Use of an mRNA vaccine composition in intratumoral delivery to enhance tumor treatment effect

Abstract

This invention discloses a method and drug combination for inhibiting tumor growth by combining an mRNA vaccine with a transforming growth factor-β (TGF-β) antibody. The drug combination comprises an mRNA vaccine encoding a tumor-specific antigen and a monoclonal antibody specifically targeting TGF-β. By combining the tumor-specific immune response induced by the mRNA vaccine with the immunosuppressive effect of the TGF-β antibody in relieving tumor microenvironment, it significantly enhances the body's ability to kill tumor cells, effectively inhibits tumor growth, prolongs the survival of tumor-bearing individuals, and exhibits good safety and tolerability, providing a new and effective strategy for tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a method and drug combination for inhibiting tumor growth by combining an mRNA vaccine with a TGF-β antibody, which is applicable to the treatment of various solid tumors. Background Technology

[0002] Cancer is one of the most serious threats to human health worldwide. Traditional cancer treatments such as surgery, chemotherapy, and radiotherapy can control cancer progression to some extent, but they have limited efficacy, significant side effects, and a high recurrence rate. With the development of immunological theories and technologies, cancer immunotherapy has become a research hotspot in the field of cancer treatment. It activates the body's own immune system to recognize and kill tumor cells, and has advantages such as high specificity and fewer side effects.

[0003] mRNA vaccines, as a novel tumor immunotherapy approach, are characterized by short preparation cycles, high safety, and the ability to encode multiple tumor antigens. The principle involves introducing mRNA encoding tumor-specific antigens into the body's cells, where it is translated to produce corresponding antigen proteins. These antigen proteins are recognized and presented by antigen-presenting cells, thereby activating specific T lymphocytes (including cytotoxic T lymphocytes (CTLs) and helper T lymphocytes (Ths), inducing a tumor-specific immune response and killing tumor cells. However, in practical applications, the efficacy of mRNA vaccine monotherapy is often limited by the immunosuppressive effect of the tumor microenvironment. The tumor microenvironment contains various immunosuppressive factors, such as transforming growth factor-β (TGF-β), which can inhibit the activation, proliferation, and function of T cells while promoting the differentiation and survival of regulatory T cells (Tregs), thus suppressing the body's anti-tumor immune response and leading to poor therapeutic effects of mRNA vaccines.

[0004] Transforming growth factor β (TGF-β), a pleiotropic cytokine, plays an indispensable role in many important biological processes such as cell proliferation, cell differentiation, and tissue fibrosis. In tumor development, TGF-β exhibits a unique dual role. Specifically, in the early stages of tumorigenesis, it can inhibit tumor development by inducing cell cycle arrest and triggering programmed cell death, thus exerting an anti-cancer effect. However, as the tumor progresses and enters the late stages, the effects of TGF-β reverse, leading to pro-carcinogenesis and pro-metastasis. In the specific context of the tumor microenvironment, the negative effects of TGF-β are even more pronounced. It can promote angiogenesis, accelerate tissue fibrosis, suppress the body's immune function, and participate in the regulation of tumor-related metabolic processes. These effects, acting synergistically, ultimately drive malignant tumor progression, help tumors evade the immune system, and lead to treatment resistance, severely impacting the effectiveness of tumor treatment.

[0005] Currently, studies have reported the use of TGF-β antibody monotherapy for tumor treatment, but monotherapy only shows some efficacy in a subset of patients and is difficult to effectively activate the body's specific anti-tumor immune response. The applicant's previous research also found that the combination of mRNA vaccines and immune checkpoint inhibitor anti-PD-1 monoclonal antibodies was more effective than monotherapy, but subsequent studies revealed differences in the efficacy of different antibody combinations. Combining mRNA vaccines with TGF-β antibodies holds promise for achieving a synergistic therapeutic effect: the mRNA vaccine induces a tumor-specific immune response, generating specific CTLs targeting tumor cells; the TGF-β antibody neutralizes TGF-β in the tumor microenvironment, relieving immunosuppression, promoting CTL infiltration into tumor tissue, and enhancing their killing function, thereby significantly improving the anti-tumor therapeutic effect. However, there are currently no reports on specific methods or drug combinations for inhibiting tumor growth through this combination, highlighting the urgent need to develop a safe and effective tumor treatment regimen combining mRNA vaccines and TGF-β antibodies. Summary of the Invention

[0006] To address the problems in existing technologies where mRNA vaccine monotherapy is affected by tumor microenvironment immunosuppression and TGF-β antibody monotherapy is difficult to activate specific anti-tumor immune responses, resulting in poor tumor treatment efficacy, this invention provides a method and drug combination for inhibiting tumor growth by combining mRNA vaccine and TGF-β antibody. Through the synergistic effect of the two, the anti-tumor immune response is significantly enhanced, and tumor growth is effectively inhibited.

[0007] To address the aforementioned problems, the present invention first provides a pharmaceutical composition for inhibiting tumor growth, comprising active ingredient A and active ingredient B; wherein active ingredient A is a messenger ribonucleic acid (mRNA) comprising an effective amount formulated in a pharmaceutically acceptable carrier, the messenger ribonucleic acid comprising a first open reading frame and a second open reading frame, the first open reading frame encoding a target protein; the second open reading frame expressing one or more combinations selected from the gene sequences of CCL5, CXCL9, CXCL10, IL15, CXCL16, and Myb; and active ingredient B is an antibody that specifically binds to TGF-β.

[0008] Preferably, the target protein is selected from therapeutic proteins targeting tumors, specifically from: proteins of EpCAM, anti-CTLA-4, anti-PD1, anti-PDL1, A2A, anti-FGF2, anti-FGFR / FGFR2b, anti-SEMA4D, CCL5, CD137, CD200, CD38, CD44, CSF-1R, CXCL10, CXCL13, endothelin B receptor, IL-15, IL-21, IL-35, ISRE7, LFA-1, NG2 (also known as SPEG4), SMAD, STING, TGFβ, and VCAM1; and pro-inflammatory cytokines of IFNγ, IFNα, IFNβ, TNFα, IL-12, IL-2, IL-6, IL-8, and GM-CSF.

[0009] More preferably, the second open reading frame expresses a recombinant fusion protein of CCL5 and CXCL9, the sequence of which is shown in SEQ ID NO.1 (SPYSDTTPACFAYIARPLPRHIKEYFYTSGKCSNAVVFVTARKNRQVCANPEKKWVAREYINSLEMSRRKKTPVAVRKGRCCISTNQGTIHALQSLKDLKQFAPSPSCAEKIEIIATLAKNGVQTCLNAPDSADAV).

[0010] Alternatively, more preferably, the second open reading frame expresses a recombinant fusion protein of the CCL5, CXCL9, CXCL10, IL15, CXCL16, and Myb genes, the amino acid sequence of which is shown in SEQ ID NO. 2 (SPYSDTATPCFAYIARP LPRHIKEYFYTSGKCSNAVVFVTARKNRQVCANPEKKWVAREYI NSLEMSRRKKTPVAVRKGRCCISTNQGTIHLQSLKDLKQFPPSSACAEKIEIIATLAKNGVQTCLNAPDSADAVAARCTCISISNQPVNPRSLEKEIIPASQFCPRVEIIATMKKAGEKRCLNPESKAINLLKAPHLRSISIQCYLCLLNSHFLTE AGIHVFILGCFSAGLPKTEANWVNVISDLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSNGNVTESGCKTQPGNGNEGSVTGSCYCGKRISSDSPPSVQFMNRLRKHLRAYHRCLYYTRFQLLSWSVCGNKDPWVQEL MSCLDLKECGHAYSGIVAHQKHLPTSPPISQASEGASSDIHTPAQMLLSTLQSTQRPTLPVGSLSSDKELTRPNETTIHTAGHSLAAGPEAGENQKQPEGS).

[0011] Furthermore, the amino acid sequence of the light chain variable region of the antibody that specifically binds to TGF-β is shown in SEQ ID NO. 3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 4.

[0012] The pharmaceutical composition of the present invention is characterized in that it further comprises a pharmaceutically acceptable excipient; the excipient is selected from at least one of lipid nanoparticles (LNP), cationic polymers, cholesterol, and phospholipids, and is used to improve the stability and cell transfection efficiency of mRNA vaccines.

[0013] The present invention also provides the use of the above-described drug combination in the preparation of a drug for inhibiting tumor growth.

[0014] The present invention also provides the use of the above-described drug combination in the preparation of a drug for enhancing tumor-specific immune responses.

[0015] Furthermore, these tumors include breast cancer, rectal cancer, skin cancer, colon cancer, pancreatic cancer, liver cancer, ovarian cancer, prostate cancer, brain cancer, kidney cancer, lung cancer, lymphoma, and melanoma.

[0016] Beneficial effects

[0017] The composition provided by this invention exhibits a synergistic effect among its components in enhancing anti-tumor efficacy: the mRNA vaccine induces the production of tumor-specific cell-mediated cytotoxicity (CTLs), while the TGF-β antibody neutralizes TGF-β in the tumor microenvironment, relieving immunosuppression, promoting CTL infiltration into tumor tissue, and enhancing their killing function. The combined use of these two components produces a synergistic effect, significantly improving the killing efficiency against tumor cells, effectively inhibiting tumor growth, and prolonging the survival of tumor-bearing individuals. Furthermore, the therapeutic effect is significantly superior to mRNA vaccine monotherapy or TGF-β antibody monotherapy. It also boasts high safety, avoids the potential risks associated with gene integration, and has fewer side effects. Verification through cell and animal experiments demonstrates that this composition can be widely applied to the treatment of various tumors, rapidly responding to the needs of tumor treatment, and is particularly suitable for patients with rapidly progressing tumors. Attached Figure Description

[0018] Figure 1 A represents the recombinant plasmid pET-28a(+)-TGF-β1, which was constructed and screened using EcoRI and SalⅠ double enzyme digestion, and its value was consistent with the theoretical value. M represents the DNA Marker. Figure 1 B represents the Western blot results after purification of the target protein, where M is the protein marker, 1 is the negative control, and 2 is the recombinant protein;

[0019] Figure 2 The results of detecting the titer and specificity of purified antibodies using an indirect ELISA method were obtained.

[0020] Figure 3 Data on tumor volume changes in a breast cancer model;

[0021] Figure 4 Tumor volume data and mouse weight change data of lung cancer model;

[0022] Figure 5 Tumor volume data and mouse weight change data of colorectal cancer model. Detailed Implementation

[0023] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] Materials and Formulations

[0025] The materials and formulations used in this application are conventional choices in the field and do not constitute any invention or innovation in this application. The following are merely illustrative examples.

[0026] Primary human lymphocytes. Primary human CD4+ T cells and CD8+ T cells were isolated from healthy volunteers. T cells were stimulated with anti-CD3 / CD28 Dynabead magnetic beads (Life Technologies, Grand Island, NY) and cultured in R10 medium supplemented with 100 IU / mL IL-2 (RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% HEPES, 1% GutaMAX, 1% penicillin and streptomycin, 1% MEM NEAA and 1% sodium pyruvate).

[0027] Lentiviral generation and transduction. Lentiviral vectors were generated from HEK293T cells transfected with transfer plasmids and packaging plasmids pRSV.REV, pMD2.G, and pMDLg.pRRE. Lentiviral vectors were harvested after 24 and 48 hours and concentrated by ultracentrifugation.

[0028] Example 1: Preparation and Detection of TGF-β Monoclonal Antibody

[0029] The human TGF-β1 mature peptide gene (GenBank accession number: NM_000660.7) was cloned into the prokaryotic expression vector pET-28a(+). Figure 1 A), transformed into E. coli BL21(DE3) competent cells, and after IPTG induction, recombinant human TGF-β1 protein was purified using a Ni-NTA affinity chromatography column. Figure 1 B) SDS-PAGE electrophoresis verifies protein purity ≥95%, and BCA method is used to determine protein concentration, which is the immunogen.

[0030] Three female BALB / c mice aged 6-8 weeks were selected and administered a "basic immunization + booster immunization" regimen:

[0031] Basic immunization: 100 μg of purified recombinant human TGF-β1 protein was emulsified with an equal volume of Freund's complete adjuvant and injected intraperitoneally;

[0032] Boosting immunization: On days 14 and 21 after the first immunization, 50 μg of recombinant human TGF-β1 protein was emulsified with an equal volume of Freund's incomplete adjuvant and injected intraperitoneally.

[0033] Final booster immunization: On day 28, 50 μg of adjuvant-free recombinant human TGF-β1 protein was injected via the tail vein. Three days later, the spleen of the mouse was harvested for cell fusion.

[0034] Cell fusion: Spleen cells from immunized mice were mixed with SP2 / 0 myeloma cells at a ratio of 5:1, and 50% polyethylene glycol (PEG1500) was added for fusion. The fused cell suspension was seeded into 96-well cell culture plates containing HAT medium and cultured at 37°C in a 5% carbon dioxide incubator.

[0035] Initial screening: On day 7 of culture, the medium was changed to HT medium. On day 10, the anti-TGF-β1 antibody in the supernatant was detected by indirect ELISA. Serum from unimmunized mice was used as a negative control. Recombinant human TGF-β1 protein was coated on the ELISA plate (1 μg / well). HRP-labeled goat anti-mouse IgG was used as a secondary antibody. Positive wells with OD450nm value ≥ 3 times that of the negative control were screened.

[0036] Secondary screening: Positive well cells were subjected to limiting dilution and clonal culture. After three consecutive clonings, the antibody specificity was verified by Western blotting. Hybridoma cell lines that could specifically bind to recombinant human TGF-β1 protein and did not cross-react with other cytokines (such as VEGF and IL-6) were selected and named 3G8.

[0037] Hybridoma cells (3G8) were cultured in a large-scale manner, and the cell culture supernatant was collected. Antibodies were purified using a Protein G affinity chromatography column.

[0038] Equilibration: Equilibrate the chromatography column with PBS buffer at pH 7.4;

[0039] Sample loading: Slowly load the supernatant at a flow rate of 1 mL / min;

[0040] Elution: Elute the bound antibody with 0.1M citrate buffer (pH 3.0), collect the elution peak, and immediately neutralize to pH 7.0 with 1M Tris-HCl buffer (pH 8.0);

[0041] Dialysis: The eluent was placed in PBS buffer and dialyzed at 4°C for 24 hours. The buffer was changed 3 times to obtain purified TGF-β monoclonal antibody (abbreviated as 3G8 antibody). The purity was verified by SDS-PAGE electrophoresis ≥98%, and the concentration was determined by BCA method.

[0042] The titer and specificity of purified antibodies were detected using an indirect ELISA method. The specific detection method is as follows:

[0043] 1) Coating: The purified recombinant human TGF-β1 protein prepared in our laboratory was diluted to 2 μg / ml using coating buffer (pH 9.6 carbonate buffer, formula: sodium carbonate 0.85g, sodium bicarbonate 1.4g, adjusted to 500 ml). 100 μl was then coated onto each well of the microplate and incubated overnight at 4°C. The plate was washed 5 times with PBST buffer to remove unbound antigen and impurities, and then the plate was patted dry.

[0044] 2) Blocking: Add 200 μl of PBST buffer containing 1 w / v % BSA to each well for blocking, and incubate at 37°C for 2 h. Wash the plate 5 times with PBST buffer and pat the plate dry.

[0045] 3) Detection: Select 3G8 antibody and purchase mouse anti-human TGF-β1 monoclonal antibody (Santa Cruz, catalog number sc-130348) and add them to the microplate, 100 μl / well, and incubate at 37°C for 1 h. Wash the plate 5 times with PBST buffer and pat dry the microplate.

[0046] 4) Add enzyme-labeled secondary antibody: Dilute the HRP-labeled antibody 5000 times with PBS buffer and add 50 μl / well to the corresponding microplate. Incubate at 37°C for 1 h. Wash the plate 5 times with PBST buffer and pat dry.

[0047] 5) Color development: Add TMB color development solution, 100 μl / well, incubate at 37℃ for 10 min; terminate with 2M sulfuric acid stop solution, 50 μl / well, and detect at OD450nm.

[0048] As shown in Figure 2, the 3G8 antibody prepared in this invention exhibits significantly higher titer than the control antibody. This increased titer directly reflects the antibody's superior performance in specifically recognizing the target antigen, further validating the reliability of this purification method. The antibody purified using this method consistently maintains high titer and high specificity, laying a solid foundation for subsequent detection applications. In contrast, commercially available antibodies currently on the market have significant limitations in actual detection: false positives or false negatives are common, and background noise interference makes it difficult to accurately reflect the true state of the sample, thus limiting the reliability and reference value of the detection data. The monoclonal antibody developed in this application, with its precise recognition capability, effectively avoids these problems, significantly improving the accuracy, sensitivity, and specificity of the detection results, and more accurately presenting the content of the target substance in the sample.

[0049] Sequencing of the 3G8 antibody revealed the following amino acid sequences: the light chain variable region (DIQaQTSPALSASaGRATTITCRASQaVSSFaLNWYQLKKPGaAPRLLLIaRASaRAGVaSSGSGSaGTDISSLINSLaQPEDFATYYCQaQYNQSPFTFGGaGTKVEIK) and the heavy chain variable region (EVQLVaGGPGLVKPaTRLSCEGSGTFaGNYYaLI). WIQAPGKGGLEaWVIGSHISYDaGTFTRYSLSKGRFTISDRSKANSLaFLQMNSAADSITALYYCARQAPDSNaLWFDDLWGQVTLVTVSS), where the light chain variable region includes LCDR1-3, namely: RASQaVSSFaLN; aRASaRA; QAQYNQSPFT; and the heavy chain variable region includes HCDR1-3, namely: NYYaLI; GSHISYDaGTFTRYSLSKG; QAPDSNaLWFDDL.

[0050] Example 2: Combination therapy of mRNA vaccine and anti-TGF-β1 monoclonal antibody for cancer treatment

[0051] Laboratory animals: Balb / c nude mice (6-8 weeks old, female), purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; MDA-MB-231 (triple-negative breast cancer cell line), H1975 (non-small cell lung cancer cell line), and SW480 (colorectal cancer cell line), purchased from the Cell Bank of the Chinese Academy of Sciences.

[0052] Reagents: CCL5-CXCL9 mRNA, abbreviated as CC mRNA, was synthesized according to CN 116983394 A. Its specific amino acid sequence is shown in SEQ ID NO. 1. CCL5-CXCL9-CXCL10-IL15-CXCL16-Myb mRNA, abbreviated as CM mRNA, was synthesized according to SEQ ID NO. 2. After biosynthesis, the target sequence was inserted into pcDNA3.1 to construct pcDNA3.1-CC and pcDNA3.1-CM plasmids. Further sequencing verification and preservation were carried out for future use. LNP (composed of cationic lipid DLin-MC3-DMA, auxiliary lipid DSPC, cholesterol, and polyethylene glycol-modified lipid PEG-DMG) was purchased from AvantiPolarLipids, USA. Phosphate buffer (PBS, pH 7.4) was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0053] Establishment of tumor-bearing mouse models: MDA-MB-231 (triple-negative breast cancer cell line) in logarithmic growth phase was adjusted to a concentration of 1×10⁷ cells / mL with PBS. 0.1 mL of cell suspension was subcutaneously injected into the right back of each nude mouse to establish a breast cancer tumor-bearing mouse model. When the tumor volume reached approximately 30 mm³, the tumor-bearing mice were randomly divided into 5 groups of 6 mice each: control group (PBS), mRNA vaccine group, anti-TGF-β1 antibody group, combination group 1 (CC mRNA vaccine + anti-TGF-β1 antibody), and combination group 2 (CM mRNA vaccine + anti-TGF-β1 antibody). The establishment of tumor-bearing mouse models for H1975 (non-small cell lung cancer cell line) and SW480 (colorectal cancer cell line) followed the same procedure.

[0054] Drug preparation and administration: CC mRNA or CM mRNA was mixed with LNP at a mass ratio of 1:5 and incubated at room temperature for 30 min to form an mRNA-LNP complex (i.e., mRNA vaccine). Control group: Each mouse was subcutaneously injected with 0.2 mL PBS every 3 days for a total of 3 injections; mRNA vaccine group: Each mouse was subcutaneously injected with mRNA vaccine (mRNA dose of 1 mg / kg) every 3 days for a total of 3 injections; Anti-TGF-β1 antibody group: Each mouse was intravenously injected with anti-TGF-β1 antibody (dose of 5 mg / kg) every 3 days for a total of 2 injections; Combination group: Each mouse was first subcutaneously injected with mRNA vaccine (dose same as the mRNA vaccine group), and one day later was intravenously injected with anti-TGF-β1 antibody (dose same as the anti-TGF-β1 antibody group), with the same dosing frequency as the single-drug group. During this period, the weight of the mice and the length and width of the tumor were measured every other day. The volume of the tumor was calculated according to the formula V=π*(length*width2) / 6. After the experiment, the mice were euthanized, the tumors were removed, and the dissected tumors were photographed and weighed.

[0055] During the experiment, the tumor size, body weight, and activity status of the mice were continuously monitored, and the tumor size was statistically analyzed. Analysis of the treatment effects on the three types of tumors revealed both commonalities and differences. In mouse models constructed from three different tumor cells, the combined use of mRNA and TGF-β1 antibody was found to be more effective than either drug alone.

[0056] Specifically, the combination of mRNA and TGF-β1 antibody significantly inhibited tumor growth. Whether using CC mRNA or CM mRNA, the combination with TGF-β1 antibody showed a clear tumor-inhibiting effect, which became more pronounced with prolonged injection time. This was mainly reflected in: Slowed tumor growth rate: As the injection time of mRNA and TGF-β1 antibody increased, the growth rate of tumor tissue in the experimental group was significantly slower than that in the control group, and the tumor volume growth curve was flatter; Enhanced tumorigenesis inhibition effect: Anatomical results at the experimental endpoint showed that in the tumor-inhibiting mice injected with mRNA, the tumor-inhibiting effect of TGF-β1 antibody was further amplified, and compared with the group without mRNA, malignant tumor proliferation was more effectively controlled; Significantly reduced tumor weight: Weighing the tumor tissue obtained from dissection revealed that the tumor weight in the experimental group was significantly lower than that in the control group, and statistical analysis confirmed that the difference between the two groups was statistically significant (p<0.05).

[0057] Meanwhile, similar to previous findings, the two mRNAs showed different effects: compared to the control group treated alone (or in combination with TGF-β1 antibody), CM mRNA exhibited significantly better tumor-suppressive effects than CC mRNA, suggesting that CM mRNA may have a stronger potential role in regulating tumor growth. In contrast, the conventional treatment control group showed less than ideal therapeutic effects, failing to significantly reduce tumor weight and size. This comparison further highlights the advantages of the "mRNA + TGF-β1 antibody" combination regimen, providing stronger support for the application of this mRNA vaccine in tumor treatment.

[0058] The outcomes also vary depending on the specific type of tumor.

[0059] In the MDA-MB-231 triple-negative breast cancer model, using tumor volume as the core evaluation indicator, the combined treatment group demonstrated a clear and significant tumor-suppressing effect: the growth rate of tumor volume was significantly slowed down. Compared to the control group, where tumor growth was not significantly inhibited and the volume continued to increase, the tumor volume in the combined treatment group mice showed a significant slowdown from the middle of the experiment, successfully inhibiting the growth of xenografts. This result indicates that the combined treatment can directly act on triple-negative breast cancer tumor tissue and intervene in its growth process. The tumor growth rate curve (Figure 3) shows that the tumor volume growth curve in the combined treatment group was extremely flat, while the curve in the control group showed a steep upward trend, with a significant difference between the two. The anatomical results at the experimental endpoint further confirmed that after injection of mRNA, the inhibitory effect of TGF-β1 antibody on the tumorigenesis process was amplified, the malignant proliferation of tumor tissue was effectively controlled, and no obvious invasive growth was observed. The tumor weight statistics showed that the tumor weight in the combined treatment group was significantly lower than that in the control group, and statistical analysis (p<0.05) excluded the interference of random factors, thus quantitatively verifying the tumor-suppressing effectiveness of the combined regimen on triple-negative breast cancer (Table 2).

[0060] Table 2. Tumor volume data (mm3) of MDA-MB-231 (triple-negative breast cancer)

[0061]

[0062] For the H1975 lung cancer model, tumor volume and mouse weight were used as dual core evaluation indicators. The combined treatment group could balance tumor suppression effect and biosafety (Table 3). In terms of tumor volume control, the combined treatment group showed an inhibitory effect on tumor growth from the early stage of the experiment. With the extension of mRNA and TGF-β1 antibody injection time, the growth rate of tumor volume slowed down significantly, and the tumor suppression effect continued to enhance. From the specific data (Figure 4), the tumor growth rate curve showed that the tumor volume growth rate of the experimental group was much slower than that of the control group, and the curve was flat. The anatomical results showed that the malignancy of the tumor tissue in the experimental group was reduced, and the tumor size was significantly smaller than that in the control group. The tumor weight statistics showed that the tumor weight of the experimental group was significantly lower than that of the control group (p<0.05), and compared with the fact that the conventional treatment control group could not reduce the tumor weight and size, the tumor suppression advantage of the combined treatment was more prominent. Throughout the experimental period, the weight of the mice in the combined treatment group increased normally and remained within the normal physiological range. No adverse reactions such as sudden weight loss or lethargy caused by treatment were observed. This result indicates that the combination of "mRNA + TGF-β1 antibody" can effectively inhibit tumor growth without causing significant damage to the mice, demonstrating good biosafety. This is crucial for subsequent clinical applications, mainly because patient tolerance directly affects the duration and final outcome of treatment. Good safety ensures that patients can complete the treatment course, thereby maximizing treatment benefits.

[0063] Table 3. Tumor volume data (mm3) and mouse body weight (g) changes in the H1975 lung cancer model.

[0064]

[0065] Regarding the SW480 colorectal cancer model, the outstanding performance of the combined treatment was: strongest tumor suppression + weight gain (Table 4). Among the three tumor models, the combined treatment effect of the SW480 colorectal cancer model was the most outstanding, mainly reflected in the following two aspects: strongest tumor suppression and best tumor control effect: using tumor volume as an indicator, the tumor volume reduction in the combined treatment group was significantly greater than that in the breast cancer and lung cancer models, and it achieved strong inhibition of tumor growth from the early stage of the experiment. By the end of the experiment, the tumor volume had been reduced to a low level, and the tumor suppression effect far exceeded that of the other two models. Its tumor growth rate was the slowest, the tumor formation inhibition effect was the strongest, and the tumor weight was the lightest, making it the most ideal model in terms of tumor suppression effect. Significant weight gain in mice indicates significant treatment benefits: unlike the breast cancer model where weight was not monitored and the lung cancer model where there was no significant decrease in weight, the combined treatment group of mice in the SW480 colorectal cancer model showed a significant upward trend in weight. The increase in mouse weight is not simply a matter of weight gain, but an important signal of improved health status. This indicates that the combined treatment regimen, while effectively inhibiting tumor growth, also improves the metabolism and nutritional status of mice, reduces the body's consumption caused by the tumor, and achieves the effect of "treating the tumor while repairing the body." This performance far exceeds the "no trend of weight gain" in the control group and is also superior to the combined treatment groups of the other two tumor models, fully demonstrating the unique advantages of this regimen in the treatment of colorectal cancer. Figure 5 ).

[0066] Table 4. Tumor volume data and mouse body weight changes in the SW480 colorectal cancer model.

[0067]

[0068] To elucidate the molecular mechanism by which mRNA inhibits tumor growth, the research team conducted multidimensional experimental analyses on dissected tumor tissues, including immunohistochemical detection, paraffin section preparation (specific procedures followed the standard procedures in the *Handbook of Molecular Biology*), and Western blot analysis. The results showed that the core mechanism of this inhibitory effect is closely related to changes in the expression of chemokines and the infiltration of immune cells within the tumor microenvironment. Specific conclusions are as follows:

[0069] In immunohistochemical experiments, compared with the control group, mice in the experimental group treated with mRNA delivery combined with TGF-β1 antibody showed significant differences in the expression of chemokines in their tumor tissues: on the one hand, the expression level of CCL5 was significantly upregulated compared with the control group; on the other hand, the expression level of CXCL9 was also significantly higher than that of the control group. To verify the reliability of this result, the study further detected the expression of related proteins using Western blot technology. The data obtained were completely consistent with the immunohistochemical conclusions, clearly confirming that the expression level of CXCL9 in tumor tissues was indeed significantly increased after treatment intervention. This finding indicates that mRNA can participate in the regulation of the tumor immune microenvironment by regulating the expression of chemokines.

[0070] In addition, the research team performed immunohistochemical staining on paraffin sections of tumor tissue and observed the distribution characteristics of CD8+ T cells under a microscope. The results showed that the number of CD8+ T lymphocytes in the tumor tissue of the experimental group mice was significantly higher than that in the control group; subsequent immunofluorescence analysis further verified this conclusion, and the data showed that the number of CD8+ T lymphocytes in the experimental group was statistically significantly higher than that in the control group.

[0071] Based on the above experimental results, the following conclusions can be drawn: mRNA induces an increase in the expression levels of chemokines such as CCL5 and CXCL9 within tumor tissues. These chemokines may recruit CD8+ T cells into the tumor tissue. This cell infiltration effect can effectively improve the immune microenvironment of tumor tissues (e.g., enhance the body's immune response against tumor cells), thereby synergistically enhancing the killing efficacy of TGF-β1 antibodies against tumor cells, ultimately achieving further inhibition of tumor growth.

[0072] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and 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 modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A pharmaceutical composition for inhibiting tumor growth, characterized in that, The product comprises active ingredient A and active ingredient B; active ingredient A is a recombinant fusion protein containing an effective amount of messenger ribonucleic acid mRNA formulated in a pharmaceutically acceptable carrier, the messenger ribonucleic acid expressing a recombinant fusion protein of CCL5, CXCL9 as shown in SEQ ID NO. 1, or expressing a recombinant fusion protein of CCL5, CXCL9, CXCL10, IL15, CXCL16, and Myb genes as shown in SEQ ID NO. 2; active ingredient B is an antibody that specifically binds to TGF-β, the amino acid sequence of the light chain variable region of the antibody being shown in SEQ ID NO. 3, and the amino acid sequence of the heavy chain variable region being shown in SEQ ID NO.

4.

2. The pharmaceutical composition according to claim 1, characterized in that, It also contains pharmaceutically acceptable excipients; said excipients are selected from at least one of lipid nanoparticles (LNP), cationic polymers, cholesterol, and phospholipids, for improving the stability and cell transfection efficiency of mRNA vaccines.

3. Use of the pharmaceutical composition of claim 1 or 2 in the preparation of a medicament for inhibiting tumor growth, wherein the tumor is selected from breast cancer, colorectal cancer, or lung cancer.

4. Use of the pharmaceutical composition of claim 1 or 2 in the preparation of a medicament for enhancing tumor-specific immune responses, wherein the tumor is selected from breast cancer, colorectal cancer, or lung cancer.

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