CXCL13 mRNA medicine and application thereof in tumor treatment

Abstract

The invention relates to a CXCL13mRNA drug and an application of the CXCL13mRNA drug in tumor treatment. The present invention provides a lipid particle comprising an ionizable lipid, a phospholipid, a cholesterol, a PEG-lipid, and a CXCL13 mRNA encapsulated within the lipid particle. The invention also provides a pharmaceutical composition comprising the lipid particle and application thereof. The medicine containing CXCL13mRNA provided by the invention is particularly suitable for treating tumors including lung cancer, colorectal cancer, breast cancer, liver cancer, pancreatic cancer, head and neck cancer, brain glioma, melanoma and the like.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals and tumor therapy, and particularly relates to mRNA drugs and their applications in tumor therapy. More specifically, this invention relates to drugs for tumor therapy and for increasing the sensitivity of tumor immunotherapy, belonging to the field of gene therapy. This invention relates to the preparation of CXCL13 mRNA encapsulated in lipid nanoparticles, and its usage. Background Technology

[0002] Chemokines CXCL13, also known as B lymphocyte chemokines, recruit specific cells through their interaction with their receptor CXCR5. In the germinal center region of secondary lymphoid organs, helper T lymphocytes (T follicular helpcells, Tfh) express CXCL13 to recruit B cells to form Tfh:B interactions, thereby promoting B cell maturation and differentiation into plasma cells that secrete high-affinity antibodies, thus promoting anti-tumor effects [1]. In addition, studies have shown that stem-like CD8 T cells expressing CXCR5, TCF7 and high levels of granzyme exist in the tumor microenvironment and can exhibit persistent anti-tumor effects through self-proliferation [2]. Furthermore, CXCL13 can serve as an important molecule for initiating and maintaining tertiary lymphoid structures (TLS). TLS, as ectopic lymphoid organs that appear near tumors, have strong anti-tumor potential and are often associated with good prognosis in clinical practice, and are often considered representative of "hot tumors" [3]. Therefore, enriching CXCL13 in the tumor microenvironment can exert anti-tumor effects by recruiting T cells and B cells and inducing the production of TLS, and can also increase the sensitivity to immune checkpoint inhibitor therapy.

[0003] Cancer treatment is currently one of the most closely watched areas in the biomedical field. Current cancer treatment methods include surgical resection, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. Among these, immunotherapy has seen rapid development in recent years, with immune checkpoint inhibitors, such as anti-PD-1 and anti-PD-L1 antibodies, achieving significant results and bringing hope for a cure for cancer. However, currently only a small percentage of patients can benefit from immune checkpoint inhibitor therapy, largely due to the low number of lymphocytes infiltrating the tumor. Therefore, it is of great significance to remodel the tumor microenvironment of "cold tumors" with low lymphocyte infiltration into "hot tumors" with high lymphocyte infiltration, thereby transforming more treatment-insensitive patients into treatment-sensitive patients.

[0004] mRNA therapy is an emerging treatment strategy. The main mechanism of action of mRNA therapy is to design and synthesize the mRNA of the target gene and deliver and release the mRNA to the tumor area through a specific delivery system [4]. mRNA therapy has many advantages. First, mRNA is relatively simple to design and synthesize, so it can be developed and put into use quickly. Second, mRNA is expressed only in the cytoplasm after being endocytosed by cells and does not enter the cell nucleus, and does not involve genome editing, so it has high safety. In addition, mRNA is easily completely degraded through the cellular physiological metabolic pathway, so it does not bring a continuous burden to the host. Finally, mRNA can theoretically encode and express various proteins and translate a large number of proteins in a short time, which reduces the amount of drugs used to a certain extent and also meets the requirements for the combined use of multiple types of mRNA [5].

[0005] Currently, mRNA therapy is mainly used for the prevention and treatment of viral infections and tumors, and several RNA vaccines have entered the clinical research stage, and some products have been officially put into clinical use, such as the mRNA vaccine against the novel coronavirus SARS-CoV-2 [6]. In tumor treatment, a relatively mature approach is to deliver mRNA encoding tumor-specific antigens to the tumor area or secondary lymphoid organs, so that dendritic cells (DCs) can phagocytose the antigens and present them to lymphocytes, especially T cells, thereby promoting lymphocyte activation and initiating the tumor-immune cycle to carry out the anti-tumor process [7]. It is worth noting that the effects of the above-mentioned immune checkpoint inhibitor therapy and mRNA vaccines depend on the infiltration of a sufficient number of lymphocytes inside the tumor, so their effects have certain limitations.

[0006] References:

[0007] [1].Cyster JG,Allen CDC.B Cell Responses:Cell Interaction Dynamicsand Decisions.Cell.2019Apr 18;177(3):524-540.doi:10.1016 / j.cell.2019.03.016.

[0008] [2].Jansen CS,Prokhnevska N,Master VAet.al.An intra-tumoral nichemaintains and differentiates stem-like CD8 T cells.Nature.2019Dec;576(7787):465-470.doi:10.1038 / s41586-019-1836-5.

[0009] [3]. Sautès-Fridman C, Petitprez F, Calderaro J et.al. Tertiary lymphoid structures in the era of cancer immunotherapy. Nat Rev Cancer. 2019 Jun;19(6):307-325. doi:10.1038 / s41568-019-0144-6. PMID:31092904.

[0010] [4]. Kon E, Ad-El N, Hazan-Halevy I et.al. Targeting cancer with mRNA-lipid nanoparticles: key considerations and future prospects. Nat Rev Clin Oncol. 2023 Nov;20(11):739-754. doi:10.1038 / s41571-023-00811-9.

[0011] [5]. Sahin U, Karikó K, Türeci mRNA-based therapeutics--developing a new class of drugs. Nat Rev Drug Discov. 2014 Oct;13(10):759-80. doi:10.1038 / nrd4278.

[0012] [6]. Fang E, Liu X, Li M, Zhang Z et.al. Advances in COVID-19 mRNA vaccine development. Signal Transduct Target Ther. 2022 Mar 23;7(1):94. doi:10.1038 / s41392-022-00950-y.

[0013] [7]. Liu C, Shi Q, Huang X, Koo S et.al. mRNA-based cancer therapeutics. Nat Rev Cancer. 2023 Aug;23(8):526-543. doi:10.1038 / s41568-023-00586-2. Summary of the Invention

[0014] One of the objectives of this invention is to address some of the pain points in cancer treatment by proposing therapeutic mRNA drugs that transform "cold tumors" into "hot tumors," increasing the sensitivity and efficacy of immune checkpoint inhibitor therapy. Currently, there are no such mRNA drugs or treatment methods.

[0015] The purpose of this invention is not only to provide a novel anti-tumor and immunotherapy sensitizing drug, but also to propose a new approach to tumor treatment, namely, to first increase the infiltration of lymphocytes inside the tumor, and then to treat with immune checkpoint inhibitors.

[0016] In some implementations, the invention described herein includes the following items.

[0017] 1. A lipid particle comprising ionizable lipids, phospholipids, cholesterol, PEG-lipids, and CXCL13 mRNA encapsulated within the lipid particle.

[0018] 2. The lipid particle according to Project 1, wherein 1) the CXCL13 mRNA includes an open reading frame encoding CXCL13, 2) the CXCL13 mRNA includes a 5' untranslated region, 3) the CXCL13 mRNA includes a 3' untranslated region, 4) the CXCL13 mRNA includes a 5' cap, and / or 5) the CXCL13 mRNA encodes the amino acid sequence of SEQ ID NO.1 or SEQ ID NO.2.

[0019] 3. The lipid particles according to item 1 or 2, wherein the ionizable lipids include one or more of DLinDMA, DLenDMA, DLin-K-C2-DMA, DLin-K-DMA, DLin-M-C2-DMA, DLin-M-C3-DMA, SM-102, L319, YSK12-C4, ALC-0315 and CL4H6, preferably including SM-102.

[0020] 4. The lipid particles according to any one of items 1-3, wherein the phospholipids include one or more of DOPE, DSPC, DUPC, DPPC, POPC, EPC, DOPC, DSPE, POPE, DOPS, DLPC, DMPC, DOPG, DPPG, phosphatidylethanolamine, DOTAP, and DGTS, preferably including DOPE.

[0021] 5. The lipid particles according to any one of items 1-4, wherein the PEG-lipids include one or more of DMG-PEG2000, PEG-DSPE, ALC-0519, PEG-DPPC, PEG-DLPE, PEG-DAG, PEG-DMPE, PEG-DAA, PEG-phospholipids, PEG-DMA and PEG-DSA, preferably including DMG-PEG2000.

[0022] 6. The lipid particles according to any one of items 1-5, having any one of the following characteristics: 1) the lipid particles have a lipid:mRNA mass ratio of 9:1 to 20:1; 2) the lipid particles are lipid nanoparticles; 3) the lipid particles have an average diameter of 30 nm to 150 nm; 4) the molar ratio of the ionizable lipids, phospholipids, cholesterol, and PEG-lipids is (30-65):(5-20):(20-50):(0.5-2); 5) The molar ratio of the ionizable lipids, phospholipids, cholesterol, and PEG-lipids is (40-60):(8-15):(30-45):(0.8-1.8), 6) the molar ratio of the ionizable lipids, phospholipids, cholesterol, and PEG-lipids is (46-55):(9-11):(35-42):(1.0-1.6), or 7) the molar ratio of the ionizable lipids, phospholipids, cholesterol, and PEG-lipids is 50:10:38.5:1.5.

[0023] 7. A pharmaceutical composition comprising any one of items 1-6 and a pharmaceutically acceptable carrier.

[0024] 8. Use of the lipid particles described in any one of items 1-6 or the pharmaceutical composition described in item 7 in the preparation of a medicament or kit for treating tumors.

[0025] 9. The lipid particles of any one of items 1-6, the pharmaceutical composition of item 7, or the use of item 8, having any one of the following characteristics: 1) the lipid particles are in a form suitable for systemic or local administration; 2) the lipid particles are in a form suitable for intratumoral administration; 3) the lipid particles are in a form suitable for intraperitoneal or nasal inhalation.

[0026] 10. The lipid particles described in any one of items 1-6, the pharmaceutical composition described in item 7, or the use described in item 8, having any one of the following characteristics: 1) the lipid particles are used in combination with other tumor therapeutic agents (e.g., tumor immunotherapy agents, immune checkpoint blockade agents, PD-1, PD-L1, CTLA4 inhibitors, especially PD-1, PD-L1 and / or CTLA4 antibodies); 2) the lipid particles are used to increase the sensitivity to other tumor therapies (e.g., tumor immunotherapy, immune checkpoint blockade therapy, PD-1, PD-L1, CTLA4 inhibitor therapy, especially PD-1, PD-L1 and / or CTLA4 antibody therapy); 3) the lipid particles are used to increase the infiltration of lymphocytes within the tumor; or 4) the lipid particles are used to treat tumors such as lung cancer, colorectal cancer, breast cancer, liver cancer, pancreatic cancer, head and neck cancer, glioma, and melanoma.

[0027] In this paper, there are no particular restrictions on CXCL13 mRNA, as long as it can encode the CXCL13 protein or its functional fragment and perform CXCL13 function. In some implementations, CXCL13 function may include one or more of the following activities: 1) binding to the CXCL13 receptor CXCR5 in target tissues (e.g., tumors), 2) recruiting B cells and / or T cells, 3) maintaining the tertiary lymphoid structure TLS, 4) transforming “cold tumors” with low lymphocyte infiltration into “hot tumors” with high lymphocyte infiltration, 5) transforming patients who are insensitive to tumor treatment into patients who are sensitive to treatment, and / or 6) promoting anti-tumor effects.

[0028] In some embodiments, the CXCL13 mRNA may include a sequence encoding an open reading frame of CXCL13. In some embodiments, the CXCL13 mRNA may also optionally include a 5' untranslated region, a 3' untranslated region, and / or a 5' cap. In some embodiments, the CXCL13 mRNA may include mRNA encoding the amino acid sequence of SEQ ID NO.1 or SEQ ID NO.2.

[0029] In this document, ionizable lipids may include lipids whose charge properties change depending on pH, such as lipids that are neutral in physiological pH but positively charged in acidic environments. In some embodiments, ionizable lipids may include any suitable pH-dependent lipids known in the art for nucleic acid delivery, particularly those suitable for efficient delivery of nucleic acids in human tissues. In some embodiments, lipid particles may have a pKa of 6.0 to 7.0, for example 6.5 to 7.0, to exhibit a positive charge under acidic pH conditions, thereby enabling them to interact with negatively charged nucleic acids to form complexes. In some embodiments, ionizable lipids may optionally include a linker and a tail, wherein the tail may include a saturated / unsaturated fatty acid chain of 8 to 20 carbon atoms, which can modulate the delivery and release of nucleic acids. In some embodiments, examples of the ionizable lipids of the present invention include one or more of, for example, DLinDMA, DLenDMA, DLin-K-C2-DMA, DLin-K-DMA, DLin-M-C2-DMA, DLin-M-C3-DMA, SM-102, L319, YSK12-C4, ALC-0315, and CL4H6. In some embodiments, the present invention has found that the ionizable lipid SM-102 exhibits a significant and excellent effect in promoting CXCL13 mRNA delivery and expression, and is therefore particularly preferred. In some embodiments, the molar ratio of the ionizable lipid such as SM-102 in the lipid can be 20% to 80%, for example, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or any range therebetween, particularly preferably 50%.

[0030] In this paper, cholesterol is used as a structural lipid for lipid particles, which can improve the stability of nanoparticles. In some embodiments, cholesterol may include derivatives with similar structural properties, examples of which include, for example, cholesterol-(4'-hydroxy)-butyl ether, 5-α-cholesterol, 5β-coagulol, 5α-cholesterol, cholesterolenone, 5α-cholesterol, etc.

[0031] In this paper, phospholipids are capable of forming a lipid bilayer and binding to the phospholipid bilayer of target cells to promote the delivery and release of nucleic acids in target tissues. In some embodiments, phospholipids may include any lipid suitable for promoting lipid particle fusion, including one or more of, for example, DOPE, DSPC, DUPC, DPPC, POPC, EPC, DOPC, DSPE, POPE, DOPS, DLPC, DMPC, DOPG, DPPG, phosphatidylethanolamine, DOTAP, and DGTS. In some embodiments, the present invention has found that DOPE interacts with CXCL13 mRNA and exhibits significantly greater promoting activity compared to other commonly used phospholipids such as DSPC, and is therefore particularly preferred. In some embodiments, the present invention has surprisingly found that including phospholipids such as DOPE can promote the specific high expression of CXCL13 mRNA in target tissues (such as tumors), thus making it particularly suitable for delivering and expressing CXCL13 mRNA to tumor tissues to exert antitumor activity. In some embodiments, the present invention, by including phospholipids such as DOPE, can increase CXCL13 mRNA expression activity by 2, 3, 4, 5, 10, or more times compared to a control (e.g., other phospholipids such as DSPC). In some embodiments, the present invention, by including phospholipids such as DOPE, can specifically increase CXCL13 mRNA expression activity by 2, 3, 4, 5, 10, 20, 50, 100, 1000, or more times in tumor tissues (e.g., compared to control tissues such as normal tissues, non-tumor tissues, and non-target tissues) compared to a control (e.g., other phospholipids such as DSPC).

[0032] In this document, PEG-lipids include conjugates of lipids and PEG, wherein PEG can extend half-life, prevent lipid particle aggregation, and act as a functional molecule to couple lipid particles to other active moieties. In some embodiments, PEG-lipids may include one or more of DMG-PEG2000, PEG-DSPE, ALC-0519, PEG-DPPC, PEG-DLPE, PEG-DAG, PEG-DMPE, PEG-DAA, PEG-phospholipids, PEG-DMA, and PEG-DSA. In some embodiments, the present invention has found that the ionizable lipid DMG-PEG2000 exhibits a significant and excellent effect in promoting CXCL13 mRNA delivery and expression, and is therefore particularly preferred. In some embodiments, the present invention, by including PEG-lipids such as DMG-PEG2000, can enhance CXCL13 mRNA expression activity compared to controls (e.g., other PEG-lipids such as DSPE-PEG).

[0033] In some embodiments, the lipid particles described herein can be prepared by any suitable method known in the art. In some embodiments, the lipid particles described herein may have a lipid:mRNA mass ratio of 9:1 to 20:1, including, for example, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or any range therebetween. In some embodiments, the lipid particles described herein may be lipid nanoparticles. In some embodiments, the lipid particles described herein may have an average diameter of 30 nm to 150 nm, including, for example, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, or any range therebetween. In some embodiments, the molar ratio of ionizable lipids, phospholipids, cholesterol, and PEG-lipids can be any suitable range, for example (30-65):(5-20):(20-50):(0.5-2), such as (40-60):(8-15):(30-45):(0.8-1.8), (46-55):(9-11):(35-42):(1 0-1.6), for example (46, 47, 48, 49, 50, 51, 52, 53, 54, 55): (9, 10, 11): (35, 36, 37, 38, 39, 40, 41, 42): (1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6), or any range therein, for example, the molar ratio of the ionizable lipids, phospholipids, cholesterol, PEG-lipids can be 50:10:38.5:1.5.

[0034] In some embodiments, the present invention provides a composition, such as a pharmaceutical composition, which may include the lipid particles containing mRNA as described herein. In some embodiments, the pharmaceutical composition may further include pharmaceutically acceptable carriers, excipients, and / or other pharmaceutical additives, such as saline, sterile water, buffered saline, glucose solution, glycerol, ethanol, lactose, sucrose, gelatin, starch, buffers, preservatives, diluents, dispersants, surfactants, etc. In some embodiments, the present invention provides a kit that may include the lipid particles described herein.

[0035] In some embodiments, the present invention provides the use of lipid particles and pharmaceutical compositions in the preparation of medicaments or kits for treating diseases such as tumors. In some embodiments, the lipid particles or medicaments may be in forms suitable for systemic administration, local administration, intratumoral administration, intraperitoneal administration, or nasal inhalation. In some embodiments, the lipid particles or medicaments may be used in combination with other tumor therapeutic agents (e.g., tumor immunotherapy agents, immune checkpoint blockade agents, PD-1, PD-L1, CTLA4 inhibitors, especially PD-1, PD-L1, and / or CTLA4 antibodies). In some embodiments, the lipid particles or medicaments may be used to increase the sensitivity to other tumor therapies (e.g., tumor immunotherapy, immune checkpoint blockade therapy, PD-1, PD-L1, CTLA4 inhibitor therapy, especially PD-1, PD-L1, and / or CTLA4 antibody therapy). In some embodiments, the lipid particles or medicaments may be used to increase the infiltration of lymphocytes within the tumor. In some embodiments, the lipid particles or medicaments may be used to treat any suitable tumor. In some embodiments, the lipid particles or medicaments may be used to treat CXCL13-related tumors. In some implementations, the lipid particles or drugs can be used to treat tumors expressing the receptor CXCR5 of CXCL13. In some implementations, the lipid particles or drugs can be used to treat lung cancer, colorectal cancer, breast cancer, liver cancer, pancreatic cancer, head and neck cancer, glioma, and melanoma, among others.

[0036] In some embodiments, the compositions of the present invention may include CXCL13 mRNA and LNP. In some embodiments, the CXCL13 mRNA may contain an open reading frame encoding CXCL13 for translation, and optionally has a 5' untranslated region and a 3' untranslated region upstream and downstream. In some embodiments, the translated amino acid sequence of CXCL13 is SEQ ID NO.1 and SEQ ID NO.2. In some embodiments, the components of the LNP may include, but are not limited to, SM102, DSPC, cholesterol, and DMG-PEG2000. In some embodiments, the administration route may include intraperitoneal administration and nasal inhalation. In some embodiments, this document provides combined immunotherapy methods comprising administration of CXCL13 mRNA and additional therapies, including but not limited to anti-PD-1, anti-PD-L1, and anti-CTLA4. In some embodiments, the amino acid sequence encoding CXCL13 can be SEQ ID NO.1 (MKFISTSLLLMLLVSSLSPVQGVLEVYYTSLRCRCVQESSVFIPRRFIDR IQILPRGNGCPRKEIIVWKKNKSIVCVDPQAEWIQRMMEVLRKRSSSTL PVPVFKRKIP, species: human) and SEQ ID NO.2 (MRLSTATLLLLLASCLSPGHGILEAHYTNLKCRCSGVISTVVGLNIIDRI QVTPPGNGCPKTEVVIWTKMKKVICVNPRAKWLQRLLRHVVQSKSLSS TPQAPVSKRRAA, species: mouse).

[0037] In some embodiments, the purified mRNA may be capped. In some embodiments, the LNP may include SM-102 mixed with 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), cholesterol, and DMG PEG2000 in a molar ratio of 50:10:38.5:1.5, dissolved in ethanol to prepare an ethanol lipid solution. In some embodiments, the mRNA may be diluted with 10 mM acetic acid at pH 5.2 to obtain an acidic dilute mRNA solution; subsequently, the ethanol lipid solution and the mRNA aqueous solution are vortexed at a volume ratio of 1:3 to prepare lipid nanoparticles, diluted with ten times the volume of PBS, centrifuged through a 100 kDa ultrafiltration centrifuge tube, and washed three times with PBS until the formulation is neutral; the lipid nanoparticle solution is then sterilely filtered (0.22 μm) to obtain a lipid nanoparticle formulation encapsulating mRNA.

[0038] In some embodiments, the treatment methods and / or uses provided by the present invention may include monotherapy with mRNA drugs or combination therapy with at least one immunotherapy (e.g., anti-PD-1, anti-PD-L1, anti-CTLA4). In some embodiments, the present invention can be used to treat tumors such as lung cancer, colorectal cancer, breast cancer, liver cancer, pancreatic cancer, head and neck cancer, glioma, and melanoma. Attached Figure Description

[0039] Figure 1 This section describes the expression level and intraperitoneal delivery targeting of lipid nanoparticles containing CXCL13 mRNA. In the figures, A is a schematic diagram of CXCL13 mRNA LNP; B shows the CXCL13 protein expression level in cells treated with CXCL13 mRNA LNP; C shows the CXCL13 protein secretion level in cells treated with CXCL13 mRNA LNP; D shows the targeting of the LNP intraperitoneal delivery system, with fluorescein as the detection signal; and E shows the proportion of tumor and organ-targeted distribution after LNP delivery.

[0040] Figure 2 The efficacy and side effects of CXCL13 mRNA in treating mouse tumors were evaluated. Figures AC represent the efficacy in treating melanoma, DF in treating lung tumors, and GI in treating breast cancer tumors. Figure J shows H&E staining of the heart, kidney, liver, lung, and spleen of mice after treatment; no side effects were observed. The scale bar in the figures is 5 μm.

[0041] Figure 3 This study illustrates the effect of persistent CXCL13 mRNA treatment on the survival of tumor-bearing mice and its efficacy in combination with immunotherapy. Figures AD show the prolongation of survival and induction of TLS in melanoma-bearing mice by persistent CXCL13 mRNA treatment. In Figure C, the blue layer represents DAPI, the green layer represents B220, and the red layer represents Cd3 in the immunofluorescence image. Figure D shows the statistical analysis of TLS. Figures EF show the effect of CXCL13 mRNA combined with immunotherapy on the survival rate of tumor-bearing mice, demonstrating that CXCL13 mRNA treatment increases the sensitivity to anti-PD1 therapy. The top left of the immunofluorescence image shows DAPI + Cd3 + B220 co-display; the top right shows Cd3 + B220 co-display; the bottom left shows Cd3; and the bottom right shows B220. The scale bar in the figures is 10 μm.

[0042] Figure 4Lung delivery of CXCL13 mRNA induces TLS. Figure A shows the targeting of lung delivery. Figure B is a schematic diagram of lung tumor treatment, and Figures C and D show the induction of lung TLS, where blue represents DAPI, green represents B220, and red represents Cd3. Figure F shows the statistical results of lung TLS, indicating that CXCL13 mRNA can induce lung TLS, suggesting its potential use in lung tumor treatment. The immunofluorescence images show DAPI+Cd3+B220 co-presented in the upper left; Cd3+B220 co-presented in the upper right; Cd3 represented in the lower left; and B220 represented in the lower right. The scale bar in the figures is 10 μm.

[0043] Figure 5 The effects of SM102 were shown, with 20%–80% of cases showing significant effects compared to no SM102 use (p<0.0001).

[0044] Figure 6 The effect of PEG is shown, with a significant effect compared to no PEG used (p < 0.0001).

[0045] Figure 7 The effects of DOPE and DSPC are shown, with DOPE showing a significant effect compared to DSPC (p < 0.0001).

[0046] Figure 8 The effects of DOPE and DSPC are shown, with DOPE demonstrating a significant improvement over DSPC.

[0047] Figure 9 The study showed the effects of DOPE and DSPC, with DOPE showing a stronger tendency to be delivered to tumors compared to DSPC, while DSPC showed stronger enrichment in the spleen compared to DOPE. Detailed Implementation

[0048] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the invention in any way. Any simple modifications, equivalent changes, and alterations made to the embodiments based on the technical essence of the present invention without departing from the spirit and essence of the invention are within the scope of the present invention.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available. The LNP composition SM-102 was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), cholesterol, and DMG PEG2000 were purchased from MedChemExpress. The plasmid was purchased from CellRay Biotech Co., Ltd., and the transcriptase was purchased from Thermo Fisher Scientific. The mouse melanoma cell line B16-F1 and the mouse breast cancer cell line 4T1 were purchased from Wuhan Pronosei Biotechnology Co., Ltd., and the mouse lung tumor cell line TC-1 was purchased from Shanghai Kanglang Biotechnology Co., Ltd. C57 / B6J and Balb / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and anti-PD-1 and anti-IgG were purchased from BioXCell.

[0050] Example 1: Synthesis and Detection of CXCL13-mRNA Lipid Nanoparticles

[0051] 1. mRNA preparation

[0052] 1.1. Plasmid linearization

[0053] The target plasmid was linearized using the restriction endonuclease AflII, and the reaction system is shown in Table 1 below:

[0054] Table 1. Enzyme digestion system for plasmid linearization

[0055] Components volume Plasmid DNA (50 ng) xμL 10×NEBuffer 50μL Restriction Enzyme 25μL Nuclease-free Water Up to 500μL

[0056] After gentle mixing, the mixture was incubated at 37°C for 4 hours. Then, it was incubated at 65°C for 10 minutes to inactivate the endonuclease. The linearized plasmid was recovered using a large-volume DNA product purification kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.), and the plasmid concentration was measured using an ultra-micro UV-Vis spectrophotometer. The plasmid was stored at -20°C for later use.

[0057] 1.2. Linearized plasmid in vitro transcription system

[0058] The T7 High Yield RNA Synthesis Kit (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.) was used for mRNA transcription. mRNA was transcribed according to the product instructions. All components except the T7 RNA Polymerase Mix were vortexed and collected briefly by centrifugation at the bottom of the tube, then stored on ice for later use. The following components were added sequentially (refer to Table 2 for details), and each component was gently mixed with a pipette and collected briefly by centrifugation. The mixture was incubated at 37°C for 4 hours. 2 μL of DNase I was added to the reaction system, and the mixture was incubated at 37°C for 15 minutes to digest the transcribed DNA template.

[0059] Table 2. In vitro transcription system of linearized plasmids

[0060]

[0061]

[0062] 1.3. Purification of mRNA

[0063] Using Dynabeads for RNA purification TM The carboxylated magnetic bead kit (purchased from Thermo Fisher Scientific) was used to purify mRNA. The specific purification steps are as follows: Prepare a 4 μg / μL working solution from the above transcription system, and add an equal volume of Dynabeads. TM RNA Binding Buffer, add 30 μL of Dynabeads to 100 μL of working solution. TM The proportion of Carboxylic Acid for RNA Purification was scaled up proportionally to the purification system. The mixture was pipetted until the magnetic beads were fully resuspended. Incubation was performed at 37°C and 1000 RPM for 10 min. The tube was placed on a magnetic rack until the solution became clear, and the supernatant was discarded. The beaded complex was resuspended in 70% ethanol and placed on a magnetic rack until the solution became clear, and the supernatant was discarded. Washing was repeated three times to remove all residual washing solution, and the tube was dried at room temperature for 10 min. The tube was removed from the magnet, diluted with an appropriate amount of RNase-free ddH2O to the desired concentration, and incubated at 37°C and 1000 RPM for 5 min. The tube was then placed on the magnet, and the supernatant was transferred to a new RNA-free tube to measure the RNA concentration.

[0064] 1.4. Capping reaction of mRNA

[0065] The purified RNA was capped using a capping kit (Vaccinia Capping Enzyme, S-adenosylmethionine (SAM) GMP-grade (32mM)), purchased from Yisheng Biotechnology (Shanghai) Co., Ltd. The specific steps are as follows: The efficiency of the capping reaction is affected by the structure of the RNA 5' end; therefore, rapid cooling with heat denaturation (heating at 65℃ for 10 min, then placing on ice for 5 min) was used to open the higher-order structure of the RNA 5' end. The following components were added sequentially, as detailed in Table 3. Reaction conditions: 37℃ for 90 min.

[0066] Table 3. Capped Reaction System

[0067]

[0068]

[0069] 1.5. Purification of mRNA

[0070] Using Dynabeads for RNA purification TM The carboxylated magnetic bead kit (purchased from Thermo Fisher Scientific) was used to purify mRNA. The specific purification steps were the same as in step 3. The mRNA concentration was measured using a spectrophotometer, and the mRNA was aliquoted and stored at -80°C for later use.

[0071] 2. Preparation of lipid nanoparticle formulations

[0072] 2.1. An ethanol lipid solution was prepared by dissolving the ionizable lipid compound SM-102 in ethanol with 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), cholesterol, and DMG-PEG2000 in a molar ratio of 50:10:38.5:1.5.

[0073] 2.2. Dilute the mRNA with 10mM acetic acid at pH 5.2 to obtain an acidic dilute mRNA solution.

[0074] 2.3. Lipid nanoparticles were prepared by vortexing an ethanol lipid solution and an mRNA aqueous solution at a volume ratio of 1:3. The mixture was then diluted with 10 times the volume of PBS, centrifuged using a 100 kDa ultrafiltration centrifuge tube, and washed three times with PBS until the formulation was neutral. Finally, the lipid nanoparticle solution was sterilely filtered (0.22 μm) to obtain the lipid nanoparticle formulation encapsulating mRNA.

[0075] 3. Expression level detection

[0076] B16-F1 cells were seeded into 12-well plates and treated with PBS, empty LNP, GFP mRNA lipid nanoparticles (containing 1 μg mRNA), and CXCL13 mRNA lipid nanoparticles (containing 1 μg mRNA) in 1 μL volumes. After incubation for 24 hours, the supernatant was collected, and the expression level of CXCL13 was detected by ELISA. Subsequently, the cells were collected, lysed, and the relevant proteins were detected by Western blot.

[0077] The results showed that CXCL13-mRNA lipid nanoparticles could successfully express CXCL13 protein and be secreted from cells.

[0078] Conclusion: CXCL13-mRNA lipid nanoparticles can be used to deliver and express CXCL13 protein.

[0079] Example 2: CXCL13-mRNA lipid nanoparticles for the treatment of B16-F1 abdominal tumors

[0080] Six-week-old C57 / B6J mice were divided into four groups of 4–10 mice each, and were injected intraperitoneally with 2 × 10⁻⁶ mg / L of iodine solution. 6 A peritoneal tumor model was established by injecting 100 μL of a B16-F1, TC1, and 4T cell suspension at a concentration of [cells / mL]. Starting from the third day of tumor bearing, mice were treated with intraperitoneal injection of CXCL13-mRNA LNP, with treatment repeated every other day for a total of three treatments. The injection methods were as follows: Group 1 received 100 μL of empty LNP; Group 2 received 100 μL of LNP containing 20 μg of CXCL13-mRNA. Mice were sacrificed on day 10, and tumors were collected, photographed, and weighed. Tumor and tumor-bearing mice were treated in the same manner, and mouse survival time was observed to evaluate the treatment effect.

[0081] The results showed that the tumors in the CXCL13-mRNA treatment group were significantly smaller than those in the empty LNP group. Tumor weighing revealed that the tumors in the CXCL13-mRNA treatment group were significantly smaller than those in the empty LNP group, indicating a certain therapeutic effect. Furthermore, immunofluorescence staining of tissue sections revealed not only abundant infiltration of T cells and B cells in the CXCL13 treatment group, but also the induction of intratumoral TLS. In addition, continuous CXCL13-mRNA LNP treatment significantly prolonged the survival of mice compared to empty LNP, indicating that CXCL13-mRNA has a significant anti-tumor effect.

[0082] Conclusion: CXCL13-mRNALNP has specific anti-tumor function, and its mechanism of action is mainly to recruit T cells and B cells to induce tumors to present an immune infiltration phenotype and form TLS to carry out anti-tumor effects, thereby prolonging the survival time of mice.

[0083] Example 3: Side effect assessment of CXCL13-mRNA lipid nanoparticles

[0084] Organ toxicity is a common side effect of therapeutic mRNA LNP drugs. To address this, we assessed the side effects of mRNA LNP drugs by evaluating changes in the physiological / pathological structure of the treated organ. PBS was used as a normal control in this section.

[0085] The results showed that H&E staining revealed no difference in the structure of the heart, liver, lungs, kidneys, and spleen in mice compared to the PBS group in the empty LNP group and the CXCL13 mRNA LNP group, indicating that there were no obvious side effects.

[0086] Conclusion: CXCL13-mRNA lipid nanoparticle therapy has no significant side effects.

[0087] Example 4: CXCL13-mRNA lipid nanoparticles enhance anti-PD-1 therapeutic effects

[0088] The combined therapy of CXCL13-mRNA LNP and anti-PD-1 was mainly used in the abdominal tumor model.

[0089] Six-week-old C57 / B6J mice were divided into four groups of five mice each, and were injected intraperitoneally with 2×10⁻⁶ mg / L of the solution. 6 A B16-F1 cell suspension at a concentration of [cells / mL] was injected in 100 μL to establish a peritoneal tumor model. Starting from the third day after tumor bearing, 20 μg of CXCL13-mRNA LNP was injected intraperitoneally every two days for a total of three injections. The following Monday, 200 μg of anti-PD-1 was injected intraperitoneally every two days for a total of three injections. The injection methods for each group were as follows: Group 1 received empty LNP and anti-Isotype; Group 2 received empty LNP and anti-PD-1; Group 3 received CXCL13-mRNA LNP and anti-Isotype; and Group 4 received CXCL13-mRNA LNP and anti-PD-1. Mice survival data were then collected.

[0090] The results showed that CXCL13-mRNALNP monotherapy had a certain anti-tumor effect. Anti-PD-1 monotherapy did not show any therapeutic effect, while the combination therapy with CXCL13-mRNALNP significantly enhanced the anti-PD-1 therapeutic effect.

[0091] Conclusion: CXCL13 mRNA treatment can significantly increase the sensitivity to anti-PD-1 therapy.

[0092] Example 5: CXCL13-mRNA lipid nanoparticles for the treatment of B16-F1 lung tumors

[0093] Six-week-old C57 / B6J mice were divided into three groups of five mice each, and injected with 2×10⁻⁶ mice via the tail vein. 6 A lung tumor model was constructed by injecting 100 μL of B16-F1 cell suspension at a concentration of [cells / mL]. Subsequently, starting on day 3 of tumor bearing, mice were treated for three weeks with tail vein injection of CXCL13-mRNA LNP, administered three times per week. The injection regimens were as follows: Group 1 received 100 μL of PBS; Group 2 received 100 μL of empty LNP; and Group 3 received 100 μL of LNP containing 20 μg of CXCL13-mRNA. Mice were then sacrificed on day 24, and lungs were collected for photographing and immunohistochemical staining.

[0094] The results showed that immunofluorescence staining of tissue sections revealed a large number of T cells and B cells infiltrating the lung tumors in the CXCL13 treatment group, and TLS structures were observed.

[0095] Conclusion: Lung delivery of CXCL13 mRNA can induce TLS within lung tumors, suggesting an increased sensitivity to lung tumor immunotherapy.

[0096] Example 6: Role of different components in CXCL13-mRNA lipid nanoparticles

[0097] This invention conducted in vivo / in vitro experiments on the lipid nanoparticle components to verify the necessity of each component, including the proportion of SM102, PEG screening, and phospholipid excipient screening. Experimental materials included C57BL / 6J mice, B16-F1 melanoma cell line, HeLa cell line, SM102, DOPE, DSPC, DMG-PEG, DSPE-PEG, cholesterol, ethanol, 10 mM acetic acid, PBS, luciferase substrate, DMEM medium, and serum-free medium. All relevant components were prepared according to the methods described in the preceding embodiments.

[0098] Specifically as follows:

[0099] 1. SM102 percentage screening:

[0100] 1.1. HeLa cells were aspirated into 96-well plates for overnight culture;

[0101] 1.2. Prepare a series of LNP solutions with different SM102 molar concentrations, while maintaining a consistent ratio of DOPE, DMG-PEG, and cholesterol. The LNP system ratios are shown in Table 4.

[0102] Table 4. Proportion of LNP system in SM102 screening

[0103]

[0104] In Table 4, SM102 concentration is 10 mg / mL, MIX is 10 mg / mL DOPE, DMG-PEG, and cholesterol solution are mixed in a volume ratio of 2:1:4, and all are ethanol phase solutions.

[0105] 1.3. The above solutions were mixed with 100 μg / mL luciferase mRNA (diluted with 10 mM acetic acid) at a volume ratio of 1:3 by shaking, and then diluted with 10 times the volume of PBS.

[0106] 1.4. Remove DMEM medium from HeLa cells, add 200 μL of serum-free medium and LNP preparation containing 250 ng luciferase mRNA to each well, and incubate overnight;

[0107] 1.5. Remove serum-free medium and add 100 μL of DMEM medium and 100 μL of luciferase substrate to each well;

[0108] 1.6. Transfer to a 96-well plate for microplate reader and measure the bioluminescence value on the microplate reader.

[0109] 2. PEG screening:

[0110] 2.1. The steps for mRNA loading, cell culture, and bioluminescence detection are as described in SM102.

[0111] 2.2. The proportions of the LNP system are shown in Table 5:

[0112] Table 5. Proportion of LNP system in PEG screening

[0113] SM102 DOPE cholesterol DMG-PEG DSPE-PEG DMG-PEG group 10 2 4 1 0 DSPE-PEG group 10 2 4 0 1 Non-PEG group 10 2 4 0 0

[0114] 2.3. Subsequently, the bioluminescence value was detected using the same method as the SM102 detection described above.

[0115] 3. Screening of phospholipid excipients (in vitro experiments):

[0116] 3.1. The steps for mRNA loading, cell culture, and bioluminescence detection are as described in SM102.

[0117] 3.2. The proportions of the LNP system are shown in Table 6:

[0118] Table 6. Proportion of LNP in the screening of phospholipid excipients

[0119] SM102 DMG-PEG cholesterol DOPE DSPC DOPE group 10 1 4 2 0 DSPC Group 10 1 4 0 2

[0120] All solutions in the table have a concentration of 10 mg / mL and are ethanol phase solutions.

[0121] 3.3. Subsequently, the bioluminescence value was detected using the same method as the SM102 detection described above.

[0122] 4. Screening of phospholipid excipients (in vivo experiments):

[0123] 4.1. 200,000 B16-F1 cells were implanted into the peritoneal cavity of 6-week-old C57BL / 6J mice.

[0124] 4.2. On day 14 of tumor bearing, mice were intraperitoneally injected with 10 μg of lipid nanoparticles composed of DOPE or DSPC encapsulating luciferase mRNA. Six hours after injection, the mice were dissected, and the tumor, liver, spleen, and lungs were removed for imaging. (The preparation method of the lipid nanoparticles is the same as described above.)

[0125] 4.3. Perform quantitative analysis on the imaging signal.

[0126] All statistical tests in this part of the study were t-tests, with p<0.05 as the statistical significance threshold.

[0127] The results showed that a 20%-80% SM102 ratio was significantly effective in screening, with the highest luciferase expression value observed at a 50% SM102 ratio. The highest luciferase expression values ​​were observed when DMG-PEG and DOPE phospholipid excipients were used. Furthermore, DOPE exhibited higher tumor targeting than DSPC.

[0128] Conclusion: The inclusion of SM102, DMG-PEG and phospholipid excipient DOPE in lipid nanoparticles is particularly beneficial for effectively improving mRNA expression, and can prepare excellent CXCL13-mRNA delivery lipid nanoparticles.

[0129] In summary, the therapeutic CXCL13-mRNALNP of this invention exhibits significant anti-tumor effects, whether used as monotherapy or in combination with immune checkpoint inhibitors. CXCL13-mRNALNP promotes the infiltration of T cells and B cells within tumors and induces the production of intratumoral TLS, transforming "cold tumors" into "hot tumors," effectively treating tumors and possessing the potential to significantly increase the number of patients benefiting from immunotherapy. This invention can be delivered via various methods, including intraperitoneal injection, intravenous injection, and nasal inhalation, to treat a variety of cancers.

[0130] The above embodiments merely illustrate several implementation methods, effects, and safety aspects of the present invention, and while the descriptions are relatively detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A lipid particle comprising ionizable lipids, phospholipids, cholesterol, PEG-lipids, and CXCL13 mRNA encapsulated within the lipid particle.

2. The lipid particles according to claim 1, wherein, 1) The CXCL13 mRNA includes an open reading frame encoding CXCL13; 2) The CXCL13 mRNA includes a 5' untranslated region; 3) The CXCL13 mRNA includes a 3' untranslated region; 4) The CXCL13 mRNA includes a 5' cap; and / or 5) The CXCL13 mRNA encodes the amino acid sequence of SEQ ID NO.1 or SEQ ID NO.

2.

3. The lipid particles according to claim 1 or 2, wherein the ionizable lipids include one or more of DLinDMA, DLenDMA, DLin-K-C2-DMA, DLin-K-DMA, DLin-M-C2-DMA, DLin-M-C3-DMA, SM-102, L319, YSK12-C4, ALC-0315 and CL4H6, preferably including SM-102.

4. The lipid particles according to any one of claims 1-3, wherein the phospholipids include one or more of DOPE, DSPC, DUPC, DPPC, POPC, EPC, DOPC, DSPE, POPE, DOPS, DLPC, DMPC, DOPG, DPPG, phosphatidylethanolamine, DOTAP, and DGTS, preferably including DOPE.

5. The lipid particles according to any one of claims 1-4, wherein the PEG-lipids include one or more of DMG-PEG2000, PEG-DSPE, ALC-0519, PEG-DPPC, PEG-DLPE, PEG-DAG, PEG-DMPE, PEG-DAA, PEG-phospholipids, PEG-DMA and PEG-DSA, preferably including DMG-PEG2000.

6. The lipid particle according to any one of claims 1-5, characterized by any one of the following: 1) the lipid particle has a lipid:mRNA mass ratio of 9:1 to 20:1; 2) the lipid particle is a lipid nanoparticle; 3) the lipid particle has an average diameter of 30 nm to 150 nm; 4) the molar ratio of the ionizable lipid, phospholipid, cholesterol, and PEG-lipid is (30-65):(5-20):(20-50):(0.5-2). 5) The molar ratio of the ionizable lipids, phospholipids, cholesterol, and PEG-lipids is (40-60):(8-15):(30-45):(0.8-1.8), 6) The molar ratio of the ionizable lipids, phospholipids, cholesterol, and PEG-lipids is (46-55):(9-11):(35-42):(1.0-1.6), or 7) The molar ratio of the ionizable lipids, phospholipids, cholesterol, and PEG-lipids is 50:10:38.5:1.

5.

7. A pharmaceutical composition comprising lipid particles according to any one of claims 1-6 and a pharmaceutically acceptable carrier.

8. Use of the lipid particles of any one of claims 1-6 or the pharmaceutical composition of claim 7 in the preparation of a medicament or kit for treating tumors.

9. The lipid particles of any one of claims 1-6, the pharmaceutical composition of claim 7, or the use of claim 8, wherein the lipid particles are characterized by any one of the following: 1) the lipid particles are in a form suitable for systemic or local administration; 2) the lipid particles are in a form suitable for intratumoral administration; or 3) the lipid particles are in a form suitable for intraperitoneal or nasal administration.

10. The lipid particles of any one of claims 1-6, the pharmaceutical composition of claim 7, or the use of claim 8, characterized by any one of the following: 1) the lipid particles are used in combination with other tumor therapeutic agents (e.g., tumor immunotherapy agents, immune checkpoint blockade agents, PD-1, PD-L1, CTLA4 inhibitors, especially PD-1, PD-L1, and / or CTLA4 antibodies); 2) the lipid particles are used to increase the sensitivity to other tumor therapies (e.g., tumor immunotherapy, immune checkpoint blockade therapy, PD-1, PD-L1, CTLA4 inhibitor therapy, especially PD-1, PD-L1, and / or CTLA4 antibody therapy); 3) the lipid particles are used to increase the infiltration of lymphocytes within the tumor; or 4) the lipid particles are used to treat tumors such as lung cancer, colorectal cancer, breast cancer, liver cancer, pancreatic cancer, head and neck cancer, glioma, and melanoma.

Images