Use of mRNA nanoparticles targeting the spleen for the preparation of an immunostimulating medicament

Abstract

The application relates to application of mRNA nanoparticles targeting spleen in preparation of immune activation drugs and belongs to the technical field of biological medicine. The mRNA nanoparticles coding T cell redirection molecules are prepared by mixing mRNA coding T cell redirection molecules and polymers. The application realizes a system for in-vivo programming of endogenous T cells and application thereof by in-vivo delivery of nucleic acid molecules, in-situ expression of T cell redirection molecules in immune organs, enabling the nucleic acid drugs coding T cell redirection molecules to preferentially enter immune organs such as spleen in-vivo, and in-situ expression of functional immune regulation proteins in the organs, so as to endow the endogenous T cells with tumor recognition, redirection killing and immune effect enhancement capacity in-vivo.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of spleen-targeting mRNA nanoparticles in the preparation of immune-activating drugs. Background Technology

[0002] T-cell redirection therapy is an important direction in current tumor immunotherapy. It mediates the specific recognition and killing of tumor cells expressing tumor-associated antigens by T cells through artificially designed antigen recognition or immune bridging molecules. Representative technologies include engineered immunomolecular technologies such as chimeric antigen receptor T-cell (CAR-T) therapy and bispecific T-cell adaptor protein therapy. These technologies have shown significant application value in hematologic malignancies and are gradually expanding to the treatment of solid tumors.

[0003] However, existing T-cell redirection therapies still have certain limitations, greatly restricting their therapeutic efficacy and safety in clinical applications. CAR-T cell therapy, in particular, relies on a complex preparation process involving in vitro T-cell isolation, gene modification, large-scale expansion, quality control, and in vivo reinfusion. This process is characterized by long preparation cycles, complex processes, and high production costs. Furthermore, it suffers from poor batch-to-batch consistency of cell products and uncontrollable in vivo expansion behavior after reinfusion. While protein-based bispecific T-cell connectives do not require in vitro cell modification and preparation, these proteins require continuous and repeated systemic administration to maintain effective drug concentrations and target receptor occupancy in vivo. This can easily trigger widespread abnormal activation of T cells throughout the body, inducing severe cytokine release syndrome and other toxic side effects, thus limiting the therapeutic window.

[0004] The rapid development of mRNA drugs and nanodelivery technology has provided a new technical route for achieving in situ expression of therapeutic immunomodulatory proteins in vivo, circumventing the shortcomings of traditional cell therapy and protein drugs. Using delivery carriers such as lipid nanoparticles, mRNA encoding T cell redirection proteins can be delivered in vivo and expressed in situ, potentially enabling in vivo reprogramming of endogenous T cells. However, current mainstream lipid nanoparticle delivery systems show a significant tendency for liver enrichment after intravenous administration, resulting in the primary expression of the target therapeutic protein in the liver. However, the liver is not the primary site for natural T cell activation, expansion, and functional programming. It cannot fully utilize the physiological microenvironment of highly enriched immune cells and orderly immune responses in secondary lymphoid organs such as the spleen, making it difficult to achieve efficient and specific in situ T cell redirection and functional activation, and thus failing to fully realize the application potential of in vivo mRNA expression technology.

[0005] In summary, the shortcomings of existing technologies are as follows: research focuses primarily on optimizing the molecular structure of T-cell retargeting and improving drug delivery efficiency, lacking a technical solution for precisely matching the expression sites of therapeutic molecules with the microenvironment of T-cell innate immune programming. Currently, there is no nucleic acid therapy strategy that can circumvent complex in vitro cell preparation processes, reduce systemic drug exposure toxicity, and simultaneously leverage the advantageous microenvironment of secondary lymphoid organs such as the spleen to efficiently and controllably achieve endogenous T-cell tumor retargeting and functional remodeling. Therefore, there is an urgent need in this field to develop a novel immune organ-targeted nucleic acid delivery and in situ expression technology system to balance the effectiveness, safety, and accessibility of tumor immunotherapy. Summary of the Invention

[0006] To address the problems of existing T-cell redirection therapies, such as reliance on in vitro cell engineering, high exposure to protein T-cell connective systems, and mismatch between expression sites and natural T-cell programming locations in conventional mRNA delivery systems, this invention provides the application of spleen-targeting mRNA nanoparticles in the preparation of immunomodulatory drugs. This invention achieves in vivo programming of endogenous T cells through the in vivo delivery of nucleic acid molecules, enabling the in situ expression of T-cell redirection molecules within immune organs. This allows nucleic acid drugs encoding T-cell redirection molecules to preferentially enter immune organs such as the spleen in vivo, and to express functional immunomodulatory proteins in situ within these organs, thereby endowing endogenous T cells with tumor recognition, redirection killing, and enhanced immune response capabilities in vivo.

[0007] This technical solution is expected to improve T cell redirection efficiency, enhance anti-tumor immune effects, and improve the balance between therapeutic efficacy and systemic toxicity without relying on in vitro cell engineering operations or maintaining high levels of systemic free protein exposure.

[0008] This invention is achieved through the following technical solution: The purpose of this invention is to provide an application of spleen-targeting mRNA nanoparticles in the preparation of immune-activating drugs. The mRNA nanoparticles encoding T-cell redirection molecules are prepared by mixing the mRNA encoding T-cell redirection molecules with a polymer.

[0009] Furthermore, the polymer is a cationic polymer PEI containing cyclic disulfide groups.

[0010] Furthermore, the structure of the polymer is as follows: .

[0011] Furthermore, the mass ratio of the mRNA encoding the T-cell redirection molecule to the polymer is 1:2 to 1:100.

[0012] Furthermore, the particle size of the mRNA nanoparticles encoding T-cell redirection molecules is 20 nm to 300 nm.

[0013] Furthermore, the dosage of the mRNA nanoparticles encoding T-cell redirection molecules is 0.20 mg / kg to 0.30 mg / kg.

[0014] Furthermore, the mRNA nanoparticles encoding T-cell redirection molecules are used once every 1 to 3 days, for a total of 1 to 3 times.

[0015] Furthermore, the dosage form of the drug is an injection.

[0016] Furthermore, the drug may also be used in combination with immune checkpoint inhibitors, chemotherapy drugs, cytokines, tumor vaccines or oncolytic viruses; or in conjunction with radiotherapy.

[0017] Furthermore, the drug also includes a pharmaceutically or pharmacologically acceptable carrier and / or salt.

[0018] Compared to traditional CAR-T cell therapy, protein-based T-cell connective systems, and conventional liver-oriented mRNA delivery protocols, this invention achieves a superior overall balance between effector molecule expression sites, T-cell in vivo arming efficiency, anti-tumor efficacy, and system safety, offering the following advantages: First, this invention enables in situ programming of endogenous T cells in vivo, avoiding the complex cell preparation processes of in vitro isolation, gene modification, expansion, quality control, and reinfusion required in traditional CAR-T cell therapy. By delivering nucleic acids encoding T cell redirection molecules to immune organs such as the spleen, this invention can directly generate T cells with tumor redirection capabilities in vivo, thereby improving treatment convenience, reproducibility, and clinical translational potential.

[0019] Secondly, this invention improves the spatial utilization efficiency of T cell redirection molecules. Unlike conventional LNPs that primarily express therapeutic proteins in the liver after delivering mRNA, this invention preferentially expresses the nucleic acids encoding T cell redirection molecules in immune organs such as the spleen, making the production site of the therapeutic protein closer to the physiological environment for natural T cell activation, proliferation, and functional shaping. Experimental results show that after in vivo administration, TCE in the spleen... + The proportion of T cells increases in a dose-dependent manner, reaching more than 20% of spleen T cells at the preferred dosage, indicating that the present invention can effectively form a high proportion of functionally redirected T cell populations in immune organs.

[0020] Furthermore, this invention enhances the antigen-dependent killing function of T cells. T cells generated after treatment with the system of this invention can recognize tumor cells expressing corresponding target antigens and induce the release of cytotoxic effector molecules, thereby achieving specific killing of target cells. In in vitro functional verification, spleen T cells from mice treated with this invention exhibited stronger cytotoxic activity against antigen-positive target cells, superior to the protein T cell connector group and the conventional LNP-mRNA delivery group, indicating that this invention can generate effector T cells that can continue to function outside of local immune organs.

[0021] Furthermore, this invention can significantly improve the in vivo anti-tumor efficacy. In solid tumor models, the treatment group of this invention showed a stable and sustained tumor growth inhibition effect, with the endpoint tumor mass or tumor burden reduced by approximately 70% compared to the control group. Simultaneously, individual tumor growth curves showed that the efficacy was more consistent among mice in the treatment group of this invention, suggesting that spleen-centered in vivo T-cell programming can more stably generate anti-tumor effects, rather than relying on random uptake of nano-formulations at the tumor site.

[0022] Furthermore, this invention has good potential for combination therapy applications. When used in combination with local radiotherapy, this invention can further enhance the anti-tumor effect. In preferred embodiments, complete remission was observed in some animals; for example, 4 out of 6 animals achieved complete remission. This indicates that this invention can be used not only as a standalone immunotherapy regimen but also as a combined enhancement strategy for radiotherapy, immune checkpoint inhibitors, chemotherapy, or other anti-tumor therapies.

[0023] Finally, this invention improves the balance between efficacy and safety. Compared to systemic administration of protein-based T-cell connectives, this invention reduces systemic exposure to free proteins in vivo by locally generating T-cell redirection molecules in immune organs such as the spleen, thereby lowering the risk of widespread T-cell activation and cytokine-related toxicity. Experimental results showed that the overall systemic cytokine levels were moderately increased after treatment with this invention, without the excessively high cytokine response observed in the protein-based T-cell connective group; simultaneously, there was no significant decrease in mouse body weight, and liver and kidney function indicators such as serum ALT, AST, and urea remained within the normal range. Histological examination of major organs showed no significant damage, indicating that this invention maintains good systemic safety while preserving antitumor efficacy.

[0024] In summary, this invention achieves in vivo T-cell programming centered on immune organs by changing the in vivo production site of T-cell retargeting molecules from peripheral circulation or liver-biased expression to in situ expression in immune organs such as the spleen. This technical solution can improve T-cell arming efficiency, enhance anti-tumor efficacy, and reduce the risk of systemic inflammation and organ toxicity while avoiding complex in vitro cell engineering operations, demonstrating good therapeutic index and clinical translation prospects. Attached Figure Description

[0025] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0026] Figure 1 This is a schematic diagram of in vivo and in vitro bioluminescence imaging (a) and Cre-Lox tdTomato reporter assay within 6 hours after intravenous injection of PBS, Luc@LNP or Luc@SPOT-NP according to the present invention (b); Figure 2 This is a diagram showing the results of in vitro SPOT-NP delivery of TCE mRNA according to the present invention; where a represents the TCE concentration in the spleen after SPOT-TCE injection. + Figure b shows the dose-dependent increase in T cells; b is an immunofluorescence image of spleens treated with TCE mAb, LNP-TCE, or SPOT-TCE, and quantitative analysis of relative TCE expression (n = 3 per group), scale bar, 50 μm; c is an image of CD31 expression in the spleen. + Blood vessels (green) and CD3 + Immunofluorescence observation of T cells (red), scale bar, 50 μm; d shows the relative mean fluorescence intensity (MFI) of TCE at specified time points after treatment, normalized to the TCE mAb group, and showing higher relative TCE signal persistence after SPOT-TCE treatment (n = 5 cells per group); e shows TCE detected by flow cytometry in spleen and blood. + T cell diagram; f represents the spleen-targeted mRNA translation model; Figure 3 This invention presents the results of SPOT-TCE-dependent spleen-mediated antigen-specific T-cell killing; where a is a schematic diagram of T-cell receptor (TCE)-mediated cytotoxicity; b is an in vitro cell lysis activity diagram of spleen T cells treated with SPOT-TCE, recombinant TCE, or LNP-TCE isolated from mice; c is a timeline of spleen collection experiments after TCE mAb / LNP-TCE / SPOT-TCE administration; d is a representative flow cytometry diagram and quantification of spleen B-cell exhaustion in treated mice (n=5 cells per group). Figure 4This is a graph showing the results of in vivo treatment and combined radiotherapy of the present invention; where a is the experimental timeline of SPOT-TCE systemic administration in 4T1-HER2 tumor-carrying mice; b is the tumor growth curve; c is the body weight monitored after treatment; d is the growth trajectory of a single tumor; e is a representative tumor image; f is the tumor body weight on day 20; g is the serum cytokine heatmap; h is the serum biochemistry of liver (ALT, AST) and kidney (urea) function; i is the H&E staining results of major organs, scale bar, 50 micrometers; j is the experimental timeline of SPOT-TCE systemic administration and combined radiotherapy in 4T1-HER2 tumor-carrying mice; k is the tumor growth curve; l is the mouse survival curve. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0028] Based on the disclosure of this invention, those skilled in the art can adjust the nucleic acid construct sequence, T cell redirection molecular structure, nanocarrier composition, dosage, frequency of administration, tumor target antigen, and indications according to actual needs, and such adjustments should all fall within the reasonable protection scope of this invention.

[0029] The "immunoorgan-targeting polymer nanocarrier" referred to in this invention refers to polymer nanoparticles capable of forming nanocomplexes with nucleic acid molecules and preferentially delivering and expressing these nanoparticles to immune organs such as the spleen after in vivo administration. In one specific embodiment, the immunoorgan-targeting polymer nanocarrier is formed by the self-assembly of a cationic polymer modified with cyclic disulfide groups and mRNA. For ease of description, this type of immunoorgan-targeting polymer nanocarrier used in implementing this invention will be abbreviated as "SPOT-NP" below.

[0030] It should be noted that in this invention, SPOT-NP is used as a preferred delivery vector for mRNA encoding T-cell redirection molecules, and its function is to achieve in situ translational expression of the mRNA in immune organs such as the spleen. The inventive point of this invention does not lie in the polymer nanocarrier material itself, but in the use of an immune organ-targeted nucleic acid delivery vector to deliver a nucleic acid construct encoding T-cell redirection molecules, thereby forming an immune organ-centric T-cell programming system in vivo.

[0031] Specifically, this invention provides the application of spleen-targeting mRNA nanoparticles in the preparation of immune-activating drugs: First, a nucleic acid construct encoding a T-cell redirection molecule. The nucleic acid construct is preferably mRNA, but can also be other nucleic acid forms capable of expressing the corresponding protein in vivo. The T-cell redirection molecule encoded by the mRNA includes, but is not limited to, bispecific T-cell connectives, trispecific T-cell connectives, T-cell activating molecules, T-cell co-stimulatory molecule fusion proteins, or other secretory immune proteins capable of regulating T-cell recognition, activation, migration, or killing functions. Preferably, the T-cell redirection molecule is a secretory-expressible bispecific T-cell connective, comprising a T-cell binding domain and a target-cell binding domain. The T-cell binding domain can be an antibody fragment or ligand fragment binding to CD3, CD28, 4-1BB, OX40, or other T-cell surface molecules; the target-cell binding domain can be an antibody fragment or ligand fragment binding to tumor-associated antigens or disease-associated target antigens. More preferably, the T cell binding domain is an anti-CD3ε single-chain antibody fragment, and the target cell binding domain is an antibody fragment targeting HER2, CD19, DLL3, EGFR, EGFRvIII, CLDN18.2, GPC3, MSLN, PSMA, BCMA, CD20, CD22, CD33, or other tumor-associated antigens.

[0032] Second, the nucleic acid construct may further include functional elements for in vivo expression and secretion. Preferably, the mRNA comprises, from the 5' end to the 3' end, a 5' cap structure, a 5' untranslated region, an open reading frame, a 3' untranslated region, and a poly(A) tail. The open reading frame may sequentially encode a secretion signal peptide, a first binding domain, a flexible linker peptide, a second binding domain, and an optional detection tag. The first binding domain may be a T cell binding domain or a target cell binding domain; the second binding domain corresponds to the first binding domain. The flexible linker peptide includes, but is not limited to, a linker peptide composed of glycine and serine, such as (Gly4Ser)n, where n is 1-6. The detection tag includes, but is not limited to, His tags, Flag tags, HA tags, or other tags that are easy to detect and purify. Through the above structural design, the nucleic acid construct, after in vivo translation, can produce a secretible, diffusible, and simultaneously binding T cell and target cell functional T cell redirection molecule.

[0033] Third, this invention provides an immune organ-targeting nanocarrier for delivering the aforementioned nucleic acid construct. The nanocarrier can form a nanocomplex with nucleic acid encoding T cell redirection molecules and is preferentially delivered to immune organs such as the spleen after in vivo administration. The nanocarrier can be a cationic polymer, an ionizable polymer, a lipid-polymer hybrid nanoparticle, polymer micelles, nanogels, or other delivery systems capable of loading nucleic acids and achieving enrichment in immune organs. Preferably, the nanocarrier is a cationic polymer nanocarrier with spleen-preferential enrichment characteristics. More preferably, the nanocarrier is a polyethyleneimine-based cationic polymer carrier containing a cyclic disulfide structure modification, capable of forming a stable nanocomplex with mRNA and achieving spleen-preferential delivery in vivo. This type of carrier can be considered a preferred embodiment of this invention, but the scope of protection of this invention is not limited to this specific carrier.

[0034] Fourth, this invention provides a pharmaceutical composition or nanoformulation comprising the above-mentioned nucleic acid construct and immune organ-targeting nanocarrier. The nanoformulation is formed by the interaction of mRNA encoding T-cell redirection molecules and the nanocarrier via electrostatic interaction, self-assembly, complexation, or nanoprecipitation. Preferably, the mass ratio of the mRNA to the carrier is 1:2 to 1:100, more preferably 1:5 to 1:50. The resulting nanoparticles preferably have a particle size of 20-300 nm, more preferably 30-200 nm, to suit in vivo administration and enrichment in immune organs. The nanoformulation may further comprise pharmaceutically acceptable buffer solutions, stabilizers, lyophilization protectants, or isotonic modifiers to improve storage stability and in vivo administration safety.

[0035] Fifth, this invention provides an in vivo T-cell programming method. The method includes: forming a nanoparticle formulation by combining a nucleic acid construct encoding a T-cell redirection molecule with an immune organ-targeting nanocarrier; intravenously injecting the nanoparticle formulation into a subject; after entering the body, the nanoparticle formulation preferentially accumulates in the spleen immune organ and is taken up by cells in the immune organ; the nucleic acid construct is released and translated into T-cell redirection molecules within the cells; the T-cell redirection molecules bind to endogenous T cells in the local environment of the immune organ, enabling the T cells to acquire tumor redirection capabilities similar to CAR-T cells; the in vivo programmed T cells further migrate to tumor or lesion tissue, recognizing and killing cells expressing corresponding target antigens.

[0036] Sixth, this invention provides the application of the above-mentioned in vivo T-cell programming system, nucleic acid construct, nano-formulation, or pharmaceutical composition in the preparation of drugs for treating tumors or immune-related diseases. The tumors include, but are not limited to, breast cancer, lymphoma, leukemia, lung cancer, small cell lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, liver cancer, ovarian cancer, prostate cancer, melanoma, glioma, and other solid tumors or hematologic malignancies expressing specific target antigens. The system can also be used in combination with radiotherapy, chemotherapy, immune checkpoint inhibitors, cytokine therapy, tumor vaccines, oncolytic viruses, or other immunomodulatory therapies to further enhance the anti-tumor immune effect.

[0037] Through the above technical solution, this invention transforms the production site of therapeutic T-cell redirection molecules from traditional peripheral system exposure or liver-biased expression to in situ expression within immune organs such as the spleen. This allows T cells to be programmed in vivo within the immune microenvironment where they are naturally activated and functionally shaped. This approach does not rely on complex in vitro cell preparation processes, nor does it require maintaining high levels of free protein system exposure. It can form a population of T cells with tumor-redirecting killing capabilities in vivo, thereby improving anti-tumor efficacy and reducing the risk of systemic toxicity.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0039] DTC 17 The preparation method of PEI polymer is disclosed in Chinese Patent CN 121758745 A.

[0040] The mRNA sequence can be found in GenBank Accession No. U17870.

[0041] BALB / c mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0042] Mice containing the loxP-STOP-loxP-tdTomato reporter element were purchased from Shanghai Southern Model Biotechnology Co., Ltd.

[0043] Example 1: Preparation of mRNA nanoformulation encoding T cell redirection molecules First, the mRNA encoding the T-cell redirection molecule was diluted to 20 μg / mL using 10 mM HEPES buffer to obtain an aqueous solution of the mRNA encoding the T-cell redirection molecule, with the buffer pH set to 5.2. Separately, the polymer component DTC was prepared. 17-PEI polymer was reconstituted with 100 mM sodium acetate aqueous solution to prepare a 10 mg / mL polymer aqueous solution. Then, an appropriate amount of the polymer aqueous solution was added to the bottom of a 1.5 mL EP tube, and the mRNA aqueous solution was quickly added using a pipette and repeatedly mixed by pipetting. The tube was then allowed to stand at room temperature for 5 min, allowing the mRNA and polymer to form an mRNA nanoparticle encoding T cell redirection molecules through electrostatic interactions and self-assembly, abbreviated as SPOT-TCE; where "SPOT" represents the immune organ-targeting polymer nanocarrier, and "TCE" represents the mRNA load encoding the T cell connector. By adjusting the mixing ratio of polymer to mRNA, mRNA nanoparticles SPOT-TCE (mCD3×HER2) and SPOT-TCE (mCD3×mCD19) with different nitrogen-to-phosphorus ratios (10:1, 6:1, 3:1) were obtained. Nanoparticles with a nitrogen-to-phosphorus ratio of 10:1 were used for subsequent experiments.

[0044] The particle size, PDI, and Zeta potential of SPOT-TCE with a nitrogen-to-phosphorus ratio of 10:1 were determined using dynamic light scattering, and the mRNA encapsulation efficiency was assessed using the RiboGreen assay. The results showed that the prepared SPOT-TCE nanoparticles could form a uniform and stable nanocomposite with a hydrated particle size of approximately 65 nm, a PDI of approximately 0.15, and a Zeta potential of approximately +15 mV. Transmission electron microscopy revealed that the obtained nanoparticles exhibited a near-spherical structure with a size of approximately 35 nm and a relatively uniform particle size distribution. The RiboGreen assay results indicated that the polymer nanocarrier could effectively encapsulate mRNA, demonstrating that this preparation method can yield mRNA nanoformulations encoding T-cell redirection molecules suitable for in vivo administration.

[0045] This embodiment demonstrates that the mRNA encoding T-cell redirection molecules can be stably assembled with immune organ-targeting polymer nanocarriers, forming the material basis of the in vivo T-cell programming system of the present invention.

[0046] Example 2: Verification of in situ mRNA translation in the spleen mediated by polymer nanocarriers.

[0047] To verify whether the immune organ-targeting polymer nanocarrier used in this invention can preferentially deliver mRNA to the spleen after intravenous administration and achieve protein expression in the spleen, an in vivo distribution experiment was conducted using mRNA encoding firefly luciferase as reporter mRNA.

[0048] Luc mRNA was respectively combined with conventional lipid nanoparticles SM102 LNP or SPOT-NP (DTC). 17 -PEI polymer) was prepared as Luc@LNP and Luc@SPOT-NP nanoformulations: Luc@SPOT-NP nanoparticles: Luc mRNA was diluted to 20 μg / mL with 10 mM HEPES buffer, and the SPOT-NP polymer component was reconstituted to 10 mg / mL with 100 mM sodium acetate solution. The two were mixed and allowed to stand at room temperature for 5 min to obtain the final product.

[0049] Luc@LNP nanoparticles: The preparation method is similar to that of Luc@SPOT-NP nanoparticles, except that SPOT-NP is replaced with SM102 LNP.

[0050] Experimental animals: Healthy BALB / c mice were used and divided into three groups: PBS, Luc@LNP, or Luc@SPOT-NP. Each group received a tail vein injection. The dosage was set to 0.375 mg / kg based on mRNA. Six hours after administration, D-fluorescein substrate was injected intraperitoneally into the mice, and the bioluminescent signal in vivo was detected using a small animal in vivo imaging system. Subsequently, the mice were sacrificed, and major organs such as the heart, liver, spleen, lungs, and kidneys were harvested for in vitro imaging. The total photon flux of each organ was quantitatively analyzed.

[0051] The experimental results showed that the PBS group showed almost no obvious bioluminescent signal; the Luc@LNP group mainly showed strong bioluminescent signal in the liver region after drug administration, suggesting that conventional LNP mRNA delivery mainly expresses reporter proteins in the liver; in contrast, the Luc@SPOT-NP group showed obvious bioluminescent signal in the spleen region after drug administration, and in vitro organ imaging also showed that the signal was mainly distributed in the spleen. Figure 1 (a) in the middle.

[0052] The above results demonstrate that the SPOT-NP delivery vector can significantly alter the organ distribution of mRNA expression in vivo, shifting mRNA expression from a liver-biased distribution mediated by conventional LNPs to an immune organ-biased distribution, such as the spleen. This proves that the present invention has a spatial basis for delivering mRNA encoding therapeutic immune proteins to the spleen and performing in situ translation and expression within the spleen.

[0053] Example 3: Cre-loxP system to verify the functional expression of mRNA in spleen immune cell subsets.

[0054] To further verify whether the mRNA delivered by SPOT-NP can be functionally expressed in a subset of spleen immune cells, the Cre-loxP tdTomato reporter system was used for detection.

[0055] The experimental animals used were mice containing the loxP-STOP-loxP-tdTomato reporter element. The mRNA encoding Cre recombinase was diluted to 20 μg / mL with 10 mM HEPES buffer, and then reacted with SPOT-NP (DTC).17 The Cre@SPOT-NP nanoformulation was prepared by rapidly mixing the PEI polymer at a predetermined ratio (10:1) and allowing it to stand for 5 min.

[0056] Cre@SPOT-NP was administered to reporter mice via tail vein injection. Mice were sacrificed 24-48 hours post-administration, and spleens were harvested to prepare single-cell suspensions. Flow cytometry was used to detect the proportion of lycopene fluorescent protein (tdTomato, GenBank accession number KJ081243) positivity in different splenic immune cell subsets. Antibodies detected by flow cytometry included CD45, CD3, B220, NK1.1, CD11c, F4 / 80, CD11b, Ly6G, and Ly6C, used to differentiate major immune cell populations such as T cells, B cells, NK cells, dendritic cells, macrophages, neutrophils, and monocytes.

[0057] like Figure 1 As shown in b in the figure. The results showed that after Cre@SPOT-NP administration, tdTomato-positive cells could be detected in various immune cell subsets of the spleen, indicating that Cre mRNA was taken up by the corresponding cells and successfully translated into Cre protein with recombinase activity, thereby cleaving the STOP element and activating tdTomato expression. The detection results showed that reporter gene expression was observed to varying degrees in B cells, T cells, NK cells, dendritic cells, macrophages, neutrophils and monocytes, with some myeloid cell subsets showing a high transfection rate.

[0058] This embodiment illustrates that the immune organ-targeting polymer nanocarrier used in this invention can not only deliver mRNA to the spleen, but also achieve functional protein expression in a subset of spleen immune cells, providing a cellular basis for the subsequent in situ generation of T cell retargeting molecules in the spleen and the realization of in vivo programming of endogenous T cells.

[0059] Example 4: Spleen-Guided Translation Platform-Mediated T-Cell Adaptor (SPOT-TCE) First, BALB / c mice were given escalating doses (0.0 μg, 2.5 μg, 5.0 μg, 10.0 μg, 20.0 μg) of SPOT-TCE (mCD3×HER2, NPK ratio of 10) intravenously. TCE levels in the spleen were quantified 24 hours later. + The frequency of T cells.

[0060] The results are as follows Figure 2 As shown in a, TCE + T cell abundance increased in a dose-dependent manner, reaching >20% of spleen T cells at the highest dose tested, with 5.0 μg (0.25 mg / kg) selected as the optimal value.

[0061] Experimental animals: Eight-week-old female BALB / c mice were used and divided into three groups: TCE mAb (mouse-derived recombinant TCE protein), LNP-TCE, and SPOT-TCE. TCE mAb was prepared by weighing the recombinant TCE protein and dissolving it in PBS. LNP-TCE was prepared with the following parameters: lipid molar ratio 50:10:38.5:1.5 (SM-102:DSPC:cholesterol:DMG-PEG2000), aqueous / organic phase flow rate ratio 3:1, lipid to mRNA mass ratio 0.05, citrate buffer pH 4.0, particle size approximately 100 nm, polydispersity index (PDI) <0.2, and stored at 4℃.

[0062] Each group was injected via tail vein. The dosage for the LNP-TCE and SPOT-TCE groups was set at 0.25 mg / kg based on mRNA, and the dosage for TCE mAb was 5 mg / kg. Mice were sacrificed 24 hours later, and their hearts, livers, spleens, lungs, and kidneys were harvested for in vitro imaging. Immunofluorescence staining analysis was performed on the spleen.

[0063] Immunofluorescence staining of TCEs in the spleen showed that the accumulation of TCEs in the spleen was significantly higher in the SPOT-TCE group than in the TCE mAb and LNP-TCE groups (see...). Figure 2 (b) Notably, SPOT-TCE treatment significantly increased CD3 levels. + T cells along CD31 + Accumulation of vascular structures (see) Figure 2 c) indicates enhanced vascular transport and activation in the spleen microenvironment. To assess the systemic persistence of the injected agent expression, this invention quantified the relative mean fluorescence intensity (MFI) of TCE in peripheral blood cells. SPOT-TCE induced a higher relative TCE signal level than LNP-TCE or TCE mAb relative to the TCE mAb group (see c). Figure 2 (d) Flow cytometry further confirmed that TCE levels in the spleen and blood increased after SPOT-TCE (mCD3×HER2) administration. + Increased T cell frequency (see...) Figure 2 (e). The model proposed in this invention demonstrates spleen-targeted mRNA translation, i.e., locally produced TCEs surrounding circulating T cells, reducing free serum TCE concentrations and increasing circulating TCEs. + The proportion of T cells.

[0064] Example 5: In vivo therapeutic effect study of SPOT-TCE Redirecting endogenous T cells to target tumor antigens requires efficient initiation and maintained target selectivity. Therefore, this study evaluated whether SPOT-TCE-induced T cells could perform precise antigen-dependent killing.

[0065] Mice were intravenously injected with PBS, TCE mAb, LNP-TCE, and SPOT-TCE, respectively. After 24 h, spleens were harvested to prepare single-cell suspensions, which were then co-incubated with target cells at different effector-to-target ratios, and the specific killing rate was detected.

[0066] like Figure 3 As shown in a, SPOT-TCE links CD3 on T cells to tumor-associated antigens (TAAs) on tumor cells, promoting physical synapse formation and inducing the release of cytotoxic mediators such as granzyme B, IFN-γ, and TNF-α. Splenic T cells were isolated from treated mice, and their in vitro activity was assessed. Spleen-derived T cells treated with SPOT-TCE (mCD3×HER2) maintained significant cytotoxic potential against antigen expression targets, exceeding that of T cells obtained from mice treated with LNP-delivered TCE mRNA or TCE mAb (see [reference needed]). Figure 3 (b) These results indicate that SPOT-TCE can generate effector T cells capable of maintaining function outside the local environment. To test whether SPOT-TCE works through secondary lymphoid activation, the spleens of mice treated with SPOT-TCE (mCD3×mCD19) were analyzed. Figure 3 c). After SPOT-TCE (mCD3×mCD19) treatment, flow cytometry showed mCD19. + B cell frequency decreased significantly (see...) Figure 3 (cd in the middle).

[0067] Next, BALB / c mice carrying 4T1-HER2 breast tumors were evaluated and compared with conventional formulations (including TCE mAb, LNP-TCE prepared by targeting liver LNP, and empty control nanoparticles Luc@SPOT-NP). Results showed that SPOT-TCE (mCD3×HER2) significantly and persistently inhibited tumor growth, while tumors in all control groups still rapidly expanded (see [link to relevant documentation]). Figure 4 (a and b in the text). It is worth noting that this effect was achieved without significant weight loss (see a and b in the text). Figure 4 (c) indicates that systemic tolerability was maintained at the effective therapeutic dose. These findings support the ability of spleen-targeted in situ expression agents to achieve tumor control levels comparable to or even exceeding those of conventional forms, while avoiding high-dose systemic exposure. Longitudinal analysis of individual tumor growth curves showed that SPOT-TCE produced homogeneous and durable control in mice, in contrast to the heterogeneous and often incomplete responses observed with TCE mAb and LNP-TCE. Figure 4(d) Endpoint analysis further showed that SPOT-TCE reduced tumor mass by approximately 70% compared to the control group, as evidenced by both tumor weight and large resection images (see d). Figure 4 (e and f in the text). These data not only demonstrate group-level efficacy but also show consistency of response, indicating that spleen-based initiation reliably generates a functional repertoire of effectors in vivo, rather than relying on random uptake at the tumor site. Multiple cytokine concentrations, including IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, CXCL1, and TNF-α, were measured using the Luminex multiplex bead immunoassay. The TCE mAb group showed the highest levels of IFN-γ, IL-2, TNF-α, and IL-12p70, the SPOT-TCE group showed intermediate increases, and the LNP-TCE group showed the lowest response (see [reference]). Figure 4 (g in the text). This hierarchical order is consistent with the pharmacokinetic differences between the different modalities: protein-dose injections produce high systemic Cmax and widespread exposure, rapidly activating circulating and tissue-resident T cells25, while in situ mRNA expression of TCE in the spleen produces a moderate systemic cytokine footprint while maintaining local T cell "arming." Notably, this response occurs without excessive induction of IL-6 or IL-10 (see g in the text). Figure 4 These cytokines (g) are often associated with cytokine release syndrome or compensatory immunosuppression in a high systemic T-cell binding environment.

[0068] To assess whether splenic restriction mRNA expression improved this therapeutic window, systemic safety indicators were monitored. Serum chemical markers of liver injury (ALT, AST) and renal stress (urea) remained within normal physiological ranges in all treatment groups (including SPOT-TCE (mCD3×HER2)). Figure 4 (h in the text). Histopathological evaluation of major organs (heart, liver, spleen, lung, kidney) showed no tissue damage or inflammatory lesions after SPOT-TCE administration (see h in the text). Figure 4 i in the text). Combined with the saved weight ( Figure 4 (c) These data indicate that SPOT-TCE (mCD3×HER2) can achieve deep anti-tumor activity without inducing significant systemic toxicity.

[0069] Experimental Methods: To further investigate the efficacy of SPOT-TCE combined with radiotherapy, a 4T1-HER2 subcutaneous tumor model was established in BALB / c mice. On day 6 after tumor cell inoculation, mice were divided into three groups: PBS group (PBS buffer injection as a control); RT group (radiotherapy to the tumor site, 6 Gy); and SPOT-TCE & RT group (radiotherapy to the tumor site, 6 Gy, followed by SPOT-TCE treatment (0.25 mg / kg) on ​​days 7, 12, and 17).

[0070] The experimental timeline for systemic administration of SPOT-TCE combined with radiotherapy in mice carrying 4T1-HER2 tumors is as follows: Figure 4 As shown in j in the figure, it can be seen that, compared with any single-agent therapy, local radiation combined with SPOT-TCE significantly increased tumor regression ( Figure 4 (k in the middle), and extended the overall survival ( Figure 4 (l in the text). Notably, 4 out of 6 mice achieved complete remission, with the tumor being permanently eradicated.

[0071] In summary, spleen-guided mRNA translation redefines the deployment of TCEs in vivo (shifting from systemic protein exposure to organ-centric effector programs), enabling precise, short-window T cell redirection and improving the therapeutic index. This invention emphasizes spleen-guided mRNA translation as a generalizable paradigm for in situ immune engineering, reprogramming immune function with spatial and temporal precision.

[0072] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of spleen-targeting mRNA nanoparticles in the preparation of immune-activating drugs, characterized in that, The mRNA nanoparticles encoding T-cell redirection molecules are prepared by mixing the mRNA encoding T-cell redirection molecules with a polymer.

2. The application according to claim 1, characterized in that, The polymer is a cationic polymer PEI containing cyclic disulfide groups.

3. The application according to claim 2, characterized in that, The structure of the polymer is shown below: 。 4. The application according to claim 1, characterized in that, The mass ratio of the mRNA encoding the T-cell redirection molecule to the polymer is 1:2 to 1:

100.

5. The application according to claim 1, characterized in that, The mRNA nanoparticles encoding T-cell redirection molecules have a particle size of 20 nm to 300 nm.

6. The application according to claim 1, characterized in that, The dosage of the mRNA nanoparticles encoding T-cell redirection molecules is 0.20 mg / kg to 0.30 mg / kg.

7. The application according to claim 1, characterized in that, The mRNA nanoparticles encoding T-cell redirection molecules are used once every 1 to 3 days, for a total of 1 to 3 times.

8. The application according to claim 1, characterized in that, The drug is in the form of an injection.

9. The application according to claim 1, characterized in that, The drug may also be used in combination with immune checkpoint inhibitors, chemotherapy drugs, cytokines, tumor vaccines or oncolytic viruses; or in conjunction with radiotherapy.

10. The application according to claim 1, characterized in that, The drug also includes pharmaceutically or pharmacologically acceptable carriers and / or salts.

Images