Use of fibrinogen alpha chain in car-t therapeutic drugs

Abstract

The application belongs to the technical field of biology and specifically relates to application of fibrinogen alpha chain in chimeric antigen receptor T cell (CAR-T) therapeutic drugs, wherein the fibrinogen alpha chain plays a direct role in maintaining tumor cell population stemness by activating a TLR4-NF-kappa B signal pathway. Meanwhile, the fibrinogen alpha chain promotes a significant increase in expression level and proportion of TROP2 in triple-negative breast cancer (TNBC) by activating the TLR4-NF-kappa B signal pathway, and shows enhanced sensitivity to targeted TROP2-CAR-T therapy. Therefore, through exogenous regulation of the fibrinogen alpha chain, up-regulation of tumor cell TROP2 expression and homogenization among tumor cell subpopulations are induced, thereby providing an important scheme for tumor stem cell targeted therapy of TNBC.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of fibrinogen α chain in CAR-T therapeutic drugs. Background Technology

[0002] Solid tumors exhibit significant heterogeneity in their cell populations and demonstrate adaptive reprogramming during tumor progression, a key factor contributing to tumor progression and drug resistance. One of the main mechanisms of tumor heterogeneity is the existence of different subclonal populations within the tumor. The concept of "adaptive reprogramming" emphasizes the changes in protein expression among tumor subclones, which can lead to drug resistance and progression in some tumor cells, including those treated with immunotherapy or targeted therapies. Previous research has focused on the modification of the tumor microenvironment by tumor cells, including the recruitment of immunosuppressive cells. However, the impact of the tumor microenvironment on tumor cell subpopulations remains to be investigated, including the influence of specific proteins secreted by tumor cells on the adaptive reprogramming of tumor cell populations.

[0003] Fibrinogen is a triad of single-chain (FGA, FGB, FGG) glycoproteins primarily produced by the liver, and its aberrant expression in tumor cells has been detected in cancer patients. Previous studies have elucidated that fibrinogen secreted by tumor cells is a potential target for improving the therapeutic effect of breast cancer. However, the impact of paracrine fibrinogen on the tumor cell population and its value for targeted therapy require further investigation. Summary of the Invention

[0004] To address the problems raised in the background art, the present invention provides the application of fibrinogen α chain in chimeric antigen receptor T cell (CAR-T) therapeutic drugs.

[0005] The technical solution of the present invention is as follows:

[0006] This invention provides the application of fibrinogen α chain in the preparation of CAR-T therapeutic drugs, wherein the fibrinogen α chain is used to prepare CAR-T therapeutic drugs that enhance TROP2 targeting in triple-negative breast cancer.

[0007] Furthermore, the drug includes a lentiviral vector encoding the fibrinogen α-chain sequence and a TROP2-CAR vector.

[0008] Furthermore, the drug also includes substances that regulate the function of fibrinogen α-chain.

[0009] Furthermore, the substance that regulates the function of fibrinogen α chain is achieved by promoting the highly uniform expression of TROP2 protein on the surface of tumor stem cells.

[0010] Fibrinogen α chain maintains the stemness of tumor stem cell populations by activating the TLR4-NF-κB signaling pathway, promoting the enrichment of tumor stem cells and the high expression of TROP2 protein, as well as the homogeneity of the tumor cell population.

[0011] In addition, fibrinogen α chains are secreted into the tumor microenvironment by secretory cell populations or stress-responsive secretory cell subsets in triple-negative breast cancer.

[0012] The present invention also provides the application of fibrinogen α chain as a biomarker for the treatment of triple-negative breast cancer.

[0013] Beneficial effects

[0014] This invention investigates how stress-responsive secretory cell subsets (SRSCs) in triple-negative breast cancer (TNBC) secrete fibrinogen α chains (FGA) into the tumor microenvironment, thereby promoting the enrichment of tumor stem cell (CSC) subsets within a broader tumor cell population. This invention demonstrates that fibrinogen α chains (FGA) play a direct role in maintaining tumor cell population stemness by activating the TLR4-NF-κB signaling pathway. Simultaneously, fibrinogen α chains, by activating the TLR4-NF-κB signaling pathway, significantly increase the expression level and proportion of TROP2 in TNBC, demonstrating enhanced sensitivity to TROP2-targeted CAR-T therapy. Therefore, by extrinsically regulating fibrinogen α chains, inducing upregulation of TROP2 expression in tumor cells and homogenization among tumor cell subsets, an important strategy for tumor stem cell-targeted therapy of TNBC is provided. Attached Figure Description

[0015] Figure 1 This image shows the distribution of FGA-positive cells in various cell clusters within breast cancer tumor tissue. In the image, A represents the classification of different cell types within the tumor tissue as shown by UMAP, and B represents the distribution of FGA-positive cells.

[0016] Figure 2 FGA is specifically expressed in the SRSCs subset of breast cancer tumor cell clusters, where A represents the classification of tumor cells in the tumor tissue as shown by UMAP, and B represents the distribution of FGA-positive cells in the tumor cell subset.

[0017] Figure 3 The distribution of tumor stem cell subpopulations in different FGA breast cancers is shown, where A is the FGA-positive group (FGA+ group) and B is the FGA-negative group (FGA- group).

[0018] Figure 4 Enrichment analysis of the NF-κB pathway in FGA-positive / negative tumor stem cells.

[0019] Figure 5 Western blot analysis was performed on MDA-MB 436 and MDA-MB 231 cells overexpressing FGA (FGA group) and control cells (vector group) to analyze the activation of the NF-κB pathway.

[0020] Figure 6 To knock down TLR4 in MDA-MB 436 and MDA-MB 231 cells overexpressing FGA, the activation of the NF-κB pathway was analyzed by Western blotting.

[0021] Figure 7 This provides evidence for the protein interaction between FGA and TLR4.

[0022] Figure 8 The changes in the ALDH1+ ratio in cells of the vector, FGA, shCtrl, and shFGA groups were detected by flow cytometry.

[0023] Figure 9 The expression levels of stem cell markers in the vector, FGA, knockdown control, and FGA knockdown group were detected by PCR.

[0024] Figure 10 To analyze the localization of TROP2 in tumor cell subpopulations using single-cell sequencing data, where A represents the classification of various cell types within the tumor tissue as shown by UMAP, and B represents the distribution of TROP2-positive cells.

[0025] Figure 11 To analyze TROP2 expression in FGA-positive (FGA+) and FGA-negative (FGA-) groups using single-cell sequencing data, A shows the classification of TROP2-positive tumor cells in tumor tissue as displayed by UMAP, B shows the distribution of TROP2-positive cells in FGA-positive tumor cell subpopulations, and C shows the distribution of TROP2-positive cells in FGA-negative tumor cell subpopulations.

[0026] Figure 12 To enrich TROP2 expression in the early differentiation stage of breast cancer cells.

[0027] Figure 13 To analyze the difference in TROP2 expression in FGA-overexpressing (A) and FGA-non-overexpressing (B) breast cancer tissues using immunohistochemistry.

[0028] Figure 14 To detect the TROP2 positivity rate of triple-negative breast cancer cells of MDA-MB 231, MDA-MB 231 / FGA, MDA-MB 436, and MDA-MB 436 / FGA by flow cytometry.

[0029] Figure 15 For the TROP2-CAR vector structural model.

[0030] Figure 16 Analysis of cell transduction efficiency targeting TROP2-CAR-T.

[0031] Figure 17 To analyze the killing efficiency of TROP2-CAR-T targeting MDA-MB 231 / FGA and MDA-MB 231 / V cells in in vitro experiments.

[0032] Figure 18 To analyze the inhibitory effect of TROP2-CAR-T on MDA-MB 231 triple-negative breast cancer xenografts overexpressing FGA in vivo experiments.

[0033] Figure 19 Quantitative analysis of the volume of MDA-MB 231 triple-negative breast cancer xenografts.

[0034] Figure 20 To analyze the inhibitory effect of TROP2-CAR-T on MDA-MB 436 triple-negative breast cancer xenografts overexpressing FGA in vivo experiments.

[0035] Figure 21 Quantitative analysis of the volume of MDA-MB 436 triple-negative breast cancer xenografts. Detailed Implementation

[0036] The following examples are intended to illustrate the present invention, and not to further limit the invention.

[0037] Experimental results

[0038] This invention systematically evaluated single-cell sequencing data from 11 cases of triple-negative breast cancer, analyzed the expression of FGA in various cell subpopulations, and found that FGA-positive cells were mainly concentrated in tumor cell clusters ( Figure 1 Further analysis of FGA expression in tumor cell subsets revealed that FGA was specifically expressed in the stress-responsive secretory cell (SRSC) subset. Figure 2 Furthermore, it was found that triple-negative breast cancer tissues in the FGA-positive group (FGA+ group) were significantly enriched with a subset of tumor stem cells (CSCs). Figure 3 By comparing differentially expressed genes in CSCs cell subpopulations from FGA-positive and FGA-negative (FGA+ / -) tumor tissues, and through pathway enrichment analysis, it was found that the FGA+ group of CSCs cell populations was significantly enriched in the positive regulatory signaling pathway of NF-κB transcription factor activity. Figure 4 ).

[0039] This invention observed activation of the NK-κB signaling pathway in FGA-overexpressing triple-negative breast cancer cells (MDA-MB 436 and MDA-MB 231), manifested as a significant increase in p-p65 and p-IκBα protein levels. Figure 5 Furthermore, knocking down TLR4 expression in triple-negative breast cancer cells overexpressing FGA resulted in a significant decrease in the phosphorylation levels of p65 and IκBα. Figure 6 This invention further demonstrates that endogenous TLR4 and FGA interact ( ). Figure 7 This indicates that FGA activates the NF-κB signal by binding to TLR4.

[0040] This invention analyzed the changes in tumor stem cells in FGA-transfected triple-negative breast cancer cells, such as... Figure 8 As shown, the proportion of ALDH1+ cells overexpressing FGA was significantly increased ( Figure 8 The proportion of ALDH1+ cells with knocked-down FGA expression was significantly reduced (vector and FGA group). Figure 8 The expression of stem cell-related markers (SOX2, POU5F1, and NANOG) in MDA-MB 231 cells overexpressing FGA was significantly increased, while the expression of stem cell-related markers (SOX2, POU5F1, and NANOG) in MDA-MB 468 cells with knocked-down FGA expression was significantly decreased. Figure 9 ).

[0041] This invention compared the expression of common CAR-T targets in FGA-positive and FGA-negative tumor tissues, identifying TROP2 as a potential candidate antigen for CAR-T cell therapy in FGA-positive triple-negative breast cancer. In FGA-positive (FGA+ group) triple-negative breast cancer tissues, TROP2 expression was concentrated in a subset of tumor cells ( Figure 10 ), and showed significantly uniform high expression in the CSCs subgroup ( Figure 11 Further analysis showed that TROP2 gene expression in FGA-positive cells was concentrated in the early differentiation stage. Figure 12 TROP2 is a potential target for targeting tumor stem cells. TROP2 immunohistochemical staining was performed on triple-negative breast cancer samples. TROP2 is mainly located in the cell membrane. TROP2 was overexpressed in FGA-treated triple-negative breast cancer samples (…). Figure 13 The positive rate of A in the sample was significantly higher than that of the non-overexpressing FGA sample. Figure 13 (B in the text), and more uniform. More importantly, as... Figure 14As shown, compared with the control group (MDA-MB 231, MDA-MB 436) that did not overexpress FGA, the positive rate of TROP2 expression in the ALDH1+ CSCs subset of the cell lines that overexpress FGA (MDA-MB 231 / FGA, MDA-MB 436 / FGA) was significantly increased.

[0042] This invention investigated the killing effect of TROP2-CAR-T cells on FGA-overexpressing triple-negative breast cancer in vitro and in vivo. A second-generation TROP2-CAR vector (structure shown in Figure 1) was used. Figure 15 As shown in the figure, CAR-T cells were constructed. Flow cytometry analysis showed that the percentage of CAR-T cells expressing CARs was approximately 64.2% (as shown in the figure). Figure 16 (This meets the experimental requirements.)

[0043] This invention investigated the in vitro killing activity of TROP2-CAR-T cells against FGA-positive triple-negative breast cancer cells. FGA-overexpressing triple-negative breast cancer cell lines (MDA-MB 231 / FGA) and control cells (non-FGA-overexpressing cells, MDA-MB231 / V) were co-cultured with TROP2-CAR-T cells for 6 hours. The results showed that approximately 35% of the triple-negative breast cancer cells in the FGA-overexpressing triple-negative breast cancer cell line were killed by TROP2-CAR-T cells at an effector-to-target ratio of 1:10, which was at least 10% higher than the effector-to-target ratio of the control group. Figure 17 ).

[0044] This invention further investigated the in vivo killing activity of TROP2-CAR-T cells against FGA-positive triple-negative breast cancer cell xenografts. The in vivo killing effect of TROP2-CAR-T cells on FGA-overexpressing triple-negative breast cancer cells was evaluated using tumor-bearing nude mice. In fact, compared to the MDA-MB 231 control group xenografts, the tumor volume of FGA-overexpressing triple-negative breast cancer cells (MDA-MB 231) was significantly reduced under the action of TROP2-CAR-T cells. Figure 18 The growth rate of the transplanted tumor was significantly slowed down. Figure 19 Similarly, a similar phenomenon was observed in a triple-negative breast cancer cell (MDA-MB 436) xenograft model. Figure 20 The growth rate of the transplanted tumor was significantly slowed down. Figure 21 ).

[0045] This invention investigates how stress-responsive secretory cell subsets (SRSCs) in triple-negative breast cancer (TNBC) secrete fibrinogen α chains (FGA) into the tumor microenvironment, thereby promoting the enrichment of tumor stem cell (CSC) subsets within a broader tumor cell population. This invention demonstrates that fibrinogen α chains (FGA) play a direct role in maintaining tumor cell population stemness by activating the TLR4-NF-κB signaling pathway. Simultaneously, fibrinogen α chains, by activating the TLR4-NF-κB signaling pathway, significantly increase the expression level and proportion of TROP2 in TNBC, demonstrating enhanced sensitivity to TROP2-targeted CAR-T therapy. Therefore, by extrinsically regulating fibrinogen α chains, inducing upregulation of TROP2 expression in tumor cells and homogenization among tumor cell subsets, an important strategy for tumor stem cell-targeted therapy of TNBC is provided. In summary, FGA / TROP2 double staining (IHC) of biopsy samples from TNBC patients can screen for individuals with high FGA expression and homogeneous TROP2, who may be potential beneficiaries of TROP-2 targeted therapy; TROP2-CAR-T is an important treatment strategy for FGA-positive triple-negative breast cancer.

[0046] The above experiments were conducted using the following experimental methods.

[0047] Experimental methods

[0048] 1. Single-cell sequencing data and analysis

[0049] Single-cell RNA sequencing data, including GEO's GSE176078 dataset and two self-test datasets, were generated in 10x Genomics format. Data were processed in R using Seurat (4.1.0): low-quality cells were filtered by gene expression, UMI count, and mitochondrial gene proportion; normalization was performed; and 2000 hypervariable genes were selected (FindVariableFeatures). The integrated data were corrected for batch effects using the CCA method (FindIntegrationAnchors and IntegrateData) to generate a unified matrix. TNBC samples were screened for analysis, retaining ER+ and HER2+ samples to compare fibrinogen α-chain (FGA) expression differences. Clustering was performed using FindClusters, and UMAP was used for dimensionality reduction visualization. Single-cell RNA sequencing data were annotated with cell clusters using SingleR (2.4.1) in R (4.3.0), and cell identity was identified by comparison with a reference dataset. Results were integrated into Seurat (4.1.0) metadata and visualized using DimPlot. Manual validation using CellMarker (2.0) was performed, and classification was corrected based on marker genes. The limma package was used to analyze differentially expressed genes (DEG) in the FGA high and low expression groups (|log2 Fold Change|>1, adjusted P<0.05). The results were plotted as a volcano plot using R (4.3.0) ggplot2, with upregulated genes highlighted in red and downregulated genes in blue. Genes were sorted by log2 Fold Change using GSEA, and enrichment scores were calculated by comparing them with the MSigDB c2.all and c5.all gene sets. The results were run using R (4.3.0) clusterProfiler (4.8.1), with adjusted significance thresholds showing P<0.05. Significant gene sets were presented as GSEA curves and tables, highlighting function and pathway.

[0050] 2. Flow cytometry

[0051] Cells were collected by centrifugation and washed twice with phosphate-buffered saline (PBS). Subsequently, cell staining, precipitation, and washing were performed according to the antibody manufacturer's instructions. Finally, cells were placed in PBS containing 2% fetal bovine serum (FBS) and incubated at 4°C for analysis using flow cytometry (BD, FACSAria II). Rabbit anti-human TROP2 antibody (Abcam ab214488) was used to detect TROP2 expression on the surface of cancer cells. CAR expression on the surface of CAR-T cells was detected using EGFR-FITC marker (ICARTAB, 21092702) and Protein L (Pierce, 2997)-streptavidin APC (Aqua Blue, Invitrogen, S21374). The following antibody combination was used for T-cell immunophenotyping: APC-labeled mouse anti-human CD3 antibody (Invitrogen, 17-0038-42), FITC-labeled mouse anti-human CD62L antibody (Invitrogen, 11-0629-42), BV605-labeled mouse anti-human CD3 antibody (BioLegend, 317321), BV510-labeled mouse anti-human CD4 antibody (BioLegend, 300546), AF700-labeled mouse anti-human CD8 antibody (BioLegend, 344724), and APC-labeled mouse anti-human CD45RO antibody (BioLegend, 304210). In addition, the proportion of ALDH1-positive cells was analyzed using the ALDEFLUOR kit (StemCell Technologies, Durham, North Carolina, USA) according to the manufacturer's instructions. Apoptosis was analyzed using the Annexin V-FITC / PI apoptosis detection kit (MultiSciences Biotech, Co., Ltd., Hangzhou, China). Data were analyzed using FlowJo V.7.6.1 software.

[0052] 3. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Detection

[0053] Total RNA was extracted from triple-negative breast cancer (TNBC) cells using RNAiso Plus (TAKARA, 9108), and the total RNA was quantified and reverse transcribed (TAKARA, RR047A). Subsequently, mRNA expression levels were analyzed using the SYBR Prime-Script PCR Kit II (TaKaRa, RR820A), and quantification was performed using the Bio-Rad CFX96 detection system (Bio-Rad). The primer sequences used in this invention are as follows:

[0054] FGA: Forward primer (Fwd) 5'-GGGCACATTTGAAGAGGTGT-3';

[0055] Reverse primer (Rev) 5'-TTGTGACCATCAGGACCAA-3'.

[0056] SOX2: Forward primer (Fwd) 5'-CTCGTGCAGTTCTACTCGTCG-3';

[0057] Reverse primer (Rev) 5'-AGCTCTCGGTCAGGTCCTTT-3';

[0058] POU5F1: Forward primer (Fwd) 5'- GTGTTCAGCCAAAAGACCATCT-3';

[0059] Reverse primer (Rev) 5'- GGCCTGCATGAGGGTTTCT-3';

[0060] NANOG: Forward primer (Fwd) 5'- TCCCGAGAAAAGATTAGTCAGCA-3';

[0061] Reverse primer (Rev) 5'- AGTGGGGCACCTGTTTAACTT-3';

[0062] GAPDH: Forward primer (Fwd) 5'-TGTGGGCATCAATGGATTTGG-3';

[0063] The reverse primer (Rev) was 5'-ACACCATGTATTCCGGGTCAAT-3'. GAPDH was used as an internal reference gene, and gene expression levels were calculated using the 2^(-ΔΔCt) relative quantification method. Each experiment was independently repeated at least three times.

[0064] 4. Immunoblotting and co-precipitation

[0065] Cells were lysed using RIPA lysis buffer (Thermo Fisher Scientific) supplemented with a mixture of protease inhibitors (Roche) and phosphatase inhibitor tablets (Roche). Total protein concentration was determined using a BCA protein assay kit (Thermo, 23225). Equal volumes of protein were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The transferred membrane was incubated overnight at 4°C with primary antibody. Subsequently, the membrane was incubated at room temperature for 1 hour with horseradish peroxidase (HRP)-labeled secondary antibody (Rockland; Beyotime) and developed using SuperSignal West Dura chemiluminescent substrate (Thermo Scientific, 34075). GAPDH was used as an internal control protein. Protein samples isolated using the nucleoplasmic protein extraction kit (P0028, Beyotime) were also subjected to Western blot analysis, with β-actin and lamin B1 used as cytoplasmic and nucleoprotein internal controls, respectively. The primary antibodies used are listed below: FGA (ab19079, Abcam), Lamin B1 (Ab16048, Abcam), β-actin (ab8227, Abcam), p65 (ab16052, Abcam), phosphorylated p65 (p-p65, ab86299, Abcam), TLR4 (Ab8376, Abcam), MyD88 (ab2064, Abcam), IκBα (ab7217, Abcam), phosphorylated IκBα (p-IκBα, ab133462, Abcam), cleaved PARP-1 (#D64E10, Cell Signaling Technology), survivin (#FL-142, Cell Signaling Technology), TROP2 (ab214488, Abcam), and GAPDH (#5174, Cell Signaling Technology).

[0066] The immunoprecipitation procedure was as follows: HEK293T cells were lysed on ice using IP lysis buffer (Thermo Fisher Scientific) for 15 minutes, which contained a mixture of protease inhibitors (Roche) and phosphatase inhibitor tablets (Roche). After centrifugation, the cell lysate was incubated overnight at 4°C with TLR4 (Ab8376, Abcam), HA (Ab9110, Abcam), or Flag (Ab205606, Abcam) antibodies. Then, 40 μL of Protein G Sepharose beads (GE Healthcare) were added, and the cells were incubated at 4°C for 3 hours. After washing the beads six times with IP lysis buffer, Western blot analysis was performed.

[0067] 5. Mouse model

[0068] Six-week-old female nude mice were used in the experiment. Control cells expressing luciferase, MDA-MB 231 cells overexpressing FGA, and MDA-MB 436 cells overexpressing FGA (1×10⁻⁶ cells) were used. 6 Cells were suspended in 50 μL PBS and mixed with 50 μL L Atrigel (Corning) and injected subcutaneously into the mammary fat pads of mice to form tumors. Tumor growth was monitored and recorded. For in vivo epirubicin (EPI) treatment experiments, mice were randomly assigned to groups and treated with epirubicin (5 mg / kg EPI every 3 days) when the tumor volume reached approximately 100 mm³. For CAR-T therapy experiments, in vivo imaging of luciferase-expressing xenografts in mice was performed before TROP2-CAR-T treatment, and mice were grouped according to the imaging results to ensure that the tumor burden was approximately equal between groups. Tumor size was measured every three days. Imaging was performed after intraperitoneal injection of luciferin (2 μg / 20 g body weight; Promega, P1043), and the tumor treatment effect was evaluated using an in vivo imaging system (BLT, AniView100). Statistical significance was determined using the log-rank (Mantel-Cox) test.

[0069] 6. TROP2-CAR vector construction and lentivirus preparation

[0070] Based on the sequence of a humanized monoclonal antibody (hRS7) targeting TROP2, this invention constructed a second-generation TROP2-CAR vector through gene synthesis. This vector contains: a TROP2-specific single-chain antibody fragment (anti-TROP2 scFv), a CD8 signal peptide, two intracellular co-stimulatory signal domains (CD3ζ and 4-1BB), a CD8 hinge region, and a transmembrane sequence. The TROP2-CAR vector, along with two helper packaging plasmids (psPAX2, pMD2.G), was co-transfected into HEK293T cells using polyethyleneimine (PEI). Cell supernatants were collected at 48 and 72 hours post-transfection, centrifuged, filtered, and concentrated using PEG8000 (Beyotime, ST483) at a 5:1 ratio. The concentrated virus was collected, and viral titers were determined using qPCR reagent (Yeasen, H7101190).

[0071] 7. Preparation and Identification of CAR-T Cells

[0072] Fresh peripheral blood was collected from healthy volunteers. Peripheral blood mononuclear cells (PBMCs) were isolated from normal donor blood using density gradient separation medium. Primary cells were cultured in 1640 medium containing interleukin-2 (IL-2). T cells were activated for 48 hours using anti-human CD3 (Miltenyi, GMP-E240 1h-1mg) and CD28 antibody (Miltenyi, GMP-E240 2h-1mg). After cell counting, the required viral load was calculated based on a multiplicity of infection (MOI) of 30. The viral load was added to 24-well plates pre-coated with RetroNectin (TaKaRa, SD3668) and incubated overnight, and centrifuged at 4°C, 3500 rpm for 60 minutes. Subsequently, the activated T cells were seeded into the culture plates, supplemented with polybrene (8 μg / mL), and centrifuged at 32°C, 3500 rpm for 60 minutes. Finally, TROP2-CAR-T cells were cultured in an incubator. CAR expression on the surface of CAR-T cells was assessed by flow cytometry. The prepared CAR-T cells were used for in vitro and in vivo cytotoxicity experiments.

[0073] 8. Lactate dehydrogenase (LDH) release experiment

[0074] Total 8×10 4MCF7 cells were seeded and cultured in 96-well plates. Subsequently, T cells were added to the 96-well plates at different effector-to-target cell ratios and incubated for 4 hours. LDH levels were measured using a lactate dehydrogenase (LDH) assay kit (Beyotime, C0017). After selecting the optimal effector-to-target ratio, cytotoxicity experiments were performed using TROP2-CAR-T cells at this optimal ratio, and LDH release assays were performed. The procedure is briefly described as follows: The mixture was centrifuged at 400×g for 5 minutes, and the supernatant was carefully aspirated. 150 μL of diluted LDH release reagent was added, and the mixture was incubated for 1 hour. The mixture was then centrifuged at 400×g for 5 minutes, and 120 μL of the supernatant was transferred to a new plate. 60 μL of LDH working solution was added, and the plate was incubated in the dark for 30 minutes. The absorbance (OD) values ​​were measured at 490 nm and 620 nm. Three replicates were set for each sample. Cytotoxicity (%) was calculated based on the OD values ​​of the samples and controls.

Claims

1. The application of a fibrinogen α chain in the preparation of CAR-T therapeutic drugs, characterized in that, Lentiviral vectors encoding the fibrinogen α-chain sequence and TROP2-CAR vectors were used to prepare CAR-T therapeutic drugs targeting TROP2 in triple-negative breast cancer.

2. The application of the fibrinogen α chain according to claim 1 in the preparation of CAR-T therapeutic drugs, characterized in that, Fibrinogen α chain maintains the stemness of tumor stem cell populations by activating the TLR4-NF-κB signaling pathway, promoting the enrichment of tumor stem cells and the high expression of TROP2 protein, as well as the homogeneity of the tumor cell population.

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