Use of bcl7a in preparation of liver cancer anti-pd-1 treatment sensitization drugs

Abstract

This invention discloses the application of a BCL7A upregulator in the preparation of a sensitizing drug for anti-PD-1 immunotherapy in liver cancer. The upregulator can upregulate BCL7A gene expression or protein activity, including BCL7Am RNA, expression vectors, recombinant proteins, etc. This invention also provides methods for screening and preparing the upregulator, as well as a pharmaceutical composition comprising the BCL7A upregulator and an anti-PD-1 antibody. Experiments have demonstrated that the BCL7A upregulator can significantly enhance the sensitivity of liver cancer cells to PD-1 inhibitors, increase CD8+ T cell infiltration and IFN-γ secretion, and in animal models, the combination therapy showed significantly better tumor suppression than single-agent therapy, reversing immune resistance. This invention provides a novel target and drug for precision immunotherapy of liver cancer.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy, specifically involving the application of the chromatin remodeling complex SWI / SNF (BAF) subunit BCL7A in enhancing the sensitivity and efficacy of liver cancer against PD-1 immunotherapy. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a highly malignant disease, and there is an urgent need for effective clinical treatment. Immune checkpoint inhibitors, represented by anti-PD-1 inhibitors, have become an important treatment for advanced HCC. However, the objective response rate of single-agent therapy is only about 20%, and most patients have primary or secondary immune resistance. Therefore, there is an urgent clinical need for effective sensitization strategies that can improve the treatment response rate.

[0003] The chromatin remodeling complex SWI / SNF (Switch / Sucrose Non-Fermentable) plays a crucial role in tumorigenesis, development, and immune regulation. Existing research indicates that some core subunits of this complex have a negative regulatory function on immunotherapy: ARID1A loss can significantly enhance tumor sensitivity to anti-PD-1 / PD-L1 immunotherapy by remodeling the tumor microenvironment; similarly, the loss or inhibition of BRG1 has been shown to improve immunotherapy efficacy. Based on these findings, a unified technical consensus has been reached: inactivating or inhibiting specific core subunits of the SWI / SNF complex can improve immunotherapy resistance and enhance anti-tumor efficacy.

[0004] However, existing technologies have obvious limitations and cognitive biases:

[0005] 1. One-sided and singular treatment strategies: Existing solutions all revolve around "functional inactivation / inhibition", which can only achieve immune sensitization by weakening the function of specific subunits. They lack positive sensitization targets and intervention strategies based on "functional activation / functional gain", thus limiting the clinical translation pathway.

[0006] 2. Common misconceptions exist: Existing studies incorrectly extrapolate the conclusions of a few subunits such as ARID1A and BRG1 to the entire SWI / SNF complex, ignoring the functional specificity and diversity of different subunits within the complex, thus forming a technical bias that "SWI / SNF subunits all negatively regulate immunotherapy".

[0007] 3. Incomplete target coverage and insufficient specificity: Research is highly focused on core catalytic or scaffold subunits such as ARID1A and BRG1, while the role of specific subunits such as BCL7A, which have significantly different structures and functions, in immunotherapy is completely unknown, making it impossible to develop precise sensitization strategies based on BCL7A.

[0008] In summary, the current single-technology approach, primarily based on "inhibiting the core subunits of SWI / SNF," cannot meet the clinical needs for precise and efficient anti-PD-1 immunotherapy in liver cancer. There is an urgent need in this field to overcome existing cognitive biases, discover novel targets with positive regulatory effects, and establish new "functional gain" immunosensitization strategies to provide new directions for liver cancer immunotherapy. Summary of the Invention

[0009] (a) Technical problems to be solved

[0010] Addressing the shortcomings of existing technologies, which focus solely on the inactivation of SWI / SNF complex subunits, suffer from cognitive bias, and lack positive sensitization targets and strategies, this invention is the first to discover and demonstrate that high expression of BCL7A can positively and specifically enhance the response of liver cancer to anti-PD-1 immunotherapy. It establishes BCL7A as a positive sensitization target for anti-PD-1 therapy in liver cancer, provides upregulators capable of upregulating BCL7A expression or activity and their screening methods, and constructs a synergistic treatment regimen combining BCL7A upregulators with anti-PD-1 antibodies. This approach is suitable for the development and clinical application of sensitizing drugs in precision immunotherapy for liver cancer.

[0011] (II) Technical Solution

[0012] In a first aspect, the present invention provides the application of a BCL7A upregulator in the preparation of a sensitizing drug for anti-PD-1 immunotherapy of liver cancer, wherein the BCL7A upregulator can upregulate the expression or activity of BCL7A.

[0013] Preferably, the BCL7A upregulator is selected from one or more of BCL7A mRNA, BCL7A expression vector, and BCL7A recombinant protein.

[0014] Preferably, the BCL7A mRNA is 5' capped, 3' polyA tailed, and modified with a nucleoside (e.g., using pseudouridine instead of uridine), and then delivered by lipid nanoparticles (LNPs). This method utilizes delivery systems such as lipid nanoparticles, polymer nanoparticles, or exosomes to encapsulate the mRNA, targeting it to the liver and tumor tissue via intravenous or intratumoral injection. The mRNA is translated into BCL7A protein in the cytoplasm, achieving rapid and transient expression.

[0015] Preferably, the expression vector is a viral vector or a non-viral vector, and the vector further contains a liver-specific promoter. Preferably, the expression vector is a lentiviral vector or an adeno-associated virus vector. The vector can carry a liver-specific promoter to enhance targeting. The vector infects tumor cells via hepatic artery perfusion or local injection, achieving long-term, stable expression of the BCL7A gene.

[0016] Preferably, the drug is a combination drug and further comprises a monoclonal antibody (such as nivolumab or pembrolizumab) that targets PD-1 or its ligand PD-L1.

[0017] In a second aspect, the present invention provides a hepatocellular carcinoma anti-PD-1 therapy sensitizing pharmaceutical composition comprising: a BCL7A upregulator and a monoclonal antibody targeting PD-1 or its ligand PD-L1; wherein the upregulator is capable of upregulating the expression or activity of BCL7A; and wherein the upregulator is used to increase the sensitivity of hepatocellular carcinoma to anti-PD-1 therapy.

[0018] Preferably, the BCL7A upregulator is delivered by lipid nanoparticles (LNPs). The lipid nanoparticles (LNPs) consist of ionizable lipids SM102, DSPC, cholesterol, and DMG-PEG2000, with a molar percentage of SM102, DSPC, cholesterol, and DMG-PEG2000 of 45-55:8-12:35-42:1-3, preferably 50:10:38.5:1.5; the BCL7A upregulator is mRNA, with a molar ratio (N / P ratio) of nitrogen in the ionizable lipid SM102 to phosphorus in the mRNA of 4:1-8:1, preferably 6:1; the BCL7A upregulator has a base sequence as shown in SEQ ID NO.4.

[0019] In the lipid nanoparticles (LNPs), only SM102 is an ionizable lipid; DSPC, cholesterol, and DMG-PEG2000 are structural phospholipids, stabilizers, and PEG-modified lipids, respectively, none of which are ionizable lipids. LNPs achieve efficient encapsulation and intracellular release by forming an N / P charge ratio with the ionizable lipid SM102 and mRNA.

[0020] The preparation method of the drug composition delivered by LNP is as follows: SM102, DSPC, cholesterol, and DMG-PEG2000 are dissolved in anhydrous ethanol to obtain an oil phase solution with a total lipid concentration of 5 mg / mL-20 mg / mL; mRNA is dissolved in citrate buffer or sodium acetate buffer (preferably pH 4.0) at pH 3.5-4.5 to obtain an aqueous phase solution with an mRNA concentration of 0.1-0.5 mg / mL; the oil phase and aqueous phase are mixed at a volume ratio of 1:3 using a microfluidic mixer, with an oil phase flow rate of 4-12 mL / min and an aqueous phase flow rate of 12-36 mL / min, allowing the lipids to self-assemble into a nanoparticle suspension. The resulting suspension is concentrated in a 100 kDa ultrafiltration centrifuge tube, and then dialyzed or tangentially filtered with Tris-HCl buffer at pH 7.4 to remove organic solvents and unencapsulated mRNA. Finally, the purified lipid nanoparticles are resuspended in Tris-HCl / sucrose buffer. The concentration of Tris-HCl was 10 mM-50 mM, the pH was 7.4±0.5, and the sucrose concentration was 8%-15% (w / v), preferably 10% (about 300 mM).

[0021] Preferably, the BCL7A upregulator and the monoclonal antibody targeting PD-1 or its ligand PD-L1 can be two separate formulations provided in a co-packaged form, or they can be a combination formulation mixed in the same formulation.

[0022] Thirdly, the present invention provides a method for screening sensitizers for anti-PD-1 therapy in liver cancer, comprising: screening sensitizers with BCL7A as the upregulation target.

[0023] Preferably, the screening method includes:

[0024] a. Construct cell / animal models in which BCL7A expression is positively correlated with anti-tumor immunity;

[0025] b. Treat with the compound to be tested;

[0026] c. Detect BCL7A upregulation and immune markers;

[0027] d. Screening to obtain positive upregulatory agents.

[0028] Fourthly, the present invention provides a method for preparing a BCL7A mRNA-LNP formulation, comprising: preparing a modified mRNA encoding BCL7A, wherein the mRNA is 5' capped, 3' polyA tailed and modified with nucleoside (e.g., using pseudouridine instead of uridine), and then encapsulated with lipid nanoparticles to obtain a liver-targeted sensitizing formulation.

[0029] Preferably, the BCL7A mRNA has the base sequence shown in SEQ ID NO.4.

[0030] (III) Beneficial Effects

[0031] This invention breaks through the conventional understanding that SWI / SNF subunits negatively regulate immunotherapy, and achieves the following core technical effects:

[0032] (1) A new paradigm of “functional gain” immunosensitization was established for the first time: overturning the existing strategy of inactivating ARID1A, BRG1 and other functions, it was discovered and confirmed that high expression of BCL7A can positively and specifically enhance the efficacy of liver cancer against PD-1 therapy, providing a new immunosensitization mechanism for immune-resistant liver cancer.

[0033] (2) Achieve immune microenvironment remodeling and drug resistance reversal: By upregulating BCL7A expression or activity, immune cold tumors can be transformed into immune hot tumors, significantly increasing the number of tumor-infiltrating CD8+ T cells and IFN-γ secretion levels, effectively reversing liver cancer's tolerance to anti-PD-1 therapy.

[0034] (3) Constructing a synergistic combination therapy: Develop a system combining BCL7A upregulators and anti-PD-1 antibodies. The synergistic effect of the two is significantly better than that of single therapy, greatly improving the anti-tumor effect and providing a more efficient combination therapy strategy for clinical practice.

[0035] (4) Provide multiple clinically applicable upregulation agent implementation plans: Establish diversified upregulation pathways, including BCL7A mRNA-LNP delivery and gene vectors, to achieve safe, efficient, and targeted activation of BCL7A function. The LNP-mRNA and other preparations have low immunogenicity, strong targeting, simple preparation process, and cost far lower than traditional cell and virus therapies, making them easy to scale up and popularize in clinical practice.

[0036] In addition, this invention provides a method for screening tumor anti-PD-1 therapy efficacy sensitizers, providing guidance for drug development to improve the responsiveness and efficacy of anti-PD-1 therapy for liver cancer. Attached Figure Description

[0037] Figure 1 This is the experimental result of Example 1, which verifies that overexpression of BCL7A can promote the response of liver cancer cells to CD8+ T cells in a mouse liver cancer model.

[0038] Figure 2 This describes the preparation process of the mRNA drug encapsulated in lipid nanoparticles (LNPs) in Example 2.

[0039] Figure 3 In Example 3, the modified mRNA encoding human BCL7A was combined with an anti-PD-1 antibody in a mouse liver cancer model to verify the therapeutic effect of this combination therapy. Detailed Implementation

[0040] To enable those skilled in the art to fully understand and reproduce the present invention, the technical solution of the present invention will be described in detail and in a non-limiting manner below with reference to specific embodiments. These embodiments cover the entire process from diagnostic and therapeutic drug preparation to efficacy verification, and the specific parameters, materials and operating steps are all based on practically feasible experimental schemes.

[0041] Example 1

[0042] This embodiment verifies in mice that overexpression of BCL7A promotes the response of liver cancer cells to CD8+ T cells. The experimental protocol is as follows:

[0043] 1. Select 6-8 week old BALB / c-nu mice with T lymphocyte deficiency and Rag1 mice lacking mature T cells and B cells. - / - Mice and immunocompetent male C57BL / 6 mice were used. The logarithmically growing mouse hepatocellular carcinoma cell line Hepa1-6 was resuspended in PBS to a concentration of 1.5 × 10^7 cells / mL. 150 μL of the cell suspension (containing 2 × 10^6 cells) was subcutaneously injected into the right side of each mouse.

[0044] 2. Experimental Grouping

[0045] When the tumor volume reached 100±20 mm³ (approximately 7-10 days after inoculation), the mice were randomly divided into 18 groups of 4 mice each, and the effects of BCL7A overexpression and BCL7A knockdown on hepatocellular carcinoma formation and immune microenvironment were evaluated.

[0046] C57BL / 6 mouse blank control group (NC): Hepa1-6-NC cells were subcutaneously injected.

[0047] C57BL / 6 mice overexpressing BCL7A (OE): Hepa1-6-OE cells were subcutaneously injected.

[0048] BALB / c-nu mouse blank control group (NC): Hepa1-6-NC cells were subcutaneously injected.

[0049] BALB / c-nu mice overexpressing BCL7A (OE): subcutaneously inoculated with Hepa1-6-OE cells.

[0050] Rag1 - / - Mouse blank control group (NC): Hepa1-6-NC cells were subcutaneously injected.

[0051] Rag1 - / - Mouse BCL7A overexpression group (OE): Hepa1-6-OE cells were subcutaneously inoculated.

[0052] C57BL / 6 mouse blank control group (PLKO.1): Hepa1-6-PLKO.1 cells were subcutaneously injected.

[0053] C57BL / 6 mice with BCL7A knockdown group 1 (shBCL7A-1): Hepa1-6-shBCL7A-1 cells were subcutaneously inoculated.

[0054] C57BL / 6 mice with BCL7A knockdown group 2 (shBCL7A-2): Hepa1-6-shBCL7A-2 cells were subcutaneously inoculated.

[0055] BALB / c-nu mouse blank control group (PLKO.1): Hepa1-6-PLKO.1 cells were subcutaneously injected.

[0056] BALB / c-nu mice with BCL7A knockdown group 1 (shBCL7A-1): Hepa1-6-shBCL7A-1 cells were subcutaneously inoculated.

[0057] BALB / c-nu mice with BCL7A knockdown group 2 (shBCL7A-2): Hepa1-6-shBCL7A-2 cells were subcutaneously inoculated.

[0058] Rag1 - / - Mouse blank control group (PLKO.1): Hepa1-6-PLKO.1 cells were subcutaneously injected.

[0059] Rag1 - / - Mouse BCL7A knockdown group 1 (shBCL7A-1): Hepa1-6-shBCL7A-1 cells were subcutaneously inoculated.

[0060] Rag1 - / - Mouse BCL7A knockdown group 2 (shBCL7A-2): Hepa1-6-shBCL7A-2 cells were subcutaneously inoculated.

[0061] The sequences of PLKO.1, shBCL7A-1, and shBCL7A-2 in the above experiment are as follows:

[0062] PLKO.1 (SEQ ID NO.1) CCTCGATGGAACATTCGAT shBCL7A-1 (SEQ ID NO.2) CATCCCTACGAATCTACAA shBCL7A-2 (SEQ ID NO.3) CTATCGAGAAAGTGCGCAA

[0063] 3. Efficacy monitoring and endpoint analysis

[0064] Tumor monitoring: Measure the major diameter (L) and minor diameter (W) of the tumor every 2 days using calipers, and calculate the tumor volume using the formula V = (L×W^2) / 2. Plot a tumor growth curve.

[0065] 4. Mechanism exploration (flow cytometry analysis)

[0066] After treatment (day 22), mice were sacrificed and tumor tissue was collected. Single-cell suspension was prepared by cutting the tumor tissue into small pieces, digesting it with a digestive solution containing collagenase IV and DNase at 37°C for 30 minutes, and then passing it through a 70μm cell sieve to obtain a single-cell suspension.

[0067] Antibody staining: Cells are stained using fluorescently labeled antibodies. Key antibodies include: CD45 (leukocyte marker), CD3 (T cells), CD8 (cytotoxic T cells), and CD4 (helper T cells).

[0068] Data acquisition was performed using a flow cytometer, and then analyzed using FlowJo software.

[0069] 5. Experimental results, such as Figure 1 As shown.

[0070] Figures A and B show the expression of related proteins in Hepa1-6 cells overexpressing and knocking down BCL7A, respectively. In Figure A, the BCL7A protein expression level in Hepa1-6-OE cells overexpressing BCL7A is significantly higher than that in Hepa1-6-NC cells. Figure B shows that the BCL7A protein expression level in Hepa1-6-PLKO.1 cells is higher than that in knockdown group 1 Hepa1-6-shBCL7A-1 cells and knockdown group 2 Hepa1-6-shBCL7A-2 cells. Figure C shows the effect of BCL7A overexpression on tumor growth morphology and tumor weight in vivo (overexpression group OE and control group NC). The results indicate that in immunocompetent C57BL / 6 mice, BCL7A overexpression significantly inhibits the tumorigenicity of liver cancer cells (OE group weight less than 0.1g, NC weight approximately 0.25g). However, in BALB / c-nu mice (overexpression group OE weight and control group NC weight both approximately 0.45g) and Rag1 mice... - / - In mice (OE and NC groups, weight approximately 0.35g each). This indicates that in T lymphocyte-deficient BALB / c-nu mice, Rag1, lacking mature T cells and B cells... - / - In mice, overexpression of BCL7A did not affect tumor size. Figure D shows the effect of BCL7A knockdown on tumor growth morphology and tumor weight in different mice (knockdown groups shBCL7A-1, shBCL7A-2 and control group PLKO.1). The results indicate that in immunocompetent C57BL / 6 mice, BCL7A knockdown significantly promoted the tumorigenicity of liver cancer cells (shBCL7A-1 group weight approximately 0.12 g, shBCL7A-2 group weight approximately 0.18 g, PLKO.1 weight approximately 0.08 g). However, in BALB / c-nu mice (shBCL7A-1, shBCL7A-2 and PLKO.1 weights approximately 0.28 g each) and Rag1...- / - In mice (shBCL7A-1, shBCL7A-2 and PLKO.1 groups, each weighing approximately 0.29 g). This indicates that Rag1 in T lymphocyte-deficient BALB / c-nu mice, lacking mature T cells and B cells... - / - In mice, BCL7A knockdown did not affect tumor size. EF plots show the flow cytometry analysis results of tumor tissues from the above groups, analyzing changes in the immune microenvironment within the tumor tissues. The analysis revealed that BCL7A overexpression significantly increased the proportion of CD45+ immune cells and CD8+ T cells; conversely, BCL7A knockdown reduced the infiltration of CD45+ and CD8+ T cells. GH plots show the results of CD8+ T cell immunofluorescence staining in tumor tissues from each group. The staining results showed that BCL7A overexpression promoted CD8+ T cell infiltration, while BCL7A knockdown (shBCL7A-1, shBCL7A-2) inhibited CD8+ T cell infiltration.

[0071] The above results confirm that BCL7A not only directly inhibits tumor growth, but more importantly, it reshapes the immune microenvironment and significantly increases the infiltration of CD8+ T cells. Driving the transformation of "cold tumors" into "hot tumors" helps improve the sensitivity of liver cancer to immune checkpoint inhibitors.

[0072] Example 2

[0073] This embodiment provides a modified mRNA encoding human BCL7A, the sequence of which is SEQ ID NO.4. This mRNA is 5' capped, 3' polyA tailed, and modified with nucleoside. The nucleoside modification uses pseudouridine to replace uridine. The mRNA is synthesized and modified using existing technologies, and then encapsulated with lipid nanoparticles (LNPs) to obtain BCL7A mRNA-LNP, thus yielding a targeted delivery sensitizing agent. The mRNA sequence represented by SEQ ID NO.4 is as follows:

[0074] .

[0075] The lipid nanoparticles (LNPs) consist of ionizable lipid SM102, DSPC, cholesterol, and DMG-PEG2000, with a molar percentage of SM102, DSPC, cholesterol, and DMG-PEG2000 of 45-55:8-12:35-42:1-3, and in this embodiment, 50:10:38.5:1.5. The molar ratio (N / P ratio) of nitrogen in the ionizable lipid SM102 to phosphorus in the mRNA is 4:1-8:1, and in this embodiment, it is 6:1.

[0076] The preparation method of the lipid nanoparticles (LNPs) encapsulating mRNA drugs is as follows: SM102, DSPC, cholesterol, and DMG-PEG2000 are dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 to obtain an oil phase solution, with a total lipid concentration of 5 mg / mL-20 mg / mL; mRNA is dissolved in citrate buffer or sodium acetate buffer (preferably pH 4.0) at pH 3.5-4.5 to obtain an aqueous phase solution, with an mRNA concentration of 0.1 mg / mL-0.5 mg / mL. The oil phase and aqueous phase are mixed at a volume ratio of 1:3 using a microfluidic mixing device, with an oil phase flow rate of 5 mL / min and an aqueous phase flow rate of 15 mL / min, allowing the lipids to self-assemble into nanoparticles. Then, after concentration in 100 kDa ultrafiltration centrifuge tubes, the nanoparticles were dialyzed with pH 7.4 Tris-HCl buffer to remove organic solvents and unencapsulated mRNA. Finally, the purified lipid nanoparticles were resuspended in Tris-HCl / sucrose buffer with a Tris-HCl concentration of 25 mM, pH 7.4 ± 0.5, and a sucrose concentration of 10% (approximately 300 mM).

[0077] like Figure 2 The figure shows the particle size and integrity of the lipid nanoparticles prepared by this method. The average particle size is 65nm-125nm, the polydispersity index is less than 0.2, and the encapsulation efficiency is greater than 85%. When stored at -80℃, sucrose as a cryoprotectant can effectively maintain the structural integrity of the nanoparticles. After freeze-thaw, the particle size does not increase significantly and the encapsulation efficiency remains stable.

[0078] Example 3

[0079] In this embodiment, the mRNA represented by SEQ ID NO.4 in Example 2, modified with a 5' cap, 3' polyA tail, and nucleoside, was combined with an anti-PD-1 antibody (mouse PD-1 monoclonal antibody, Bio X Cell, clone number: 29F.1A12, catalog number: BE0273) in a mouse hepatocellular carcinoma model to verify the therapeutic effect of this combination therapy in an immunocompetent mouse model of hepatocellular carcinoma. The combination therapy methods included delivering the mRNA and mouse PD-1 monoclonal antibody using an LNP, or directly delivering the mRNA and mouse PD-1 monoclonal antibody without using an LNP. Blank controls were provided for "injection of PBS only with an equal volume of LNP" and "injection of PBS only". The specific experimental protocol is as follows:

[0080] 1. Select 6-8 week old male C57BL / 6 mice. Take the Hepa1-6 mouse hepatocellular carcinoma cell line in the logarithmic growth phase and resuspend it in PBS to 5×10^6 cells / mL. Subcutaneously inject 100μL of cell suspension (containing 2×10^6 cells) into the right side of each mouse.

[0081] 2. Experimental grouping and dosing regimen

[0082] When the tumor volume grows to 100±20mm³ (approximately 7-10 days after inoculation), the mice are randomly divided into 8 groups of 5-6 mice each.

[0083] Blank control group 1 (NC): PBS (equal volume to other groups) was injected via the tail vein, and IgG2a isotype control antibody (mouse IgG2a monoclonal antibody, Bio X Cell, clone number: 2A3, catalog number: BE0) was injected intraperitoneally.

[0084] Blank control group 2 (LNP-NC): PBS and an equal volume of LNP were injected via the tail vein, and IgG2a control antibody was injected intraperitoneally.

[0085] The single-drug anti-PD-1 group (αPD-1) was administered PBS via tail vein injection and anti-mouse PD-1 antibody, 200 μg / dose, via intraperitoneal injection, once every 3 days for a total of 4 times.

[0086] The single-drug anti-LNP-PD-1 group (LNP-αPD-1) was administered PBS via tail vein injection and LNP-encapsulated anti-mouse PD-1 antibody via intraperitoneal injection. The anti-mouse PD-1 antibody dose was 200 μg, administered every 3 days for a total of 4 times.

[0087] The single-drug BCL7A mRNA group (BCL7A): BCL7A mRNA (lipid-free LNP delivery vector) prepared in Example 2 was injected via tail vein at a dose of 0.3 mg mRNA / kg body weight twice on days 1 and 4; IgG2a control antibody was injected intraperitoneally.

[0088] The single-drug BCL7A mRNA-LNP group (LNP-BCL7A) was administered via tail vein injection of BCL7A mRNA-LNP prepared in Example 2 at a dose of 0.3 mg mRNA / kg body weight, twice on days 1 and 4; and intraperitoneal injection of IgG2a control antibody.

[0089] The combined treatment group (BCL7A mRNA + αPD-1) received BCL7A mRNA via tail vein injection (without LNP) and anti-PD-1 antibody via intraperitoneal injection.

[0090] The combined treatment group (LNP-BCL7A mRNA+αPD-1) received BCL7A mRNA-LNP (liposome modified) via tail vein injection and anti-PD-1 antibody via intraperitoneal injection.

[0091] The dosage and frequency of administration of BCL7A mRNA and anti-PD-1 antibody were the same in all the above-mentioned treatment groups.

[0092] Dosage and time are the same as for Group mRNA

[0093] 3. Efficacy monitoring and endpoint analysis

[0094] Tumor monitoring: Measure the major diameter (L) and minor diameter (W) of the tumor every two days using calipers, and calculate the tumor volume using the formula V=(L×W^2) / 2. Plot a tumor growth curve.

[0095] 4. Mechanism exploration (flow cytometry analysis)

[0096] After treatment (day 22), mice were sacrificed and tumor tissue was collected. Single-cell suspension was prepared by cutting the tumor tissue into small pieces, digesting it with a digestive solution containing collagenase IV and DNase at 37°C for 30 minutes, and then passing it through a 70μm cell sieve to obtain a single-cell suspension.

[0097] Antibody staining: Cells are stained using fluorescently labeled antibodies. Key antibodies include: CD45 (leukocyte marker), CD3 (T cells), CD8 (cytotoxic T cells), CD4 (helper T cells), and IFN-γ (staining after cell stimulation and cell rupture).

[0098] Data acquisition was performed using a flow cytometer, and then analyzed using FlowJo software.

[0099] 5. Experimental results, such as Figure 3 As shown.

[0100] Figure A shows the statistical analysis of tumor growth morphology and volume in vivo (BCL7A monotherapy and anti-PD-1 monotherapy). The left image is a photograph of the xenografts in C57BL / 6 mice, comparing the tumor size of the control group (NC), the BCL7A monotherapy group, the anti-PD-1 antibody monotherapy group, and the BCL7A+PD-1 combination group. The right image is a bar chart of tumor weight. The NC group (negative control) had the largest tumor volume / weight (approximately 0.3g). Both the BCL7A monotherapy group and the anti-PD-1 antibody monotherapy group showed some inhibition of tumor growth, with tumor weights of 0.2g and 0.21g, respectively, but the tumor burden remained high. The combination group (BCL7A+PD-1) had the smallest tumor volume / weight, only 0.13g, demonstrating a significantly better inhibitory effect than any single-drug group. This indicates that BCL7A and anti-PD-1 antibodies have a synergistic anti-tumor effect in vivo, and combined treatment can significantly inhibit the proliferation of liver cancer xenografts, with an effect greater than the sum of its parts (1+1>2).

[0101] Figure B illustrates the in vivo efficacy validation of BCL7A mediated by the LNP delivery system. The LNP-NC group (blank control), LNP-BCL7A group, and LNP-BCL7A+PD-1 group all utilized the LNP delivery system. Results showed that the tumor in the LNP-NC group (blank control) grew rapidly and had the largest volume. The LNP-BCL7A group, using LNP-delivered BCL7A alone, showed a significant tumor-suppressing effect. The combination of LNP-BCL7A and PD-1 resulted in the highest tumor inhibition rate, with a statistically significant difference. (p < 0.001). This confirms that the LNP vector can effectively deliver BCL7A to exert its therapeutic effect in vivo, and that LNP-BCL7A combined with anti-PD-1 antibody is a highly effective and safe synergistic in vivo treatment regimen.

[0102] Figures C and D show flow cytometry analysis of tumor-infiltrating immune cells (T cell killing and infiltration). Figures C and D are IFN-γ+-FTIC-CD8+ dot plots, designed to detect the function of cytotoxic T cells (CD8+ T cells). The results showed that the CD8+ T cell function in the combination therapy group (BCL7A+PD-1 or LNP-BCL7A+PD-1) was significantly higher than that in the single-drug groups (BCL7A, LNP-BCL7A, αPD-1, LNP-αPD-1) and the blank control group (NC or LNP-NC). This indicates that upregulating BCL7A can recruit and activate CD8+ cytotoxic T cells in the tumor microenvironment, and that BCL7A enhances the efficacy of anti-PD-1 therapy by relieving immunosuppression through increased T cell infiltration and function.

[0103] Figure E shows the expression levels of immune-related signaling pathway proteins detected by Western blotting, illustrating the expression levels of immune and apoptosis-related proteins (BCL7A, β-actin, IRF3, STAT1, p-STAT1, STING, GAS, GAPDH) in tumor tissue. The results showed that the BCL7A protein level was significantly increased in the LNP-BCL7A treatment group, validating successful delivery. Furthermore, the LNP-BCL7A treatment group significantly upregulated the expression of key immune factors such as p-IRF3, p-STING, STING, IRF3, and p-STAT1, and significantly increased the levels of downstream effector molecules (such as c-GAS). This indicates that the molecular mechanism of BCL7A sensitization involves activation of the STING-IRF3-STAT1 innate immune pathway. Activation of this pathway is the core molecular basis for inducing inflammatory responses in the tumor microenvironment, recruiting T cells, and reversing immune resistance.

[0104] The above experiments demonstrate that BCL7A upregulators (such as LNP-BCL7A mRNA), by activating the STING / IRF3 / STAT1 signaling pathway, not only directly inhibit tumor growth but, more importantly, reshape the immune microenvironment, significantly increasing the infiltration and activation of CD8+ T cells. This transformation from "cold tumor" to "hot tumor" allows anti-PD-1 antibodies to exert their killing effect more effectively, ultimately achieving synergistic effects.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of chromatin remodeling factor BCL7A upregulators in the preparation of sensitizing drugs for anti-PD-1 immunotherapy in liver cancer, characterized in that, The BCL7A upregulator can upregulate the expression or activity of BCL7A.

2. The application according to claim 1, characterized in that, The BCL7A upregulator is selected from one or more of BCL7A mRNA, BCL7A expression vector, and BCL7A recombinant protein.

3. The application according to claim 2, characterized in that, The BCL7A mRNA is 5' capped, 3' polyA tailed, and nucleoside modified, and delivered by lipid nanoparticles (LNPs). The LNPs consist of ionizable lipids SM102, DSPC, cholesterol, and DMG-PEG2000, with a molar percentage of 45-55:8-12:35-42:1-3. The BCL7A mRNA has the base sequence shown in SEQ ID NO.

4.

4. The application according to claim 2, characterized in that, The expression vector is a viral vector or a non-viral vector, and the vector also contains a liver-specific promoter.

5. The application according to claim 4, characterized in that, The expression vector is a lentiviral vector or an adeno-associated virus vector.

6. The application according to claim 1, characterized in that, The drug is a combination drug and also contains a monoclonal antibody that targets PD-1 or its ligand PD-L1.

7. A drug composition for enhancing the sensitivity of hepatocellular carcinoma to anti-PD-1 therapy, characterized in that, It includes: BCL7A upregulator and a monoclonal antibody targeting PD-1 or its ligand PD-L1; the upregulator is capable of upregulating the expression or activity of BCL7A; the upregulator is used to increase the sensitivity of liver cancer to anti-PD-1 therapy.

8. A method for screening sensitizers for anti-PD-1 therapy in liver cancer, characterized in that, include: Sensitizers were screened with BCL7A as the upregulation target.

9. The screening method according to claim 8, characterized in that, include: a. Construct cell / animal models in which BCL7A expression is positively correlated with anti-tumor immunity; b. Treat with the compound to be tested; c. Detect BCL7A upregulation and immune markers; d. Screening to obtain positive upregulatory agents.

10. A method for preparing a BCL7A mRNA-LNP formulation, comprising: A modified mRNA encoding BCL7A was prepared. The mRNA was 5' capped, 3' polyA tailed, and nucleoside modified, and then encapsulated with lipid nanoparticles to obtain a targeted delivery sensitizing agent. The BCL7A mRNA has the base sequence shown in SEQ ID NO.4.

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