Combined vaccine based on Th1 / Th2 immune dynamic balance as well as preparation method and application of combined vaccine

Abstract

The invention provides a combined vaccine based on Th1 / Th2 immune dynamic equilibrium as well as a preparation method and application of the combined vaccine. Subcutaneous delivery of interleukin 12 (IL12) and antigen (such as ovalbumin OVA) mRNA is adopted in the early stage of tumor occurrence, so that specific antigen recognition and T cell immune response of an organism are activated; when immune response enters a function decline stage, the tumor microenvironment is regulated through intratumor delivery of interleukin 4 (IL4) mRNA, the metabolic activity of T cells is maintained, and the depletion of the T cells is delayed. Through space (subcutaneous / intra-tumor) and time (early stage / middle stage) separated delivery of different immune factors, 'start-steady state maintenance 'type immune stylization regulation and control are realized, antigen-specific CTL reaction and persistent anti-tumor effect are remarkably enhanced, and the problem of immune bias caused by independent use of the antigen-specific CTL reaction and the persistent anti-tumor effect is solved.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically tumor immunotherapy technology, and specifically relates to a combined vaccine based on Th1 / Th2 immune dynamic balance, its preparation method, and its application. Background Technology

[0002] CD4 in the tumor microenvironment (TME) + T cell Th1 / Th2 subset differentiation and macrophage M1 / M2 polarization are core factors determining the direction of the immune response and therapeutic sensitivity. As CD4... + The two classic subsets of T helper cells, Th1 and Th2, exhibit a complementary and dynamically balanced relationship in the immune response. Th1 cells dominate the cellular immune response, primarily by secreting cytokines such as interferon-γ (IFN-γ) and interleukin-2 (IL2) to enhance CD8+. + The cytotoxic effects of T cells promote the polarization of macrophages towards the M1 phenotype, thereby exerting significant antiviral and antitumor effects. In contrast, Th2 cells primarily regulate humoral immunity, and the cytokines they secrete, such as IL-4, IL-5, IL-10, and IL-13, play important roles in anti-parasitic infections, allergic reactions, and immune homeostasis regulation. In the tumor microenvironment, Th2 immune responses have long been considered associated with immunosuppression and tumor progression; therefore, most current immunotherapy strategies focus on enhancing Th1 responses.

[0003] In cytokine therapy, Th1-type cytokines such as IL-12 have been extensively studied, as they can enhance dendritic cell (DC) activation and promote CD8 activation. + T cell expansion and IFN-γ secretion induce potent anti-tumor immunity. However, traditional recombinant protein or viral vector administration suffers from short half-life, unstable expression, and high systemic toxicity, limiting its clinical application. Meanwhile, tumor antigen vaccines (such as OVA model antigens) can induce specific immune responses, but their immunogenicity is limited, often requiring combination with immune adjuvants or cytokines to enhance efficacy.

[0004] In recent years, the rapid development of mRNA delivery technology has provided new solutions for tumor immunotherapy. mRNA has advantages such as not integrating into the genome, the ability to programmatically express multiple immune factors, and ease of preparation, enabling multi-target combined immune regulation. However, current research largely focuses on the activation of a single immune factor or a single immune step; for example, mRNA-OVA vaccines can express antigens in vivo and activate CD8. + T cells, but their immunogenicity is limited, so CD8 +Insufficient T cell expansion leads to short-lived tumor suppression, rapid cell depletion, and poor therapeutic efficacy. For example, IL-12 strongly activates dendritic cells (DCs) and critical leukocytes (CTLs), resulting in an IFN-γ-driven Th1 bias. In the absence of antigens, immunity lacks specificity, and high doses of IL-12 often cause systemic toxicity (cytokine storm, liver damage). Co-administration with antigens further accelerates T cell overactivation and premature depletion. Continuous or high-dose IL-12 stimulation often leads to overactivation of the immune system and a cytokine storm, while simultaneously causing effector T cells to rapidly enter a state of depletion under continuous antigen stimulation (e.g., PD-1 and TIM-3 upregulation), thus limiting its long-term efficacy and safety.

[0005] Therefore, there is currently a lack of systematic regulatory strategies for Th1 / Th2 immune balance. How to utilize mRNA platforms to achieve synergistic expression of cytokines and antigens and precisely regulate the dynamic balance of type I and type II immune responses is an unsolved problem in tumor immunotherapy. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention designs a phased immune strategy of "interleukin-12 initiation - interleukin-4 homeostasis maintenance". In the early stage, IL12 binds to tumor antigens (such as OVA-mRNA) to strongly activate dendritic cells and CD8. + T cells establish a high-intensity Th1-type anti-tumor response; subsequently, when the immune response enters the exhaustion or decline phase, IL4-mRNA is locally delivered to remodel the tumor microenvironment, maintain T cell metabolism and survival, thereby prolonging the duration of the immune response and enhancing overall anti-tumor efficacy. This strategy achieves a dynamic division of labor between pro-inflammatory and regulatory signals over time, overcoming the limitations of single-cytokine therapy and providing a new approach to programmed immune regulation for cytokine-mRNA vaccines. One objective of this invention is to provide a combined tumor immunotherapy vaccine prepared from interleukin-12 (IL12), interleukin-4 (IL4), and an antigen; a second objective is to provide a staged delivery method for the combined vaccine; and a third objective is to provide an application of the combined vaccine in tumor drug preparation and immunotherapy.

[0007] To achieve the above objectives, the present invention employs the following technical solution: On one hand, the present invention provides a combination vaccine for immunotherapy, the combination vaccine comprising at least one type II immunomodulator selected from IL4, IL10 or their functional variants.

[0008] Furthermore, the combined vaccine also includes at least one type I immunizing agent, which is selected from IL12, IL2 or their functional variants.

[0009] Furthermore, the combined vaccine also includes tumor antigens.

[0010] Furthermore, the type I immunomodulator is selected from IL12 mRNA, encoded by the nucleotide sequence shown in SEQ ID NO.5; the type II immunomodulator is selected from IL4 mRNA, encoded by the nucleotide sequence shown in SEQ ID NO.6; and the tumor antigen is ovalbumin or its immunogenic fragment.

[0011] Traditional IL12 therapy can strongly activate anti-tumor immune responses, but the sustained high-intensity immune stimulation can lead to side effects such as T cell depletion and cytokine storms, limiting the sustainability of its efficacy. Conversely, IL4, a typical type II cytokine, while possessing anti-inflammatory and tissue repair functions, is biased towards Th2 responses, and its use alone can easily weaken anti-tumor immunity. Based on the dynamic balance reasoning of type I and type II immune responses, this invention proposes a sequential immune regulation strategy of "IL12 initiation → IL4 homeostasis maintenance," which achieves immunity through the spatial (subcutaneous / intratumoral) and temporal (early / mid-stage) separation and delivery of different immune factors.

[0012] First, regarding the timing of delivery, since a successful adaptive immune response requires following the natural rhythm of "initiation-expansion-contraction-memory," this invention initially delivers IL12 mRNA and tumor antigen mRNA subcutaneously to strongly activate dendritic cells (DCs), inducing the production of high quantities of antigen-specific CD8. + T cells are activated and driven to polarize towards highly lethal Th1 / CTLs, laying a strong foundation for anti-tumor immunity. After the immune response reaches its peak (approximately 72 hours), IL4 mRNA is delivered intratumorally to the tumor. At this point, IL4 no longer antagonizes initial activation but acts on the already activated T cell population, preventing them from progressing to terminal exhaustion through metabolic reprogramming and other mechanisms, prolonging their lifespan and functional persistence, and promoting the formation of memory T cells, thereby extending the duration of the immune response. The sequential delivery of IL12 followed by IL4 ensures synergistic effects, rather than signal cancellation due to simultaneous action. From a toxicological perspective, this also avoids the potential inhibitory effect of IL4 at the peak of immune activation, ensuring the effectiveness of the attack.

[0013] Secondly, in terms of spatial delivery, IL12 and tumor antigen mRNA are first injected subcutaneously, followed by intratumoral injection of IL4 mRNA. This achieves spatial complementarity between peripheral immunogenic activation and local immune environment remodeling, improving efficacy and reducing systemic toxicity. The reason for using this delivery method is that the subcutaneous region is rich in antigen-presenting cells such as dendritic cells (DCs). After subcutaneous injection of IL12 and tumor antigen, the antigen is captured by DCs and can migrate to draining lymph nodes, thereby activating a large number of naive T cells and efficiently initiating systemic immunity. Then, IL4 is directly injected into the tumor, allowing it to act at high concentrations on tumor-infiltrating T cells and macrophages, directly improving their functional state without excessively interfering with the systemic immune status. This strategy ensures both the "breadth" of the immune response and the "depth" and "precision" of the attack.

[0014] This invention selects the typical type I cytokine IL12 and type II cytokine IL4 for combined application, achieving a dynamic balance between immune activation and homeostasis through a sequential dosing strategy. This breaks through the traditional understanding that type I and type II immune responses are antagonistic and mutually exclusive. This strategy not only reflects the inherent dynamic synergistic law of the immune system in terms of mechanism, but also provides a generalizable approach for combined treatment of type I / II immune responses. The experimental results of this invention also fully demonstrate that the IL12+IL4 combination regimen achieved a cure rate of approximately 80% in animal models, significantly superior to any single component; flow cytometry analysis confirmed that the combined group showed a significant increase in CD8 infiltrating tumors. + The increased number and enhanced function of T cells (increased IFN-γ secretion) directly validates the remarkable effectiveness of the "initiation-maintenance" strategy in inducing deep and durable anti-tumor immune responses.

[0015] On the other hand, the present invention provides lipid nanoparticles for immunotherapy, including combination vaccines as described in any of the preceding claims.

[0016] The delivery schemes for the combined vaccines provided by this invention include, but are not limited to, lipid nanoparticles, plasmid DNA, viral vectors, or protein delivery systems. As long as they follow the core immune rhythm principle of "activation first, then balancing" and can achieve the same inventive objective, namely, to achieve efficient, sustained activation and controllable regulation of anti-tumor immunity without increasing toxic side effects, they are still within the scope of protection of this invention.

[0017] In another aspect, the present invention provides the use of the combined vaccine or lipid nanoparticles as described above in the preparation of a drug for treating tumors, wherein a type I immunomodulator and a tumor antigen are administered first, followed by the administration of a type II immunomodulator.

[0018] Furthermore, the type II immunomodulator is administered within 48-120 hours after the administration of the type I immunomodulator and the tumor antigen.

[0019] Furthermore, the type I immunomodulator and tumor antigen are administered via subcutaneous injection, while the type II immunomodulator is administered via intratumoral injection.

[0020] In another aspect, the present invention provides the use of IL4 in preparing a combination vaccine to improve the efficacy of tumor treatment, the combination vaccine comprising IL12 mRNA and IL4 mRNA, wherein the IL12 mRNA is encoded by the nucleotide sequence shown in SEQ ID NO.5 and the IL4 mRNA is encoded by the nucleotide sequence shown in SEQ ID NO.6.

[0021] Furthermore, the combined vaccine also includes tumor antigens.

[0022] The present invention has the following beneficial effects: 1. Breaking away from the traditional approach of simply enhancing the type I (Th1) response in immunotherapy, this approach proactively incorporates the type II (Th2) factor, which is traditionally considered to have antagonistic effects, into the treatment system. It proposes a new immunotherapy paradigm of "type I initiation and type II maintenance," reshaping the two from an antagonistic relationship to a synergistic one. This combined approach achieves a cure rate of approximately 80%, significantly inhibits tumor growth, and has a far superior efficacy compared to single therapy. 2. The timing of combined use of IL12 and IL4 was clarified for the first time. IL12+ antigen was injected subcutaneously first, followed by intratumoral injection of IL4 after the peak of immune activation (48-120 hours). This sequential administration avoided signal conflicts or excessive inflammation that might result from simultaneous stimulation. 3. A spatially segmented delivery strategy of "subcutaneous + intratumoral" was adopted. Subcutaneous injection is used to efficiently initiate systemic immunity, while intratumoral injection is used to precisely reshape the local immune microenvironment. Spatial drug delivery minimizes systemic exposure of IL12 and reduces the risk of systemic inflammatory storm. At the same time, the action of IL4 is localized, avoiding the side effect of systemic Th2 shift. Attached Figure Description

[0023] Figure 1 Particle size characterization of LNP-mRNA.

[0024] Figure 2 The results are from a quantitative ELISA experiment.

[0025] Figure 3 This is the experimental protocol for the combined treatment group.

[0026] Figure 4 The average tumor growth curves of mice in different treatment groups are shown.

[0027] Figure 5 The curves showing the weight changes of mice in each group throughout the entire experimental period are shown.

[0028] Figure 6 The survival time of mice in each group.

[0029] Figure 7 The tumor growth of individual mice in each experimental group was shown.

[0030] Figure 8 The image shows the results of flow cytometry analysis of local immune cells in the lymph nodes.

[0031] Figure 9 Flow cytometry analysis of CD8 in mouse tumors + The functional status of T cells. Detailed Implementation

[0032] To make the inventive objectives, technical solutions, and beneficial effects of this application clearer, the following description, in conjunction with embodiments, further illustrates this application. It should be understood that the embodiments described are for illustrative purposes only and are not intended to limit the scope of the application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this description.

[0033] The following examples are provided to facilitate a better understanding of the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0034] Example 1: mRNA synthesis of cytokines IL12 and IL4 (1) Construction of target carrier a) Download the CDR region sequences of OVA, IL12, and IL4, optimize the codons, and have the DNA strands synthesized by the company (Genewiz). b) Design primers and amplify the desired interleukin target fragment using PCR (verified by electrophoresis, gel extraction and recovery) to obtain a target fragment with a partial vector framework. Digest the vector with enzymes (verified by electrophoresis—purification) and perform Gibson assembly. The optimized UTR region and interleukin CDS region can then be assembled into a complete plasmid for expression.

[0035] The 5′-UTR sequence used to construct the plasmid is shown in SEQ ID NO.1, the 3′-UTR sequence is shown in SEQ ID NO.2, the CDS region sequence of IL4 is shown in SEQ ID NO.3, the CDS region sequence of IL12 is shown in SEQ ID NO.4, and the CDS region sequence of OVA is shown in SEQ ID NO.7.

[0036] (2) Plasmid linearization a) Transform the constructed plasmid, pick single clones for culture, and use the plasmid extraction kit to extract the plasmid. After extraction, determine the concentration and verify it by agarose gel electrophoresis. b) Select a unique restriction enzyme site located at the end of the target fragment for restriction enzyme digestion to linearize it. Verify whether the restriction enzyme digestion is complete by agarose gel electrophoresis. After the restriction enzyme digestion is completed, purify the linearized plasmid.

[0037] (3) In vitro transcription of mRNA Using linearized plasmids as templates, in vitro transcription was performed via co-transcriptional capping. Taking a 20 μL reaction system as an example, the amplification system is shown in Table 1.

[0038] Table 1 Reaction System

[0039] The mRNA sequence of IL4 after transcription is shown in SEQ ID NO.5, the mRNA sequence of IL12 is shown in SEQ ID NO.6, and the mRNA sequence of OVA is shown in SEQ ID NO.8.

[0040] Example 2: Preparation of mRNA-lipid nanoparticles (LNP) (1) Composition of mRNA-LNP a) Cationic / ionizable lipids (as the main mRNA binding and encapsulation component): 1-octylnonyl-8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]octanoate (SM102); b) Auxiliary phospholipids (used to construct lipid bilayer structures): Distearate lecithin (DSPC); c) Cholesterol (used to regulate membrane stability and fluidity); d) PEGylated lipids (for imparting plasma circulation stability to particles and inhibiting nonspecific adsorption): 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000); e) Phosphate buffer system or isotonic buffer (as aqueous phase carrier); Table 2. Lipid materials required for preparing LNP-mRNA from 1 μg mRNA.

[0041] (2) Preparation of LNP-mRNA a) Weigh the amount of lipid components required for 1 μg mRNA according to the formula shown in Table 2, and dissolve it in 10 μl of ethanol to form a lipid stock solution; b) Dissolve 1 μg of mRNA in a 20 mM sodium citrate solution to form an aqueous solution; c) The aqueous solution and lipid stock solution were vortexed at a volume ratio of 2:1 to encapsulate mRNA into LNP through a spontaneous assembly / nanoparticle formation process. d) Use a 100k ultrafiltration tube to ultrafilter and bring the prepared mRNA-LNP to a fixed volume; e) Characterize and quality control the final formulation.

[0042] Experimental results are as follows Figure 1 As shown, the three mRNA-LNP formulations (OVA, IL4, and IL12) all formed stable and uniform nanoparticle systems with average particle sizes concentrated in the range of 120-140 nm, which is a typical size range that can efficiently enter cells.

[0043] Example 3: LNP-in vitro transfection To determine the dosage of the three mRNA-LNP agents in subsequent treatment, this invention first conducted an in vitro transfection experiment to analyze the expression of IL4 and IL12. The experimental procedure is as follows: (1) In vitro transfection Select healthy B16-OVA cells, at a ratio of 2 × 10⁶. 5 Seed cells at a density of 100 cells per well into a 12-well plate. When the cell density reaches 60%-70%, transfect the encapsulated IL12 / IL4 mRNA-LNP into the cell plate at a density of 1 μg per well. Collect the cell supernatant after 24 hours.

[0044] (2) ELISA was used to determine the expression levels of IL12 and IL4. After collecting the cell supernatant, centrifuge to remove cell debris. This experiment uses the Boster ELISA kit. Dilute the sample at two concentration gradients (100-fold and 500-fold) to ensure the cytokine concentration is within the detection range. Further procedures are performed according to the instruction manual. a) Add the sample and standard, react at 37°C for 90 min, discard the liquid without washing; b) Add biomarker antibody, react at 37°C for 60 min, and wash 3 times with 1X washing buffer; c) Add ABC, react at 37°C for 30 min, and wash 5 times with 1X washing buffer; d) React TMB at 37℃ for 15-20 min; e) Add stop solution and measure the OD value at 450 nm using an ELISA reader.

[0045] Experimental results are as follows Figure 2As shown, after transfecting cells with mRNA-LNP in vitro, the expression level of IL12 in the supernatant was approximately 7-8 times that of IL4, indicating a difference in translation efficiency between the two after mRNA transfection. This result provides a basis for determining the subsequent combined drug administration ratio. Considering that IL4 can regulate the immune microenvironment and alleviate the inflammatory response caused by excessive T cell activation under certain conditions, we set the dosage of IL12 and IL4 to 1 μg and 5 μg, respectively, in subsequent animal experiments to maintain immune activation and homeostasis, ensuring that the immunomodulatory effect of IL4 was within the auxiliary range rather than an inhibitory level.

[0046] Example 4: Construction and Treatment of Tumor Mouse Models This invention further verifies the therapeutic effects of the three mRNA-LNPs prepared in the above embodiments by constructing a mouse model of tumors. The specific experimental protocol is as follows: (1) Constructing a mouse model of tumor a) Tumor cells and animals B16-OVA melanoma cells (3rd generation passaged cells) were used in the experiment. Cells were collected when they reached approximately 70% confluence for modeling. After washing with PBS, the cells were counted and resuspended in PBS to a fixed concentration to ensure a consistent injection volume for each experimental subject. Cell viability in each mouse was confirmed to be above 95% by trypan blue staining before inoculation.

[0047] b) Tumor implantation method C57BL / 6 mice were randomly divided into groups of five, and B16-OVA cells were subcutaneously inoculated into the groin area. Each mouse received 1.5 × 10⁻⁶ cells. 5 One animal was vaccinated. After vaccination, the animals were fed routinely and monitored daily for tumor formation and overall health.

[0048] c) Treatment initiation criteria When the tumor area reaches approximately 30mm 2 When calculating the product of the major and minor axes, subsequent treatment protocols were initiated. All procedures were conducted in accordance with laboratory animal ethics requirements and approved by the institution's animal ethics committee.

[0049] (2) Treatment plan When the tumor area reaches approximately 30mm 2 At the time of administration, mouse weight and tumor size were recorded, and mice were randomly grouped according to tumor volume to ensure consistent baseline tumor size across groups. Treatment began after grouping, with all injections administered subcutaneously via the base of the tail. The treatment regimens were as follows: a) Combined treatment group: 1. OVA + IL12 + IL4 treatment group: First treatment: Seven days after tumor inoculation, the first injection was a combination of IL12 (1 μg / animal) and OVA (10 μg / animal) administered subcutaneously; 72 hours later, the second injection was IL4 (5 μg / animal) administered intratumorally. Second treatment: Repeat the treatment strategy 7 days after the first injection of the first treatment.

[0050] 2. OVA + IL12 treatment group: First treatment: Seven days after tumor inoculation, the first injection was a combination of IL12 (1μg / animal) and OVA (10μg / animal) administered subcutaneously. Second treatment: Repeat the treatment strategy 7 days after the first injection of the first treatment.

[0051] The experimental protocol for the combination therapy group is as follows: Figure 3 As shown.

[0052] b) Control group: 3. PBS control group: Administered an equal volume of PBS.

[0053] 4. OVA control group: OVA (10 μg / animal) was administered only.

[0054] During treatment, the mice's body weight, tumor growth, and general health status were continuously monitored.

[0055] (3) Tumor monitoring and treatment efficacy evaluation a) During treatment, the long and short diameters of the mouse tumors were measured every other day using calipers, and the tumor area was calculated. The mouse weight and general health status were also recorded.

[0056] b) When the area of ​​a single tumor exceeds 150 mm 2 If the mouse shows significant discomfort (such as a weight loss of more than 20% or a significant decrease in activity), euthanasia will be performed in accordance with animal experimentation ethics guidelines to reduce unnecessary suffering.

[0057] c) All mice were followed up until the endpoint was reached. Tumor growth curves and overall survival were recorded and analyzed for each group of mice during the experiment to evaluate treatment efficacy. Experimental results are as follows: Figure 4-7 As shown.

[0058] Figure 4 The results showed that tumors grew rapidly in the PBS and OVA groups, while the IL12+OVA group, although able to slow tumor proliferation, still showed a significant growth trend; however, the IL12+OVA+IL4 group, which combined local intratumoral injection of IL4, almost completely inhibited tumor growth, and the tumor area was significantly smaller than the other three groups. Figure 5As can be seen, the body weight of each group remained within ±10% of the initial body weight, without significant decrease or fluctuation, indicating that the combined mRNA administration (IL12+OVA+IL4) did not cause significant systemic toxicity or inflammatory side effects, and had good safety and tolerability.

[0059] like Figure 6 As shown, mice in both the PBS and OVA groups died within 20-25 days; some mice in the IL12+OVA group had prolonged survival, but the survival rate was only 40% within 30-40 days; while mice in the IL12+OVA+IL4 group had significantly prolonged survival, with a survival rate of 80% at day 40, and some individuals showed no tumor recurrence at the end of the observation period. These results indicate that the three-component combined treatment strategy designed in this invention not only effectively inhibits tumor growth but also significantly improves overall survival, exhibiting a long-term immune protective effect.

[0060] Figure 7 The results showed that the tumor growth curves in the PBS and OVA groups generally increased rapidly; some mice in the IL12+OVA group showed tumor suppression, but some relapses still occurred; while the tumor curves of all mice in the IL12+OVA+IL4 group were close to the horizontal line with almost no significant growth. This indicates that the combination therapy of IL12+OVA+IL4 has a stable and consistent anti-tumor effect among individuals and good reproducibility.

[0061] (4) Immunoflow cytometry analysis This embodiment further performs immune flow cytometry analysis on mice that received the above four treatment regimens to detect the expression of immune cells. The specific methods are as follows: a) 72 hours after the first subcutaneous injection of IL12+OVA (i.e., before the second injection), draining lymph nodes (dLNs) of mice were collected, and the activation status of dendritic cells (CD11c) was detected by flow cytometry. + CD86 + ) and CD4 + / CD8 + Changes in the proportion of T cells were used to assess the effectiveness of peripheral immune initiation.

[0062] b) 72 hours after intratumoral injection of IL4 mRNA-LNP (i.e., before the second treatment), tumor tissue was harvested for immune cell infiltration analysis. The experimental results are as follows: Figure 8-9 As shown.

[0063] Figure 8 The results showed that the IL12+OVA group significantly increased CD4 levels. + T cells and CD8 +The proportion of T cells in tumors, while simultaneously promoting the expression of the co-stimulatory molecule CD86 on the surface of dendritic cells (DCs), suggests that IL12 can effectively enhance antigen presentation and initial T cell activation. Figure 9 The results also showed that, compared with the PBS or OVA group, the IL12+OVA group significantly increased CD8. + T cells and their IFN-γ + With T-bet + The number of subsets; and after the addition of IL4 (IL12+OVA+IL4 group), the number and proportion of these effector T cells further increased, especially IFN-γ. + CD8 + T cells increased, and TIM-3 was detected. + PD-1 + The increased population of double-positive T cells indicates that IL4, under appropriate conditions, did not weaken the immune response; on the contrary, it enhanced the IL12-induced cytotoxic response by improving T cell survival and functional durability. This result verifies that the IL12-IL4 synergistic mechanism designed in this invention promotes the maintenance of CTL function.

[0064] In summary, this invention, through a local treatment strategy combining IL12 and IL4 delivered via mRNA-LNP, achieves significant tumor suppression and survival prolongation while ensuring safety. IL12 is responsible for activating antigen presentation and T cell responses, while IL4 maintains T cell function and microenvironmental balance in the later stages. Their synergistic effect effectively avoids T cell exhaustion and maintains a sustained cytotoxic response, providing a new technical approach for the application of mRNA cytokine combination therapy in tumor immunotherapy.

[0065] Example 5: Comparison of the effects of combined therapy with different cytokines In addition to combined treatment with IL12 and IL4, this invention also proposes the use of other type I and type II cytokines in combination for maintaining immune homeostasis and anti-inflammatory balance. Therefore, this embodiment further compares the effects of specific combined use of type I and type II cytokines. First, a mouse tumor model was constructed using the same method as in the above embodiment. When the tumor area reached approximately 30 mm²... 2 Mice were divided into groups and treated, with 5 mice in each group. The treatment grouping regimen was as follows: a) Combined treatment group: 1. OVA + IL12 + IL4 treatment group: First treatment: Seven days after tumor inoculation, the first injection was a combination of IL12 (1 μg / animal) and OVA (10 μg / animal) administered subcutaneously; 72 hours later, the second injection was IL4 (5 μg / animal) administered intratumorally. Second treatment: Repeat the treatment strategy 7 days after the first injection of the first treatment.

[0066] 2. OVA + IL2 + IL4 treatment group: First treatment: Seven days after tumor inoculation, the first injection was a combination of IL2 (1μg / animal) and OVA (10μg / animal) administered subcutaneously; 72 hours later, the second injection was IL4 (5μg / animal) administered intratumorally. Second treatment: Repeat the treatment strategy 7 days after the first injection of the first treatment.

[0067] 3. OVA + IL12 + IL10 treatment group: First treatment: Seven days after tumor inoculation, the first injection was a combination of IL12 (1μg / animal) and OVA (10μg / animal) administered subcutaneously; 72 hours later, the second injection was IL10 (5μg / animal) administered intratumorally. Second treatment: Repeat the treatment strategy 7 days after the first injection of the first treatment.

[0068] 4. OVA + IL2 + IL10 treatment group: First treatment: Seven days after tumor inoculation, the first injection was a combination of IL2 (1μg / animal) and OVA (10μg / animal) administered subcutaneously; 72 hours later, the second injection was IL10 (5μg / animal) administered intratumorally. Second treatment: Repeat the treatment strategy 7 days after the first injection of the first treatment.

[0069] In the above treatment regimen, the method for synthesizing IL2 and IL10 mRNA is as described in Example 1, and the preparation of the corresponding mRNA-LNP is as described in Example 2.

[0070] b) Control group: 5. PBS control group: Administered an equal volume of PBS.

[0071] 6. OVA control group: OVA (10 μg / animal) was administered only.

[0072] During treatment, mouse body weight, tumor growth, and general health status were continuously monitored, and survival rates were recorded. On the third day after treatment, changes in immune cells within the tumor were analyzed by flow cytometry. Table 3 shows the results, where survival rate is the survival rate of mice at day 40, and the percentage of each immune cell type is represented by a percentage per 5 × 10⁻⁶ cells. 5 The percentage of cells.

[0073] Table 3. Survival rate and immunological analysis results of mice in each group.

[0074] Survival results showed that, similar to Example 4, all mice in the PBS and OVA groups died by day 40. Some mice in the OVA + IL2 group had prolonged survival, with a survival rate of 80% by day 40; while mice in groups 1-4 had a survival rate of 60%-80% by day 40, indicating that combined treatment with type I and type II cytokines significantly prolonged mouse survival. Regarding the proportion of immune cells, compared to the PBS or OVA groups, the OVA + IL12 + IL4, OVA + IL2 + IL4, OVA + IL12 + IL10, and OVA + IL2 + IL10 groups all significantly increased CD8+. + T cells, IFN-γ + CD8 + T cells and TIM-3 + PD-1 + The number of double-positive T cells indicates that adding appropriate amounts of type II cytokines such as IL4 or IL10 can improve T cell survival and functional persistence, and enhance the cytotoxic response induced by type I cytokines. However, the effects of different cytokines used in combination vary. Based on the results in Table 3, the combined treatment regimen of OVA + IL12 + IL4 showed the best effect. Furthermore, monitoring of tumor growth revealed that the tumor area in the combined treatment groups of type I and type II cytokines (groups 1-4) was significantly lower than that in the control group, with group 1 showing the best effect in inhibiting tumor growth. These results indicate that combined treatment with type I and type II cytokines synergistically enhances the body's immune function, inhibits tumor growth, and prolongs the survival of mice, producing a synergistic effect. Among these, the combined vaccine of IL12 + IL4 showed the best therapeutic effect.

[0075] Example 6: Verification of the necessity of the time-sequential / spatialized drug delivery method provided by the present invention To verify the scientific validity and synergistic effect of the time-sequential and spatial drug delivery proposed in this invention, this embodiment further examines the impact of drug delivery method on therapeutic efficacy.

[0076] I. The Necessity of "IL12 First, Then IL4" The treatment groups for tumor-bearing mice included the PBS group, the OVA group, the OVA+IL12+IL4 sequential treatment group, and the OVA+IL12+IL4 simultaneous treatment group. The treatment regimens for each group are as follows: 1. OVA+IL12+IL4 sequential treatment group: First treatment: Seven days after tumor inoculation, the first injection was a combination of IL12 (1 μg / animal) and OVA (10 μg / animal) administered subcutaneously; 72 hours later, the second injection was IL4 (5 μg / animal) administered intratumorally. Second treatment: Repeat the treatment strategy 7 days after the first injection of the first treatment.

[0077] 2. OVA + IL12 + IL4 simultaneous treatment group: First treatment: Seven days after tumor inoculation, a combination of IL12 (1 μg / animal) and OVA (10 μg / animal) was injected subcutaneously, and IL4 (5 μg / animal) was injected intratumorally. Second treatment: Repeat the treatment strategy 7 days after the first injection of the first treatment.

[0078] 3. PBS group: Administered an equal volume of PBS 4. OAV group: OVA (10μg / animal) was administered only. Tumor growth, mouse weight, and survival were continuously monitored during treatment. Results showed that all mice in the simultaneous treatment group and the two control groups died within days 20-25, and their tumors grew rapidly. In contrast, tumor growth in the sequential treatment group was completely suppressed, with no significant change in tumor area, and the survival rate of mice in the sequential treatment group exceeded 80% within days 30-40. This result indicates that when using the same cytokines for combined treatment, only by administering IL12 and OVA first, followed by IL4, can a significant therapeutic effect be achieved. This is because IL12 and IL4 are drivers of CD4+. + Key factors for T cell differentiation: IL-12 promotes Th1 cell differentiation, while IL-4 promotes Th2 cell differentiation. These two pathways inhibit each other intracellularly. When dendritic cells (DCs) receive these two opposing signals simultaneously, their maturation and polarization become ambiguous, failing to effectively polarize towards either Th1 or Th2, thus reducing their ability to activate and direct T cells. However, administering IL-12 first, followed by IL-4, results in IL-12 strongly activating DCs and promoting naïve CD8+ differentiation. + T cells are activated into effector cytotoxic T lymphocytes and proliferate in large numbers. Once the immune response is successfully activated and reaches its peak, effector T cells begin to deplete. At this point, IL-4 intervenes to maintain and promote the metabolism of activated T cells, thereby prolonging the duration of the immune response. Therefore, only by adopting a sequential treatment regimen of "IL-12 first, then IL-4" can the treatment effect be significantly improved.

[0079] II. The Necessity of "Subcutaneous Injection of IL12 + Intratumoral Injection of IL4" The tumor-bearing mice were divided into three treatment groups: PBS group, OAV group, and OVA+IL12+IL4 combined treatment group. The treatment regimens for the PBS group and OAV group were the same as those described above. The OVA+IL12+IL4 combined treatment group was administered via the following two injection methods: Method 1 First treatment: Seven days after tumor inoculation, the first injection was a combination of IL12 (1 μg / animal) and OVA (10 μg / animal) administered subcutaneously; 72 hours later, the second injection was IL4 (5 μg / animal) administered intratumorally. Second treatment: Repeat the treatment strategy 7 days after the first injection of the first treatment.

[0080] Method 2 First treatment: Seven days after tumor inoculation, the first injection was a combination of IL12 (1μg / animal) and OVA (10μg / animal) administered intratumorally; 72 hours later, the second injection was IL4 (5μg / animal) administered subcutaneously. Second treatment: Repeat the treatment strategy 7 days after the first injection of the first treatment.

[0081] Monitoring results in mice showed that, consistent with the above findings, all mice in the control group died within 20-25 days, and all mice in the combined treatment group using method two also died by day 20. During this period, the tumors in all three groups grew rapidly, with a similar growth trend. Only the treatment regimen using method one significantly inhibited tumor growth and prolonged the survival of mice (the survival rate still reached 80% on day 40). This result indicates that the treatment method using method two was ineffective. IL12 mRNA is mainly used to induce systemic Th1 responses. Subcutaneous injection can promote the uptake and migration of local antigen-presenting cells (such as dendritic cells) to lymph nodes, triggering systemic immune activation. IL4 mRNA is used to regulate the local tumor microenvironment; therefore, intratumoral injection can more directly act on local tumor immune cells and reduce the side effects of systemic immunosuppression. If the administration method is swapped, and IL12 mRNA is directly injected into vascularized tumor tissue, it is very easy for a large number of inflammatory factors (such as IFN-γ and TNF-α) to enter the bloodstream rapidly, which may cause a severe systemic inflammatory response syndrome. On the other hand, systemic administration of IL-4 causes the body's immune system to shift towards Th2, which may suppress the body's anti-tumor Th1 immunity and may cause unnecessary side effects, thereby reducing safety and the body's immune capacity.

[0082] In conclusion, only by first injecting IL12 mRNA subcutaneously and then injecting IL4 mRNA intratumorally, through the synergistic effect of the two in terms of timing and space, can a significant synergistic effect be achieved in inhibiting tumor growth and improving the body's immunity.

[0083] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any modifications and alterations made by those skilled in the art without departing from the spirit and scope of the present invention shall still fall within the protection scope of the present invention.

[0084] sequence list SEQ ID NO:1: 5′-UTR ACTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCGCCACC SEQIDNO:2: 3′-UTR CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAA GCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACC SEQ ID NO:3: IL4-CDS ATGGGGTTGAACCCCCAGTTGGTTGTCATACTCCTTTTTTTCCTTGAATGTACACGCTCCCATATTCACGGTTGCGATAAGAATCACCTTAGAGAAATAATCGGGATACTGAATGAGGTTACCGGAGAGGGGACTCCATGTACTGAGATGGATGTTCCCAATGTATTGACCGCAACTAAAAATACTACAGAGTCAGAACTCGTTTGTAGAGCTTCAAAGGTACTGAGGATCTTTTATCTCAAGCACGGAAAAACCCCCTGCTTGAAGAAAAATTCTAGCGTCCTTATGGAACTTCAAAGATTGTTCAGGGCCTTTCGGTGCTTGGATAGTTCCATCAGTTGCACTATGAACGAAAGCAAGAGTACATCTTTGAAGGATTTTCTTGAGAGCTTGAAGTCCATAATGCAAATGGACTATAGT SEQ ID NO:4: IL12-CDS SEQ ID NO:5: IL4-mRNA ACTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCGCCACCATGGGGTTGAACCCCCAGTTGGTTGTCATACTCCTTTTTTTCCTTGAATGTACACGCTCCCATATTCACGGTTGCGATAAGAATCACCTTAGAGAAATAATCGGGATACTGAATGAGGTTACCGGAGAGGGGACTCCATGTACTGAGATGGATGTTCCCAATGTATTGACCGCAACTAAAAATACTACAGAGTCAGAACTCGTTTGTAGAGCTTCAAAGGTACTGAGGATCTTTTATCTCAAGCACGGAAAAACCCCCTGCTTGAAGAAAAATTCTAGCGTCCTTATGGAACTTCAAAGATTGTTCAGGGCCTTTCGGTGCTTGGATAGTTCCATCAGTTGCACTATGAACGAAAGCAAGAGTACATCTTTGAAGGATTTTCTTGAGAGCTTGAAGTCCATAATGCAAATGGACTATAGTtaaCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA SEQ ID NO:6: IL12-mRNA SEQ ID NO:7: OVA-CDS SEQ ID NO:8: OVA-mRNA

Claims

1. A combination vaccine for immunotherapy, characterized in that, The combination vaccine includes at least one type II immunizing agent selected from IL4, IL10, or their functional variants.

2. The combination vaccine of claim 1, wherein It also includes at least one type I immunomodulator, which is selected from IL12, IL2 or their functional variants.

3. The combination vaccine of claim 2, wherein It also includes tumor antigens.

4. The combination vaccine of claim 3, wherein The type I immunomodulator is selected from IL12 mRNA and encoded by the nucleotide sequence shown in SEQ ID NO.5; the type II immunomodulator is selected from IL4 mRNA and encoded by the nucleotide sequence shown in SEQ ID NO.6; the tumor antigen is ovalbumin or its immunogenic fragment.

5. Lipid nanoparticles for immunotherapy, characterized in that, Includes the combination vaccine as described in any one of claims 1-4.

6. The use of the combination vaccine as described in any one of claims 1-4 or the lipid nanoparticles as described in claim 5 in the preparation of a medicament for treating tumors, characterized in that, First administer type I immunomodulators and tumor antigens, then administer type II immunomodulators.

7. The use as described in claim 6, characterized in that, The type II immunomodulator is administered within 48-120 hours after the administration of the type I immunomodulator and the tumor antigen.

8. The use as described in claim 7, characterized in that, The type I immunomodulator and tumor antigen are administered via subcutaneous injection, while the type II immunomodulator is administered via intratumoral injection.

9. The use of IL4 in the preparation of combination vaccines to improve the efficacy of tumor treatment, characterized in that, The combined vaccine comprises IL12 mRNA and IL4 mRNA, wherein the IL12 mRNA is encoded by the nucleotide sequence shown in SEQ ID NO.5 and the IL4 mRNA is encoded by the nucleotide sequence shown in SEQ ID NO.

6.

10. The use as described in claim 9, characterized in that, The combined vaccine also includes tumor antigens.

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