Application of gpc3 and gpc3 car-t in diagnosis and treatment of idiopathic pulmonary fibrosis

Abstract

The present application relates to the application of GPC3 and GPC3-CAR-T in the diagnosis and treatment of idiopathic pulmonary fibrosis. The present application confirms the up-regulated expression of GPC3 in the lung fibroblasts of idiopathic pulmonary fibrosis patients in clinical cases, and confirms the up-regulation of GPC3 gene and protein in the lung fibrosis mouse model in animal experiments, indicating that it can be used as a diagnostic marker for idiopathic pulmonary fibrosis. GPC3 CAR-T cells are also prepared, and the killing effect of GPC3 CAR-T on primary lung fibroblast cells is confirmed, indicating that the application of CAR T targeting GPC3 may be a very potential treatment for idiopathic pulmonary fibrosis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical biotechnology, specifically to the application of GPC3 and GPC3 CAR-T in the diagnosis and treatment of idiopathic pulmonary fibrosis. Background Technology

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic fibrotic interstitial pneumonia with an unclear pathogenesis and poor prognosis. It primarily affects patients over 50 years of age, with men more frequently affected than women. There are approximately 3 million IPF patients worldwide. Currently, only pirfenidone and nintedanib are available for IPF, primarily working by slowing the decline in lung function, thus delaying IPF progression. However, their efficacy is limited and their tolerability is poor. The disease burden of IPF on individuals, families, society, and public health resources is significant. IPF is a fibroproliferative disease characterized by chronic damage to alveolar epithelial cells and abnormal tissue repair, leading to the formation of fibroblastic foci. These fibroblastic foci are mainly composed of fibroblasts and myofibroblasts. Myofibroblasts are the activated form of fibroblasts, which can cause basement membrane disruption, promote epithelial cell apoptosis, and prevent subsequent reepithelialization. Simultaneously, they lead to excessive deposition of extracellular matrix, resulting in pulmonary fibrosis, lung structural destruction, and loss of lung function.

[0003] In recent years, several drugs for treating idiopathic pulmonary fibrosis (IPF) have been used clinically, significantly alleviating the progression of IPF, improving clinical symptoms, and enhancing patients' quality of life. Pirfenidone is currently considered the most effective antifibrotic drug, possessing anti-inflammatory, anti-fibrotic, anti-proliferative, TGF-β-inhibiting, and collagen-synthesizing effects. It has demonstrated clear therapeutic efficacy in clinical trials and is worthy of widespread clinical application. Nintedanib has effective anti-fibrotic and anti-inflammatory effects and can inhibit the proliferation, migration, and transformation of fibroblasts into myofibroblasts. This drug can slow the decline in lung function, thereby alleviating disease progression. N-acetylcysteine ​​has antioxidant and free radical scavenging effects, inhibiting excessive proliferation and differentiation of pulmonary fibroblasts and lymphocytes, reducing the production of inflammatory mediators, and inhibiting collagen synthesis, thus playing a role in preventing disease progression in pulmonary fibrosis. Glucocorticoids are the main traditional treatment for pulmonary fibrosis. Low-dose oral glucocorticoid therapy or combination therapy with other drugs has some efficacy in treating IPF, but glucocorticoids have significant side effects. Currently, treating IPF remains a medical challenge. Clinically, individualized treatment is necessary based on each patient's condition. Existing drugs and treatments can only alleviate IPF symptoms and slow disease progression; they cannot fundamentally reverse the disease or cure it. Most research on IPF treatments has failed to achieve satisfactory results. Therefore, finding new methods to slow down or even reverse the progression of IPF and providing effective treatments for IPF patients is currently a key focus of research in idiopathic pulmonary fibrosis.

[0004] Glypican-3 (GPC3) is a cell membrane protein modified with two heparan sulfate (HS) chains. It is highly expressed in over 70% of hepatocellular carcinoma (HCC) and other solid tumors such as squamous cell carcinoma of the lung, but shows no significant expression in normal tissues, including the liver. GPC3 is involved in various processes, including cell growth, differentiation, and migration. The GPC3 gene is located in a region of the human chromosome, and the encoded protein has a relative molecular mass of approximately 70 kDa and consists of 580 amino acids. Currently, GPC3 is a prominent target for targeted therapy in liver cancer, and therapies targeting GPC3 include: conjugation to toxin immunotoxin, CTL, TCR-T, and CAR-T therapy.

[0005] CAR-T cells have become a hot topic in adoptive immunotherapy for tumors in the past five years, combining the advantages of cell immunotherapy and antibody immunotherapy: (1) More accurate treatment, because CAR-T cells use the mechanism of antigen-antibody binding to avoid immune escape of tumor cells, that is, to overcome the immune escape of tumor cells by downregulating MHC molecule expression and reducing antigen presentation, making it impossible for tumor cells to escape; (2) More accurate multi-targeting, CAR can utilize both tumor protein antigens and glycolipid non-protein antigens, expanding the range of tumor antigen targets, and the action of CAR-T cells is not limited by MHC; (3) Wider tumor killing range, many tumor cells express the same tumor antigens, and once the CAR gene targeting a certain tumor antigen is constructed, it can be widely used; (4) More durable tumor killing effect, CAR-T cells have immune memory function and can survive in the body for a long time. That is to say, these T cells do not need to be continuously administered like chemotherapy drugs. The cells are continuous, dynamic and constantly dividing drugs, and a single injection provides long-lasting protection. Due to these advantages, CAR-T cells have already been used clinically to treat neurocytoma, B-cell leukemia, B-cell lymphoma, melanoma, lung cancer, glioma, ovarian cancer, and other diseases. Previous studies have suggested that genetic ablation of mouse fibroblasts induced by cardiac injury leads to significant regression of cardiac fibrosis and improvement in cardiac function. In connective tissue proliferative tumor models, some studies have successfully targeted and ablated activated fibroblasts in the tumor stroma using CAR-T cells. Jonathan A. Epstein's team published an article in Nature entitled "Targeting cardiac fibrosis with engineered T cells," using a mouse model of cardiac disease to determine that genetically modified T cells can effectively target activated cardiac fibroblasts. This innovative result not only opens the door for CAR-T targeting other forms of cardiac fibrosis but also potentially opens the door for immunotherapy of many extracardiac fibrotic diseases.

[0006] Chinese patent CN112226506A, published on January 15, 2021, discloses a diagnostic biomarker for idiopathic pulmonary fibrosis (IPF), CCT6A, and its application in the preparation of diagnostic or prognostic tools. This invention belongs to the field of medical diagnostic biomarkers and tools. It achieves clinical diagnosis by detecting the expression of the CCT6A gene and its expression products. This study found high levels of CCT6A in the serum of IPF patients and high expression of CCT6A in the lung fibroblasts of IPF patients, indicating that CCT6A may regulate fibroblast activity during pulmonary fibrosis, further regulating the process of pulmonary fibrosis. This provides a non-invasive method for the early diagnosis of IPF in clinical practice and is suitable for further clinical application. However, there are currently no reports on the application of GPC3 and GPC3 CAR-T in the diagnosis and treatment of IPF. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by providing the application of GPC3 and GPC3 CAR-T in the diagnosis and treatment of idiopathic pulmonary fibrosis.

[0008] On the one hand, the present invention provides the application of the GPC3 gene or protein as a diagnostic marker in the preparation of diagnostic reagents or kits for idiopathic pulmonary fibrosis.

[0009] As a preferred example, the reagent or kit is used to detect the expression level of the GPC3 gene or protein, which is highly expressed in idiopathic pulmonary fibrosis tissue.

[0010] Secondly, a diagnostic kit for idiopathic pulmonary fibrosis is provided, the kit comprising reagents for detecting the expression level of the GPC3 gene or protein.

[0011] Thirdly, the application of reagents for detecting the expression level of the GPC3 gene or protein in the preparation of diagnostic kits for idiopathic pulmonary fibrosis is provided.

[0012] Fourthly, a specific chimeric antigen receptor targeting GPC3 is provided, wherein the specific chimeric antigen receptor targeting GPC3 comprises a signal peptide, an antigen-binding domain, an extracellular hinge region, a transmembrane domain, and an intracellular signaling domain connected in series.

[0013] As a preferred example, the nucleotide sequence of the GPC3-targeting chimeric antigen receptor gene is shown in SEQ ID NO.1;

[0014] The signal peptide is a CD8 leader, and its nucleotide sequence is shown in SEQ ID NO.2;

[0015] The antigen-binding domain GC33 scFv has the nucleotide sequence shown in SEQ ID NO.3;

[0016] The extracellular hinge region shown is CD8, and its nucleotide sequence is shown in SEQ ID NO.4;

[0017] The transmembrane domain is CD8, and its nucleotide sequence is shown in SEQ ID NO.5;

[0018] The intracellular signaling domains are CD28, 4-1BB, and CD3-zata, and their nucleotide sequences are shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively.

[0019] Fifthly, an immune cell is provided that expresses the specific chimeric antigen receptor targeting GPC3 as described in any one of claims 5-7.

[0020] In a sixth aspect, a pharmaceutical composition is provided, the composition comprising the immune cells of claim 8.

[0021] In a seventh aspect, an application is provided in which any of the above-described GPC3-targeting chimeric antigen receptors, immune cells, or pharmaceutical compositions are used in the preparation of a drug for treating idiopathic pulmonary fibrosis.

[0022] The advantages of this invention are:

[0023] Experiments verified that GPC3 is upregulated in IPF and closely related to the occurrence and development of fibrosis, and is specifically expressed on the surface of lung fibroblasts. A marker molecule for IPF was defined, and CAR-T cells targeting this molecule were constructed, providing an effective method and drug for the prevention and treatment of idiopathic pulmonary fibrosis. This study shows promising application prospects. Attached Figure Description

[0024] Appendix Figure 1 GPC3 is upregulated in IPF patients. (A) UMAP plot of cell populations in lung tissue samples from all IPF patients and controls in a single-cell transcriptome sequencing dataset. (B) Violin plot of GPC3 expression in fibroblasts. (C) Dot plot of GPC3 and other fibrosis markers expression in individual cells. Colors represent the average expression level of the protein, with closer to yellow indicating higher expression levels; the size of the dot indicates the proportion of cells expressing the protein, with larger dots indicating more expressing cells.

[0025] Appendix Figure 2GPC3 upregulation in a BLM-induced mouse pulmonary fibrosis model. (A) Kaplan-Meier survival curve. (B) Weight change over 15 days. (C) Lung coefficient: lung wet weight (g) / body weight (g). (D) Hematoxylin-eosin staining (HE), Masson and Sirius Red staining of lung tissue pathology. (E) Ashcroft score. (F) Collagen area. (G) Western blotting of internal reference protein (GAPDH), GPC3 and fibrosis markers in lung tissue. (H, I) Relative quantification of WB gray values ​​in G.

[0026] Appendix Figure 3 This is a diagram of the third-generation GPC3 CAR model, which incorporates GPC3.

[0027] Appendix Figure 4 This is a structural diagram of the GPC3 CAR expression vector.

[0028] Appendix Figure 5 The positive rates of Mouse GPC3 CAR-T and Human GPC3 CAR-T were measured.

[0029] Appendix Figure 6 The expression of CD69, IL-2, and IFN-γ after co-culturing Mouse GPC3 CAR-T cells with mouse lung fibroblasts and Human GPC3 CAR-T cells with human lung fibroblasts was investigated, as well as the killing effect of effector cells on the corresponding target cells (mouse lung fibroblasts and human lung fibroblasts).

[0030] It should be noted that the figures in this application have been processed in grayscale, and the descriptions of the figures above are descriptions of the original experimental results figures. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0032] Example 1 Clinical Analysis

[0033] 1.1 GPC3 is upregulated in IPF patients and is specifically expressed on lung fibroblasts.

[0034] Lung tissue samples were collected from 32 IPF patients and 28 healthy controls, and single-cell RNA sequencing (scRNA seq) datasets (GSE136831) were analyzed from the tissue samples. The results showed that GPC3 expression was upregulated in IPF patients compared to the control group. Figure 1 B); and GPC3 is mainly expressed in lung fibroblasts, with very low expression levels in other types of lung cells. Figure 1 C).

[0035] Example 2 Animal Model Experiment

[0036] 1.1 GPC3 is upregulated in a mouse model of bleomycin (BLM)-induced pulmonary fibrosis.

[0037] Bleomycin-induced mouse model is an internationally recognized in vivo model of idiopathic pulmonary fibrosis. The in vivo mouse model used 7-week-old female C57BL / 6 mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). We established the idiopathic pulmonary fibrosis model by a single intratracheal infusion of BLM (10 mg / kg). Simultaneously, a control group of mice of the same strain, sex, and age was constructed by intratracheal infusion of the same volume of PBS. Samples were collected on day 15.

[0038] The 15-day mortality rate in the IPF group was 33.33% (3 / 9). Figure 2 A) In the IPF group, the body weight of mice decreased and stabilized at a low level within 5 days, significantly lower than that of the control group. Figure 2 B). The lung coefficient in the IPF group was significantly higher than that in the control group ( Figure 2 C). Lung pathology in the IPF group showed pulmonary fibrosis characteristics such as alveolar septal fibrosis, fibrotic masses, and collagen fiber hyperplasia. Figure 2 DF). In the IPF group, GPC3 levels were significantly elevated ( Figure 2 G, 2I).

[0039] Example 3: Construction of GPC3 CAR Lentiviral Expression Vector

[0040] This invention designs a CAR sequence containing the GC33-scFv gene, based on the widely reported and applied GC33 sequence, and incorporates gene fragments from the CD8 transmembrane region and the intracellular signaling regions of CD28, 4-1BB, and CD3zata. Following the DNA coding sequence of GC33, the entire expression cassette was synthesized by Beijing Qingke Biotechnology Co., Ltd., digested with enzymes, and inserted between the EcoRI-BamHI sites of the pCDH-MCS-T2A-copGFP-MSCV viral vector to obtain the GPC3 CAR. After confirming correct sequencing, the plasmid was extracted using a Promega plasmid extraction kit. Transfection was performed on Lenti-X cells using the pSPAX2, pMD2G viral packaging system and CaCl2 transfection reagent. After virus harvesting, purification was carried out.

[0041] The specific chimeric antigen receptor targeting GPC3 is composed of CD8 leader, GC33 scFv, CD8 hinge region and transmembrane region, CD28 intracellular signaling region, 4-1BB intracellular signaling region, and CD3-zata intracellular signaling region connected in series. Figure 3-4 The nucleotide sequences of the gene encoding the specific chimeric antigen receptor targeting GPC3 are shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding the CD8 leader is shown in SEQ ID NO.2; the nucleotide sequence of the gene encoding GC33 scFv is shown in SEQ ID NO.3; the nucleotide sequence of the gene encoding the hinge region of CD8 is shown in SEQ ID NO.4; the nucleotide sequence of the gene encoding the transmembrane region of CD8 is shown in SEQ ID NO.5; the nucleotide sequence of the gene encoding the intracellular signaling region of CD28 is shown in SEQ ID NO.6; the nucleotide sequence of the gene encoding the intracellular signaling region of 4-1BB is shown in SEQ ID NO.7; and the nucleotide sequence of the gene encoding the intracellular signaling region of CD3-zata is shown in SEQ ID NO.8.

[0042] Example 4: Preparation and Identification of CAR-T Cells

[0043] 1. Preparation of Mouse GPC3 CAR-T cells

[0044] Mouse CD3 was isolated from the spleen of 4-week-old female C57BL / 6 mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). + T cells were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum and 100 U / mL IL-2. T cell activation beads coated with α-CD3 / α-CD28 antibodies were added at a T cell to magnetic bead ratio of 1:2 and gently mixed. The following day, CD3+ cells in mice activated for 24 hours were counted. +T cells, cell density adjusted to 1×10⁶ 6 Add 6 μg / mL polybrene and 100 U / mL recombinant mouse IL-2 (purchased from R&D Company) to each well. Add the mixed cell and virus suspension to each well, centrifuge at 2000×g for 90 min, and then incubate overnight. After centrifugation and medium change, continue culturing, supplementing with IL-2 every 2 days at a dose of 3 × 10⁻⁶. 5 The cells were passaged at a density of cells / mL. The constructed CAR-T cells were named MouseGPC3 CAR-T group (M-GPC3 CAR-T), while uninfected mouse T cells were used as a negative control (MT).

[0045] 2. Preparation of Human GPC3 CAR-T cells

[0046] Peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll-Hypaque density gradient centrifugation method. Peripheral blood from healthy volunteers was diluted with a proportional amount of room-temperature equilibrated DPBS. Human lymphocyte separation medium was added to centrifuge tubes, and the diluted blood was slowly poured along the tube wall onto the surface of the separation medium, creating a clear interface between the two liquids. The volume ratio of anticoagulated blood, DPBS, and separation medium was 1:1:1. Centrifugation was performed using a horizontal centrifuge at 2500 rpm for 20 minutes at room temperature. After centrifugation, due to differences in cell specific gravity, different cell types separated into four layers: the top layer was plasma; the second layer was a hazy layer containing lymphocytes and monocytes (PBMCs); the third layer was human lymphocyte separation medium; and the bottom layer consisted of heavier erythrocytes and granulocytes. Carefully aspirate the cells from the cloud layer into a new centrifuge tube, add three volumes of DPBS, centrifuge at 400×g for 10 min, discard the supernatant, and the remaining cells are PBMCs. Then use MojoSort... TM Primary T cells were isolated using a CD3+ T cell negative sorting kit (purchased from Biolegend), achieving a purity of over 99%. Cells were cultured and resuspended in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (2 × 10⁶ cells / year). 6 After 24 hours, IL-2 (300 IU / mL) and anti-CD3 antibody (50 ng / mL) were added. After 72 hours, half of the T cells were infected with GPC3-CAR lentivirus at a multiplicity of infection (MII) of 8 PFU / cell. The infected T cells (5 × 10⁻⁶ cells / mL) were then further inoculated. 5 The other half of the T cells were cultured as the Human GPC3 CAR Lentiviral Infection Group (H-GPC3CAR-T) and the other half were cultured as the Uninfected Group (HT).

[0047] 3. CAR-T cell positivity rate detection

[0048] T cells from the GPC3 CAR lentivirus-infected group and the uninfected group were collected into 15 mL centrifuge tubes. After centrifugation at 200×g for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL of PBS. After centrifugation at 200×g for 5 min, the supernatant was discarded, and the cells were resuspended in 0.2 mL of PBS (5×10⁶ cells / mL). 6 After the cells were counted at 100 cells / mL, the green fluorescence expression of T cells was detected by flow cytometry. T cells expressing green fluorescence were considered to be positive for GPC3 CAR expression.

[0049] The results showed that the positive rate of Mouse GPC3 CAR transfection was 58.7% ( Figure 5 a) The positive rate of Human GPC3CAR transfection was 26.0% ( Figure 5 b).

[0050] Example 5: In vitro detection of target cell activation of CAR-T cells

[0051] 1. Flow cytometry detection of CD69 molecule expression on the surface of CAR-T cells

[0052] Effector cells M-GPC3 CAR-T and mouse pulmonary fibroblasts (MPF) 1x10 5 / wells co-cultured in 24-well plates, 1x10 H-GPC3 CAR-T cells and human lung fibroblasts (HPF) 5 Cells were co-cultured in 24-well plates, with a separate T-cell culture group serving as a negative control. Each well was supplemented with 1 mL of RPMI 1640 medium containing 10% FBS and incubated at 37°C for 24 h. Cells were collected in 1.5 mL EP tubes, centrifuged at 300 × g for 5 min at room temperature, and the supernatant was discarded. 100 μL of antibody-labeled system was added to each sample, with each reaction system containing 1 μL of CD69-APC antibody (purchased from BD) and 99 μL of PBS. After incubation at room temperature in the dark for 30 min, the cells were washed twice with PBS, and CD69 expression was detected by flow cytometry.

[0053] result( Figure 6 a) and 6e) indicate that, compared with control T cells, M-GPC3 CAR-T and H-GPC3 CAR-T can be significantly activated by target cells MPF and HPF, respectively.

[0054] 2. Detection of CAR-T cell secretion cytokine levels using ELISA.

[0055] After CAR-T cells and target cells were co-incubated at a 1:1 effector-target ratio for 24 h, the cell supernatant was transferred to Eppendorf tubes, centrifuged at 300×g for 10 min, and the supernatant was collected. Following the instructions of the ELISA cytokine assay kit (purchased from BD), the levels of CAR-T cell-secreted cytokines murine IL-2 (mIL-2), murine interferon-γ (mIFN-γ), human IL-2 (hIL-2), and human interferon-γ (hIFN-γ) were detected using a double-sandwich assay. After substrate development, D values ​​were read at wavelengths of 490 nm and 570 nm using an ELISA reader. The D values ​​from D490 nm to D570 nm were used as calibration D values. The concentrations of the corresponding cytokines in each sample were then calculated based on a standard curve.

[0056] Figure 6 bc indicates that GPC3-overexpressing Mouse CAR-T cells released more IL-2 and IFN-γ during co-culture with mouse lung fibroblasts. Figure 6 The fg results showed that GPC3-overexpressing Human CAR-T cells also released more IL-2 and IFN-γ during co-culture with human lung fibrosis cells, suggesting that GPC3-overexpressing CAR-T cells may exert a greater anti-fibrotic effect.

[0057] 3. Annexin-V labeling method for detecting the cytotoxic effect of CAR-T cells on target cells

[0058] MT and M-GPC3 CAR-T cells were co-cultured with Celltrace Yellow (Thermo Fisher Scientific) labeled target cells (MPF) at effector-to-target ratios of 2:1, 1:1, 1:2, and 1:4 in 24-well plates, with five replicates for each ratio. HT and H-GPC3 CAR-T cells were co-cultured with target cells (HPF) using the same procedure. The culture medium used was RPMI 1640 medium containing 10% fetal bovine serum. After plating, the 24-well plates were incubated at 37°C for 24 hours. Apoptotic cells were labeled with 50 μL of Annexin-V (BD) and 200 μL of Bounding Burrer was added after 30 minutes for analysis.

[0059] The results are as follows Figure 6 As shown, mouse lung fibroblasts and human lung fibroblasts co-cultured with GPC3 CA-T cells exhibited higher lysis rates. Figure 6(d, 6h) Compared with control T cells, GPC3 CA-T cells specifically promoted apoptosis of lung fibroblasts at a lower effective-to-target ratio, indicating that GPC3-overexpressing CAR-T cells did indeed enhance their cell-killing effect. This suggests that the GPC3 CAR molecule design is reasonable and lays the foundation for the treatment of IPF.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. Application of a kit for detecting GPC3 gene or protein expression levels in the preparation of a diagnostic kit for idiopathic pulmonary fibrosis.

Images