A CAR-T cell drug targeting two tumor antigens simultaneously and its application

Abstract

The present invention relates to the field of cell drugs, specifically to a CAR-T cell drug that simultaneously targets two tumor antigens and its application. The tumor is solid glioma, and the CAR-T cell drug can reduce the risk of tumor recurrence. Traditional single-target CAR-T cell therapy may result in unsatisfactory efficacy when treating certain types of glioblastoma due to tumor heterogeneity and antigen escape mechanisms. However, through the dual-target strategy of the present invention, CAR-T cells can simultaneously recognize and bind two different tumor-associated antigens, thereby reducing the possibility of tumor cell escape and improving the treatment effect. By simultaneously targeting interleukin-13 receptor alpha 2 and NKG2D ligands, the anti-tumor activity of CAR-T cells is significantly enhanced. The dual-target CAR-T strategy provides a novel and effective means for the treatment of glioblastoma and has broad clinical application potential. The dual-target strategy may provide new ideas in improving treatment effect and reducing off-target toxicity.

Description

Technical Field

[0001] The present invention relates to the field of cell drugs, and specifically to a CAR-T cell drug that simultaneously targets two tumor antigens and its application. Background Art

[0002] CAR-T therapy, that is, chimeric antigen receptor T cell immunotherapy, uses genetic engineering technology to reprogram the patient's immune T cells in vitro so that they can recognize the antigens on the surface of tumor cells, and then these cells are infused back into the patient's body for treatment. In this way, T cells can accurately recognize and kill tumor cells in a major histocompatibility complex (MHC)-independent manner. In the past decade, CAR-T therapy has made great progress in the field of tumor treatment, treating previously incurable blood tumors. Scientists have also been working hard to apply CAR-T therapy to other cancer types other than blood cancers. However, CAR-T cell therapy technology still faces some problems in the treatment of solid tumors. After CAR-T cells successfully enter solid tumors such as glioblastoma (GBM), they will encounter two main obstacles: ① The inhibitory and damaging effects of immunosuppressive cells and inhibitory molecules such as TGF-β, IL-6, IL-10, IL-23, etc. in the glioma immune microenvironment on CAR-T cells. A lot of inhibitory leukocytes will also accumulate in GBM, such as regulatory T cells, myeloid-derived suppressor cells, tumor-associated macrophages, etc., which will thus play an inhibitory role on CAR-T cells; ② Interference with tumor antigen recognition, mainly caused by glioma heterogeneity and insufficient specific antigens. These are all important reasons for the failure of CAR-T cell function. Different pathological types of glioblastoma express different types of tumor antigens. Therefore, the selection of appropriate tumor antigens is crucial for the efficacy of CAR-T cells.

[0003] In glioblastoma, the expression of IL-13Rα2 is positively correlated with the glioblastoma grade, and high expression of IL-13Rα2 is associated with poor prognosis. IL13Rα2 is one of the two subunits of the cytokine IL-13 receptor. IL-13 binds to IL13Rα1 with low affinity, and then IL13Rα1 forms a heterodimer with IL-4Rα, activating the STAT6 signaling pathway and inducing apoptosis. Based on the above findings, targeted therapy and immunotherapy against IL-13Rα2 have been carried out. It has been reported that patients with recurrent glioblastoma have achieved good results after CAR-T cell therapy targeting IL-13Rα2, with no toxic side effects after treatment, a reduction in tumor volume, and an increase in the content of immune factors and immune cells in cerebrospinal fluid. In summary, IL-13Rα2, as a specific target of CAR-T cells, can achieve the guiding effect of killing glioblastoma cells and plays a crucial role in the research of treating glioma.

[0004] NKG2D is an activating receptor expressed on the surface of NK cells and CD8+ T cells, which plays an important role in innate immunity and is involved in the recognition of virus-infected cells and the killing of tumor cells by NK cells. Multiple studies have shown that ligands of NKG2D (NKG2DL), such as MICA / B, are highly expressed in glioma cells, especially in high-grade gliomas (such as glioblastoma), and the expression is more significant. It has been found that the high expression of NKG2DL is associated with a poor prognosis in glioma patients, which may be due to the fact that tumor cells with high expression of NKG2DL are more invasive or more likely to escape immune surveillance. Previous studies have shown that targeting and killing cancer stem cells (CSC) can effectively inhibit the occurrence of glioblastoma and prolong the survival of glioma mice. It has been reported that a large amount of NKG2D ligand ((KG2DL) is found in glioblastoma stem cells ((SC). Therefore, NKG2D-specific CAR-T cells can be used for treatment. At the same time, NKG2DL is also an ideal target for the treatment of glioma.

[0005] Single-target CAR-T cells may not be able to recognize and kill all tumor cells. As the treatment progresses, tumor cells may survive through antigen escape mechanisms, leading to the recurrence of residual tumors. Multi-target CAR-T cells can target multiple antigens, reducing the possibility of antigen escape, maintaining long-term treatment effects, and reducing non-specific attacks on normal cells and the occurrence of side effects. The efficacy of dual-target CAR-T cell therapy has been verified in some clinical trials, and the safety of dual-target CAR-T cells is also better than that of single-target CAR-T cells, with lower incidences of severe cytokine release syndrome (CRS) and neurotoxicity (NT). The main CAR structures used for dual-target CAR-T cell therapy include: single-target CAR mixtures, bivalent tandem CARs, bivalent circular CARs, and bicistronic CARs. These innovative designs provide new hopes and possibilities for the application of CAR-T cell therapy in complex tumors such as glioma.

[0006] For the first time in the present invention, by simultaneously targeting IL13Rα2 and NKG2D ligands, the aim is to more effectively recognize and attack glioma cells, improve the specificity and efficiency of treatment, overcome tumor heterogeneity and antigen escape, and enhance tumor killing ability. This innovative design is expected to bring new hopes and better treatment effects to glioma patients. Summary of the Invention

[0007] The object of the present invention is to provide a CAR-T cell drug that simultaneously targets two tumor antigens and its application, aiming to more effectively recognize and attack glioma cells, improve the specificity and efficiency of treatment, overcome tumor heterogeneity and antigen escape, and enhance tumor killing ability, so as to solve the problems raised in the above-mentioned background technology.

[0008] According to the first aspect of the present invention, there is provided a CAR-T cell drug that simultaneously targets two tumor antigens and its application, and the CAR-T cell drug simultaneously targets IL13 receptor α2 and NKG2D ligand;

[0009] The dual-target chimeric antigen receptor of the CAR-T cell drug includes:

[0010] A signal peptide connected in sequence: human CD8α signal peptide, CD8α leader;

[0011] IL13 (E13Y) mutant targeting IL13Rα2;

[0012] Linker;

[0013] NKG2D extracellular region targeting NKG2D ligand: NKG2D ECD;

[0014] CD8α hinge region: human CD8α hinge region, CD8α Hinge;

[0015] Transmembrane region: human CD8α transmembrane region, CD8α transmembrane;

[0016] Costimulatory factor and intracellular signal peptide: human CD3ζ intracellular signal, CD3ζ signal.

[0017] According to the second aspect of the present invention, there is provided a method for constructing a dual-target chimeric antigen receptor, including the following steps:

[0018] S1. Vector linearization: The plasmid pLL3.7-NKG2D-CAR stored in the laboratory is linearized by double digestion with NheⅠ and EcoRⅠ. The digestion system includes: 1 μL of 10× buffer; 1 μg of plasmid pLL3.7-NKG2D-CAR; 1 μL of Nhe I enzyme; 1 μL of EcoR I enzyme, and ddH2O is added to make up to 10 μL;

[0019] S2. PCR amplification of the linked fragment sequence: Design the ((4S)3 linker sequence on the primers used to amplify the extracellular region fragment of NKG2D and the IL13(E13Y) fragment, and obtain two large fragments containing the CD8α signal peptide - IL13(E13Y) - linker sequence and the extracellular region of NKG2D - CD8α hinge region - transmembrane region - costimulatory factor and intracellular signal peptide respectively through PCR amplification;

[0020] S3. Gel recovery and plasmid recombination: Using the method of homologous recombination, perform seamless cloning on the recovered large fragment of vector pLL3.7 - NKG2D - CAR and the two small fragments obtained by PCR amplification in step S2 using a one - step cloning kit;

[0021] S4. Transformation: Transfer the ligation product into DH5α competent cells. After ice - bathing for 30 min, perform heat shock at 42 °C, then ice - bathe for another 5 min to obtain the transformation product. Add the transformation product to 1 mL of liquid LB medium without antibiotics, culture it on a shaker at 37 °C for 1 h, centrifuge to obtain a precipitate, resuspend the precipitate with 100 μL of LB medium, and spread it onto an LB plate with Amp resistance for overnight culture;

[0022] S5. Identification: Select positive clones for sequencing and double - digestion for identification. Double - digestion with NheI and EcoR I yields fragments of 6841 bp and 1548 bp. After correct sequencing, extract the plasmid of the bacterial cells using an endotoxin - free large - scale plasmid extraction kit.

[0023] Furthermore: The conditions for the digestion reaction in step S1 are: digestion temperature is 37 °C, digestion time is 30 min, and the total volume of the digestion system is 10 μL.

[0024] Furthermore: In step S4, the heat - shock time is 45 s.

[0025] According to the second aspect of the present invention, there is provided an application of a CAR - T cell drug targeting two tumor antigens simultaneously in anti - tumor activity, and the tumor is solid glioma

[0026] Furthermore: The CAR - T cell drug can reduce the risk of tumor recurrence.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] 1. In the treatment of certain types of glioblastoma, the traditional single - targeted CAR - T cell therapy may lead to unsatisfactory curative effects due to tumor heterogeneity and antigen escape mechanisms. However, through the dual - targeted strategy of the present invention, CAR - T cells can simultaneously recognize and bind two different tumor - associated antigens, thereby reducing the possibility of tumor cell escape and improving the treatment effect;

[0029] 2. By simultaneously targeting IL13 receptor α2 (IL13Rα2) and NKG2D ligands, the anti-tumor activity of CAR-T cells was significantly enhanced. The dual-target CAR-T strategy provides a novel and effective means for the treatment of glioblastoma, with broad clinical application potential. The dual-target strategy may provide new ideas for improving the treatment effect and reducing off-target toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the plasmid structure of the dual-target chimeric antigen receptor CAR of the present invention;

[0031] Figure 2 Schematic diagram of the enzymatic digestion identification of the recombinant plasmid of the present invention;

[0032] Figure 3 Schematic diagram of the lentivirus titer determination of the present invention;

[0033] Figure 4 Schematic diagram of the detection of the CAR positive rate of the present invention;

[0034] Figure 5 Schematic diagram of the killing of U251-luciferase (high expression of IL13 ligand) of the present invention;

[0035] Figure 6 Schematic diagram of the killing of PANC-1-luciferase (high expression of NKG2D ligand) of the present invention;

[0036] Figure 7 Schematic diagram of the killing of U87-luciferase (high expression of NKG2D ligand and IL13 ligand) of the present invention;

[0037] Figure 8 Schematic diagram of the secretion of GzmB with U251 cells (high expression of IL13 ligand) as target cells of the present invention;

[0038] Figure 9 Schematic diagram of the secretion of GzmB with PANC-1 cells (high expression of NKG2D ligand) as target cells of the present invention;

[0039] Figure 10 Schematic diagram of the secretion of GzmB with U87 as target cells (high expression of NKG2D ligand and IL13 ligand) of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention will be described in detail below in conjunction with the embodiments shown in the accompanying drawings. It should be noted, however, that these embodiments are not intended to limit the present invention, and any equivalent transformation or substitution in terms of function, method, or structure made by those of ordinary skill in the art based on these embodiments shall fall within the protection scope of the present invention.

[0041] Please refer to Figure 1-10 , and the specific implementation process of the present invention will be elaborated below through the preparation of a CAR-T cell drug that simultaneously targets two tumor antigens and multiple experiments.

[0042] 1. Plasmid construction:

[0043] The dual-target chimeric antigen receptor includes: a signal peptide (human CD8α signal peptide, CD8α leader), an IL13 (E13Y) mutant targeting IL13Rα2, a linker, an NKG2D extracellular domain (NKG2D ECD) targeting NKG2D ligand, a CD8α hinge region (human CD8α hinge region, CD8α Hinge), a transmembrane region (human CD8α transmembrane region, CD8α transmembrane), a co-stimulatory factor, and an intracellular signal peptide (human CD3ζ intracellular signal, CD3ζ signal), which are connected in sequence. As Figure 1 shown, the specific construction process:

[0044] (1) Vector linearization: The plasmid pLL3.7-NKG2D-CAR stored in the laboratory was linearized by double digestion with NheⅠ and EcoRⅠ. The conditions for the digestion reaction were: the digestion temperature was 37 °C, the digestion time was 30 min, and the digestion system (total volume 10 μL) included: 1 μL of 10× buffer; 1 μg of plasmid pLL3.7-NKG2D-CAR; 1 μL of Nhe I enzyme; 1 μL of EcoR I enzyme, and ddH2O was added to make up to 10 μL.

[0045] (2) PCR amplification of the linker sequence: The (G4S)3 linker sequence was designed on the primers used for amplifying the NKG2D extracellular domain fragment and the IL13 (E13Y) fragment, and two large fragments containing the CD8α signal peptide-IL13 (E13Y)-linker sequence and the NKG2D extracellular domain-CD8α hinge region-transmembrane region-co-stimulatory factor and intracellular signal peptide were obtained by PCR amplification.

[0046] (3) Gel recovery and plasmid recombination: Using the method of homologous recombination, the recovered large fragment of the vector pLL3.7-NKG2D-CAR and the two small fragments obtained by PCR amplification in the previous step were subjected to seamless cloning using a one-step cloning kit;

[0047] (4) Transformation: Transfer the ligation product into DH5α competent cells. After ice-bathing for 30 min, heat-shock at 42 °C for 45 s, then ice-bathe for another 5 min to obtain the transformation product. Add the transformation product into 1 mL of liquid LB medium without antibiotics, culture it on a shaker at 37 °C for 1 h, centrifuge to obtain the precipitate, resuspend the precipitate with 100 μL of LB medium, and spread it onto an LB plate with Amp resistance for overnight culture.

[0048] (5) Select positive clones for sequencing and double digestion for identification. After double digestion with NheI and EcoR I, fragments of 6841 bp and 1548 bp are obtained. The identification results are as Figure 2 shown. After correct sequencing, extract the plasmid of the bacterial cells using an endotoxin-free large plasmid extraction kit according to the method described in the instruction manual.

[0049] 2. Lentiviral vector plasmid packaging:

[0050] Select 293T cells with a density of about 80%. The packaging system is as follows: for each dish, the system contains 8 μg of the target plasmid, 6.5 μg of psPAX2, 3.5 μg of pMD2.G, 50 μL of PEI, and DMEM basal medium is supplemented to 1 mL. After evenly adding the mixed system to 293T cells, change the medium with 1% DMEM medium at 6 - 8 h. Collect the supernatant containing the lentiviral vector plasmid at 48 h and 72 h after medium change. After centrifugation at 4000 g for 10 min, filter the supernatant through a 0.22-μm filter membrane to obtain the filtrate containing the lentiviral vector plasmid. Transfer the filtrate containing the lentiviral vector plasmid into an ultracentrifugation tube and centrifuge at 15000 rpm at 4 °C for 2 h. After centrifugation, discard the supernatant, and dissolve the precipitate with 400 μL of X-VIVO complete medium at 4 °C overnight.

[0051] 3. Determine the lentiviral titer:

[0052] Prepare 293T cells in good growth condition, seed them in a 24-well plate at a density of 2×105 cells / well, add different viruses to different wells at volumes of 1 μL, 2 μL, and 4 μL respectively. Place the cell culture plate in an incubator at 37°C and 5% CO2 for 48 hours. Centrifuge to collect the infected cells, discard the supernatant, resuspend the pellet with 100 μL of PBS buffer, and add 0.3 μL of APC anti-human NKG2D antibody and PE anti-human IL-13 antibody to the corresponding viruses respectively. Stain at 4°C in the refrigerator for 30 min, add 1 mL of PBS buffer and then centrifuge. Finally, resuspend the pellet with 250 μL of PBS buffer and transfer it into a flow tube for flow cytometry detection. Analyze with FlowJo V10 software: Use uninfected HEK-293T cells as a negative control for gating, and analyze the proportion of CAR-positive expressing cells after infecting HEK-293T cells with different volumes of virus concentrates. The titer detection results are as Figure 3 shown. Select samples with a positive rate of about 20%-30% to calculate the virus titer (Titer).

[0053] Calculation formula: Titer (TU / mL) = (number of positive cells × dilution factor) / volume of inoculated virus (mL).

[0054] The results are as follows: The titer of LV-NKG2D-CAR is 1.2×108 TU / mL, the titer of LV-IL13-CAR is 0.5×108 TU / mL, and the titer of LV-IL13-NKG2D-CAR is 0.5×108 TU / mL.

[0055] 4. Preparation of CAR-T cells:

[0056] Isolate peripheral blood mononuclear cells (PBMCs) from the donor, activate T cells using anti-CD3 and anti-CD28 antibodies, and continue to culture the activated T cells in a medium containing IL-2; Prepare virus suspensions for each virus at a ratio of MOI = 10. Calculate the required virus volume according to the virus titer, add the calculated virus suspension to 5×105 T cells in a 24-well plate, mix well, and continue to culture the T cells in a medium containing IL-2. Usually incubate in an incubator at 37°C and 5% CO2 for 24-48 hours. Detect the expression of CAR molecules by flow cytometry to determine the infection efficiency. The flow cytometry procedure is the same as above. The positive rate is as Figure 4 shown.

[0057] 5. Comparison of the in vitro anti-tumor effects of CAR-T cells:

[0058] In this experiment, the expressions of NKG2DL and IL13Rα2 on the existing cell lines in the laboratory were first detected. For example, U251-LUC cells were finally selected as the cell line with IL13Rα2+NKG2DL-. Due to resource limitations, PANC-1-LUC cells were used as the substitute for the IL13Rα2-NKG2DL+ glioma cell line, and U87-LUC was used as the IL13Rα2+NKG2DL+ cell line. Then, cytotoxicity assays were performed on these three cell lines respectively. Four effector groups, namely MOCK T, NKG2D-CAR-T, IL13-CAR-T, and TanCAR-T (IL13-NKG2D-CAR-T), were set up in this experiment. Three effector-to-target ratios of 1:1, 2:1, and 4:1 were set for each group, and three replicates were performed for each gradient to exclude errors caused by accidental factors. 39 wells were selected from a 96-well low-attachment plate for the experiment. 7.8×105 U251-LUC cells were taken and resuspended in 3.9 mL of X-VIVO complete medium. 100 μL of the target cell suspension was added to each well. 2×104 effector T cells were added to the 1:1 effector-to-target ratio group, 4×104 effector T cells were added to the 2:1 effector-to-target ratio group, and 8×104 effector T cells were added to the 4:1 effector-to-target ratio group. The total volume in the well was made up to 200 μL. The above seeding process was repeated with PANC-1-LUC cells and U87-LUC cells. The 96-well low-attachment plate was placed in an incubator at 37 °C for 16 h. After 16 h, the liquid in each well was taken out, labeled, centrifuged, and resuspended in 100 μL of firefly luciferase reporter gene cell lysis buffer. The samples were transferred to a white detection plate, 20 μL of firefly luciferase detection reagent was added, and the absorbance value was measured on a luminometer. The cell killing efficiency = [(control well of only target cells - experimental well) / control well of only target cells]×100%. The killing results are shown in Figure 5 、 6 and 7. The results showed that the dual-target chimeric antigen receptor could not only specifically recognize tumor cells expressing either of the two single targets, NKG2DL or IL13Rα, but also recognize tumor cells co-expressing NKG2DL or IL13Rα. The dual-target CAR-T had stronger anti-tumor activity, could avoid immune escape of positive tumor cells with low-abundance antigen expression, and thus reduced the recurrence risk.

[0059] 7. Detection of the activation index GzmB of CAR-T cells:

[0060] Add 1×105 tumor cells to each well of a 48-well plate, add 3×105 CAR-T cells according to an effector-to-target ratio of 3:1, make up the volume to 400 μL, place it in an incubator, add a Golgi blocker after culturing at 37 °C for 12 h, transfer the cells into a 1.5 mL centrifuge tube after 4 h, remove the supernatant after centrifugation, add 100 μL of fixation and permeabilization agent for 1 h, wash once with 1 mL of PBS buffer, resuspend with 100 μL of PBS buffer, add 0.3 μL of Anti-GzmB antibody for staining for 30 min, wash with 1 mL of buffer, and resuspend the cell pellet with 250 μL for flow cytometry analysis. The results are shown in Figure 8 、 9 and 10. It can be seen from the figure that for target cells highly expressing the IL13 ligand or the NKG2D ligand, the expression level of GzmB in dual-target CAR-T cells is equivalent to that in IL13-CAR-T cells ( Figure 8 ) or NKG2D-CAR-T cells ( Figure 9 ), respectively. However, when co-cultured with target cells highly expressing both the NKG2D ligand and the IL13 ligand, the expression level of GzmB in dual-target CAR-T cells is significantly higher than that in single-target CAR-T cells, indicating that dual-target CAR-T cells can be more highly activated by simultaneously recognizing two different tumor-associated antigens, which may be the reason for its stronger antitumor effect.

[0061] Finally, it should be noted that in the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "vertical", "upper", "lower", "horizontal", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention.

[0062] In the description of the present invention, it should also be noted that unless otherwise clearly specified and defined, the terms "set", "installed", "connected", "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0063] The above content further elaborates on the present invention in combination with specific embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, which should all be regarded as falling within the protection scope determined by the claims submitted for the present invention.

[0064] Attachment: Gene Sequence

[0065]

[0066]

Claims

1. A CAR-T cell drug that simultaneously targets two tumor antigens, characterized in that, The CAR-T cell drug targets both IL13 receptor α2 and NKG2D ligand simultaneously; The dual-target chimeric antigen receptor of the CAR-T cell drug comprises: A signal peptide connected in sequence: CD8αleader, and the sequence encoding the CD8αleader is SEQ ID NO:1; An IL13 mutant targeting IL13Rα2, and the sequence encoding the IL13 mutant is SEQ ID NO:3; A linker fragment linker, and the sequence encoding the linker fragment linker is SEQ ID NO:5; The extracellular region of NKG2D targeting NKG2D ligand: NKG2DECD, and the sequence encoding the NKG2DECD is SEQ ID NO:7; The CD8α hinge region: CD8αHinge, and the sequence encoding the CD8αHinge is SEQ ID NO:9; The transmembrane region: CD8αtransmembrane, and the sequence encoding the CD8αtransmembrane is SEQ ID NO:11; A co-stimulatory factor, and the sequence encoding the co-stimulatory factor is SEQ ID NO:13; and An intracellular signal peptide: CD3ζsignal, and the sequence encoding the CD3ζsignal is SEQ ID NO:15.

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