Application of fibrinogen alpha chain in CAR-T (Chimeric Antigen Receptor T) therapeutic drug

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a fibrinogen alpha chain in a chimeric antigen receptor T cell (CAR-T. The fibrinogen alpha chain plays a direct role in maintaining the dryness of a tumor cell group by activating a TLR4-NF-kappa B signal channel. Meanwhile, the fibrinogen alpha chain promotes the expression level and proportion of TROP2 in triple negative breast cancer (TNBC) to be remarkably increased by activating a TLR4-NF-kappa B signal channel, and the enhanced sensitivity to a targeted TROP2-CAR-T therapy is shown. Therefore, by exogenous regulation of the fibrinogen alpha chain, up-regulation of tumor cell TROP2 expression and homogenization among tumor cell subgroups are induced, and an important scheme is provided for tumor stem cell targeted therapy of TNBC.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to application of fibrinogen alpha chain in CAR-T therapeutic drugs. BACKGROUND

[0002] Tumor cell populations of solid tumors exhibit significant heterogeneity and show adaptive reprogramming in tumor progression, which is an important cause of 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 between tumor subclones, which can make some tumor cells resistant to drug treatment and progress, including immunotherapy or targeted therapy drugs. Previous studies have focused on the modification of tumor cells to the tumor microenvironment, including the recruitment of immunosuppressive cells. However, the influence of the tumor microenvironment on tumor cell subpopulations still needs to be further studied, including the influence of specific proteins secreted by tumor cells on the adaptive reprogramming of tumor cell populations.

[0003] Fibrinogen is a three-chain (FGA, FGB, FGG) glycoprotein mainly produced by the liver, and its uneven expression and abnormal expression in tumor cells are also detected in tumor patients. Previous studies have shown that fibrinogen secreted by tumor cells is a potential target for improving the therapeutic effect of breast cancer. However, the influence of tumor cell paracrine fibrinogen on tumor cell populations and its targeted therapy value need to be further explored. SUMMARY

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

[0005] The technical scheme of the application is as follows: The application provides application of fibrinogen alpha chain in preparation of CAR-T therapeutic drugs, and the fibrinogen alpha chain is used for preparing CAR-T therapeutic drugs for enhancing TROP2 targeting in triple-negative breast cancer.

[0006] Further, the drug comprises a lentiviral vector of a fibrinogen alpha chain coding sequence and a TROP2-CAR vector.

[0007] Further, the drug further comprises a substance for regulating the function of the fibrinogen alpha chain.

[0008] Further, the substance for regulating the function of the fibrinogen alpha chain is achieved by promoting the highly uniform expression of TROP2 protein on the surface of tumor stem cells.

[0009] Fibrinogen alpha chain maintains the stemness of tumor stem cell population by activating TLR4-NF-κB signaling pathway, promotes enrichment of tumor stem cells and high expression of TROP2 protein and homogeneity of tumor cell population.

[0010] In addition, the fibrinogen alpha chain is secreted by a cell population with a secretion function or a stress response secretory cell subpopulation in triple negative breast cancer into the tumor microenvironment.

[0011] The application also provides a use of fibrinogen alpha chain as a therapeutic triple negative breast cancer response marker.

[0012] Beneficial effects The present application studies that the stress response secretory cell subpopulation (SRSCs) in triple negative breast cancer (TNBC) secretes fibrinogen alpha chain (FGA) into the tumor microenvironment, thereby promoting enrichment of tumor stem cell (CSC) subpopulation in a wider tumor cell population; the present application proves that fibrinogen alpha chain (FGA) plays a direct role in maintaining the stemness of tumor cell population by activating TLR4-NF-κB signaling pathway. At the same time, fibrinogen alpha chain promotes a significant increase in the expression level and proportion of TROP2 in triple negative breast cancer (TNBC) by activating TLR4-NF-κB signaling pathway, showing enhanced sensitivity to targeted TROP2-CAR-T therapy. Therefore, by exogenous regulation of fibrinogen alpha chain, up-regulation of tumor cell TROP2 expression and homogeneity among tumor cell subpopulations are induced, which provides an important scheme for tumor stem cell targeted therapy of TNBC. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 FIG. 1 is a distribution of FGA positive cells in various cell clusters in breast cancer tumor tissue, wherein A is a UMAP display of classification of various cells in tumor tissue, and B is a distribution of FGA positive cells.

[0014] Figure 2 FIG. 3 is a specific expression of FGA in SRSCs subpopulation in breast cancer tumor cell clusters, wherein A is a UMAP display of classification of tumor cells in tumor tissue, and B is a distribution of FGA positive cells in tumor cell subpopulation.

[0015] Figure 3 FIG. 4 is a distribution of tumor stem cell subpopulation in different FGA breast cancer, wherein A is an FGA positive group (FGA+ group), and B is an FGA negative group (FGA- group).

[0016] Figure 4 FIG. 5 is an NF-κB pathway enrichment analysis in tumor stem cells in FGA positive / negative groups.

[0017] Figure 5Western blot analysis of the activation of the NF-κB pathway in MDA-MB 436 and MDA-MB 231 cells overexpressing FGA (FGA group) and control cells (vector group).

[0018] Figure 6 Western blot analysis of the activation of the NF-κB pathway in MDA-MB 436 and MDA-MB 231 cells overexpressing FGA (FGA group) and control cells (vector group).

[0019] Figure 7 Protein interaction evidence of FGA and TLR4.

[0020] Figure 8 Flow cytometry detection of the proportion of ALDH1+ cells in the vector, FGA, shCtrl, and shFGA groups.

[0021] Figure 9 PCR detection of the expression levels of stem cell markers in the vector, FGA, knockdown control, and FGA knockdown groups.

[0022] Figure 10 Single-cell sequencing data analysis of TROP2 localization in tumor cell subgroups, where A is the UMAP display of the classification of various cells within the tumor tissue, and B is the distribution of TROP2-positive cells.

[0023] Figure 11 Single-cell sequencing data analysis of TROP2 expression in FGA-positive (FGA+ group) and FGA-negative (FGA- group) groups, where A is the UMAP display of the classification of TROP2-positive tumor cells within the tumor tissue, B is the distribution of TROP2-positive cells in the FGA-positive tumor cell subgroup, and C is the distribution of TROP2-positive cells in the FGA-negative tumor cell subgroup.

[0024] Figure 12 TROP2 expression is enriched in the early differentiation of breast cancer cells.

[0025] Figure 13 Immunohistochemical analysis of TROP2 expression differences in FGA-overexpressing (A) and non-FGA-overexpressing (B) breast cancer tissues.

[0026] Figure 14 Flow cytometry detection of the TROP2-positive rate of triple-negative breast cancer cells in MDA-MB 231, MDA-MB 231 / FGA, MDA-MB 436, and MDA-MB 436 / FGA.

[0027] Figure 15 Targeting TROP2-CAR vector structure model.

[0028] Figure 16 Cell transduction efficiency analysis for targeting TROP2-CAR-T.

[0029] Figure 17 In vitro test analysis of the killing efficiency of targeting TROP2-CAR-T on MDA-MB 231 / FGA, MDA-MB 231 / V cells.

[0030] Figure 18 In vivo experiment analysis of the inhibitory effect of targeting TROP2-CAR-T on MDA-MB 231 triple negative breast cancer transplanted tumor overexpressing FGA.

[0031] Figure 19 MDA-MB 231 triple negative breast cancer transplanted tumor volume quantification analysis.

[0032] Figure 20 In vivo experiment analysis of the inhibitory effect of targeting TROP2-CAR-T on MDA-MB 436 triple negative breast cancer transplanted tumor overexpressing FGA.

[0033] Figure 21 MDA-MB 436 triple negative breast cancer transplanted tumor volume quantification analysis. DETAILED DESCRIPTION

[0034] The following examples are intended to illustrate the present application, but not to further limit the present application.

[0035] Experimental results The present application systematically evaluates 11 cases of triple negative breast cancer single cell sequencing data, analyzes the expression of FGA in various cell subgroups, and finds that FGA positive cells are mainly concentrated in tumor cell clusters (TCCs) (Fig. 1). Figure 1 Further, the expression of FGA in tumor cell subgroups is analyzed, and it is found that FGA is specifically expressed in stress response secretory cell (SRSCs) subgroup (Fig. 2). Figure 2 In addition, it is found that triple negative breast cancer tissues in FGA positive group (FGA+ group) are significantly enriched in tumor stem cell (CSCs) subgroup (Fig. 3). Figure 3 By comparing the differential genes of CSCs cell subgroup in FGA positive group and negative group (FGA+ / -) tumor tissues, and pathway enrichment analysis, it is found that the CSCs cell group of FGA+ group is significantly enriched in the positive regulation signal pathway of NF-κB transcription factor activity (Fig. 4). Figure 4

[0036] The present application observes the activation of NF-κB signaling pathway in triple negative breast cancer cells (MDA-MB 436 and MDA-MB 231) overexpressing FGA, which is manifested as a significant increase in p-p65 and p-IκBα protein levels (Fig. 5). Figure 5 ​). Further, knockdown of TLR4 expression in triple negative breast cancer cells overexpressing FGA, the phosphorylation levels of p65 and IκBα were significantly decreased ( Figure 6 ). The present application also further proved that endogenous TLR4 combined with FGA ( Figure 7 ), indicating that FGA activated NF-κB signal by binding to TLR4.

[0037] The present application analyzed the changes of tumor stem cells in FGA transfected triple negative breast cancer cells, as shown in Figure 8 , it was found that the proportion of ALDH1+ in FGA overexpressing cells was significantly increased ( Figure 8 ), while the proportion of ALDH1+ in cells with FGA expression knocked down was significantly reduced ( Figure 8 ) in shCtrl and shFGA groups. 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 FGA expression knocked down was significantly reduced ( Figure 9 ).

[0038] The present application compared the expression of common CAR-T target points in FGA positive and negative tumor tissues, and identified TROP2 as a potential candidate antigen for CAR-T cell treatment of FGA positive triple negative breast cancer. In FGA positive (FGA+ group) triple negative breast cancer tissues, TROP2 expression was concentrated in a subpopulation of tumor cells ( Figure 10 ), and showed significant uniform high expression in CSCs subpopulation ( Figure 11 ). Further analysis showed that TROP2 gene expression in FGA positive cells was concentrated in early differentiation ( Figure 12 ), which was a potential target for targeting tumor stem cells. Triple negative breast cancer samples were subjected to TROP2 immunohistochemical staining, and TROP2 was mainly located in the cell membrane. The positive rate of TROP2 in FGA overexpressing triple negative breast cancer samples ( Figure 13 ) was significantly higher than that in FGA non-overexpressing samples ( Figure 13 ), and was more uniform. More importantly, as shown in Figure 14 , compared with the control group (MDA-MB 231, MDA-MB 436) without overexpressing FGA, the positive rate of TROP2 expression in the ALDH1+ CSCs subpopulation of FGA overexpressing cell lines (MDA-MB 231 / FGA, MDA-MB 436 / FGA) was significantly increased.

[0039] The present application studies the killing effect of TROP2-CAR-T cells on FGA overexpressing triple negative breast cancer in vitro and in vivo. CAR-T cells are constructed using a second generation TROP2-CAR vector (structure as shown in Figure 15 ). Figure 16 ), which meets the experimental requirements.

[0040] The present application detects the in vitro killing activity of TROP2-CAR-T cells on FGA positive triple negative breast cancer cells. After co-culturing FGA overexpressing triple negative breast cancer cell line (MDA-MB 231 / FGA) and control cells (non-FGA overexpressing cells, MDA-MB 231 / V) with TROP2-CAR-T cells for 6 hours, it is found that about 35% of triple negative breast cancer cells in the FGA overexpressing triple negative breast cancer cell line are killed by TROP2-CAR-T cells at an effector target ratio of 1:10, which is at least about 10% higher than that of the control cells ( Figure 17 ).

[0041] The present application further detects the in vivo killing activity of TROP2-CAR-T cells on FGA positive triple negative breast cancer cell transplanted tumors. Tumor-bearing nude mice are used to evaluate the in vivo killing effect of TROP2-CAR-T cells on triple negative breast cancer overexpressing FGA. In fact, compared with the MDA-MB 231 control group transplanted tumor, the triple negative breast cancer cell (MDA-MB 231) overexpressing FGA tumor volume is significantly reduced under the action of TROP2-CAR-T cells ( Figure 18 ), and the growth rate of the transplanted tumor is significantly slowed down ( Figure 19 ). Similarly, similar phenomena are also observed in the triple negative breast cancer cell (MDA-MB 436) transplanted tumor model ( Figure 20 ), and the growth rate of the transplanted tumor is significantly slowed down ( Figure 21 ).

[0042] The present application studies that the stress response secretory cell subpopulation (SRSCs) in triple negative breast cancer (TNBC) secretes fibrinogen alpha chain (FGA) into the tumor microenvironment, thereby promoting enrichment of the cancer stem cell (CSC) subpopulation in a wider tumor cell population; the present application proves that fibrinogen alpha chain (FGA) plays a direct role in maintaining the stemness of the tumor cell population by activating the TLR4-NF-κB signaling pathway. At the same time, fibrinogen alpha chain promotes a significant increase in the expression level and proportion of TROP2 in triple negative breast cancer (TNBC) by activating the TLR4-NF-κB signaling pathway, showing enhanced sensitivity to targeted TROP2-CAR-T therapy. Therefore, through exogenous regulation of fibrinogen alpha chain, induction of up-regulation of tumor cell TROP2 expression and homogenization between tumor cell subpopulations provides an important scheme for tumor stem cell targeted therapy of TNBC. In summary, FGA / TROP2 double staining (IHC) is performed on biopsy samples of TNBC patients to screen for FGA high expression and TROP2 homogenization population, which can be used as a potential benefit population for targeted TROP-2 therapy; TROP2-CAR-T is an important treatment strategy for FGA positive triple negative breast cancer.

[0043] The above experiments were carried out by the following experimental methods.

[0044] Experimental methods 1. Single cell sequencing data and analysis Single-cell RNA sequencing data includes GSE176078 from GEO and two self-sequencing datasets, all generated in 10x Genomics format. Processed in R using Seurat (4.1.0): filter low-quality cells by gene expression, UMI count, and mitochondrial gene proportion, NormalizeData, select 2000 variable genes (FindVariableFeatures). Integrate data to correct batch effect using CCA method (FindIntegrationAnchors and IntegrateData), generate unified matrix. Screen TNBC sample analysis, retain ER+ and HER2+ samples for comparison of fibrinogen alpha chain (FGA) expression differences. Cluster using FindClusters, UMAP dimensionality reduction visualization. Single-cell RNA sequencing data annotated cell clusters using SingleR (2.4.1) of R (4.3.0), identify cell identity by comparing with reference datasets. Results integrated into Seurat (4.1.0) metadata, visualized using DimPlot. Combined with manual verification of CellMarker (2.0), corrected classification according to marker genes. Use limma package to analyze differentially expressed genes DEG (|log2 Fold Change|>1, adjusted P<0.05) between FGA high and low expression groups, results plotted as volcano plot using R (4.3.0) ggplot2, up-regulated genes marked red, down-regulated genes marked blue. GSEA ranked genes by log2 Fold Change, compared MSigDB c2.all and c5.all gene sets, calculated enrichment scores. Run using R (4.3.0) clusterProfiler (4.8.1), significance threshold adjusted P<0.05. Significant gene sets presented as GSEA curves and tables, highlighting functions and pathways.

[0045] 2. Flow cytometry 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 instruction. Finally, cells were incubated in PBS containing 2% fetal bovine serum (FBS) at 4°C and detected using a flow cytometer (BD, FACSAria II). Rabbit anti-human TROP2 antibody (Abeam ab214488) was used to detect TROP2 expression on the surface of cancer cells. EGFR-fluorescein isothiocyanate (FITC) marker (ICARTAB, 21092702) and Protein L (Pierce, 2997)-streptavidin APC (Aqua Blue, Invitrogen, S21374) were used to detect CAR expression on the surface of CAR-T cells. The following antibody combinations were 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 an ALDEFLUOR kit (StemCell Technologies, Durham, North Carolina, USA) according to the manufacturer's instructions. Cell apoptosis was analyzed using an Annexin V-FITC / PI apoptosis detection kit (MultiSciences Biotech, Co., Ltd., Hangzhou, China). Data were analyzed using FlowJo V.7.6.1 software.

[0046] 3. Quantitative Reverse Transcription PCR (qRT-PCR) detection 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, the mRNA expression level was analyzed using a SYBR Prime-Script PCR Kit II (TaKaRa, RR820A), and quantitatively detected using a Bio-Rad CFX96 detection system (Bio-Rad). The primer sequences used in the present application are as follows: FGA: forward primer (Fwd) 5'-GGGCACATTTGAAGAGGTGT-3'; reverse primer (Rev) 5'-TTGTGACCATCAGGACCAA-3'.

[0047] SOX2: forward primer (Fwd) 5'-CTCGTGCAGTTCTACTCGTCG-3'; reverse primer (Rev) 5'-AGCTCTCGGTCAGGTCCTTT-3'; POU5F1: forward primer (Fwd) 5'-GTGTTCAGCCAAAAGACCATCT-3'; reverse primer (Rev) 5'-GGCCTGCATGAGGGTTTCT-3'; NANOG: forward primer (Fwd) 5'-TCCCGAGAAAAGATTAGTCAGCA-3'; reverse primer (Rev) 5'-AGTGGGGCACCTGTTTAACTT-3'; GAPDH: forward primer (Fwd) 5'-TGTGGGCATCAATGGATTTGG-3'; reverse primer (Rev) 5'-ACACCATGTATTCCGGGTCAAT-3'. GAPDH was used as an internal reference gene, and the gene expression amount was calculated using a 2^(-ΔΔCt) relative quantification method. Each experiment was independently repeated at least three times.

[0048] 4. Immunoblotting and co-immunoprecipitation Cells were lysed with RIPA lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitor tablet (Roche). Total protein concentration was determined using BCA protein concentration assay kit (Thermo, 23225). Equal amount of protein was separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The transferred membrane was incubated with primary antibody at 4°C overnight. Then, the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibody (Rockland; Beyotime) at room temperature for 1 hour and developed using SuperSignal West Dura chemiluminescent substrate (Thermo Scientific, 34075). GAPDH was used as the internal control protein. Protein samples isolated using nuclear and cytoplasmic protein extraction kit (P0028, Beyotime) were also subjected to immunoblot analysis with β-actin and Lamin B1 as the internal control for cytoplasmic and nuclear proteins, respectively. The list of primary antibodies used is as follows: FGA (ab19079, Abeam), Lamin B1 (Ab16048, Abeam), β-actin (ab8227, Abeam), p65 (ab16052, Abeam), phosphorylated p65 (p-p65, ab86299, Abeam), TLR4 (Ab8376, Abeam), MyD88 (ab2064, Abeam), ΙκΒα (ab7217, Abeam), phosphorylated ΙκΒα (p-IκΒα, ab133462, Abeam), cleaved PARP-1 (#D64E10, Cell Signaling Technology), survivin (#FL-142, Cell Signaling Technology), TROP2 (ab214488, Abeam), and GAPDH (#5174, Cell Signaling Technology).

[0049] Co-immunoprecipitation was performed as follows: HEK293T cells were lysed on ice for 15 min with IP lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitor tablet (Roche). After centrifugation, the cell lysate was incubated with TLR4 (Ab8376, Abeam), HA (Ab9110, Abeam) or Flag (Ab205606, Abeam) antibodies at 4°C overnight. Then, 40 μL Protein G Sepharose beads (GE Healthcare) were added and incubated at 4°C for 3 h. After washing the beads with IP lysis buffer for six times, immunoblotting analysis was performed.

[0050] 5. Mouse model Six-week-old female nude mice were used in the experiment. Control cells expressing luciferase, MDA-MB 231 cells overexpressing FGA, MDA-MB 436 cells overexpressing FGA (1 x 10 6 The cells were suspended in 50 μL PBS and mixed with 50 μL Matrigel (Corning) and injected into the subcutaneous part of the mammary fat pad of the mice to form tumors. The tumor growth was monitored and recorded. For the in vivo Epirubicin (EPI) treatment experiment, when the tumor volume reached about 100 mm3, the mice were randomly divided into groups and treated with Epirubicin (5 mg / kg EPI, once every 3 days). For the CAR-T treatment experiment, before receiving TROP2-CAR-T treatment, the mice were imaged in vivo for the transplanted tumors expressing luciferase, and the mice were grouped according to the imaging results to ensure that the tumor loads were approximately equal among the groups. The 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). The statistical significance p-value was determined using the time series test log-rank (Mantel-Cox) test.

[0051] 6. Construction of TROP2-CAR vector and preparation of lentivirus With reference to the sequence of humanized monoclonal antibody (hRS7) targeting TROP2, the second generation of TROP2-CAR vector was constructed by gene synthesis. The vector comprises: TROP2-specific single-chain antibody fragment (anti-TROP2 scFv), CD8 signal peptide, two intracellular co-stimulatory signaling domains (CD3ζ and 4-1BB), CD8 hinge region and transmembrane sequence. The TROP2-CAR vector was co-transfected with two helper packaging plasmids (psPAX2, pMD2.G) into HEK293T cells using polyethyleneimine (PEI). The cell supernatant was collected at 48 hours and 72 hours after transfection, and after centrifugal filtration, PEG8000 (Beyotime, ST483) was added at a ratio of 5:1 for concentration. The concentrated virus was collected, and the virus titer was determined using QPCR reagent (Yeasen, H7101190).

[0052] 7. Preparation and identification of CAR-T cells Fresh peripheral blood of healthy volunteers was collected. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of normal donors using density gradient separation solution. The primary cells were cultured in 1640 medium containing interleukin-2 (IL-2). T cells were activated using anti-human CD3 (Miltenyi, GMP-E2401h-1mg) and CD28 antibodies (Miltenyi, GMP-E2402h-1mg) for 48 hours. After cell counting, the required virus amount was calculated according to the multiplicity of infection (MOI) = 30. The virus solution was added to a 24-well plate previously coated with RetroNectin (TaKaRa, SD3668) and incubated overnight, and centrifuged at 4°C, 3500 rpm for 60 minutes. Subsequently, the activated T cells were inoculated into the culture plate and supplemented with polybrene (8 μg / mL), and centrifuged at 32°C, 3500 rpm for 60 minutes. Finally, the TROP2-CAR-T cells were placed in an incubator for culture. The expression of CAR on the surface of CAR-T cells was evaluated by flow cytometry. The prepared CAR-T cells were used for in vitro and in vivo cytotoxicity experiments.

[0053] 8. Lactate dehydrogenase (LDH) release experiment A total of 8 x 10 4MCF7 cells were seeded in 96-well plates for incubation. Subsequently, T cells were added to the 96-well plates at different effector to target ratios for incubation for 4 hours. The lactate dehydrogenase (LDH) release kit (Beyotime, C0017) was used to determine the LDH level. After selecting the optimal effector to target ratio, the TROP2-CAR-T cells were used for cytotoxicity experiments at the optimal ratio, and the LDH release kit was used for detection. Briefly, the mixture was centrifuged at 400 x g for 5 minutes, and the supernatant was carefully aspirated. 150 μL of diluted LDH release reagent was added, and incubated in the incubator for 1 hour. Subsequently, the mixture was centrifuged at 400 x g for 5 minutes, and 120 μL of supernatant was transferred to a new plate. 60 μL of LDH working solution was added, and incubated in the dark for 30 minutes. The absorbance (OD) value was determined at 490 nm and 620 nm wavelengths. Three replicate wells were set for each sample. The cytotoxicity (%) was calculated according to the OD value of the sample and the control.

Claims

1. The application of a fibrinogen α chain in the preparation of CAR-T therapeutic drugs, characterized in that, Fibrinogen α chain is used to prepare CAR-T therapy drugs that target 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, The drug includes a lentiviral vector encoding the fibrinogen α-chain sequence and a TROP2-CAR vector.

3. The application of the fibrinogen α chain according to claim 2 in the preparation of CAR-T therapeutic drugs, characterized in that, The drugs also include substances that regulate the function of fibrinogen α-chain.

4. The application of the fibrinogen α chain according to claim 3 in the preparation of CAR-T therapeutic drugs, characterized in that, The substance that regulates the function of fibrinogen α chain achieves this by promoting highly uniform expression of TROP2 protein on the surface of tumor stem cells.

5. 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.

6. The application of the fibrinogen α chain according to claim 1 in the preparation of CAR-T therapeutic drugs, characterized in that, Fibrinogen α-chains are secreted into the tumor microenvironment by secretory cell populations or stress-responsive secretory cell subsets in triple-negative breast cancer.

7. The application of a fibrinogen α chain as a biomarker for the treatment of triple-negative breast cancer.

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