CAR-gamma delta T cell of autotaxis immune cell adapter and preparation method of CAR-gamma delta T cell

Abstract

The invention relates to a CAR-gamma delta T cell of a self-secreting immune cell adapter and a preparation method of the CAR-gamma delta T cell. The CAR-gamma delta T cell is prepared from a gamma delta T cell, the gamma delta T cell contains a nucleotide sequence for coding a SynNotch receptor and a nucleotide sequence for coding an effector, the SynNotch receptor comprises a transmembrane core module and an intracellular regulation and control module, the intracellular regulation and control module is connected with the C end of the transmembrane core module, and the C end of the intracellular regulation and control module is connected with the C end of the transmembrane core module. The intracellular regulation module comprises a DNA binding domain and a transcriptional regulation structural domain, and the transmembrane core module can be cut by gamma-secretase to separate the intracellular regulation module; the nucleotide sequence encoding the effector includes an upstream activation sequence capable of specifically binding to the DNA binding domain, and nucleotide fragments encoding an immune cell adapter, a self-cleaving peptide, and a T cell immune activator.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, and in particular to a CAR-γδT cell that acts as an autocrine immune cell connector and its preparation method. Background Technology

[0002] Chimeric antigen receptors (CARs) are engineered receptors initially transduced into T cells to produce CAR-T cell therapy, which has achieved remarkable results in treating hematologic malignancies. However, this therapy currently has some significant limitations, including the following: 1) In one-third of patients receiving CAR-T cell therapy, significant toxicities directly related to the induction of a robust immune effector response occur. The most common immune-mediated toxicities are cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). CRS typically begins with fever and constitutional symptoms such as rigidity, malaise, and anorexia. In severe cases, CRS manifests as other features of a systemic inflammatory response, including hypotension, hypoxia, and / or organ dysfunction. ICANS typically presents as toxic encephalopathy, initially identified as difficulty speaking, confusion, developmental delays, aphasia, impaired fine motor skills, and somnolence. In more severe cases, seizures, motor weakness, cerebral edema, and coma have been observed. Most patients with clinical features of ICANS have prior symptoms of CRS. Although the clinical features of ICANS are easily identifiable, its pathophysiology remains poorly understood. Recent animal models have shown that endothelial cell activation and disruption of the blood-brain barrier lead to direct neuronal damage, as well as the effects of various pro-inflammatory cytokines. These life-threatening CAR-T cell-related toxicities greatly limit the widespread application of this technology.

[0003] 2) Currently marketed CAR-T products are all derived from autologous cells. According to disclosed clinical trials, the time from leukocyte collection to infusion in CAR-T therapy is mostly around 10-25 days. Although the basic protocols for current cell therapy development are similar, the industry has not yet developed a mature and fully acceptable CAR-T production process. Many CAR-T products rely on manual operation, which is very time-consuming and labor-intensive, difficult to scale up, and has a high failure rate. The entire CAR-T preparation process is very complex, while the optimization of its production process is relatively slow. Furthermore, the helper cells from different patients can vary greatly, such as macrophages and monocytes. However, the safety, purity, efficacy, and uniformity of cell therapy products are crucial for quality control throughout all stages of CAR-T production, which also increases the cost of CAR-T therapy.

[0004] 3) The therapeutic effect of CAR-T cell therapy in most solid tumors remains unsatisfactory. On the one hand, the heterogeneity of solid tumor cell antigens and the lack of tumor-specific antigens (TSA) make it difficult for CAR-T to accurately target and kill tumor cells. Tumor antigens in solid tumors are mostly heterogeneous, not only showing differential distribution within the same lesion, but also differing in expression between primary and metastatic tumors. TSA, which is expressed only on tumor cells, is an ideal target for anti-tumor therapy. However, due to the extreme scarcity of TSA and its predominance within cells, targeting tumor-associated antigens (TAAs), which are expressed in both tumor cells (high expression) and normal tissues (low expression), faces potential off-target effects. To date, TAA targets in solid tumors such as non-small cell lung cancer have shown heterogeneity in intensity and distribution, lacking true tumor-specific antigens, which greatly limits the clinical efficacy of CAR-T.

[0005] 4) Insufficient angiogenesis within solid tumors and the tumor microenvironment (TME) rich in extracellular matrix form a physical barrier, affecting CAR-T cell infiltration and tumor antigen recognition. Furthermore, CAR-T cells that successfully infiltrate tumor tissue to exert their immune effect still need to overcome multiple obstacles, including metabolic disorders, immunosuppressive factors, and immunosuppressive cells. These not only significantly inhibit the function of therapeutic T cells but also promote tumor cell proliferation, angiogenesis, and migration by secreting pro-angiogenic and anti-inflammatory factors. Finally, the inherent negative regulatory mechanisms of T cells lead to T cell activation inhibition and even functional exhaustion. These factors all contribute to the poor efficacy of CAR-T cell therapy in solid tumors and the failure of clinical trials. Summary of the Invention

[0006] The purpose of this invention is to disclose a CAR-γδT cell with an autocrine immune cell engager (ICE), its preparation method, and its application, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0007] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of this invention is to provide a CAR-γδT cell. The CAR-γδT cell is prepared from γδT cells, and the γδT cell contains a nucleotide sequence encoding a synthetic Notch (SynNotch) receptor and a nucleotide sequence encoding an effector. The SynNotch receptor includes a transmembrane core module and an intracellular regulatory module, the intracellular regulatory module being connected to the C-terminus of the transmembrane core module. The intracellular regulatory module includes a DNA-binding domain and a transcriptional regulatory domain. The transmembrane core module can be cleaved by γ-secretase to separate the intracellular regulatory module. The nucleotide sequence encoding the effector includes an upstream activation sequence capable of specifically binding to the DNA-binding domain, and nucleotide fragments encoding ICE, a self-cleaving peptide, and a T-cell immune activating factor.

[0008] After the SynNotch receptor binds to its ligand, the intracellular regulatory module is cleaved by γ-secretase and other enzymes. The isolated intracellular regulatory module then enters the nucleus to regulate gene expression—binding to the upstream activation sequence encoding the effector, causing γδT cells to secrete ICE and T cell immune activating factors.

[0009] This invention utilizes SynNotch to release the intracellular regulatory module and activate effector expression. Its core advantage lies in achieving antigen-dependent conditional expression, precise spatiotemporal regulation, and multiple safety controls. If, instead, T cells are edited to directly express ICE and T cell immune activating factors, the efficacy and safety would significantly decrease due to problems such as continuous activation, off-target toxicity, and functional exhaustion.

[0010] In some embodiments of the first aspect of the present invention, the DNA-binding domain is selected from Gal4 or LexA; preferably, the transcriptional regulatory domain is selected from VP64, KRAB, tTa, or ZFHD1. The intracellular regulatory module is based on the natural Notch receptor, with the intracellular region replaced by combinations such as Gal4-VP64, LexA-VP64, and Gal4-KRAB. Upon binding to the target antigen, similar to the natural Notch activation mechanism, the SynNotch receptor can undergo cleavage of the induced transmembrane core module, thereby releasing the intracellular regulatory module into the cell nucleus, where it binds to homologous upstream activation sequences to activate the expression of the regulated target gene.

[0011] In some embodiments of the first aspect of the invention, the upstream activation sequence is selected from UAS or LexAop. GAL4 / UAS is a gene expression regulatory system present in yeast, and LexA / LexAop is a group of transcriptional regulatory systems widely present in prokaryotes. After the upstream activation sequence binds to the DNA-binding domain, it is influenced by the upstream transcriptional regulatory domain, thereby initiating the expression of downstream proteins. The link between the ICE and the T-cell immune activating factor is cleaved by the self-cleaving peptide, allowing it to be released extracellularly.

[0012] In some embodiments of the first aspect of the present invention, the T-cell immune activating factor is a soluble receptor. The soluble receptor is selected from sIFN-γ, sIL-2, sIL-4, or sIL-17.

[0013] In some embodiments of the first aspect of the present invention, the SynNotch receptor further includes an extracellular recognition module, the extracellular recognition module comprising a first single-chain variable fragment (scFv) structure; the ICE comprises a second single-chain variable fragment (scFv) structure. The extracellular recognition module of the present invention replaces the extracellular region structure of the natural Notch receptor with a first scFv structure that targets and recognizes TAAs expressed or secreted by tumor cell membranes. The ICE is connected via a flexible linker to form a second scFv structure. In conjunction with the TCR of γδT cells, it can specifically recognize non-peptide phosphorylated antigens, such as isopentenyl pyrophosphate (IPP) metabolized by tumor cells, to achieve spontaneous activation and killing. The CAR-γδT cells have a three-target design and can survive in the tumor environment under non-TAA-specific activation.

[0014] In some embodiments of the first aspect of the present invention, the first scFv structure and the second scFv structure target any two of the following: CD19, BCMA, GPC3, mesothelin, EGFRvRIII, Claudin 18.2, B7-H3, ErbB2, GM3, GD2, GD3, CD20, CD22, gp100, NYESO-1, carbonic anhydrase IX, WT1, carcinoembryonic antigen (CEA), CA-125, MUC-1, MUC-3, epithelial tumor antigen, MAGE type antigen, BAGE, GAGE, XAGE1B, CTAG2, CTAG1, SSX2, LAGE1, or HER2. As a preferred embodiment targeting lung cancer, the target of the first scFv structure is the non-specific antigen CEA for lung cancer, and the target of the second scFv structure is the driving target of lung cancer, HER2 (human epidermal growth factor receptor-2).

[0015] For non-small cell lung cancer, in some embodiments of the first aspect of the present invention, the nucleotide sequence encoding the SynNotch receptor includes a CD8α signal peptide, a first scFv structure targeting CEA, a transmembrane core module cleavable by ADAM metalloproteinases and γ-secretase, and an intracellular regulatory module containing the activation domains of the GAL4 gene and VP64 protein; its nucleotide sequence is shown in SEQ ID No:1. The nucleotide sequence encoding the effector includes a UAS sequence element specifically binding to GAL4, a second scFv structure targeting HER2, a flexible linker, and CD58 ICE, a self-cleaving T2A peptide, and sIL-2; its nucleotide sequence is shown in SEQ ID No:2.

[0016] A second aspect of the present invention provides a method for preparing the CAR-γδT cells. The preparation method specifically includes the following steps: 1) Construct a recombinant viral vector containing a nucleotide sequence encoding the SynNotch receptor and a nucleotide sequence encoding the effector; 2) Obtain activated γδT cells, transduce the recombinant viral vector into the γδT cells, and screen to obtain cells that stably express the SynNotch receptor and the effector, which are the CAR-γδT cells.

[0017] In some embodiments of the second aspect of the present invention, the nucleotide sequence encoding the SynNotch receptor is shown in SEQ ID No:1, and the nucleotide sequence encoding the effector is shown in SEQ ID No:2. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the SynNotch receptor described in Example 1; Figure 2 This is a schematic diagram of the effector structure described in Example 1; Figure 3 This is a curve showing the increase in the number of CAR-γδT cells during the preparation process in Example 4; Figure 4 This is a graph showing the changes in cell viability during the preparation of CAR-γδT cells in Example 4; Figure 5 This is the original flow cytometry image of T lymphocyte activation detection in the CAR-γδT cell co-culture experiment in Example 5; Figure 6 This is a bar chart comparing the activation of T lymphocytes in the CAR-γδT cell co-culture experiment in Example 5; Figure 7The CAR-γδT cell co-culture experiment in Example 5 was used to detect TNF-α secretion by ELISA. Figure 8 This is a detection diagram of CAR-γδT cells regulated by CEA to express ICE-linked HER2 in Example 5; Figure 9 This is a flow cytometry diagram of CAR-γδT cells as target immune cells in Example 5; Figure 10 This is a bar chart showing the concentration of IL-2 expressed by CAR-γδT cells after CEA activation, as described in Example 5. Figure 11 This is a flow cytometry statistical bar chart of the expression levels of CEA secreted by lung cancer cell lines A549 and H1975 and the expression level of HER-2 membrane in Example 6; Figure 12 This is a bar chart comparing the killing effects of CAR-γδT cells on A549 and H1975 cells as described in Example 6; Figure 13 This is a statistical graph showing the cytotoxic and proliferative effects of CAR-γδT cells combined with immune cells on H1975, as described in Example 7. Figure 14 The CAR-γδT cells described in Example 7 synergistically activate CD3 in an in vitro H1975 tumor cell killing model. + CD4 + T lymphocytes and CD3 - CD56 + Statistical graph of NK cell expansion; Figure 15 This is a statistical graph showing the synergistic enhancement of inflammatory cytokine production by CAR-γδT cells in an in vitro H1975 tumor cell killing model, as described in Example 7. Figure 16 This is a graph showing the change in body weight of tumor-bearing mice after administration of the CAR-γδT cells described in Example 8; Figure 17 This is a comparison of the number of tumor-bearing mice surviving at different time points after drug administration in Example 8. Detailed Implementation

[0019] Unless otherwise specified, the molecular biology experimental methods described in the following examples were performed in accordance with Molecular Cloning: A Laboratory Manual (3rd Edition) or the method and product instructions. Unless otherwise specified, the biological materials used in these methods are commercially available.

[0020] The reagents and culture media involved in the following examples include: The complete culture medium for T lymphocyte activation is shown in Table 1. After preparation, it should be temporarily stored at 4°C.

[0021] Table 1 - Complete Culture Medium Formula for T Lymphocyte Activation

[0022] The cell cryopreservation solution is prepared as shown in Table 2. After preparation, it should be stored at 4°C in the dark.

[0023] Table 2 - Cell cryopreservation solution formulations

[0024] Example 1: Design of a CAR-γδT cell with specific killing effect on solid tumors of lung cancer.

[0025] The CAR-γδT cells achieve three-target binding to solid tumors in non-small cell lung cancer through the Synnotch receptor, effector, and TCR inherent in γδT cells, so that they are released only locally in the TME, reducing systemic exposure-induced CRS and immunosuppression.

[0026] (1) Synnotch receptor The structural composition of the Synnotch receptor is as follows: Figure 1 As shown, it includes CD8α leader, Anti-CEA scFv, NotchCore, and Gal4-VP64.

[0027] 1) The CD8α leader element encodes the CD8α signal peptide located at the N-terminus of the mature protein, which guides the protein to cross the membrane and is cleaved after transmembrane expression.

[0028] 2) The Anti-CEA scFv element encodes the first scFv structure. It references the heavy chain variable region (VH) and light chain variable region (VL) of a CEA-specific antibody that specifically recognizes and binds to target proteins, forming an scFv via a flexible linker, enabling the SynNotch receptor expressing this structure to target CEA-positive cells or secreted proteins.

[0029] 3) The Notch Core encodes the transmembrane core module, which is designed with reference to the natural Notch receptor. When the first scFv structure on the SynNotch receptor binds to the target antigen, the transmembrane core module undergoes induced transmembrane region cleavage, thereby releasing the intracellular regulatory module into the cell nucleus, where it binds to a homologous upstream activation sequence to activate the expression of the nucleotide sequence encoding the effector.

[0030] 4) Gal4-VP64 encodes the intracellular regulatory module, its sequence structure integrating the GAL4 / UAS gene expression regulatory system and the VP64 protein activation domain. GAL4 is a transcription factor that specifically recognizes and binds to the UAS sequence. Typically, the GAL4 gene is placed under the control of a specific promoter to ensure expression in specific times and cell types. The intracellular transcribed region of the protein encoded by this sequence is released after cleavage of the inducible transmembrane core module. Since GAL4 is a transcription regulator, its binding domain (BD) binds to the UAS sequence in the effector element sequence, and its activity domain (AD) and promoter region bind to induce gene expression. Simultaneously, this protein structure includes the VP64 protein activation domain. This protein can bind to the gene promoter region under gRNA-mediated binding and activate gene transcription by binding to PolII through the VP64 activation domain.

[0031] The complete nucleotide sequence encoding the Synnotch receptor is shown in SEQ ID No:1. The nucleotide sequences of the upstream and downstream primers used to amplify the nucleotide sequence encoding the Synnotch receptor are as follows: upstream primer PR.1: 5'-ATGGCCTTACCAGTGACCGC-3' (SEQ ID No:3); downstream primer PR.2: 5'-GGATGACTTTGACCTGGACATGCTC-3' (SEQ ID No:4).

[0032] (2) Effector The structural composition of the effector is as follows Figure 2 As shown, it includes UAS, Anti-HER2 scFv, Linker, CD58, T2A, and sIL-2.

[0033] 1) The UAS element is not expressed and is located upstream of the target gene. When the intracellular regulatory module described in the SynNotch receptor is released and binds to UAS, the sequence is activated, thereby initiating the transcription of downstream genes.

[0034] 2) The Anti-HER2 scFv element encodes the second scFv structure. The expressed second scFv structure serves as part of the immune cell connective, enabling immune cells, especially T lymphocytes and NK cells, to target HER2-positive cells, thereby allowing immune cells to specifically kill HER2-positive lung cancer tumor cells.

[0035] 3) The Linker component encodes the flexible connector in the immune cell connector, which connects the second scFv structure with the CD58 structure to achieve bispecificity of the connector.

[0036] 4) The CD58 primitive encodes the CD58 extracellular domain. This CD58 extracellular domain enables the immune cell adjuvant to bind to the natural ligand CD2 expressed on the surface of T / NK cells. The immune cell adjuvant not only promotes adhesion and recognition between autoimmune cells and HER2-positive cells, but also induces T / NK cell activation and proliferation. The expressed immune cell adjuvant protein enables immune cells, especially T lymphocytes and NK cells, to target HER2-positive cells, thereby activating and specifically killing HER2-positive lung cancer tumor cells.

[0037] 5) T2A peptide is a short peptide derived from the virus that allows the protein or amino acid sequence translated from the nucleic acid sequence of subsequent effector elements to be cleaved intracellularly and released extracellularly.

[0038] 6) sIL-2 is derived from an artificially modified IL-2 coding sequence. This sequence is located downstream of the effector component T2A and is released into the extracellular space via self-cleavage during nucleic acid translation. The translated, expressed, and released sIL-2 into the tumor microenvironment can support the expansion and survival of immune cells.

[0039] The complete nucleotide sequence encoding the effector is shown in SEQ ID No:2. The nucleotide sequences of the upstream and downstream primers used to amplify the nucleotide sequence encoding the effector are as follows: upstream primer PR.3: 5'-GATATTCAGATGACTCAGTCTCCTAGCTCT-3' (SEQ ID No:5); downstream primer PR.4: 5'-CTCACACGGAAGCGCAGA-3' (SEQ ID No:6).

[0040] Example 2: Plasmid production.

[0041] (1) After designing the nucleotide sequences encoding the SynNotch receptor and the effector, the DNA sequences were synthesized by Yiming (Suzhou) Cell Biotechnology Co., Ltd. The synthesized DNA was delivered using ultra-low temperature freezing of the DNA solution. Simultaneously, the SynNotch receptor original sequence transduction plasmid (SEQ ID No:7) and the effector original sequence transduction plasmid (SEQ ID No:8) were synthesized by Genscript Biotech Co., Ltd. After synthesis, they were transduced into TransStbl3 Chemically Competent Cell bacteria and delivered by Genscript.

[0042] The packaging plasmid psPAX2 (Plasmid #12260) and the envelope plasmid pMD2.G (Plasmid #12259) were both plasmid products whose sequences were publicly available and directly purchaseable from a bioinformatics database (https: / / www.addgene.org / ). The corresponding plasmid sequences SEQ ID No:7 and SEQ ID No:8 were synthesized by GenScript Biotech Inc. After synthesis, they were transduced into TransStbl3 Chemically Competent Cells for delivery.

[0043] (2) The steps for competent bacteria to amplify the above plasmids include: 1) Prepare LB Agar solid medium (preferably with an agarose concentration of 2%) and LB liquid medium. The prepared medium should be sterilized by moist heat before use.

[0044] 2) The delivered competent bacteria containing different plasmids were rewarmed, transferred to liquid culture medium for rewarming and dissolution, and after inoculation and recovery, they were incubated at 37 ℃ inverted for 16-20 h. The culture medium was observed to be turbid.

[0045] 3) Aseptically transfer the cultured bacterial solution into LB Agar solid medium, incubate at 37 ℃ inverted for 16-20 h, and store at 4 ℃ after small colonies are observed.

[0046] 4) Under aseptic conditions, add ampicillin to the LB liquid culture medium prepared above at a volume ratio of 1:1000 and mix well to form LB screening culture medium.

[0047] 5) Under aseptic conditions, select single clones from solid culture medium and inoculate them into liquid LB selection medium. Amplify them at 37 ℃ and 225 rpm for 5 h in a constant temperature shaker until the medium becomes turbid.

[0048] 6) Optionally, take the positive bacterial culture at 12,000 rpm, centrifuge for 3 min, resuspend the precipitate in 50% glycerol, freeze and store at -80℃ for long-term use in the next production.

[0049] 7) The positive bacterial culture was centrifuged at 12,000 rpm for 3 min for subsequent plasmid production.

[0050] (3) Positive competent bacteria were subjected to large-scale plasmid extraction using the TIANGEN endotoxin-free plasmid extraction kit (enhanced version) (DP120). The specific steps included: 1) Complete the preparation of the working solution according to the instructions, including adding RNase A (10 mg / mL) to solution P1.

[0051] 2) After thoroughly centrifuging the bacterial cell precipitate of the engineered plasmid to remove all supernatant, add solution P1 and vortex to mix, then add an equal volume of solution P2.

[0052] 3) Thorough inversion is to allow the bacterial cells to fully lyse and leave at room temperature for 5 minutes, then add solution P2 and thoroughly invert to mix until a white flocculent precipitate appears.

[0053] 4) Let the plasmid suspension stand at room temperature for 10 min, centrifuge at 8228×g for 10 min to settle the white flocculent matter, pour the plasmid suspension into filter CS1 to recover the filtrate.

[0054] 5) After adding equilibration solution BL to the adsorption column CP6, centrifuge at 8228×g for 10 min and discard the effluent.

[0055] 6) Add the plasmid suspension to the endotoxin-free solution ER and mix until it turns a clear yellow color. Add 0.2 times the volume of isopropanol, mix well, and transfer to the equilibrated adsorption column CP6. Centrifuge at 8228×g for 2 min at room temperature and discard the effluent.

[0056] 7) Add buffer ED to the CP6 adsorption column that has been loaded with samples, centrifuge at 8228×g at room temperature for 2 min, discard the eluent, and repeat the above operation twice.

[0057] 8) Add washing buffer PW to the loaded adsorption column CP6, centrifuge at room temperature (8228×g) for 2 min, discard the effluent, and repeat the above operation twice.

[0058] 9) Centrifuge the CP6 adsorption column at 8228×g for 2 min at room temperature, discard the effluent, and allow it to air dry. Suspend the column and add elution buffer TB dropwise to the adsorption membrane. Let it stand at room temperature for 5 min.

[0059] 10) The concentration and purity of the extracted plasmid solution were detected using the Qubit 4 fluorescence instrument nucleic acid protein fluorescence quantitative system, and the Qubit dsDNA high sensitivity (HS) kit (32851) was used for detection.

[0060] 11) Centrifuge the adsorption column at 8228×g for 5 min at room temperature, collect the eluent, aliquot and transfer to -20℃ for storage or use directly for constructing recombinant target plasmids or for lentiviral transfection vectors—SynNotch receptor sequence transduction plasmid, effector element sequence transduction plasmid, envelope plasmid pMD2.G (SEQ ID No:9) and packaging plasmid psPAX2 (SEQ ID No:10).

[0061] Example 3: Lentiviral production.

[0062] Based on second-generation lentiviral packaging technology, HEK293T cells (purchased from ATCC) were used as packaging cells. The plasmid transfection protocol was based on the common principle of transfection using polyethylenimine (PEI) cationic polymers. PEI40000 (also known as PEIMAX) was selected for plasmid co-transfection. The corresponding transfection system was Polyethylenimine Linear (PEI) MW40000 (rapid lysis) linear PEI transfection reagent (fast-dissolving type) MW40000 (brand: YEASEN, catalog number: 40816ES02 / 40816ES03). Before use, follow the instructions for use (Gibco). TM Opti-MEM TM I-reduced serum culture medium was dissolved and prepared into a stock solution of 1 mg / mL.

[0063] (1) Transfection: According to every 75cm 2 Add 1 mL of the transfection base mix; the corresponding systems are shown in Table 3. Table 3 - Transfection System

[0064] After dissolving the corresponding plasmid DNA products in the two mixed systems above using serum-reduced medium, 38 μL of PEI MW40000 at a concentration of 1 mg / mL was added to each 1 mL and vortexed. The mixture was then allowed to stand at room temperature for 30 min to obtain the transfection mix.

[0065] 1) After thoroughly washing the co-transfected HEK293T cells with PBS buffer to remove residual FBS, Opti-MEM was added. TM I-reduced serum medium was washed once and cultured for 2 hours.

[0066] 2) Remove all liquid and add the two prepared transfection mixes separately, at a ratio of 75 cm⁻¹. 2 Add 1 mL of transfection mix solution, mix well, and incubate at 37 °C with 5% CO2 for 8 h.

[0067] 3) After 8 hours of transfection, pour out the transfection mixture at 75 cm intervals. 2 After washing the mixture with 10 mL of PBS, discard the PBS. Completely culture HEK293T cells at 37 °C with 5% CO2 for 72 h.

[0068] 4) According to every 75 cm 2 Add 15 mL of complete culture medium and collect all culture supernatant. Centrifuge at 300×g for 6 min to remove detached cells, and then recover all supernatant to produce viral vectors.

[0069] (2) Virus titer detection: The lentivirus product is designed to infect T lymphocytes; therefore, Jukart cells are used as the target cells for infection titer detection. Viral titers are obtained after infecting the target cells via reverse transcription and qRT-PCR. Specific steps include: 1) After transfection, the cultured cells were recovered and washed twice by centrifugation with PBS at 300×g for 6 min. The cell pellet was then treated with Trizol reagent (Invitrogen). TM Total RNA was extracted from the sample (product number: 15596026).

[0070] 2) RNA extracted with chloroform was used to perform reverse transcription using the RevertAid RT kit (Thermo Scientific). TM (Catalog number: K1691) The cDNA products of each experimental group were obtained by reaction.

[0071] 3) qRT-PCR was performed using the cDNA products of each group as templates, with actin as an internal control gene in each group. Specifically, three qRT-PCR replicates were set up for each experimental group, with the corresponding primers added according to the lentivirus used in the experiment: upstream primer PR.1, downstream primer PR.2, upstream primer PR.3, and downstream primer PR.4. The corresponding reaction systems were constructed for amplification. Three replicates of actin were included for each viral dilution gradient.

[0072] 4) The titer value is determined based on the difference in Ct values ​​between the internal control actin well and the experimental groups at each dilution ratio. Generally, a difference of 2 or more in Ct values ​​is considered statistically significant. The titer is calculated based on the lowest dilution ratio where a difference in Ct value was detected, indicating that at least one infectious viral particle is present at that dilution ratio.

[0073] 5) Calculate the lentivirus titer—IU (Interval Unit) / mL—based on the dilution ratio of the smallest infectious viral particle, using the following formula: IU / mL = (C*N*D*1000) / V, C = Average number of lentiviral integration copies per genome. D = virus dilution factor. N = the number of cells at the time of infection (approximately 2 × 10⁻⁶) 5 cells), V = the volume of diluted virus added.

[0074] Example 4: Target cell production.

[0075] (1) Activation of allogeneic γδT cells 1) T lymphocytes were isolated and purified from immune cells collected from peripheral blood of healthy adults after epidemiological testing confirmed that there was no risk, to obtain γδT cells.

[0076] 2) Based on the cell count results, add γδT cell activation complete medium preheated to 37 ℃ to a concentration of 1×10⁻⁶. 6 Cells / mL were seeded into cell culture flasks.

[0077] 3) Add the corresponding cell activating factors, including isopentenyl pyrophosphate (IPP) (brand: Sigma-Aldrich, catalog number: 00297-10MG) at a concentration of 5 μmol / L, zoledronic acid (brand: MedChemExpress, catalog number: 118072-93-8) at a concentration of 5 μmol / L, or IL-15 (brand: ACRO, catalog number: IL5-H5215-20ug) at a concentration of 50 ng / mL.

[0078] 4) The cells were placed in a 37 ℃, 5% CO2 incubator and cultured for 48 h.

[0079] (2) Nucleotide transduction encoding the SynNotch receptor 1) After washing and recovering the activated γδT cells, dilute and combine them, take 30 μL of the sample for counting, and retain 5 × 10⁻⁶ cells. 5 Cells were used as the Mock-T control group cells at a concentration of 5 × 10⁶. 5 Cells / mL were seeded in culture flasks, activated with γδT cells, and cultured in complete culture medium for quality control.

[0080] 2) Centrifuge the remaining cells at 300×g for 10 min (acceleration 9, deceleration 9), and discard the supernatant. Resuspend the cell pellet in T lymphocyte activation complete medium to 3×10⁻⁶ cells / min based on the cell count results. 6 cells / mL density.

[0081] 3) Based on the total number of cells and the viral titer, take out the corresponding number of SynNotch receptor transfection lentiviruses from the ultra-low temperature freezer and thaw them at 4 °C.

[0082] 4) Based on the cell number, take Polybrene (hexadimethrine bromide) (10 mg / mL) (brand YEASEN, catalog number: 40804ES76) and mix it with T lymphocyte activation complete culture medium at a volume ratio of 1:500 to prepare Polybrene working solution.

[0083] 5) Transfection is performed by adding 50 μL of lentivirus solution for every 400 μL of cell suspension. Mix the prepared Polybrene working solution with the SynNotch receptor transfection lentivirus and then gently add it to the surface of the remaining cell suspension.

[0084] 6) After adding polybrene to the mixture of virus and cells, reduce shaking and place it in a 37 ℃, 5% CO2 incubator for 24 h to obtain the corresponding SynNotch-γδT cells.

[0085] (3) Nucleotide transduction of coding effectors 1) After washing and recovering the SynNotch-γδT cells, dilute and combine them, take 20 μL of the sample for counting, and retain 5 × 10⁻⁶ cells. 5 Cells were used as the SynNotch-γδT control group cells at a concentration of 5 × 10⁻⁶. 5 Cells / mL were seeded in culture flasks and cultured with T lymphocyte activation complete medium for quality control.

[0086] 2) Centrifuge the remaining cells at 300×g for 10 min (acceleration 9, deceleration 9), and discard the supernatant. Resuspend the cell pellet in T lymphocyte activation complete medium to 3×10⁻⁶ cells / min based on the cell count results. 6 cells / mL density.

[0087] 3) Based on the total number of cells and the viral titer, take out the corresponding number of effector transfection lentiviruses from the ultra-low temperature freezer and thaw them at 4 ℃.

[0088] 4) Based on the cell number, take Polybrene (hexadimethrine bromide) (10 mg / mL) (brand YEASEN, catalog number: 40804ES76) and mix it with T lymphocyte activation complete culture medium at a volume ratio of 1:500 to prepare Polybrene working solution.

[0089] 5) Transfection is performed by adding 50 μL of lentivirus solution for every 400 μL of cell suspension. Mix the prepared Polybrene working solution with the effector transfection lentivirus and then gently add it to the surface of the remaining cell suspension.

[0090] 6) After adding polybrene to the virus-cell mixture, reduce shaking and incubate at 37 ℃ in a 5% CO2 incubator for 24 h. Obtain the corresponding Synnotch-CAR-ICEγδT cells.

[0091] (4) Termination of transfection The Synnotch-CAR-ICE γδT cells were added to an equal volume of complete T lymphocyte activation medium and mixed thoroughly to dilute the virus solution. After dilution, the cells were incubated at 37 °C in a 5% CO2 incubator for 48 h. All cells were then collected and centrifuged at 300 × g for 10 min (acceleration 9, deceleration 6), and the supernatant was discarded. The cell pellets were resuspended in complete T lymphocyte activation medium to a final volume of 1 × 10⁻⁶ cells based on cell count results. 6 Cells / mL density. After replenishment of fluid, cytokines, including isopentenyl pyrophosphate, were added to the target concentration of 5 μmol / L according to the size of the cell culture system. The cell suspension was seeded into culture flasks and cultured continuously at 37 ℃ in a 5% CO2 incubator for 48 h to obtain the CAR-γδT cells.

[0092] (5) Quality testing using Mock-T cells and SynNotch-γδT cell expansion The aforementioned γδT cells and SynNotch-γδT cells were not subsequently subjected to viral transduction and were continuously cultured with fluid replacement. Growth was observed under a microscope every 48 hours, and cell clusters were gently agitated to disperse them. A 30 μL sample was taken to count cell number and viability.

[0093] After each count, the cell clumps were evenly dispersed, and the cells were resuspended to 0.5 × 10⁻⁶ cells in T lymphocyte activation complete culture medium. 6 cells / mL to 1×10 6 Cells / mL density. Cytokines, including isopenten pyrophosphate, were added during the culture process to the target concentration of 5 μmol / L, depending on the size of the culture system.

[0094] Cell suspensions were seeded into cell culture flasks and continuously cultured at 37°C in a 5% CO2 incubator. The total culture time was 16 to 21 days.

[0095] (6) Expansion of the CAR-γδT cells The CAR-γδT cells were seeded in breathable cell culture flasks or bags and placed in a 37 ℃, 5% CO2 incubator. Growth was observed under a microscope every 48 h, and cell clusters were gently agitated to disperse them. A 2 mL sample was taken each time to count the cells and to determine the purity of the target cells using flow cytometry.

[0096] After counting, the cell clumps were evenly dispersed, and the cells were resuspended to 0.5 × 10⁻⁶ cells in complete T lymphocyte expansion culture medium. 6 cells / mL to 1×10 6Cells / mL density. After replenishing the culture medium, add cytokines, including isopentenyl pyrophosphate (IPP) (brand: Sigma-Aldrich, catalog number: 00297-10MG), to the target concentration of 5 μmol / L according to the size of the culture system. Seed the cell suspension into cell culture flasks and incubate at 37 ℃ in a 5% CO2 incubator for 16 days of subsequent expansion culture.

[0097] Cell count, such as Figure 3 As shown, changes in cell viability are as follows: Figure 4 As shown, the above method can achieve large-scale expansion of CAR-γδT cells in vitro, producing off-the-shelf drug formulations that meet therapeutic needs for disease treatment and achieving the goals set in the technology design.

[0098] (7) Formulation and cryopreservation of the CAR-γδT cells Cells continuously expand for more than 16 days and the total number is greater than 1×10⁻⁶. 9 After collecting the cells, all cells were recovered and combined. The recovered cells were centrifuged at 300×g for 10 min (acceleration 9, deceleration 9), the supernatant was discarded, and the cells were resuspended in a compound electrolyte injection containing 1% human serum albumin for injection, called Pepta-A. The cell suspension was centrifuged again at 300×g for 10 min (acceleration 9, deceleration 6), the supernatant was discarded, and the cells were resuspended in a compound electrolyte injection containing 1% human serum albumin for injection to a final volume of 200 mL.

[0099] The recovered CAR-γδT cell suspension was sampled in a 30 μL volume for cell counting. The recovered cells were centrifuged at 300×g for 10 min (acceleration 9, deceleration 9), the supernatant was discarded, and the cells were resuspended in cell cryopreservation medium to a final volume of 4×10⁻⁶. 6 cells / mL.

[0100] Cell suspension was drawn using a syringe and filled into CryoMACS Freezing Bag 50 (Brand: Miltenyi, Germany, Catalog No.: 200-074-400), with each bag containing at least 25 mL of cell suspension. The remaining 2 mL of cells was retained for sterility testing. The sealed cells were then processed using Thermo Scientific. TM CryoMed TM After being programmed to -80°C using a controlled-rate cryogenic freezer, the cells are temporarily stored in a liquid nitrogen tank for cryopreservation until use in treatment. Each bag contains one unit of the CAR-γδT cells.

[0101] Example 5: Example of using the CAR-γδT cells.

[0102] (1) Study of the CAR-γδT cell in vitro model The CAR-γδT cells were prepared according to the above embodiments. On day 20 of in vitro cell culture after all gene transfections were completed, samples of Mock-T control group cells and CAR-γδT cell suspensions were taken and subjected to in vitro mixed lymphocyte culture reaction to preliminarily assess the risk of allogeneic cell rejection during treatment.

[0103] Peripheral blood was collected from three healthy individuals, and mononuclear cells were prepared by Ficoll density gradient centrifugation. After washing three times with PBS, the cells were incubated at 37 °C with 5% CO2 for 2 hours to allow cell adhesion. The suspended cells were then collected as reaction cells. The reaction cell volume was adjusted to 1 × 10⁻⁶ cells / mL. 6 Cells / mL density was measured using CellTrace. TM CFSE Cell Proliferation Kit (Brand: Invitrogen) TM Product code: C34570. CFSE-labeled peripheral blood mononuclear cells were used as reactants. CAR-γδT cells and Mock-T cells were used as stimulants. Stimulant-reactant ratios were 1:1, 1:2, 1:5, 1:10, and 1:20, respectively. PBMCs from different donors served as positive controls, seeded at the same effector-target ratio. The culture system contained 100 IU / mL of exogenously supplemented IL-2.

[0104] Cells were cultured continuously for 96 hours. All cells were recovered, and CD4+ in the cells was analyzed by flow cytometry after antibody labeling of target cells. + T lymphocytes (CD3) + CD4 + The activation and proliferation of CAR-γδT cells were analyzed, along with the secretion of the inflammatory cytokine TNF-α in the reaction system. The immune response of the reacting cells to the CAR-γδT cells was used to assess the risk of immune rejection when using this technology in a xenograft setting.

[0105] Research results are as follows Figures 5 to 7 As shown, the prepared CAR-γδT cells were co-cultured with immune cells from different donors at different effector-to-target ratios to simulate the immune response after the product enters the recipient's body, in order to evaluate its risk of immune rejection. Results showed that, using allogeneic immune cells as reactants, no significant immune cell expansion (proliferation index, PI) or inflammatory cytokine secretion (TNF-α content in the reaction system) was observed at any of the different effector-to-target ratios. The experimental group data were significantly lower than those of the positive control group. This suggests that the CAR-γδT cells obtained by this technology do not induce significant rejection reactions in in vitro studies, and the risk of allogeneic use is low.

[0106] (2) The expression and secretion of ICE by the CAR-γδT cells after CEA stimulation The CAR-γδT cells prepared using the above-described embodiments were used. On day 20 of in vitro cell culture, after all gene transfections were completed, the SynNotch-γδT control group cells and the CAR-γδT cell suspensions were collected, respectively.

[0107] With 5×10 6 After seeding at a density of cells / mL, carcinoembryonic antigen (CEA) – CEACAM-5 / CD66e protein was added to the culture system to a target final concentration of 100 ng / mL. Each group of cells was cultured for 48 h in basal medium free of cytokines, animal serum, and their derivatives. The culture supernatant was then collected, and cells and debris were thoroughly removed and temporarily stored at -20℃.

[0108] Peripheral blood from healthy individuals was collected and monocytes were prepared by Ficoll density gradient centrifugation. After washing three times with PBS, the cells were incubated at 37°C with 5% CO2 for 2 hours to allow them to adhere. The suspended cells were then collected as the reaction cells. The reaction cell volume was adjusted to 1×10⁻⁶ cells / mL. 7 The cells / mL density was determined by adding the previously collected culture supernatant and HER2 protein (name: PE-Labeled Human Her2 / ErbB2 Protein, His Tag (Site-specific conjugation, brand: ACR0, catalog number: HE2-HP2E3-25tests) to the reactants and culturing for 12 h.

[0109] Recycle all cells greater than 1×10 6 Cells were dispersed into a cell suspension, diluted with PBS, centrifuged at 1500 rpm for 6 min (acceleration 9, deceleration 9) to remove all culture medium components, and resuspended. The cells were then incubated with the following detection working solutions at 4 °C for 30 min. The added flow cytometry antibodies / proteins had been diluted and dissolved according to the manufacturer's instructions.

[0110] After staining, the cell suspension was diluted with PBS and centrifuged at 1500 rpm for 6 min (acceleration 9, deceleration 9) to remove the staining solution. Flow cytometry was used to detect the percentage of HER2-binding protein in CD3-positive T lymphocytes to assess the characteristics of CAR-γδT cells activated by CEA to express HER2-CD2-specific ICE binding to allogeneic immune cells.

[0111] Test results as follows Figure 8 , Figure 9As shown, the obtained CAR-γδT cells, after CEA stimulation, secrete a protein targeting HER2 and CD2-expressing positive cells. The CAR-γδT cells expressed the corresponding ICE in the culture supernatant, which was then collected and added to fluorescently labeled HER2 and allogeneic immune cells. Flow cytometry analysis showed that 69.3% of the CD3+ in this in vitro model... + T lymphocytes can specifically bind to the HER2 protein. This suggests that the prepared CAR-γδT cells can express the ICE protein, which meets the design goals.

[0112] (3) The IL-2 expression and secretion of the CAR-γδT cells after CEA stimulation The CAR-γδT cells prepared using the above-described embodiments were used. Suspensions of SynNotch-γδT cells, Mock-T control cells, and the CAR-γδT cells were collected on day 20 of in vitro cell culture after all gene transfections were completed.

[0113] With 5×10 6 After seeding at a density of cells / mL, carcinoembryonic antigen (CEA) – CEACAM-5 / CD66e protein was added to the culture system to a target final concentration of 100 ng / mL. Each group of cells was cultured for 48 h in basal medium free of cytokines, animal serum, and their derivatives. The culture supernatant was then collected, and cells and debris were thoroughly removed and temporarily stored at -20℃. The IL-2 content in the culture supernatant was detected using the absin Human IL-2 ELISA Kit (catalog number: abs510001-96T).

[0114] Test results as follows Figure 10 As shown, the CAR-γδT cells, after being activated by CEA, can express and secrete IL-2 protein, achieving the design goal of the invention.

[0115] Example 6: Verification that CAR-γδT cells have the specific killing ability against lung cancer cells that highly express CEA and HER2.

[0116] Previous studies have shown that CEA expression in lung cancer cell lines (A549, H1975, H460, SK-MES-1) and normal lung epithelial cells BEAS-2B can be detected using Western blot. Results showed that CEA expression in adenocarcinoma cells H1975 was significantly higher than in BEAS-2B, A549, H460, and SK-MES-1 cells. Real-time RT-qPCR results were consistent with Western blot results (cited from "Clinical Relevance Analysis of Combined Detection of Estrogen and Tumor Markers in the Assessment of Diagnosis and Prognosis of Non-Small Cell Lung Cancer"). RT-PCR was used to detect HER2 mRNA expression in lung adenocarcinoma cell lines A549, H1299, PC9, HCC827, H1650, and H1975. RT-PCR results showed that HER2 expression levels varied among different non-small cell lung cancer cell lines, with H1975 showing the highest HER2 expression and A549 showing the lowest. The average HER2 expression level in H1975 cells was 18.87 times that in A549 cells (cited from "The Role of HER2 in EGFR-mutant Advanced Lung Adenocarcinoma"). Therefore, lung cancer cell lines A549 and H1975 were selected as target cells to verify the effectiveness of the described logic gate activation strategy for CAR-γδT cells.

[0117] Both the A549 and H1975 cell lines were purchased from Hefei Wanwu Biotechnology Co., Ltd. After being revived and continuously passaged to the target number, they were inoculated for subsequent research. BDGolgiPlug was added to the culture system. TM Protein transport inhibitors (containing BFA) block the transport of secreted proteins, including CEA, in A549 and H1975 cells. Incubate for 6 hours.

[0118] Cells were digested with 0.1% trypsin, washed by centrifugation, and then used to the target quantity. The recovered cells were washed by centrifugation and then used with APC Mouse Anti-Human HER-2 / neu (brand: BD). TM (Catalog number: 340554) The cell membrane surface was labeled with HER2 protein. Flow cytometry was then used to detect the expression of HER2 protein in different tumor cell lines. After extracellular staining, the cells were washed and processed using eBioscience. TM Permeabilization (Thermo Fisher, catalog number: 00-5123-43) was used for membrane permeabilization. Intracellular proteins were treated with PE Mouse Anti-Human CD66 (BDPharmingen). TMThe tumor cells (catalog number: 551480) were labeled with CEA protein. Flow cytometry was then used to detect the expression of CEA protein in different tumor cell lines.

[0119] Two types of tumor cells identified by flow cytometry were recovered, washed to remove all culture medium components, and their viability was confirmed to be greater than 95% by cell counting. The cells were then resuspended to 2 × 10⁻⁶ cells / mL. 7 Cells were inoculated at a density of 1 × 10⁶ cells / mL in PBS. The cells were stained using the PKH67 Green Fluorescent Cell Linker Mini Kit (Sigma-Aldrich, catalog number: MINI67-1KT). Staining conditions were a cell density of 1 × 10⁶ cells / mL. 7 Incubate at 1 mL of 1 cell / mL solution in the dark at room temperature for 2–4 min. After labeling, add serum-containing complete culture medium preheated to 37 °C to terminate labeling. Centrifuge and wash twice, then resuspend to 1 × 10⁻⁶ cells / mL. 6 cells / mL.

[0120] The CAR-γδT cells prepared using the above-described embodiments were used. Suspensions of both the Mock-T control group cells and the CAR-γδT cells were collected on day 20 of in vitro cell culture after all gene transfections were completed.

[0121] The recovered cells were co-cultured with the two types of tumor cells mentioned above, with effector cell to target cell ratios of 1:1, 2:1, and 5:1, respectively. Each cell group was initially seeded with 2 × 10⁶ target cells. 6 Cells were cultured in each well of a 24-well plate. Then, corresponding effector cells were seeded. Finally, the complete culture medium corresponding to the target tumor cells was added to 1 mL. The cells were cultured at 37°C with 5% CO2 in the dark for 8 h. Cells from each well were then recovered and added to eBioscience. TM 7-AAD active staining solution (brand: Invitrogen) TM (Catalog No.: 00-6993-50) was incubated at 4 ℃ for 30 min and stained in the dark. Subsequent cell suspensions were analyzed by flow cytometry to determine the proportion of viable target cells in different tumor cells, in order to investigate the killing ability of the CAR-γδT cells against lung cancer cell lines with different CEA and HER-2 expression levels.

[0122] Test results as follows Figure 11 and Figure 12As shown, both the A549 and H1975 lung cancer cell lines expressed the targets CEA and HER2. Specifically, the expression level of CEA protein in the H1975 cell line was significantly higher than that in the A549 cell line, while the membrane expression level of HER2 was significantly higher in the H1975 cell line than in the A549 cell line. This can be used to verify the bispecific logic gate activation strategy of CAR-γδT cells.

[0123] The CAR-γδT cells described above showed significantly higher cytotoxicity against the H1975 cell line, which highly expresses both CEA and HER2, than the A549 cell line, which expresses both targets at low levels. Compared with data from unmodified Mock-T cells, the CAR-γδT cells exhibited bispecific cytotoxicity, recognizing CEA protein and initiating ICE expression to target and kill the HER2-positive target lung cancer cell line, H1975, thus fulfilling the objectives of the technical design.

[0124] Example 7: CAR-γδT cells combined with other immune cells support continuous expansion.

[0125] The CAR-γδT cells prepared using the above-described embodiments were subjected to a mixed lymphocyte culture reaction in vitro on day 20 after all gene transfections were completed, i.e., Mock-T control group cells and CAR-γδT cell suspensions were sampled to preliminarily evaluate the combined effect of activated autologous immune cells in killing lung cancer cells during treatment.

[0126] Peripheral blood was collected from three healthy individuals, and mononuclear cells were prepared by Ficoll density gradient centrifugation. After washing three times with PBS, the cells were incubated at 37 °C with 5% CO2 for 2 hours to allow cell adhesion. The suspended cells were then collected as reaction cells. The reaction cell volume was adjusted to 1 × 10⁻⁶ cells / mL. 6 Cells / mL density was measured using CellTrace. TM CFSE Cell Proliferation Kit (Brand: Invitrogen) TM (Catalog number: C34570). CFSE-labeled peripheral blood mononuclear cells were used as reactants.

[0127] After recovering and washing the pre-cultured H1975 lung cancer cell line to remove all culture medium components, cell counts were performed to confirm a viability greater than 95%, and the cells were resuspended at 2 × 10⁻⁶ cells / mL. 7 Cells / mL in PBS. CellTrace was used. TM Far-infrared cell proliferation kit (brand: Invitrogen) TM Cells (catalog number: C34564) were stained. Staining conditions were a cell density of 1×10⁻⁶. 7Incubate at 1 mL of 1 cell / mL solution in the dark at room temperature for 2–4 min. After labeling, add serum-containing complete culture medium preheated to 37 °C to terminate labeling. Centrifuge and wash twice, then resuspend to 1 × 10⁻⁶ cells / mL. 6 cells / mL.

[0128] The three cell types were co-cultured by seeding, initially at a seeding density of 1×10⁶ cells per well. 6 Using H1975 cells as target cells, 2 × 10⁶ reaction cells were then seeded. 6 Cells / well, and finally seed each well with 2 × 10 CAR-γδT cells. 5 Cells. The total volume of the experimental culture system was 2 mL. Wells containing only peripheral blood mononuclear cells without the aforementioned CAR-γδT cells or Mock-T cells served as blank control groups. The cells were cultured at 37 ℃ in the dark with 5% CO2 for 72 h.

[0129] After culture, all cells were recovered, and the cell suspension was analyzed by flow cytometry to detect the proliferation coefficient of tumor target cells—analyzing the proliferation peak of far-infrared cell proliferation dyes to study and evaluate the killing effect of the CAR-γδT cells and receptor immune cells on lung cancer cells.

[0130] After culture, all cells were recovered, and the CD4+ in the cells was analyzed by flow cytometry after antibody labeling of the target cells. + T lymphocytes (CD3) + CD4 + ) and NK cells (CD3) - CD56 + The activation and proliferation of CAR-γ T cells were analyzed, along with the secretion of the inflammatory cytokine IFN-γ in the reaction system. The immune response of the reacting cells in the presence of effector cells was used to assess the synergistic activation of the receptor-mediated immune system by CAR-γδT cells.

[0131] Test results as follows Figures 13 to 15 As shown, the CAR-γδT cells were used in an in vitro model to synergistically inhibit the proliferation of H1975 lung cancer cell lines, which highly express both CEA and HER2, in conjunction with allogeneic immune cells. Simultaneously, analysis revealed the presence of NK cells and CD4+ in this model. + T lymphocytes are activated and proliferate, and the proportion of immune cells secreting the inflammatory cytokine IFN-γ, which has an inhibitory effect on tumors, increases. This demonstrates that, in addition to their self-killing function, the CAR-γδT cells secrete ICE, which can specifically activate allogeneic immune cells, and the IL-2 they express supports the expansion and survival of immune cells. Thus, they exert a synergistic anti-tumor effect. Therefore, the CAR-γδT cells prepared by this technical solution meet the purpose of the technical design and have a therapeutic effect on lung cancer in vitro.

[0132] Example 8: Verify that the CAR-γδT cells have therapeutic effects and can survive for a long time in a xenograft animal model.

[0133] Forty-five 6-8 week old NSG mice (weighing 18-22 g) were subcutaneously inoculated with H1975 lung cancer tumor cell line after one week of acclimatization. Each mouse was inoculated with 5 × 10⁻⁶ cells. 6 Tumor cells were identified, and the animals' condition was closely monitored. Tumor volume in mice was measured every three days using calipers. Tumors were classified as tumor cells when they reached 100 mm². 3 Mice were randomly grouped according to body weight and tumor size, and then infused with the CAR-γδT cells or control Mock-T cells via the tail vein. Detailed administration methods, dosages, and routes are shown in Table 4. Mouse body weight and overall survival were recorded every 72 hours after administration.

[0134] Table 4 - Administration Methods, Dosage, and Routes of Administration

[0135] Test results as follows Figure 16 , Figure 17 The figures show the body weight and survival rate of animals in a subcutaneous xenograft animal model of lung cancer treated with the CAR-γδT cells. Based on the long-term survival of the animals, it is demonstrated that the cells described in this technique can have a therapeutic effect in vivo, treating or improving the condition of H1975 lung cancer cell tumor-bearing animals and prolonging the survival period with the disease. Simultaneously, the changes in body weight of the surviving animals indicate that the condition is relatively stable after treatment with this technique, with slowed tumor growth and fewer complications. This suggests that the CAR-γδT cells have development potential in lung cancer treatment, and their in vivo therapeutic effect can achieve the objectives of the technical design.

[0136] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A CAR-γδT cell, characterized in that, γδT cells contain nucleotide sequences encoding a synthetic Notch receptor and an effector. The synthetic Notch receptor comprises a transmembrane core module and an intracellular regulatory module. The intracellular regulatory module is connected to the C-terminus of the transmembrane core module and includes a DNA-binding domain and a transcriptional regulatory domain. The transmembrane core module can be cleaved by γ-secretase to separate the intracellular regulatory module. The nucleotide sequence encoding the effector includes an upstream activation sequence that specifically binds to the DNA-binding domain, as well as nucleotide fragments encoding immune cell adaptors, self-cleaving peptides, and T-cell immune activating factors.

2. The CAR-γδT cells according to claim 1, characterized in that, The DNA binding domain is selected from Gal4 or LexA; preferably, the transcriptional regulatory domain is selected from VP64, KRAB, tTa or ZFHD1.

3. The CAR-γδT cells according to claim 2, characterized in that, The upstream activation sequence is selected from UAS or LexAop.

4. The CAR-γδT cells according to claim 1, characterized in that, The T-cell immune activating factor is a soluble receptor; preferably, the T-cell immune activating factor is selected from sIFN-γ, sIL-2, sIL-4 or sIL-17.

5. The CAR-γδT cells according to claim 1, characterized in that, The synthetic Notch receptor further includes an extracellular recognition module, which includes a first single-stranded variable fragment structure; the immune cell connector includes a second single-stranded variable fragment structure.

6. The CAR-γδT cells according to claim 5, characterized in that, The first single-chain variable fragment structure and the second single-chain variable fragment structure target any two of the following: CD19, BCMA, GPC3, mesothelin, EGFRvRIII, Claudin 18.2, B7-H3, ErbB2, GM3, GD2, GD3, CD20, CD22, gp100, NYESO-1, carbonic anhydrase IX, WT1, carcinoembryonic antigen, CA-125, MUC-1, MUC-3, epithelial tumor antigen, MAGE type antigen, BAGE, GAGE, XAGE1B, CTAG2, CTAG1, SSX2, LAGE1, or HER2.

7. The CAR-γδT cells according to claim 1, characterized in that, The nucleotide sequence encoding the synthetic Notch receptor is shown in SEQ ID No:

1.

8. The CAR-γδT cells according to claim 7, characterized in that, The nucleotide sequence encoding the effector is shown in SEQ ID No:

2.

9. The method for preparing CAR-γδT cells according to any one of claims 1 to 8, characterized in that, Including the following steps: 1) Construct a recombinant viral vector containing a nucleotide sequence encoding the synthetic Notch receptor and a nucleotide sequence encoding the effector; 2) Obtain activated γδT cells, transduce the recombinant viral vector into the γδT cells, and screen to obtain cells that stably express the synthetic Notch receptor and the effector, which are the CAR-γδT cells.

10. The preparation method according to claim 8, characterized in that, The nucleotide sequence encoding the synthetic Notch receptor is shown in SEQ ID No:1, and the nucleotide sequence encoding the effector is shown in SEQ ID No:2.

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