Car-t cell having good blood-brain barrier permeability, product, and use

Abstract

Provided are a CAR-T cell having good blood-brain barrier permeability, a product, and a use. On the basis of the research on CAR-T cell therapy for central nervous system diseases, provided are a CAR-T cell having good blood-brain barrier permeability and a corresponding drug, for use in treating or relieving central nervous system diseases. Such a CAR-T cell uses a B cell maturation antigen as a target, and highly expresses a CXCR3 chemokine receptor and CCL1, CCL3 and CCL4 chemokines. Also provided is a product for testing and determining the blood-brain barrier permeability of the CAR-T cell, e.g., a probe, a reagent, and a kit, thereby accurately testing and determining the blood-brain barrier permeability of a certain CAR-T cell.

Description

CAR-T cell with good blood-brain barrier permeability, product and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of central nervous system disease treatment, in particular to a CAR-T cell with good blood-brain barrier permeability, and application in detecting the blood-brain barrier permeability of the CAR-T cell for treating central nervous system diseases. BACKGROUND

[0002] CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) treatment, i.e. chimeric antigen receptor T cell immunotherapy, belongs to a kind of immunotherapy. CAR-T treatment is to collect and separate T cells from patient blood, to genetically modify and then culture and expand in vitro, and finally to reinfuse into the patient's body, so as to target the clearance of target cells and thus exert its unique therapeutic effect. At present, CAR-T treatment is mainly used for clinical treatment of patients with malignant hematological diseases and malignant tumors, such as relapsed / refractory acute B lymphoblastic leukemia, conventional treatment ineffective large B cell non-Hodgkin lymphoma, relapsed / refractory malignant lymphoma and relapsed / refractory multiple myeloma, etc., and has shown significant efficacy and sustained durability. Due to its ability to directly induce target cell death and its own expansion characteristics, CAR-T therapy has been recommended as a second-line treatment within one year after initial treatment of B-cell lymphoma.

[0003] However, although CAR-T therapy has shown therapeutic effectiveness in various types of solid tumors and hematological tumors, the therapeutic effect of CAR-T treatment in CNS diseases has not yet achieved satisfactory results due to the presence of the blood-brain barrier (BBB) in the central nervous system (CNS) with its special structure. The blood-brain barrier is a dynamic barrier existing between the CNS and the blood circulation system, which strictly regulates the material transport between blood and cerebrospinal fluid, provides a stable internal environment for the realization of neural function, and at the same time protects the CNS from damage by various harmful substances, but also becomes a barrier that the therapeutic drugs for CNS-related diseases have to face. Many drugs are eliminated because they cannot reach an effective dose through the blood-brain barrier. In the treatment of CNS diseases, including central nervous system leukemia, brain tumors such as glioblastoma, encephalitis, and neuromyelitis optica spectrum disease, the ability of drugs to effectively penetrate the blood-brain barrier is highly required.

[0004] In the process of treating central nervous system diseases with CAR-T cells, it is very important for CAR-T cells to effectively penetrate the blood-brain barrier into the central nervous system to exert their targeted therapeutic effects. Therefore, in the treatment of CNS diseases, it is urgent to develop a reliable CAR-T cell with good blood-brain barrier permeability. SUMMARY

[0005] The present application provides a CAR-T cell with good blood-brain barrier permeability for treating central nervous system diseases, and a product for detecting the blood-brain barrier penetration ability of the CAR-T cell and related applications, which are specifically achieved by the following technologies.

[0006] A CAR-T cell with good blood-brain barrier permeability, which is used for treating or improving central nervous system diseases, targets B-cell maturation antigen, and highly expresses CXCR3 chemokine receptor and CCL1, CCL3 and CCL4 chemokines.

[0007] The present application proves that the above-mentioned CAR-T cell can effectively penetrate the blood-brain barrier. This CAR-T cell has good chemotactic properties and the ability to activate chemotaxis-related pathways.

[0008] Further, the CAR-T cell is derived from PBMC separated from peripheral blood.

[0009] The present application also provides a method for detecting the blood-brain barrier penetration ability of the above-mentioned CAR-T cell without the purpose of disease detection and diagnosis, which is judged by detecting the expression level of CXCR3 chemokine receptor and CCL1, CCL3 and CCL4 chemokines in cerebrospinal fluid; or detecting and analyzing the chemotactic properties of the CAR-T cell based on single-cell transcriptome sequencing data (such as obtained by using 10xgenomics platform) to make a judgment.

[0010] A product for detecting the blood-brain barrier penetration ability of the above-mentioned CAR-T cell for treating or improving central nervous system diseases, which is used for detecting the expression level of CXCR3 chemokine receptor and CCL1, CCL3 and CCL4 chemokines in cerebrospinal fluid.

[0011] Further, the product includes reagents or probes for detecting the expression level of CXCR3 chemokine receptor and CCL1, CCL3 and CCL4 chemokines

[0012] Further, the product is a detection kit, test strip or detection chip.

[0013] The application also provides the application of the CAR-T cell with good blood-brain barrier permeability as described above in the preparation of a drug for targeted treatment of central nervous system diseases.

[0014] Further, the central nervous system disease is a neuromyelitis optica spectrum disorder.

[0015] Compared with the prior art, the application has the advantages that the application provides a method for detecting the blood-brain barrier permeability of the CAR-T cell, and the detection accuracy is high.

[0016] The application also constructs a CAR-T cell product with high expression of CXCR3 chemokine receptors and CCL1, CCL3 and CCL4 chemokines, and proves the effect of effectively penetrating the blood-brain barrier. The CAR-T cell has good chemotaxis characteristics and the ability to activate chemotaxis-related pathways.

[0017] Therefore, the CAR-T cell prepared by the method provided by the application has good blood-brain barrier permeability, and is beneficial to the CAR-T cell to play a more efficient role in the treatment of central nervous system diseases, especially neuromyelitis optica spectrum disorders (NMOSD). BRIEF DESCRIPTION OF DRAWINGS

[0018] Fig. 1 is a diagram of the CAR copy number in cerebrospinal fluid at different times after CAR-T treatment;

[0019] Fig. 2 is a diagram of the expression level of chemokines and receptors of the CAR-T cell that effectively penetrates the blood-brain in CD4+ T cells;

[0020] Fig. 3 is a diagram of the expression level of chemokines and receptors of the CAR-T cell that effectively penetrates the blood-brain in CD8+ T cells;

[0021] Fig. 4 is a diagram of cell marker grouping screening 3 months after CAR-T treatment;

[0022] Fig. 5 is a diagram of the proportion of cell groups 3 months after CAR-T treatment;

[0023] Fig. 6 is a diagram of the difference in chemotaxis score of CAR-T cells in peripheral blood and cerebrospinal fluid;

[0024] Fig. 7 is a diagram of the difference in expression level of chemokines and receptors of CAR-T cells in peripheral blood and cerebrospinal fluid;

[0025] Fig. 8 is an enrichment diagram of the up-regulation of chemotaxis-related pathways of CAR-T cells in cerebrospinal fluid;

[0026] Fig. 9 is a diagram of the expression level of CXCR3 and the decrease level of SBCMA in cerebrospinal fluid. DETAILED DESCRIPTION

[0027] The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0028] The following specific embodiments select neuromyelitis optica spectrum disorder (NMOSD) as a representative of central nervous system (CNS) diseases for testing. Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory autoimmune demyelinating disease in the central nervous system (CNS), mainly manifested as optic neuritis and acute transverse myelitis-related symptoms such as visual impairment, limb numbness and weakness, movement disorders, and autonomic nervous function abnormalities. The pathogenesis of NMOSD is mainly that plasma cells produce aquaporin 4 (AQP4) antibodies, which cross the blood-brain barrier, bind to AQP4 antigens on astrocytes, and ultimately cause damage to astrocytes, oligodendrocytes, demyelination, and neuronal loss through mechanisms such as antibody-dependent cellular cytotoxicity and complement-dependent cellular cytotoxicity.

[0029] The clinical research subjects selected in the following specific embodiments are 5 patients with refractory or recurrent aquaporin 4 (AQP4) serum antibody positive NMOSD treated with anti-BCMA CAR-T cells. These patients obtained cerebrospinal fluid samples from NMOSD patients at baseline and 3 months after CAR-T cell infusion, respectively.

[0030] At the same time, 5 control subjects matched with the NMOSD subjects in terms of age and gender were selected, including 1 patient with primary headache, 3 patients with non-inflammatory peripheral neuropathy, and 1 patient with idiopathic intracranial hypertension. Blood and cerebrospinal fluid samples were collected from these control subjects. With the approval of the Shanghai Jiaotong University Institutional Review Board, the patients in the related research of the following specific embodiments provided informed consent documents for the use of their infusion products, blood, and cerebrospinal fluid samples.

[0031] In the following specific embodiments, cerebrospinal fluid samples were obtained from NMOSD patients at baseline and 3 months after CAR-T product infusion, respectively.

[0032] The CAR-T cells provided and backed up by the following specific embodiments are CAR-T cells targeting B-cell maturation antigen (BCMA) and capable of high expression of CXCR3 chemokine receptor and CCL1, CCL3 and CCL4 chemokines. The CAR-T cell product is prepared by transfecting autologous T cells with a lentivirus as a gene vector, and the CAR contains a fully human scFv, a CD8a hinge and transmembrane, a 4-1BB co-stimulation and a CD3ζ activation domain. The research code of the CAR-T cell is CT103A, which is prepared by Nanjing Tundevik Biotechnology Co., Ltd.

[0033] Specifically, the method for obtaining the CAR-T cells provided by the following specific embodiments is:

[0034] (1) First, the patient's white blood cells are separated using CD3 microbeads (Miltenyi Biotec) according to the manufacturer's protocol;

[0035] (2) Then, the separated white blood cells are activated in CTS TM OpTmizer TM medium (Gibco) with 20mM L-GlutaMAX TM (Gibco) and 200IU / mL recombinant human IL-2 (SL PHARM) using Dynabeads TM human T activator CD3 / CD28 (Invitrogen).

[0036] (3) The CAR-T cells obtained in step (2) are transfected with autologous T cells using a lentivirus as a gene vector, and the CAR contains a fully human scFv, a CD8a hinge and transmembrane, a 4-1BB co-stimulation and a CD3ζ activation domain.

[0037] The transfected cells are washed and beaded at day 5, and then cultured in G-Rex (Wilson Wolf). By day 10-11, the CAR-T cells are collected, washed and suspended in a cryopreservation solution.

[0038] (4) Finally, the prepared CAR-T cells are aliquoted into cryobags (Miltenyi Biotec) and frozen to -90°C using ThermoFisher's 7451TF CRF#4. The final product is stored in a gas phase liquid nitrogen tank (BIOBANK-22K) below -130°C.

[0039] Test Example 1: Collection of CAR-T cells that can effectively cross the blood-brain barrier

[0040] 1. Sample collection preparation and single-cell RNA sequencing

[0041] (1) Collect the cerebrospinal fluid samples of NMOSD patients at baseline, 1 month and 3 months after infusion, and control subjects using sterile centrifuge tubes.

[0042] (2) Centrifuge the collected cerebrospinal fluid samples at 300xg for 10 min at 4°C to obtain precipitated cells.

[0043] (3) Resuspend the precipitated cells with 60 μl of sterile phosphate buffer, and load the single-cell suspension into the 10X Chromium platform for machine operation. Use Cell Ranger to demultiplex and align the raw RNA data and antibody tag sequences.

[0044] Use the Seurat v4.2.0 software package in R to analyze the feature-barcode matrix generated from this pipeline for subsequent single-cell analysis operations.

[0045] 2. Detection of CAR gene copy number in cerebrospinal fluid

[0046] Droplet digital PCR (ddPCR) technology can detect the copy number of CAR gene in cerebrospinal fluid and evaluate the ability of CAR-T cells to cross the blood-brain barrier. This embodiment uses ddPCR technology to absolutely quantify the copy number of CAR gene.

[0047] The specific method includes:

[0048] (1) (1) Add lysis buffer to the collected blood samples, then use the blood genomic DNA extraction kit (51104, Qiagen) to extract sample DNA at the designated time point, and measure the CAR copy number of each sample using ddPCR.

[0049] (2) Use ddPCR to determine the copy number of CAR gene. The probe and primer target the scFV sequence. Specifically, the primer sequence of the CAR gene is as follows:

[0050] Forward primer: 5'-cagcaaaaatacgacctcctcact-3'; as shown in SEQ ID NO. 1;

[0051] Reverse primer: 5'-tggtgctgcctttgatctca-3'; as shown in SEQ ID NO. 2;

[0052] The primer sequence of the control group is as follows:

[0053] Forward primer: 5'-ggcggtggtcctggagtact-3'; as shown in SEQ ID NO. 3;

[0054] Reverse primer: 5'-agaggcctttggctttcttctt-3'; as shown in SEQ ID NO. 4;

[0055] The probe sequence is: 5'-VIC-acccgccacaagc-3', as shown in SEQ ID NO. 4 (without fluorescent label).

[0056] (3) Determine the copy number by real-time fluorescence quantitative PCR with reference to the standard curve. Perform a no-template control reaction using nuclease-free water.

[0057] The specific PCR program is: 50℃, 2min, cycle 1 time; 95℃, 10min, cycle 1 time; 95℃, 15s, cycle 45 times; 60℃, 60s, cycle 1 time; finally keep at 4℃.

[0058] 3. Detection of the expression level of the copy number of CAR gene in cerebrospinal fluid

[0059] The present application detects the expression level of the copy number of CAR gene in cerebrospinal fluid of 5 NMOSD CAR-T subjects at baseline, 1 month after product infusion, and 3 months after product infusion.

[0060] The detection results are shown in Figure 1, in which Baseline represents the copy number of CAR gene in the cerebrospinal fluid of the subject at baseline, 1m represents the copy number of CAR gene in the cerebrospinal fluid of the subject 1 month after product infusion, and 3m represents the copy number of CAR gene in the cerebrospinal fluid of the subject 3 months after product infusion.

[0061] It can be seen that 1 month after product infusion, the expression level of CAR gene in the cerebrospinal fluid of CAR-T subjects is significantly up-regulated, which indicates that CAR-T cells infused peripherally at this time have crossed the blood-brain barrier into the cerebrospinal fluid. However, 3 months after product infusion, the detection copy number suggests that the CAR-T cells in the cerebrospinal fluid of some patients have decreased to 0, but in some patients, the cerebrospinal fluid still maintains a high level.

[0062] The detection results of Figure 1 show that in the central nervous system cerebrospinal fluid, CAR-T cells with good blood-brain barrier characteristics can maintain a high level of copy number for a long time after infusion. Such CAR-T cells can effectively penetrate the blood-brain barrier, thereby exerting their key therapeutic effect in the central nervous system.

[0063] Test Example 2: Determination of high expression marker in CAR-T cells that effectively penetrate the blood-brain barrier

[0064] By single-cell differential gene analysis through R studio software, we analyzed and compared the differential genes between CAR-T cells that effectively penetrated the blood-brain barrier (CSF Exist) and CAR-T cells that could not effectively penetrate the blood-brain barrier (CSF noExist).

[0065] The analysis results are shown in Figures 2-3, and the results show that:

[0066] (1) In Figure 2, among CD4+ T cells, the expression of CXCR3 and CXCR5 chemokine receptors, and CCL1, CCL3 and CCL4 chemokines was significantly higher in CAR-T cells that could still penetrate the blood-brain barrier and reach the cerebrospinal fluid after three months of reinfusion.

[0067] (2) In Figure 3, among CD8+ T cells, the expression of CXCR3 chemokine receptor and CCL1, CCL3 and CCL4 chemokines was significantly higher in CAR-T cells that could still penetrate the blood-brain barrier and reach the cerebrospinal fluid after three months of reinfusion.

[0068] The results of Figures 2-3 show that the high expression of CXCR3 chemokine receptor and CCL1, CCL3 and CCL4 chemokines can promote CAR-T cells to continuously penetrate the blood-brain barrier and thus reach the cerebrospinal fluid to exert their effects. Therefore, CXCR3 chemokine receptor and CCL1, CCL3 and CCL4 chemokines can be used as biomarkers of CAR-T cells that effectively penetrate the blood-brain barrier for the development of CAR-T cells with good blood-brain barrier permeability.

[0069] Test Example 3: CAR-T cells with good blood-brain barrier permeability have cell chemotaxis properties and high expression of chemokines and their receptors

[0070] 1. By mapping single-cell RNA-seq to 5' CAR sequences, this test example identified a total of 287 CAR-T cells (i.e., all CAR-T cells) at 3 months, including 150 cells from 5 peripheral blood samples and 137 cells from 5 cerebrospinal fluid samples.

[0071] 2. Re-clustering single-cell RNA-seq data, according to the marker expression of CAR-T cells 3 months after reinfusion of CAR-T products, further confirmed four different clusters (0, 1, 2 and 3), as shown in Figure 4, and distinguished cells from peripheral blood and cerebrospinal fluid.

[0072] As shown in FIG. 5, Blood represents CAR-T cells in peripheral blood samples, CSF represents CAR-T cells in cerebrospinal fluid samples, and the ordinate represents the percentage of each cluster. By analyzing the characteristics of gene expression of different cell clusters, we found that the proportion of 0 cluster cells in the cerebrospinal fluid was significantly higher than that of blood cells. This indicates that 0 cluster cells can effectively penetrate the blood-brain barrier into the central nervous system cerebrospinal fluid.

[0073] Further, by applying the classical T cell chemotaxis scoring tool, we compared the chemotactic properties of CAR-T cells in peripheral blood and cerebrospinal fluid. The results are shown in FIG. 6, where the ordinate represents the T cell chemotaxis score. The left side of the figure represents the chemotaxis score of CAR-T cells in peripheral blood samples, and the right side represents the chemotaxis score of CAR-T cells in cerebrospinal fluid samples. The results of FIG. 6 show that cells in the cerebrospinal fluid exhibit enhanced chemotactic properties (P<0.0001). This suggests that CAR-T cells with high expression of chemokines and their receptors can effectively penetrate the blood-brain barrier and enter the central nervous system.

[0074] Based on the single-cell RNA sequencing analysis results of Test Example 1, we compared the differential gene expression of peripheral blood and cerebrospinal fluid cells. As shown in FIG. 7, the left side represents down-regulated differential genes, and the right side represents up-regulated differential genes. Among the up-regulated differential genes, the expression of chemokine receptors CXCR3 and CCR5 in cells in the cerebrospinal fluid was significantly up-regulated, and the expression of chemokines CCL4 and CXCL16 was also significantly increased. This suggests that CAR-T cells with high expression of chemokines and their receptors can effectively penetrate the blood-brain barrier and enter the central nervous system.

[0075] Further, by single-sample gene set enrichment analysis (GSEA), we analyzed the activation of chemotaxis-related pathways of CAR-T cells that can effectively penetrate the blood-brain barrier. The analysis results are shown in FIG. 5, which shows that whether CD4+ CAR-T cells or CD8+ CAR-T cells, CAR-T cells that can effectively penetrate the blood-brain barrier show significant up-regulation in chemotaxis-related pathways. This indicates that CAR-T cells with good blood-brain barrier permeability can up-regulate chemotaxis-related pathways, thereby entering the central nervous system to exert their targeted therapeutic effects.

[0076] Test Example 4: Evaluation of the central nervous system treatment efficacy of CAR-T cells with good blood-brain barrier permeability

[0077] BCMA is a transmembrane glycoprotein in the tumor necrosis factor superfamily, mainly expressed on the surface of mature B lymphocytes. Soluble B-cell maturation antigen (sBCMA) is directly shed from membrane BCMA and is closely related to damage to the central nervous system.

[0078] The present application uses a human BCMA / TNFRSF17 ELISA kit (R&D Systems, DY193) to detect the expression level of sBCMA in the cerebrospinal fluid of the subjects at the baseline stage and after the CAR-T cell product is returned, and calculates the decrease level of sBCMA.

[0079] By comparing the expression level of CXCR3 in CD4+T cells and CD8+T cells and the corresponding sBCMA decrease level of the subjects, as shown in the results of Figure 9, the subjects who return the CAR-T cell product with good blood-brain barrier permeability have a higher expression level of CXCR3, and the decrease degree of sBCMA is higher, which indicates that the CAR-T cell product with good blood-brain barrier permeability can enhance the efficacy of CAR-T cells in central nervous system autoimmunity.

[0080] The above specific embodiments describe the implementation of the present application in detail, but the present application is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concepts of the present application, various simple modifications and changes can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A CAR-T cell with good blood brain barrier permeability, characterized in that, The CAR-T cell is used for treating or improving central nervous system diseases, the CAR-T cell targets B cell maturation antigen and highly expresses CXCR3 chemokine receptor and CCL1, CCL3 and CCL4 chemokines.

2. The CAR-T cell with good blood-brain barrier permeability according to claim 1, characterized in that, The CAR-T cell is derived from PBMC separated from peripheral blood.

3. A method of detecting the blood-brain barrier permeability of the CAR-T cell of claim 1 or 2, which is not for the purpose of disease detection and diagnosis, characterized by, The judgment is made by detecting the expression level of CXCR3 chemokine receptor and CCL1, CCL3 and CCL4 chemokines in cerebrospinal fluid; or the chemotactic property of the CAR-T cell is detected and analyzed based on single-cell transcriptome sequencing data.

4. A product for detecting the blood brain barrier permeability of CAR-T cells, characterized in that, The CAR-T cell is the CAR-T cell of claim 1 or 2, and the product is used for detecting the expression level of CXCR3 chemokine receptor and CCL1, CCL3 and CCL4 chemokines in cerebrospinal fluid.

5. The product of claim 4, wherein, The product comprises reagents or probes for detecting the expression level of CXCR3 chemokine receptor and CCL1, CCL3 and CCL4 chemokines.

6. The product according to claim 4 or 5, characterized in that, The product is a detection kit, test strip or detection chip.

7. Use of the CAR-T cell according to claim 1 or 2, characterized in that A drug for preparing a targeted treatment or improvement of central nervous system diseases.

8. Use according to claim 7, characterized in that, The central nervous system disease is a neuromyelitis optica spectrum disease.

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