An mRNA-lipid nanoparticle delivery system encoding an anti-b7h3 / cd3 bispecific antibody and its use in treating breast cancer

Abstract

The application discloses an mRNA-LNP delivery system for coding anti-B7H3 / CD3 bispecific antibody and application thereof in treatment of breast cancer, and particularly relates to an optimized mRNA sequence for coding anti-B7H3 / CD3 bispecific antibody and a lipid nanoparticle (LNP) delivery system. The application solves the problems of weak specificity and short half-life of existing therapies in the treatment of breast cancer, and provides a novel immunotherapy capable of efficiently activating endogenous T cells to kill breast cancer cells. Single intravenous injection can realize in-vivo sustained expression of BiTE, and the half-life is about 72 hours, which is significantly longer than that of recombinant BiTE (about 2 hours), and repeated administration is not needed. B7H3 is highly expressed in breast cancer cells and lowly expressed in normal tissues, so that the off-target effect is reduced; the LNP has good biocompatibility and does not cause obvious damage to normal organs. The system can efficiently activate endogenous T cells to specifically kill breast cancer cells through LNP delivery of the optimized mRNA, and solves the problems of poor specificity and short half-life of traditional therapies.

Description

Technical Field

[0001] This invention belongs to the field of medical technology and relates to an mRNA-LNP delivery system encoding an anti-B7H3 / CD3 bispecific antibody and its application in the treatment of breast cancer. Background Technology

[0002] (1) Current status and limitations of bispecific antibody therapy

[0003] In recent years, immunotherapy based on bispecific T-cell binders (BiTEs) has shown great potential in the treatment of malignant tumors. BiTE molecules have two antigen-binding arms: one binds to a specific antigen on the surface of tumor cells, and the other binds to a marker antigen (such as CD3) on the surface of effector T cells. This activates effector cells and recruits T cells to form cytolytic synapses with tumor cells, exerting a specific killing effect. However, this protein-based immunotherapy faces several challenges in the treatment of solid tumors: First, BiTE proteins have an extremely short half-life in vivo, usually requiring long-term, continuous intravenous infusion to maintain effective blood drug concentrations, which not only leads to high treatment costs but also reduces patient compliance; second, protein drugs have poor stability in vivo and are difficult to effectively penetrate dense solid tumor tissue, limiting their anti-tumor efficacy.

[0004] (2) Advantages of mRNA-LNP technology

[0005] To overcome the aforementioned shortcomings of protein drugs, in vitro transcription of mRNA (IVT-mRNA) technology has attracted considerable attention as an alternative strategy. mRNA offers advantages such as simple production processes, rapid development, no risk of genomic integration, and the elimination of the need for complex recombinant protein purification facilities. However, naked mRNA molecules are extremely unstable, easily degraded by RNases widely present in the body, and struggle to cross cell membranes to enter the cytoplasm for translation. Therefore, developing efficient delivery systems is crucial. Currently, lipid nanoparticles (LNPs) are the most clinically mature mRNA delivery system. The successful approval of the SARS-CoV-2 mRNA vaccines developed by Pfizer / BioNTech and Moderna has fully validated the safety and efficacy of the LNP-mRNA platform. Nevertheless, current mRNA delivery systems for cancer treatment still require further optimization to improve their targeting and expression efficiency in tumor tissues, while reducing potential hepatotoxicity or immunogenicity.

[0006] (3) Triple-negative breast cancer and B7H3 target

[0007] Breast cancer is the most common malignant tumor among women worldwide. Triple-negative breast cancer (TNBC, which is negative for estrogen receptor, progesterone receptor, and HER2) accounts for approximately 15%-20% of all breast cancers. TNBC is characterized by its high invasiveness, high recurrence rate, and poor prognosis. Furthermore, due to the lack of clear targeted therapies, clinical treatment options are very limited, mainly relying on chemotherapy. There is an urgent need to develop novel immunotherapies.

[0008] B7 homolog 3 protein (B7H3, also known as CD276) is a member of the B7 immune checkpoint protein family. It has been shown to be abnormally overexpressed in various solid tumors, but its expression is extremely low or absent in normal tissues. Particularly in breast cancer, overexpression of B7H3 is closely associated with tumor growth, metastasis, and poor prognosis, and it helps tumor cells evade immune surveillance. This makes B7H3 an ideal specific target for the treatment of triple-negative breast cancer.

[0009] (4) Technical requirements of the present invention

[0010] While monoclonal antibodies or CAR-T therapies targeting B7H3 are in preclinical or clinical research stages, research on using LNPs to deliver mRNA encoding B7H3×CD3 BiTE for breast cancer treatment is still in its early stages. In existing technologies, how to screen for specific mRNA sequence structures with highly efficient killing effects on breast cancer cells and combine them with optimized LNP vectors to achieve long-term drug expression and tumor suppression in vivo is a pressing technical challenge that needs to be solved. Based on this, this invention aims to provide a novel LNP-mRNA drug that, through in situ expression of anti-B7H3 bispecific antibodies in vivo, offers a safe, efficient, and cost-effective new approach for the treatment of triple-negative breast cancer.

[0011] Disadvantages of existing technology:

[0012] (1) Limitations of protein drugs: Recombinant BiTE protein has a very short half-life, poor patient compliance, and is difficult to penetrate deep into solid tumors.

[0013] (2) Differences in sequence and efficacy: Although the B7H3 target is known, different scFv (single-chain antibody) linker sequences, linker designs, and untranslated region (UTR) optimizations can significantly affect mRNA translation efficiency and the final cell-killing ability. Existing technologies have not disclosed specific sequence designs that have the best killing activity for breast cancer cell lines (such as MDA-MB-231 and MCF-7).

[0014] (3) Indication gap: Existing LNP@BiTE-mRNA research mainly focuses on hematologic malignancies or melanomas, and lacks systematic pharmacodynamic data in breast cancer models. Summary of the Invention

[0015] The purpose of this invention is to provide an mRNA-LNP delivery system encoding an anti-B7H3 / CD3 bispecific antibody and its application in the treatment of breast cancer. Specifically, it provides an optimized mRNA sequence encoding an anti-B7H3×CD3 bispecific antibody, combined with a lipid nanoparticle (LNP) delivery system. This invention addresses the problems of low specificity and short half-life in existing breast cancer treatments, providing a novel immunotherapy that can efficiently activate endogenous T cells to kill breast cancer cells.

[0016] To achieve the objective of this invention, the following technical solution is adopted: a bispecific antibody encoding anti-B7H3 / CD3, characterized in that it is a B7H3×CD3 BiTE encoding sequence; its sequence is shown in the table below:

[0017]

[0018]

[0019]

[0020] The present invention demonstrates the specific targeting effect of the B7H3×CD3 BiTE coding sequence in the preparation of breast cancer therapeutic drugs.

[0021] This invention provides a method for preparing mRNA-loaded LNPs, comprising the following steps: dissolving a lipid mixture (LNP) in anhydrous ethanol as the organic phase; dissolving B7H3×CD3 mRNA in 10 mM pH 3.0 citrate buffer as the aqueous phase; the organic phase N / aqueous phase P volume ratio is 6; using a microfluidic mixing device, the flow rate ratio of the aqueous phase to the organic phase is set to 3:1, with the aqueous phase at 9 mL / min and the organic phase at 3 mL / min, for a total flow rate of 12 mL / min; after mixing via the microfluidic device, the mixture is dialyzed and filtered to obtain mRNA-loaded LNPs (abbreviated as LNP@BiTE-mRNA).

[0022] The application of the mRNA-loaded LNPs prepared by the above-described preparation method of the present invention in the preparation of breast cancer therapeutic drugs.

[0023] The application of the LNP-mRNA delivery system encoding an anti-B7H3 / CD3 bispecific antibody described above in this invention for the efficient expression of BiTE in vivo includes in vitro cell killing and in vivo anti-tumor therapy.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1. Low production cost: mRNA is synthesized through in vitro transcription, which is a simple and flexible process that does not require complex recombinant protein purification procedures;

[0026] 2. High transfection efficiency: The LNP delivery system can efficiently deliver mRNA to the cytoplasm;

[0027] 3. Long-lasting therapeutic effect: BiTE can be continuously expressed in the body with a single intravenous injection, with a half-life of about 72 hours, which is significantly longer than that of recombinant BiTE (about 2 hours), eliminating the need for repeated administration;

[0028] 4. High specificity and low toxicity: B7H3 is highly expressed in breast cancer cells and lowly expressed in normal tissues, reducing off-target effects; LNP has good biocompatibility and does not cause significant damage to normal organs.

[0029] 5. Excellent stability: LNP@BiTE-mRNA can be stably stored at 4℃ for 1 month, which facilitates transportation and clinical application.

[0030] 6. Linker replacement: (G4S) n linkers of different lengths (n=1-3) can be used to replace the original 5 amino acid linkers, while still ensuring the spatial conformation and binding activity of B7H3 scFv and CD3 scFv;

[0031] 7. LNP component adjustment: A small amount of targeting molecules can be added to the LNP to further improve the targeted delivery efficiency;

[0032] mRNA modification optimization: Other nucleoside modifications can be used to replace N1-methylpseuuridine, which can also reduce the immunogenicity of mRNA and improve expression efficiency. Attached Figure Description

[0033] Figure 1 mRNA design for B7H3 targeting bispecific T cell adaptors.

[0034] Figure 2 In vitro functional assessment of mRNA encoding B7H3-TCE.

[0035] Figure 3 In vitro validation of the efficacy of LNP@BiTE-mRNA.

[0036] Figure 4 This is an in vitro comparison of circRNA-TCE versus linear mRNA.

[0037] Figure 5 In vitro screening of LNP formulations.

[0038] Figure 6 To assess the in vitro efficacy and systemic safety of LNP-circRNA formulations.

[0039] Figure 7 Transcriptomic landscape of T cells versus linear mRNA in response to circRNA-TCE.

[0040] Figure 8 The in vivo anti-tumor efficacy in humanized xenotransplantation models.

[0041] Figure 9 Mechanism / Schematic Diagram (drawn using SIDER).

[0042] Figure 10 Agarose gel electrophoresis analysis of MGA271, 376.96, TE9 and CD3 mRNA.

[0043] Figure 11 To screen different IRES elements for translation efficiency.

[0044] Figure 12 3′UTR sequence optimization was performed to enhance circRNA translation efficiency.

[0045] Figure 13 This study compares the transfection efficiency and protein expression of eGFP circRNA and linear mRNA.

[0046] Figure 14 This study investigated T cell-dependent cytotoxicity mediated by circRNA-TCE at different effector-target ratios.

[0047] Figure 15 Representative microscopic images of T cell-mediated cytotoxicity.

[0048] Figure 16 The cytotoxic activity of HEK293T cells transfected with 500 ng circRNA in conditioned medium against multiple cell types was determined.

[0049] Figure 17 To demonstrate the dose-dependent and target-specific cytotoxicity of circRNA-TCE induced in vitro.

[0050] Figure 18 Characterization of the physicochemical properties of LNP formulations.

[0051] Figure 19 For in vitro protein expression: kinetic curves of TCE secretion into culture supernatant in cells transfected with different LNPs within 48 hours.

[0052] Figure 20 circRNA-TCE exhibited excellent serum stability and sustained protein expression both in vitro and in vivo.

[0053] Figure 21To monitor mouse body weight in single-dose and multiple-dose safety studies.

[0054] Figure 22 Representative H&E stained images of tumor sections harvested at the end of the study show extensive necrosis in the TCE treatment group. Detailed Implementation

[0055] The present invention will be further described below through specific embodiments.

[0056] Example 1: Preparation of mRNA encoding B7H3×CD3 BiTE

[0057] 1. Design the mRNA coding sequence of B7H3×CD3 BiTE: including secretory peptide sequence, B7H3 scFv sequence (also known as B7H3 specific single-chain antibody fragment (scFv) sequence), (G4S) linker sequence (also known as linker sequence), and CD3scFv sequence (also known as CD3 specific single-chain antibody fragment (scFv) sequence), wherein the linker is a peptide composed of 5 amino acids (G4S);

[0058] The synthesis of the above mRNA was performed via in vitro transcription. The co-transcriptional capping reaction was carried out in a 20 μL system containing 1× transcription buffer (40 mM Tris-HCl (pH 8.0), 20 mM MgCl2, 2.5 mM TCEP, 2 mM spermidine), 500 ng IVT template, 2.5 U / μL T7 RNA polymerase, 0.01 U / μL inorganic pyrophosphatase, 5 mM each of ribonucleotides, and 4 mM cap structure analog (CAG Trimer (m 7 G(5')ppp(5')(2'OMeA)pG), purchased from Novizan. The transcription reaction was incubated at 37°C for 2 hours, followed by treatment with deoxyribonuclease I at a final concentration of 0.04 U / μL at 37°C for 30 minutes. The synthesized mRNA was purified using magnetic beads. The above mRNA can also be purchased directly from APExBio.

[0059]

[0060] 3. In vitro transcription synthesis of mRNA: Using the optimized coding sequence cDNA as a template, in vitro transcription was performed using the T7 High Yield RNA Transcription Kit. After transcription, the mRNA was modified by capping and poly (A) tailing. The reaction system was 20 μL, and the reaction was incubated at 37°C for 4 hours to obtain the in vitro transcribed mRNA.

[0061] 4. Purification: The in vitro transcribed mRNA obtained in step 3 was precipitated with 8M LiCl, the precipitate was collected by centrifugation, washed with 75wt% ethanol, dried and dissolved in RNase-free water, and the purity was verified by agarose electrophoresis. The A260 / A280 ratio was 1.8-2.0 as determined by Nanodrop.

[0062] Table 1: mRNA coding sequence information of B7H3×CD3 BiTE

[0063]

[0064]

[0065]

[0066] In this invention, all sequences are assembled from known sequences, and then we use the circular RNA method to make them applicable.

[0067] Example 2: Preparation and characterization of LNPs loaded with mRNA

[0068] 1. Preparation of LNP (lipid mixture): The organic phase of LNP (lipid mixture) was prepared by molar ratio of 50 mol% SM-102, 10 mol% 1,2-distearate phosphatidylcholine (DSPC), 38.5 mol% cholesterol (Cholesterol), and 1.5 mol% DMG-PEG-2000; the 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid-1-octylnonyl ester is abbreviated as "SM-102"; the 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 is abbreviated as (DMG-PEG-2000).

[0069] 2. Microfluidic synthesis:

[0070] The lipid mixture (LNP) obtained in step 1 was dissolved in anhydrous ethanol as the organic phase; the B7H3×CD3 mRNA obtained in Example 1 was dissolved in 10 mM citrate buffer (pH 3.0) as the aqueous phase (organic phase N / aqueous phase P ratio was 6). A microfluidic mixing device was used, with the flow rate ratio of aqueous phase to organic phase set at 3:1 (aqueous phase 9 mL / min, ethanol phase (organic phase) 3 mL / min, total flow rate 12 mL / min). After mixing through the microfluidic device, the mixture was dialyzed and filtered to obtain the mRNA-loaded LNP (abbreviated as LNP@BiTE-mRNA).

[0071] 3. Dialysis and characterization:

[0072] LNP solutions loaded with mRNA were dialyzed overnight in 1×PBS at 4°C to remove ethanol and replace the buffer.

[0073] Particle size was determined using dynamic light scattering.

[0074] mRNA encapsulation efficiency was determined using the RiboGreen method.

[0075] 4. Application of LNP@BiTE-mRNA in Breast Cancer Treatment

[0076] 1. In vitro validation: LNP@BiTE-mRNA was co-incubated with breast cancer cell lines (such as MDA-MB-231, MCF-7) and human primary T cells to activate T cells and induce them to kill breast cancer cells;

[0077] 2. In vivo validation: 1.5 mg / kg LNP@BiTE-mRNA was intravenously injected into subcutaneous breast cancer tumor model animals (such as NOG mice) to express B7H3×CD3 BiTE in vivo and exert anti-tumor effects.

[0078] 3. Characterization: Particle size, PDI, and zeta potential were measured using a Zetasizer Nano ZS90 microscope; LNP morphology was observed using transmission electron microscopy (TEM); and mRNA loading efficiency was verified using the Ribogreen assay.

[0079] 4. Stability test: LNP@BiTE-mRNA was stored at 4℃ and samples were taken at 0, 7, 14, 21 and 35 days to measure particle size, zeta potential and transfection efficiency.

[0080] Table 2. Results of particle size distribution, PDI, Zeta potential, and mRNA encapsulation efficiency of LNP@BiTE-mRNA

[0081] 1 94.87 0.09901 -3.161 92.01 2 92.6 0.1026 -4.389 93.42 3 91.67 0.1008 -5.245 90.15

[0082] Example 3: In vitro antitumor activity detection

[0083] 1. Cell culture: Breast cancer cell lines MDA-MB-231-Luc and MCF-7-Luc and human primary T cells were cultured at 37°C and 5 wt% CO2. T cells were activated with CD3 and CD28 antibodies and IL-2.

[0084] 2. Transfection and co-incubation: 293T cells were transfected with different concentrations of LNP@BiTE-mRNA prepared in Example 2, and the supernatant was collected; the supernatant was co-incubated with T cells and breast cancer cells at E:T=5:1;

[0085] 3. Killing effect detection: Luciferase luminescence reading (RLU) was used to detect the survival of target cells and calculate the T cell-mediated killing rate.

[0086] Example 4: In vivo antitumor activity detection

[0087] 1. Model establishment: 2×10⁻⁶ mg / L was injected orally into the mammary glands of NOG mice. 6 MDA-MB-231-Luc cells were used to establish a breast cancer xenograft model.

[0088] 2. Treatment regimen: When the tumor volume reached approximately 100 mm³, the following groups were administered the drug: control group (physiological saline), recombinant BiTE group, and LNP@BiTE-mRNA group prepared in Example 2, with 10 animals in each group; the dose of LNP@BiTE-mRNA group was 1.5 mg / kg;

[0089] 3. Detection indicators: The body weight and tumor volume of mice were measured regularly; after the experiment, the mice were sacrificed, the tumor tissue was collected, and HE staining analysis was performed.

[0090] The specific test results are as follows:

[0091] Figure 1 mRNA design for a B7H3-targeting bispecific T-cell adaptor. Figure: (A) Schematic diagram of three candidate anti-B7H3 conjugates (MGA271, 376.96, and TE9), fused to the CD3 binding domain via flexible peptide linkers. The scFv architecture (VL and VH domains) is illustrated. (B) Modular architecture of the nucleoside-modified linear mRNA platform for delivery, comprising a 5' cap, 5' UTR, CDS, 3' UTR, and a poly(A) tail. (Created on BioRender.com)

[0092] Figure 2In vitro functional assessment of mRNA encoding B7H3-TCE. Figures: (A) Schematic diagram of the TDCC experimental workflow. (B) Summary of EC50 and maximum killing percentage against MDA-MB-231 cells. (C, D) Dose-response cytotoxicity curves for B7H3-high expressing MDA-MB-231-luc cells (C) and B7H3-low expressing MCF-7-luc cells (D) at E:T=5:1. (E, F) ELISA detection of IFN-γ (E) and TNF-α (F) concentrations in the co-culture supernatant. Data are mean ± SEM (n=3). Statistical significance was determined by two-way ANOVA: *p<0.05, **p<0.01, ***p<0.0001.

[0093] Figure 3 In vitro validation of LNP@BiTE-mRNA efficacy and T cell activation. Figures: (A) Experimental design using LNP@BiTE-mRNA to treat ICR mouse serum to validate in vivo TCE expression. (B) Summary of mouse serum TDCC parameters (EC50 and maximum kill %). (C, D) Cytotoxicity curves against MDA-MB-231-luc (C) and MCF-7-luc (D). (E, F) IFN-γ and TNF-α secretion profiles induced by serum TCE expression. (G, H) Quantitative analysis (G) and representative flow cytometry plots (H) of CD69 expression on human CD8+ T cells after 12 hours of co-culture. Data are mean ± SEM (n=3). Two-way ANOVA: *p<0.05, **p<0.001, ***p<0.0001; ns indicates not significant.

[0094] Figure 4For the comparative evaluation of circRNA-TCE and linear mRNA, circRNA is circular RNA, which is linear mRNA converted into circular form, making it more stable than linear mRNA. Figure: (A) Schematic diagram of in vitro functional comparison. HEK293T cells were electroporated with linear mRNA or circRNA encoding B7H3-TCE. Secretory proteins in the supernatant were collected and co-cultured with human PBMCs and luciferase-expressing MDA-MB-231 cells at E:T=5:1 for 48 hours. (B) Dose-dependent in vitro cytotoxicity. Comparative cytotoxicity curves of T cells redirected from the supernatant of HEK293T cells transfected with a specified amount of circRNA or linear mRNA against MDA-MB-231-luc cells. (C, D) ELISA detection of IFN-γ (C) and TNF-α (D) concentrations in the co-culture supernatant. Data are mean ± SEM (n=3). Two-way ANOVA: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns indicates not significant.

[0095] Figure 5 In vitro screening and functional validation of LNP formulations for TCE delivery. Figure: (A) Schematic diagram of LNPs loaded with mRNA. (BD) Transfection efficiency (B), mean fluorescence intensity (C), and representative flow cytometry plots (D) showing the encapsulation of enhanced green fluorescent protein (eGFP) mRNA by LNP-1, LNP-2, and LNP-3 in HEK293T cells 24 hours after transfection. (EH) Dose-response cytotoxicity curves (E) and cytokine secretion (G, H) in PBMC co-culture. Data are mean ± SEM (n=3). Two-way ANOVA: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0096] Figure 6The in vitro functional potency and systemic safety of the LNP@BiTE-circRNA formulation. Figure: (A) Experimental workflow for assessing in vivo TCE expression potency using LNP-treated ICR mouse serum. (B) Dose-dependent T cell-mediated cytotoxicity of serum-derived TCE at different concentrations in MDA-MB-231-luc cells. (C, D) ELISA detection of IFN-γ (C) and TNF-α (D) concentrations in co-culture supernatant. (EH) Serum biochemical analysis of renal biomarkers [BUN (E), CREA (F)] and liver enzymes [ALT (G), AST (H)] (n=3) 48 hours after intravenous injection of PBS or the specified LNP formulation. (I) Representative H&E-stained histological sections of the heart, liver, spleen, lung, and kidney, showing no histopathological changes detected in any treatment group. Scale bar, 50 μm. Data are mean ± SEM. Two-way ANOVA (BD); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0097] Figure 7 Transcriptomic landscape of circRNA-TCE and linear mRNA-redirected T cells. Figure: (A) KEGG pathway enrichment analysis of differentially expressed genes. Bubble size represents gene number, color indicates significance level (-log10 P-adjust). (B) Heatmap showing relative expression of genes related to T cell activation, effector cytokines, and chemokines. (C) DEG heatmap involved in metabolic reprogramming, highlighting upregulation of glycolysis and solute carrier family genes in the circRNA-TCE group. (D) Volcano plot of overall DEG distribution. Key effectors and activating genes are highlighted. Red dots: upregulated genes; blue dots: downregulated genes; gray dots: insignificant genes (FDR < 0.05, |log2FC| > 1).

[0098] Figure 8To demonstrate the potent antitumor efficacy and enhanced T cell activation of systemically delivered B7H3-targeting circRNA-TCE LNP in humanized xenograft mice. Figures: (A) Schematic diagram of a single-dose experiment. (B) Tumor growth curves comparing the antitumor efficacy of B7H3-CD3 antibody, linear mRNA-TCE, and circRNA-TCE. (CE) Tumor-infiltrating lymphocyte characteristics: flow cytometry quantification of human CD3+ T cell counts (C), CD8+ T cell frequency (D), and CD8+ T cell PD-1 expression levels (E) in the tumor microenvironment (day 24). (F) Schematic diagram of the experimental design for a multiple-dose experiment in a humanized TNBC xenograft model. (G) Dose-dependent tumor suppression: tumor volume measurement in mice treated with different doses (1 μg vs 5 μg) of circRNA-TCE LNP compared to the control group. (H, I) Quantitative analysis of IFN-γ expression in human CD8+ T cells in the spleen (H) and representative flow cytometry images (I), showing dose-dependent T cell activation. Data are presented as mean ± SEM (n=5 per group). Tumor growth was analyzed using two-way ANOVA, and T cell analysis was performed using one-way ANOVA. *p<0.01, **p<0.001, ***p<0.0001; ns indicates no significance.

[0099] Figure 9 A schematic diagram illustrating the stable and sustained in vivo production of the B7H3 × CD3 T cell adjuvant via LNP@BiTE-circRNA delivery, leading to T cell redirection and potent antitumor activity against B7H3-positive triple-negative breast cancer.

[0100] Figure 10 Agarose gel electrophoresis analysis of representative denaturing agarose gel electrophoresis images of MGA271, 376.96, TE9, and CD3 mRNAs showed the in vitro transcripts of three B7H3-targeting TCE variants (MGA271-TCE, 376.96-TCE, and TE9-TCE) and a control mRNA containing only CD3. All transcripts showed clear, monodisperse bands without obvious tailing or degradation, indicating the successful synthesis of high-quality, intact mRNA transcripts suitable for subsequent functional evaluation.

[0101] Figure 11 Screening for translation efficiency of different IRES elements

[0102] NanoLuciferase activity guided by various internal ribosome entry site (IRES) candidate sequences (HCV, HRV, EMCV, EV71, and CVB3) was compared on the circRNA platform. Relative luminescent units (RLUs) were measured 24 h after electroporation of HEK293T cells with circRNA encoding NanoLuciferase. CVB3 (marked in red) was identified as the dominant IRES with the highest translational output and was used in the final circRNA-TCE construct. Data are presented as mean ± SD (n = 3).

[0103] Figure 12 Optimization of the 3′UTR sequence to enhance circRNA translation efficiency

[0104] NanoLuciferase activity was compared within a circRNA framework guided by four different 3′ untranslated region (UTR) variants. The optimal UTR configuration, combined with a 40-nucleotide poly(A) spacer sequence to minimize interference from residual exon sequences on the ribosome, was used in all subsequent circRNA-TCE constructs. Data are presented as mean ± SD (n = 3).

[0105] Figure 13 Comparison of transfection efficiency and protein expression of eGFP circRNA and linear mRNA.

[0106] (a) Flow cytometry analysis of the transfection efficiency (%) of 500 ng eGFP-encoded circRNA and nucleoside-modified linear mRNA in HEK293T cells, measured 24 h after electroporation. (b) Mean fluorescence intensity (MFI) of eGFP, reflecting the protein expression level of a single cell. (c) Representative flow cytometry contour plots showing the gating strategies and the percentage of eGFP-positive cells in each group. Both RNAs achieved transfection efficiencies exceeding 90%, and there was no significant difference in eGFP expression levels. Data are expressed as mean ± SD (n = 3).

[0107] Figure 14 T cell-dependent cytotoxicity mediated by circRNA-TCE at different effector-to-target ratios

[0108] CD8 +T cell killing assays of MDA-MB-231-luc cells in the presence of circRNA-TCE. Conditioned medium was obtained from the supernatant of HEK293T cells 24 h after electroporation with 500 ng circRNA-TCE. Bar charts show the percentage of tumor cell lysis at specified effector-to-target ratios (E:T, 1:1 to 20:1). The circRNA-TCE platform induced significant cell lysis activity at all tested effector-to-target ratios. Data are presented as mean ± SD (n = 3).

[0109] Figure 15 Representative microscopic images of T cell-mediated cytotoxicity

[0110] MDA-MB-231 cells expressing eGFP and CD8 + Representative bright-field (left) and fluorescence (right) images of T cells co-incubated with conditional supernatant (E:T = 5:1). The supernatant was collected 24 h after electroporation of HEK293T cells with 500 ng linear mRNA or circRNA. Compared with the linear mRNA group and the control group, the circRNA-TCE group showed significantly lower eGFP fluorescence intensity and cell density, indicating stronger T cell-mediated cell lysis activity. Scale bar: 10 μm.

[0111] Figure 16 Cytotoxic activity of HEK293T cells transfected with 500 ng circRNA against multiple cell types in conditioned medium.

[0112] The lead B7H3-targeting TCE exhibited target-dependent cytolytic activity against a range of B7H3-expressing tumor cell lines and primary human cells. This TCE induced strong lysis in B7H3-positive tumor cells (MDA-MB-231: 53.1%; A549: 14.6%), while showing very low cytotoxicity against primary human fibroblasts (2.7%) and primary human hepatocytes (6.8%). These data support its favorable preliminary safety profile, indicating limited off-target activity against normal cells.

[0113] Figure 17 To investigate the in vitro induced dose-dependent and target-specific cytotoxicity of circRNA-TCE.

[0114] Dose-response cytotoxicity curves of circRNA-TCE against MDA-MB-231-luc cells at specified RNA doses (0–1000 ng). Target specificity was confirmed by blocking assays: pre-incubation with soluble B7H3 protein significantly reduced circRNA-TCE-mediated killing. Furthermore, the B7H3-scFv monovalent control (lacking the CD3 binding domain) failed to induce detectable tumor cell lysis, confirming that T cell redirection is strictly dependent on dual binding of B7H3 and CD3. Data are presented as mean ± SEM (n = 3).

[0115] Figure 18 Physicochemical characterization of LNP formulations; Figure: (a) Hydrodynamic diameter distribution curves of LNP-1 (MC3), LNP-2 (ALC-0315), and LNP-3 (SM-102) determined by dynamic light scattering (DLS). Tables summarize the mean diameter, polydispersity index (PDI), and zeta potential of each formulation. LNP-3 exhibits the most desirable characteristics, with a diameter of 145.5 ± 4.9 nm and a PDI of 0.26 ± 0.03. (b) Representative transmission electron microscopy (TEM) image of the lead formulation LNP-3, showing a uniform spherical morphology. Scale bar: 100 nm. Data are expressed as mean ± SD (n = 3).

[0116] Figure 19 For in vitro protein expression: kinetic curves of TCE secretion into culture supernatant in cells transfected with different LNPs within 48 hours.

[0117] HEK293T cells were treated with LNP-1, LNP-2, or LNP-3 containing B7H3-TCE mRNA. Supernatants were collected at specified time points (0, 8, 16, 24, and 48 h post-transfection), and TCE concentration was quantified by ELISA. LNP-3 (SM-102) exhibited the best kinetic characteristics, reaching a peak secretion level at 48 h significantly higher than LNP-1 and LNP-2, and was therefore selected as the lead formulation for in vivo studies. Data are presented as mean ± SD (n = 3).

[0118] Figure 20circRNA-TCE exhibited excellent serum stability and durable protein expression both in vitro and in vivo; Figure: (a) Serum stability: Quantification of the relative transcript stability of circRNA-TCE and m¹Ψ-modified linear mRNA-TCE after incubation at 37 °C in RPMI 1640 medium containing 10% FBS by RT-qPCR. Half-life of circRNA-TCE (t 1 / 2 (a) CircRNA-TCE secretion: 6.0 h, which is 2.4 times that of modified linear mRNA (2.5 h). (b) In vitro protein expression: kinetic curves of TCE secretion in the supernatant of cells transfected with circRNA or modified linear mRNA over 48 hours. circRNA-TCE levels steadily increased throughout the observation period, while linear mRNA-TCE expression peaked early and then plateaued. (c) In vivo protein expression: Pharmacokinetic curves of serum TCE concentrations after a single systemic administration of 5 μg LNP-encapsulated circRNA or modified linear mRNA to mice. The circRNA plateau reached a significantly higher peak serum concentration at later time points and maintained a higher and more stable level, demonstrating superior pharmacokinetic characteristics.

[0119] Figure 21 Monitoring of mouse body weight in single-dose (a) and multiple-dose (b) safety studies; Figure: (a) Single-dose study: Body weight changes in NOG mice receiving PBS, B7H3-CD3 antibody, linear mRNA-TCE LNP, or circRNA-TCE LNP (5 μg, intravenously) during treatment. (b) Multiple-dose study: Body weight changes in mice receiving PBS, CD3-LNP (1 μg), or low-dose (1 μg) or high-dose (5 μg) circRNA-TCE LNP on days 14, 17, and 20. No significant weight loss or deterioration of general health was observed in any treatment group compared to the PBS control group, indicating that the LNP-formulated circRNA-TCE was well-tolerated at all tested doses. Data are presented as mean ± SEM (n = 5 / group).

[0120] Figure 22 Representative H&E stained images of tumor sections harvested at the end of the study, showing extensive necrosis in the TCE treatment group, scale bar 100 μm.

[0121] Histopathological evaluation of xenograft tumors harvested on day 24. Control group tumors (PBS and CD3-LNP groups) showed typical central spontaneous necrosis with peripheral viable tumor tissue remaining. In contrast, tumors in the circRNA-TCE treatment group exhibited a diffuse immune-mediated destruction pattern, characterized by dense mononuclear cell infiltration, numerous apoptotic bodies, nuclear fragmentation, and significant loss of tissue structure. These findings provide direct histological evidence for T cell-mediated tumor regression. Scale bar: 100 μm.

[0122] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. It is neither necessary nor possible to exhaustively describe all embodiments here. However, obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A bispecific antibody encoding anti-B7H3 / CD3, characterized in that: It is a B7H3×CD3 BiTE encoded sequence; its sequence is shown in the table below: ； ； ； 。 2. The specific targeting effect of the B7H3×CD3 BiTE coding sequence as described in claim 1 in the preparation of breast cancer therapeutic drugs.

3. A method for preparing mRNA-loaded LNPs, comprising the following steps: dissolving a lipid mixture (LNP) in anhydrous ethanol as the organic phase; dissolving B7H3×CD3 mRNA in 10 mM pH 3.0 citrate buffer as the aqueous phase; the volume ratio of organic phase N / aqueous phase P is 6; using a microfluidic mixing device, setting the flow rate ratio of aqueous phase to organic phase to 3:1, the flow rate of aqueous phase to 9 mL / min, the flow rate of organic phase to 3 mL / min, and the total flow rate to 12 mL / min; after mixing through the microfluidic device, dialyzing and filtering are performed to obtain mRNA-loaded LNPs (abbreviated as LNP@BiTE-mRNA).

4. The use of the mRNA-loaded LNP prepared by the method of claim 3 in the preparation of a breast cancer treatment drug.

5. The application of the LNP-mRNA delivery system encoding an anti-B7H3 / CD3 bispecific antibody as described in claim 1 for the efficient in vivo expression of BiTE, including in vitro cell killing and in vivo anti-tumor therapy.

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