccaatgccgcatccgtgctgcagtgggccaaaaagggttactacaccatgaagagtaacctggtgatgctggagaacggcaaacagctgacagtgaagagagagggcctgtactacgtgtacactcaggtgacattttgcagcaaccgggagcctagcagtcagagaccattcatcgtgggcctgtggctgaaacctagctccggcagcgagcgcattctgctgaaggctgccaacactcattcttcctcccagctgtgcgagcagcagagcgtgcacctgggcggcgtgttcgagctgcaggccggcgcctccgtgttcgtcaacgtgaccgaggcctctcaggtgatccacagggtgggattctcctcctttggactgctgaagctg

[0039] The specific sequence of SEQ ID NO.4 is as follows:

[0040] MIETYSQPSPRSVATGLPASMKIFMYLLTVFLITQMIGSVLFAVYLHRRLDKVE

[0041] EEVNLHEDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKKENSF

[0042] EMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVK

[0043] REGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQ

[0044] SVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSSFGLLKL

[0045] Wherein, the mRNA and amino acid sequence encoding IL-36γ protein preferably include SEQ ID NO.5

[0046] and the sequence shown in SEQ ID NO.6; in the present invention, the specific sequence of SEQ ID NO.5 is as follows:

[0047] atgttcagcaagcacccttttagcacacacatctctggaagagagacccctgattttggcgaggtgttcgatctggatcagcaggtgtggatctttaggaaccaggccctggtgaccgtgcct agaagccacagagtgacacctgtgagcgtgacaattctgccttgtaagtaccctgagtctctggaacaggacaagggcatcgctatctacctgggaatccagaatcctgacaagtgtctgttt tgtaaggaggtgaatggccaccctacacttctgctgaaggaggagaagatcctggacctgtatcaccaccctgagcctatgaagccatttctgttctatcacacaagaacaggaggaacatct acatttgagagcgtggcctttcctggccactacatcgcctcttccaagaccggaaaccctattttcctgacatctaagaagggcgagtactacaacatcaacttcaacctggacatcaagagc

[0048] The specific sequence of SEQ ID NO.6 is as follows:

[0049] MFSKHPFSTHISGRETPDFGEVFDLDQQVWIFRNQALVTVPRSHRVTPVS

[0050] VTILPCKYPESLEQDKGIAIYLGIQNPDKCLFCKEVNGHPTLLLKEEKILDLYHHPEP

[0051] MKPFLFYHTRTGGTSTFESVAFPGHYIASSKTGNPIFLTSKKGEYYNINFNLDIKS

[0052] The present invention provides a cytokine in an mRNA dosage form, comprising mRNA encoding CD80 protein.

[0053] In the present invention, the mRNA and amino acid sequence encoding the CD80 protein preferably include the sequences shown in SEQ ID NO.7 and SEQ ID NO.8, respectively; in the present invention, the specific sequence of SEQ ID NO.7 is as follows:

[0054] atggcctgtaactgtcagctgatgcaggacacccctctgctgaagtttccttgtcctaggctgatcctgttgttcgtgctgctgatcagactgtctcaggtgagctctgatgtggatgagcagctgtctaagagcgtgaaggataaggtgctgctgccatgtagatataacagcccacacgaagatgagtctgaggatagaatctattggcagaagcatgataaggtggtgctgtccgtgatcgccggcaagctgaaggtgtggcctgagtataagaacaggacactgtatgataatacaacatattctctgatcatcctcggcctggtgctgtctgatagaggtacatacagctgtgtggtgcagaagaaggagcggggcacctacgaggtgaagcacctggccctggtgaagctgtccatcaaggccgacttttccacccctaacatcaccgagagcggcaaccctagcgccgacaccaagcggatcacctgcttcgctagcggcggcttccctaagccaaggttcagctggctggagaacggcagagagctgcctggcatcaacaccaccatctcccaggaccccgagagcgagctgtacaccatcagcagccagctggacttcaacaccaccaggaaccacaccatcaagtgcctgatcaagtacggcgacgcccacgtgagcgaggacttcacctgggagaagcctcctgaggacccccccgacagcaagaacaccctggtgctgttcggcgccggcttcggcgccgtgatcaccgtggtcgtgatcgtcgtgatcatcaagtgcttctgcaagcacaggagctgtttcagaaggaacgaggccagcagagagaccaacaacagcctgaccttcggccctgaggaggccctggccgagcagaccgtgttcctg

[0055] The specific sequence of SEQ ID NO.8 is as follows:

[0056] MACNCQLMQDTPLLKFPCPRLILLFVLLIRLSQVSSDVDEQLSKSVKDKVLLP

[0057] CRYNSPHEDESEDRIYWQKHDKVVLSVIAGKLKVWPEYKNRTLYDNTTYSLIILGL

[0058] VLSDRGTYSCVVQKKERGTYEVKHLALVKLSIKADFSTPNITESGNPSADTKRITCF

[0059] ASGGFPKPRFSWLENGRELPGINTTISQDPESELYTISSQLDFNTTRNHTIKCLIKYG

[0060] DAHVSEDFTWEKPPEDPPDSKNTLVLFGAGFGAVITVVVIVVIIKCFCKHRSCFRRN

[0061] EASRETNNSLTFGPEEALAEQTVFL

[0062] The present invention provides a cytokine in an mRNA dosage form, comprising mRNA encoding CD86 protein.

[0063] In the present invention, the mRNA and amino acid sequence encoding CD86 protein preferably include the sequences shown in SEQ ID NO.9 and SEQ ID NO.10 respectively; in the present invention, the specific sequence of SEQ ID NO.9 is as follows:

[0064] atggaccctagatgtacaatgggcctggccatcctgatcttcgtgaccgtgctgctgatcagcgatgccgtgagtgtggaaacccaggcctattttaatggaaccgcctatctgccttgtccctttacaaaggctcagaatattagcctgtctgagctggtggtgttctggcaggatcagcagaagctggtgctgtacgagcattatctgggcacagagaagctggacagcgtgaacgccaagtacctgggaagaacctctttcgatagaaataactggaccctgagactgcacaacgtgcagatcaaggacatgggcagctacgactgtttcatccagaagaagccccctaccggcagcatcatcctgcagcagaccctgaccgagctgagcgtgatcgccaacttcagcgagccagagatcaagcttgcccagaacgtgaccggcaacagcggcatcaacctgacctgtaccagcaagcagggccaccctaagcccaagaagatgtacttcctgatcaccaacagcaccaacgagtacggcgacaacatgcagatcagccaggacaacgtgacagagctgttcagcatctccaactccctgagcctgagcttccccgacggcgtgtggcacatgaccgtggtgtgcgtgctggagaccgagagcatgaagatcagctccaagcccctgaacttcacccaggagttcccttctcctcagacctactggaaggagatcaccgccagcgtgaccgtggccctgctgctcgtgatgctgctgatcatcgtgtgccacaagaagcccaaccagccctccaggcctagcaataccgcctccaagctggagagggacagcaacgccgacagagagaccatcaacctgaaggagctggagcctcagatcgccagcgccaagcctaacgccgagSEQ ID NO. 10 The specific sequence is as follows:

[0065] MDPRCTMGLAILIFVTVLLISDAVSVETQAYFNGTAYLPCPFTKAQNISLSELV

[0066] VFWQDQQKLVLYEHYLGTEKLDSVNAKYLGRTSFDRNNWTLRLHNVQIKDMGS

[0067] YDCFIQKKPPTGSIILQQTLTELSVIANFSEPEIKLAQNVTGNSGINLTCTSKQGHPKP

[0068] KKMYFLITNSTNEYGDNMQISQDNVTELFSISNSLSLSFPDGVWHMTVVCVLETES

[0069] MKISSKPLNFTQEFPSPQTYWKEITASVTVALLLVMLLIIVCHKKPNQPSRPSNTASK

[0070] LERDSNADRETINLKELEPQIASAKPNAE

[0071] The present invention provides a cytokine in an mRNA dosage form, comprising mRNA encoding a CD70 protein.

[0072] In the present invention, the mRNA and amino acid sequence encoding the CD70 protein preferably include the sequences shown in SEQ ID NO.11 and SEQ ID NO.12, respectively; in the present invention, the specific sequence of SEQ ID NO.11 is as follows:

[0073] atgcctgaggaaggcagaccttgtccatgggtgagatggtctggcacagcttttcagagacagtggccttggctgctgctggtggtgtttattacagtgttttgttgttggtttcattgttctggactgctgtctaagcagcagcagaggctgctggagcacccagagcctcacaccgccgagctgcagctgaacctgaccgtgcctcggaaggaccctaccctgagatggggcgccggccccgccctgggcaggagcttcacccacggccctgagctggaggagggccacctgagaatccaccaggacggcctgtaccggctgcacatccaggtgaccctggccaactgcagcagccctggcagcaccctgcagcacagggccaccctggccgtgggcatctgctcccctgccgcccacggcatcagcctgctgaggggcaggttcggccaggactgcaccgtggccctgcagaggctgacctacctggtgcacggggacgtgctgtgtaccaacctgaccctgcccctgctgcctagccggaacgctgacgagacctttttcggcgtgcagtggatctgccct

[0074] The specific sequence of SEQ ID NO.12 is as follows:

[0075] MPEEGRPCPWVRWSGTAFQRQWPWLLLVVFITVFCCWFHCSGLLSKQQQRL

[0076] LEHPEPHTAELQLNLTVPRKDPTLRWGAGPALGRSFTHGPELEEGHLRIHQDGLYR

[0077] LHIQVTLANCSSPGSTLQHRATLAVGICSPAAHGISLLRGRFGQDCTVALQRLTYLV

[0078] HGDVLCTNLTLPLLPSRNADETFFGVQWICP

[0079] The present invention combines mRNA encoding 4-1BBL with mRNA encoding CD40L and mRNA encoding IL-36γ to achieve significant synergistic effects. In the present invention, the concentrations of mRNA encoding 4-1BBL, CD40L and IL-36γ proteins in the composition are equal.

[0080] The present invention also provides another composition in the form of mRNA, including three or four of mRNA encoding CD80, mRNA encoding CD86, mRNA encoding CD40L and mRNA encoding IL-36γ. In the present invention, the composition preferably includes mRNA encoding CD80, mRNA encoding CD40L and mRNA encoding IL-36γ; the composition preferably includes mRNA encoding CD86, mRNA encoding CD40L and mRNA encoding IL-36γ; the composition preferably includes mRNA encoding CD80, mRNA encoding CD86 and mRNA encoding CD40L; the composition preferably includes mRNA encoding CD80, mRNA encoding CD86 and mRNA encoding IL-36γ. The present invention combines mRNA encoding CD80, mRNA encoding CD86, mRNA encoding CD40L and mRNA encoding IL-36γ to have a certain synergistic effect. In the present invention, the concentrations of mRNA encoding CD80, CD86, CD40L and IL-36γ proteins in the composition are equal.

[0081] The present invention also provides another composition in the form of mRNA, including two or three of mRNA encoding CD70, mRNA encoding CD40L and mRNA encoding IL-36γ. In the present invention, the composition preferably includes mRNA encoding CD70 and mRNA encoding CD40L; the composition preferably includes mRNA encoding CD70 and mRNA encoding IL-36γ; the composition preferably includes mRNA encoding CD40L and mRNA encoding IL-36γ. The present invention combines mRNA encoding CD70 with mRNA encoding CD40L and mRNA encoding IL-36γ, which has a certain synergistic effect. In the present invention, the concentrations of mRNA encoding CD70, CD40L and IL-36γ proteins in the composition are equal.

[0082] The present invention also provides a method for preparing the cytokine mRNA, comprising the following steps:

[0083] (1) synthesizing and transcribing a DNA fragment of the mRNA of the active ingredient in the cytokine combination, and cloning the DNA fragment into an expression plasmid to obtain a recombinant plasmid;

[0084] (2) transferring the recombinant plasmid into host cells to obtain recombinant cells, extracting plasmids from the expanded recombinant cells, and performing PCR amplification using the extracted plasmids as templates to obtain DNA templates for in vitro expression of mRNA;

[0085] (3) constructing an RNA in vitro synthesis system including the DNA template to perform in vitro synthesis of mRNA to obtain the active ingredient mRNA.

[0086] Preferably, the RNA in vitro synthesis system comprises the following components in 1600 μl:

[0087] RNA-free water 440μL;

[0088] 7.5 mM ATP 160 μL;

[0089]

[0090] Preferably, the conditions for in vitro synthesis of the RNA are 36-38° C. for 8-12 hours.

[0091] On the other hand, the present invention also provides a lipid nanoparticle loaded with any one of the aforementioned mRNA molecules of the present invention.

[0092] The mRNA encapsulated in lipid nanoparticles is protected from enzymatic degradation during the delivery process and is efficiently delivered into cells, where it is released and translated into therapeutic protein.

[0093] The lipid nanoparticles can be lipid nanoparticles known in the art. For example, the lipid nanoparticles described in CN114380724A can be used.

[0094] According to a specific embodiment of the present invention, in the cytokine mRNA combination drug of the present invention, the lipid nanoparticles include mRNA and lipids, wherein the lipids include:

[0095] (1) one or more of positively charged lipids and / or ionizable lipids;

[0096] (2) neutral helper lipids;

[0097] (3) Cholesterol;

[0098] (4) PEG-modified lipids.

[0099] According to a specific embodiment of the present invention, in the cytokine mRNA combination drug of the present invention, the nitrogen-phosphorus molar ratio of the positively charged lipids and / or ionizable lipids to the mRNA is 5:1 to 20:1.

[0100] According to a specific embodiment of the present invention, in the cytokine mRNA combination drug of the present invention, in the lipid nanoparticles, based on the total molar amount of lipids as 100%, the molar ratio of each lipid component is:

[0101] Positively charged lipids or ionizable lipids 46% to 50%;

[0102] Neutral auxiliary lipids 5% to 10%;

[0103] Cholesterol 38.5%-48%;

[0104] PEG-modified lipids 0-3%.

[0105] According to a specific embodiment of the present invention, in the cytokine mRNA combination drug of the present invention, in the lipid nanoparticles, the ionizable lipids include but are not limited to: one or more of 4-(N,N-dimethylamino)butyric acid (diaoleyl) methyl ester ((Dlin-MC3-DMA), SM-102, ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), the positively charged lipids include but are not limited to: one or more of DOTMA and DOTAP; the neutral auxiliary lipids include but are not limited to: one or more of DSPC, DOPE, and DSPE; the PEG-modified lipids include but are not limited to: one or more of methoxypolyethylene glycol ditetradecyl acetamide (ALC-0159) and DMG-PEG.

[0106] The present invention also provides an application of a composition of an mRNA dosage form in the treatment of tumors. The present invention does not specifically limit the type of the tumor, and any common tumor type in the art can be used. In the specific implementation of the present invention, mouse colon cancer is taken as an example. In the specific implementation of the present invention, it is preferred that an mRNA dosage form encoding 4-1BBL, CD40L and IL-36γ is introduced into a tumor-bearing mouse as a composition, an mRNA dosage form encoding CD80, CD86, CD40L and IL-36γ is introduced into a tumor-bearing mouse as a composition, and an mRNA dosage form encoding CD70, CD40L and IL-36γ is introduced into a tumor-bearing mouse as a composition.

[0107] The present invention does not specifically limit the dosage form of the drug, and the conventional dosage form in the art can be used. In the specific implementation of the present invention, the drug is an injection preparation, and the injection preparation preferably uses physiological saline as a solvent, and the naked mRNA is directly dissolved in physiological saline, and more preferably the mRNA is encapsulated in LNP and then dissolved in physiological saline. In the specific implementation of the present invention, the drug is preferably introduced into the tumor-bearing mouse, and the introduction is preferably injection, and the injection site is preferably intratumoral injection. When the drug is a naked mRNA solution, the injection dose is preferably 80μL~120μL, more preferably 100μL, and the concentration of the injected mRNA composition is preferably 150μg / mL~250μg / mL, more preferably 200μg / mL; when the drug is a lipid nanoparticle solution, the injection dose is preferably 30μL~80μL, more preferably 50μL, and the concentration of the injected mRNA composition is preferably 100μg / mL~200μg / mL, more preferably 200μg / mL. The frequency of the injection is preferably three times a week.

[0108] In the present invention, the uracil in the composition of the mRNA dosage form is preferably replaced with pseudouracil. In the specific mRNA synthesis process of the present invention, replacing uracil with pseudouracil can further enhance the stability of mRNA in vivo, prolong its half-life, increase the expression rate of the target protein, and reduce the immunogenicity of mRNA.

[0109] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0110] Example 1: Synthesis of mRNA

[0111] The constructed expression plasmid was amplified with DNA template. The reaction system is as follows: 1μL of F primer, 1μL of R primer, 1μL of DNA template (concentration is generally 1ng / μL), 25μL of Prime STAR MAX high-fidelity PCR enzyme, and 50μL of enzyme-free water (this is one reaction system, and different number of reactions can be configured according to needs). After configuration, centrifuge and mix in a microcentrifuge, place in a gene amplifier for PCR reaction, and the general reaction procedure is as follows: pre-denaturation at 98℃ for 30s; denaturation at 98℃ for 10s, annealing at 54℃ for 20s, extension at 72℃ for 30s, 35 cycles; and final extension at 72℃ for 10min.

[0112] After the reaction is completed, the PCR product is mixed with DNA loading buffer and spotted on the gel well. After electrophoresis at 120V for 50 minutes, confirm whether the size of the target band is correct and whether there are any mixed bands. If it meets the standard, the target band gel is cut and the gel is recovered. The recovered sample is identified by agarose gel electrophoresis and its concentration is determined by Nanodrop.

[0113] The IVT system is configured in the medical clean bench. The IVT system is as follows:

[0114]

[0115] According to the experimental requirements, the reaction system can be scaled up in equal proportions. After preparation, mix with a microcentrifuge, then place in a thermostat, react at 37°C for 10 hours, add 120μL DNase I to each tube, mix upside down, react at 37°C for 1 hour, and then use for subsequent experiments. mRNA purification is performed using a kit. The kit purification refers to the Thermo fisher MEGAclear Transcription Clean-Up Kit instructions.

[0116] Example 2: Preparation of LNP-mRNA

[0117] The combined mRNA was dissolved in the aqueous phase, and the cations, DSPC, cholesterol, and PEG lipids (46.3:9.4:42.7:1.6, refer to patent document CN114380724A) were dissolved in the organic phase. The two-phase solution was injected into the two inlet channels of the preparation system. The synthesis of nucleic acid lipid nanoparticles was completed by rapid mixing of the two phases. The collected samples were stored in PBS, mixed upside down, and placed at 4°C. A small portion of the sample was taken to test the encapsulation efficiency, particle size, and concentration. These test indicators are routine experimental operations, and the results are shown in Table 1 below:

[0118] Table 1: LNP-mRNA index test results

[0119]

[0120] Example 3: Anti-tumor effect of cytokine mRNA composition in mouse colon cancer model First, a mouse colon cancer tumor model was established, and the specific steps were as follows:

[0121] 1. CT26.WT cell culture: CT26.WT (mouse colon cancer cells) were cultured in RPMI 1640 medium containing 10% FBS and subcultured 1 to 2 times a week at a subculture ratio of 1:3 or 1:6. During subculture, the medium was aspirated, the cell layer was rinsed with 2 ml of 0.25% trypsin, and then the trypsin was aspirated, the cells were digested in an incubator for 1 to 3 minutes, and fresh medium was added to resuspend the cells.

[0122] 2. Collection of CT26.WT cells: Digest the cells in logarithmic phase with trypsin, resuspend in complete medium and count.

[0123] 3. Cell inoculation: 6-8 week old female BALB / c mice (purchased from Beijing Weitong Lihua) were raised in a ventilated cage under SPF conditions and maintained with a 12 h light and 12 h dark cycle. The cell density was adjusted to 5×10 6 / mL, 100 μL was inoculated subcutaneously on the back of each mouse.

[0124] The tumor volume of mice was measured after 5 days, and the tumor volume of mice reached 20-30mm 3 When the mice were about 100 μg / mL, they were divided into groups according to the tumor volume, with 8 mice in each group, for a total of 4 groups. The first group was a normal saline group, the second group was a LNP-mRNA composition containing mRNA encoded by SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5, the third group was a LNP-mRNA composition containing mRNA encoded by SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.3 and SEQ ID NO.5, and the fourth group was a LNP-mRNA composition containing mRNA encoded by SEQ ID NO.11, SEQ ID NO.3 and SEQ ID NO.5. The above compositions were dissolved in normal saline to prepare injection preparations, and the total volume of the injection preparation was 50 μL, and each cytokine injected into each mouse was 2 μg. The drug was administered 3 times a week via intratumoral injection for a total of 2 weeks. The tumor volume and body weight of the mice were measured before each administration, and the tumor volume and body weight of the mice were measured regularly after the end of administration. As Figure 1 As shown in Table 2, the tumor volume of mice in the 4-1BBL+CD40L+IL-36γ cytokine mRNA combination group grew slowly over 15 days, reaching just 200 mm on day 15. 3 The tumor volume of the CD80+CD86+CD40L+IL-36γ group, the CD70+CD40L+IL-36γ group, and the saline group grew faster within 15 days, and the tumor volume even exceeded 1000mm on day 15. 3From the results, the combination of 4-1BBL+CD40L+IL-36γ significantly reduced the tumor volume of mice. 4-1BBL promotes the activation and migration of monocytes, stimulates T cell proliferation and induces effective anti-tumor immune response. CD40L activates T cells as a co-stimulatory signal molecule for T cell activation. IL-36γ can activate monocytes and macrophages, and can also activate T cells as a co-stimulatory molecule. The three cytokines enhance the body's immunity through the joint action of activating T cells, thereby achieving the purpose of reducing or even eliminating tumors. Figure 2 As shown in Table 3, there was no significant change in body weight of mice administered with the cytokine mRNA composition relative to that of mice administered with normal saline, indicating that the cytokine mRNA composition had no significant toxicity.

[0125] Table 2: Average tumor volumes of mice in different drug groups at different times (unit: mm 3 )

[0126]

[0127] Table 3: Average weight of mice in different drug groups at different times (unit: g)

[0128]

[0129] It can be seen from the above examples that the mRNA dosage form of the cytokine composition provided by the present invention has significant advantages over traditional protein and antibody drugs in terms of efficacy, biosafety, synthesis process, cost, etc.

[0130] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A cytokine mRNA combination drug, characterized in that: The combination drug comprises mRNA encoding 4-1BBL protein, mRNA encoding CD40L protein and mRNA encoding IL-36γ protein.

2. The cytokine mRNA combination drug according to claim 1, characterized in that: The mRNA encoding 4-1BBL, CD40L and IL-36γ proteins were at equal concentrations.

3. The cytokine mRNA combination drug according to claim 1, characterized in that: The mRNA encoding the 4-1BBL protein includes the nucleotide sequence shown in SEQ ID NO.1 or a nucleotide sequence having at least 80% homology thereto.

4. The cytokine mRNA combination drug according to claim 1, characterized in that: The mRNA encoding CD40L protein includes the nucleotide sequence shown in SEQ ID NO. 3 or a nucleotide sequence having at least 80% homology thereto.

5. The cytokine mRNA combination drug according to claim 1, characterized in that: The mRNA encoding the IL-36γ protein includes the nucleotide sequence shown in SEQ ID NO.5 or a nucleotide sequence having at least 80% homology thereto.

6. The cytokine mRNA combination drug according to claim 1, characterized in that: Each mRNA in the combination drug is encapsulated by lipid nanoparticles respectively.

7. The cytokine mRNA combination drug according to claim 1, characterized in that: The combined drug is a liquid preparation.

8. The cytokine mRNA combination drug according to claim 7, characterized in that: The solvent of the liquid preparation is physiological saline.

9. Use of the cytokine mRNA combination drug according to any one of claims 1 to 8 in the preparation of a drug for treating or preventing tumors.

10. The use according to claim 9, characterized in that: The tumor is melanoma, non-small cell lung cancer, gastric cancer, liver cancer, breast cancer or colorectal cancer, preferably colon cancer.

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