TCR-T cell and application thereof in preparation of product for treating chronic virus infection

Abstract

The invention relates to a TCR-T cell and application thereof in preparation of a product for treating chronic virus infection, a method for preparing the TCR-T cell comprises the steps of knocking out a KLF2 gene in the TCR-T cell and knocking out the KLF2 gene in the TCR-T cell to obtain an improved TCR-T cell, so that the in-vivo accumulation capacity, the killing function and the chronic infection pathogen removal efficiency of the cell can be remarkably improved, and the TCR-T cell can be used for treating chronic virus infection. And a new strategy is provided for cell therapy of chronic virus infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunotherapy, in particular to a TCR-T cell and application thereof in preparing a product for treating chronic viral infection. BACKGROUND

[0002] Chronic infection refers to the long-term existence of a pathogen in the host body, which is difficult to eliminate. Common chronic infection pathogens include hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and some Mycobacterium tuberculosis, etc. Such infections often lead to long-term immune stress, tissue damage and immune function disorder of the body, and pose a serious threat to public health. During the process of chronic infection, CD8 + T cells, as cytotoxic T lymphocytes, are important immune effector cells for eliminating virus-infected cells. However, under the background of chronic infection, CD8 + T cells often experience "functional exhaustion", which is manifested as decreased killing function, reduced cytokine secretion and persistent expression of inhibitory receptors. Therefore, improving the number and function of CD8 + T cells is crucial for effectively controlling chronic infection. One of the promising methods is adoptive cell therapy (ACT).

[0003] Adoptive cell therapy is a treatment strategy that reinfuses immune cells expanded or modified in vitro into patients to enhance the immune system's ability to recognize and eliminate specific pathogens or tumors. This therapy was first applied to antiviral therapy and has made breakthroughs in tumor immunotherapy in recent years. According to the source and processing method of cells, adoptive cell therapy mainly includes tumor infiltrating lymphocyte therapy (TILs), T cell receptor engineered T cell therapy (TCR-T), chimeric antigen receptor T cell therapy (CAR-T) and virus-specific T cell therapy (VST), etc. Through these methods, a large number of CD8 + T cells with enhanced function and strong targeting ability can be obtained for targeted elimination of specific antigen-positive infected cells or tumor cells.

[0004] In recent years, adoptive cell therapy has been gradually applied to the study of chronic infection, and has shown certain prospects in the treatment of chronic hepatitis B, HIV infection and Epstein-Barr virus-related diseases. However, the application of this therapy in chronic infection still faces many challenges. First, the immune environment caused by chronic infection is strongly suppressed, which may limit the long-term survival and function of adoptive T cells. Second, it is still difficult to reverse the "functional exhaustion" state of T cells, and exogenous T cells may also gradually lose activity. SUMMARY

[0005] This invention aims to at least partially solve one of the technical problems in the related art. Therefore, one object of this invention is to provide a TCR-T cell and its application in the preparation of products for treating chronic viral infections, thereby enhancing the in vivo accumulation, killing function, and clearance capacity of T cells from chronic infectious pathogens.

[0006] Therefore, in one aspect of the present invention, a method for preparing TCR-T cells with enhanced killing ability against chronic infectious pathogens is proposed, comprising the following steps: knocking out the KLF2 gene in TCR-T cells.

[0007] According to the present invention, a method for preparing TCR-T cells with enhanced killing ability against chronic infectious pathogens is disclosed. This method involves knocking out the KLF2 gene in TCR-T cells to obtain modified TCR-T cells, which exhibit significantly improved in vivo accumulation capacity, killing function, and clearance efficiency against chronic infectious pathogens. Experimental data show that the modified TCR-T cells obtained by this method can effectively reduce the viral load of chronic infectious pathogens in mouse serum and organs, providing a new strategy for cell therapy of chronic viral infections.

[0008] Furthermore, the method for preparing TCR-T cells with enhanced killing ability against chronic infectious pathogens proposed in the above embodiments of the present invention may also have the following additional technical features: Optionally, the knockout method includes gene editing methods based on the CRISPR / Cas9 system.

[0009] Optionally, the following steps are also included: Step 1: Design an sgRNA targeting the KLF2 gene, construct a plasmid containing the sgRNA, and obtain the sgKLF2 plasmid; Step 2: The sgKLF2 plasmid is packaged into a retrovirus to obtain the sgKLF2 retrovirus; Step 3: Use the sgKLF2 retrovirus to transduce TCR-T cells expressing Cas9 protein to obtain TCR-T cells with enhanced killing ability against chronic infectious pathogens.

[0010] Optionally, the chronic infection pathogen includes: chronic hepatitis B virus, human immunodeficiency virus, chronic lymphocytic choroid plexus meningitis virus, or chronic hepatitis C virus.

[0011] In a second aspect of the invention, the present invention proposes the above-described method for preparing TCR-T cells with enhanced killing ability against chronic infectious pathogens.

[0012] According to the present invention, when the TCR-T cells are adopted into mice, they can recognize chronically infected viral strains and perform clonal expansion to kill virus-infected cells; the TCR-T cells have a stronger T cell accumulation advantage; enhance the cytokine secretion level of the TCR-T cells; and effectively reduce viral load.

[0013] In a third aspect, the present invention proposes the application of the above-described TCR-T cells in the preparation of biomaterials for treating chronic viral infections.

[0014] In a fourth aspect, the present invention proposes the use of the above-described TCR-T cells in the preparation of a medicament for treating chronic viral infections.

[0015] Optionally, the TCR-T cells can recognize chronic infectious pathogens and kill pathogen-infected cells; the TCR-T cells enhance the secretion levels of cytokines TNF-α and IFN-γ and enhance the expression level of Granzyme B.

[0016] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of a plasmid according to an embodiment of the present invention; Figure 2 This invention relates to the changes in the fold increase of TCR-T cells in the KLF2 knockout group and the control group at different time points after chronic infection, according to an embodiment of the present invention. Figure 3 The absolute number of TCR-T cells expressing TNF-α+IFN-γ+ detected at specific time points after chronic infection according to embodiments of the present invention; Figure 4 The expression levels of Granzyme B in TCR-T cells of the KLF2 knockout group and the control group after chronic infection according to embodiments of the present invention; Figure 5 The amount of live virus in the serum of mice after chronic infection according to an embodiment of the present invention infects Vero cells; Figure 6 The viral load in different organs of mice after chronic infection according to embodiments of the present invention. Detailed Implementation

[0018] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0019] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0020] The test materials used in this invention are all commercially available products and can be purchased on the market; unless otherwise specified, the experiments involved are all conventional experimental methods.

[0021] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0022] Example 1: Packaging of sgNTC and sgKLF2 retroviruses Construction of sgNTC and sgKLF2 plasmids: Referring to the plasmid sequences of pMSCV-IRES-GFP II (pMIG II, addgene #52107) and lentiGuide-Puro (addgene #52963), the pMSCV-U6sgRNA(BbsI)-PGK-GFP vector was synthesized. The vector sequence is shown in SEQ ID NO: 1. The synthesized plasmids were then used... BbsI After enzyme digestion, the sequence of non-targeting guide RNA (sgNTC) (caccgATGACACTTACGGTACTCGTSEQ ID NO: 2) or the sequence of KLF2 guide RNA (sgKLF2) (caccgGGGAGGCCCGTAGTGCAACGSEQ ID NO: 3) is inserted to construct a retroviral vector expressing sgNTC and sgKLF2. A schematic diagram of the plasmid is shown below. Figure 1 .

[0023] (1) Resuscitation of 293T cell line: Take the 293T cell line preserved from liquid nitrogen or -80℃, thaw the cells in a 37℃ water bath, add 1 mL of preheated DMEM medium and transfer to a 1.5 mL centrifuge tube. Centrifuge at 300 rpm for 2 min, remove the supernatant by aspirating pump, add 1 mL of DMEM medium to resuspend thoroughly, and transfer to a culture dish containing 10 mL of DMEM medium. Mix well by cross-shaking. Change the medium once after 48 h.

[0024] (2) When the cell density reaches approximately 70-90%, cell passage can be performed: Aspirate the supernatant culture medium using a suction pump, slowly add 1 mL of preheated PBS along the side of the culture dish until it is full, and wash away any remaining culture medium by tilting the dish up and down. Slowly add 1 mL of preheated 0.05% Trypsin solution along the side of the culture dish and incubate at 37°C until all cells have detached from the culture medium. At this point, add 1 mL of DMEM culture medium to stop the digestion. Transfer the liquid to a new 15 mL tube and centrifuge at 300 rpm for 2 min. Resuspend the cells in 1 mL of DMEM culture medium and seed them into a new 10 cm culture dish at a 1:4 ratio. After culturing for about 2 days, prepare to package the virus.

[0025] (3) 293T cell seeding: 293T cells were seeded at a density of 1.2 × 10⁶ cells per well. 6 One cell was seeded into a 6-well plate, and 2 mL of DMEM medium was added to each well.

[0026] (4) Change medium: After 24 h, the cell density is about 80-90%. Remove the supernatant and replace it with 2 mL of fresh DMEM medium.

[0027] (5) Virus packaging can be performed approximately 2 hours after replacing the culture medium: Add 2 μg of sgNTC and sgKLF2 plasmid, 2 μg of pCL-Eco helper packaging plasmid (add gene #12371), and 200 μL of Opti-MEM reagent to a 1.5 mL centrifuge tube. After vortexing the above components, slowly add 8 μL of PEI transfection plasmid in a spiral motion and mix thoroughly by pipetting. After standing for 15 min, evenly drop the mixture onto the surface of the 6-well plate approximately 2 hours after replacing the culture medium and mix in a cross shape. After 10-12 h, remove the culture medium containing the transfection reagent and replace it with fresh T cell culture medium.

[0028] (6) Collecting the virus: Collect the virus supernatant 48 h and 72 h from the virus packaging as the starting point.

[0029] Example 2: T cell isolation, viral transduction, culture, and adoptive transfer P14 TCR-T mice that identify the LCMV CL13 gp33 epitope were purchased from Jackson Lab (037394-JAX).

[0030] Cas9-expressing mice (IMSR_JAX:024858) were crossed with P14 TCR-T mice expressing the LCMV CL13 gp33 epitope to obtain Cas9-expressing P14 TCR-T mice. At 6-10 weeks of age, unactivated TCR-T cells were isolated from the spleen and peripheral lymph nodes of these Cas9-expressing P14 TCR-T mice using an antigen-free CD8α+ T cell isolation kit (BioLegend, 480043) and magnetic bead purification technology, according to the kit instructions (BioLegend, 480043). One million purified TCR-T cells were activated in vitro for 18 h with plate-bound anti-CD3 (10 μg / mL; Bio-X-Cell) and anti-CD28 (5 μg / mL; Bio-X-Cell) antibodies.

[0031] Transduction of sgNTC or sgKLF2 retroviruses (obtained in Example 1) was performed using a centrifugation-infection method. The transduction conditions were: centrifugation at 900 g for 3 h at 25°C, with the addition of 10 μg / mL of hexamethylene bromide polybrene, followed by incubation at 37°C and 5% CO2 for 3 h. After washing away the cells, they were cultured for 3 days with mouse IL-7 (final concentration 2.5 ng / mL; PeproTech) and mouse IL-15 (final concentration 5 ng / mL; PeproTech). Cells expressing fluorescent proteins were sorted using a BD FACSAria™ Fusion sorter to obtain KLF2 gene-deficient TCR-T cells and non-deficient TCR-T cells. 10,000 TCR-T cells were then adoptively injected into recipient C57BL / 6 mice via tail vein.

[0032] Chronic viral infection: 3 hours after adoptive transfer, mice were infected with LCMV CL13 via intravenous injection (2 × 10⁶ cells per mouse). 6 pfu). LCMV CL13 was a kind gift from Professor Xiao Nengming's research group at Xiamen University.

[0033] Viral titer determination: 100 μL of whole blood samples were collected from LCMV CL13-infected mice on days 5, 7, and 12. After standing at room temperature for 2 hours, the samples were centrifuged at 4000 rpm, and the supernatant serum was collected. On days 7 or 12 post-infection, spleen, kidney, and liver samples were collected. Cells were homogenized using a tissue homogenizer to release the virus, which was then serially diluted and added to Vero cell culture medium. After 24 hours, the cell layers were fixed in 4% formaldehyde in phosphate-buffered saline, permeated with 0.5% Triton X-100 in balanced salt solution, and stained overnight at 4° with 1 μg / mL VL-4 antibody (Bioxcell), which targets the LCMV nucleoprotein (NP). After primary antibody staining, the cells were washed with 0.5% Triton X-100 and then stained with 1 μg / mL FITC-labeled anti-rat secondary antibody at room temperature for one hour to visualize LCMV-positive cells. Viral titer was determined by counting fluorescence focusing units under a fluorescence microscope.

[0034] The results are as follows Figure 2 As shown, Figure 2 To investigate the ploidy change of KLF2 knockout (sgKLF2) TCR-T cells relative to the control group (sgNTC) at specific time points after chronic infection using flow cytometry, we used flow cytometry to detect the ploidy change of KLF2 knockout (sgKLF2) TCR-T cells relative to the control group (sgNTC). KLF2-deficient TCR-T cells and non-deficient TCR-T cells were adoptively transferred to mice. LCMV-CL13 virus was then injected via the tail vein, and the number of accumulated TCR-T cells in the spleen of the mice was analyzed at days 5, 7.5, 12, and 30 post-infection. TCR-T cell expansion is considered an effective strategy for controlling chronic viral infection. The results showed that after chronic viral infection, KLF2 gene deletion significantly increased the accumulation of TCR-T cells in the spleen, and this accumulation continued until the late stage of infection (day 30).

[0035] The results are as follows Figure 3 and Figure 4 As shown, Figure 3 To quantify the absolute number of TCR-T cells expressing TNF-α+IFN-γ+ in the control group and KLF2 knockout group at specific time points using flow cytometry. Figure 4To quantify Granzyme B levels in the control and KLF2 knockout groups at specific time points using flow cytometry. TNF-α and IFN-γ are two important pro-inflammatory cytokines; their co-expression usually indicates that CD8⁺ T cells are in a highly activated effector state, possessing better antiviral and antitumor capabilities. Granzyme B (GzmB) is another effector molecule for CD8⁺ T cell function. It can enter target cells, activate the caspase cascade, and rapidly induce DNA breaks in target cells, leading to rapid apoptosis. In chronic infection, persistent antigen stimulation may lead to CD8⁺ T cell exhaustion, resulting in reduced secretion of TNF-α, IFN-γ, and GzmB. Figure 3 and Figure 4 It can be seen that, compared with the control group cells, knocking out KLF2 significantly increased the absolute number of TNF-α+IFN-γ+ cells, and the average fluorescence intensity of GzmB was also significantly increased, indicating that KLF2 deficiency enhances the killing function of TCR-T cells.

[0036] The results are as follows Figure 5 and Figure 6 As shown, Figure 5 To quantify the ability of live virus in mouse serum to infect Vero cells using immunofluorescence detection. Figure 6 To quantify live virus in different organs of mice using immunofluorescence technology, KLF2 gene-deficient TCR-T cells were found to provide better protection against chronic viral infection. Continuous monitoring of viral titers in the serum of mice that received adoptive transfer showed that recipient mice receiving KLF2-deficient TCR-T cells better controlled viral load in LCMV CL13. Figure 5 The viral load in different organs was also examined, and it was found that KLF2-deficient TCR-T cells significantly suppressed viral load in the liver, spleen, and kidneys. Figure 6 ).

[0037] In summary, according to embodiments of the present invention, adoptive transfer of KLF2-deficient TCR-T cells into mice enables them to recognize chronically infected viral strains (e.g., LCMV CL13, clonal lymphocytic choroidal meningitis virus clone 13) and perform clonal expansion, killing virus-infected cells; KLF2-deficient TCR-T cells exhibit a stronger T cell accumulation advantage; KLF2 deficiency enhances the cytokine secretion level of TCR-T cells; and KLF2-deficient antigen-specific T cells effectively reduce viral load.

[0038] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0039] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing TCR-T cells with enhanced killing ability against chronic infectious pathogens, characterized in that, Includes the following steps: Knock out the KLF2 gene in TCR-T cells.

2. The method as described in claim 1, characterized in that, The knockout methods include gene editing methods based on the CRISPR / Cas9 system.

3. The method as described in claim 1, characterized in that, Includes the following steps: Step 1: Design an sgRNA targeting the KLF2 gene, construct a plasmid containing the sgRNA, and obtain the sgKLF2 plasmid; Step 2: The sgKLF2 plasmid is packaged into a retrovirus to obtain the sgKLF2 retrovirus; Step 3: Use the sgKLF2 retrovirus to transduce TCR-T cells expressing Cas9 protein to obtain TCR-T cells with enhanced killing ability against chronic infectious pathogens.

4. The method as described in claim 1, characterized in that, The chronic infection pathogens include: chronic hepatitis B virus, human immunodeficiency virus, chronic lymphocytic choroid plexus meningitis virus, or chronic hepatitis C virus.

5. A TCR-T cell with enhanced killing ability against chronic infectious pathogens prepared by the method of any one of claims 1-4.

6. The use of the TCR-T cells as described in claim 5 in the preparation of biomaterials for treating chronic viral infections.

7. The use of the TCR-T cells as described in claim 5 in the preparation of a medicament for treating chronic viral infections.

8. The application as described in claim 6 or 7, characterized in that, The TCR-T cells can recognize chronic infectious pathogens and kill pathogen-infected cells; the TCR-T cells enhance the secretion levels of cytokines TNF-α and IFN-γ and enhance the expression level of Granzyme B.

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