Construction and application of reverse cytokine receptor of targeted transforming growth factor TGF-beta

Abstract

The invention discloses construction of a reverse cytokine receptor of a targeted transforming growth factor TGF-beta and application of the reverse cytokine receptor. The constructed reverse cytokine receptor of the targeted transforming growth factor TGF-beta comprises an extracellular fragment, a transmembrane fragment and an intracellular fragment which are sequentially connected, the extracellular fragment is a human TGFBR1 extracellular fragment or a human TGFBR2 extracellular fragment, and the intracellular fragment is a human KIR3DS1 intracellular fragment or a rodent KIR3DS1 intracellular fragment. The transmembrane segment is a transmembrane segment from any transmembrane protein and can be used for immune cell modification, so that the negative regulation function of a TGF-beta signal channel on immune cells is reversed, and the inhibition effect of TGF-beta on the immune cells is converted into the activation effect.

Description

[Technical field]

[0001] The present invention relates to the technical field of immunotherapy, in particular to the technical field of construction and application of reverse cytokine receptor targeting transforming growth factor TGF-β. [Background technology]

[0002] In recent years, with the rapid development and interweaving of fields such as oncology, immunology and molecular biology, tumor immunotherapy has made remarkable progress in basic research and clinical application, bringing about major changes in cancer treatment; taking chimeric antigen receptor T cell immunotherapy (CAR-T) as an example, this therapy has achieved remarkable success in the treatment of hematological tumors, especially in patients with drug-resistant B-cell acute lymphoblastic leukemia, with a total remission rate of up to 93%; CAR-T therapy can transfer genetic material with specific antigen recognition domains and T cell activation signals into T cells through gene modification technology, so that T cells can directly bind to specific antigens on the surface of tumor cells and be activated, directly killing tumor cells by releasing perforin and granzyme B, and at the same time recruiting endogenous immune cells in the human body to kill tumor cells by releasing cytokines, thereby achieving the purpose of treating tumors, and can also form immune memory T cells, thereby obtaining a specific long-term anti-tumor mechanism.

[0003] The application of CAR-T cells in solid tumors is also being explored (currently, as many as 22 different targets have been used to treat solid tumors, even more than the targets for hematological tumors); however, the preliminary results of many clinical trials have not met expectations (their efficacy is significantly lower than that of hematological tumors); this is mainly due to the heterogeneity of solid tumors and the immunosuppressive properties of the tumor microenvironment, which makes it impossible for CAR-T cells to effectively eliminate all tumor cells; the more complex tumor microenvironment exhibited by solid tumors compared to hematological tumors not only promotes tumor proliferation and metastasis, but also enables tumor cells to evade host immune surveillance and resist immunotherapy; studies have shown that immunosuppressive factors (such as PGE2, TGF-β, IL-4 and IL-10, etc.), suppressive immune cells (such as Treg, MDSCs and TAMs, etc.), immunosuppressive signals (such as PD-1 / PD-L1, CTLA-4 and TIM3, etc.) and metabolism-related factors (such as hypoxia and lack of glucose and amino acids, etc.) in the immunosuppressive microenvironment can inhibit the anti-tumor function of immune cells through multiple mechanisms.

[0004] At present, a variety of strategies have been proposed to improve the activity of immune cells in the immunosuppressive microenvironment, such as designing chimeric antigen receptors targeting immune checkpoints and combining monoclonal antibodies targeting immunosuppressive factors. Among them, modifying cytokine receptors is an effective method. This method usually combines the extracellular structure of inhibitory receptors with the intracellular part of stimulatory receptors to form an inverted cytokine receptor, so that the immunosuppressive factor activates the immune cell after binding to the corresponding receptor. Studies have shown that this strategy has certain effects in improving the activity of CAR-T cells in the tumor microenvironment, such as Wang Y and Leen AM (Wang Y, Jiang H, Luo H, Sun Y, Shi B, Sun R, Li Z. An IL-4 / 21 Inverted Cytokine Receptor Improving CAR-T Cell Potency in Immunosuppressive Solid-Tumor Microenvironment [J]. Frontiers in Immunology, 2019, 10: 1691 and Leen AM, Sukumaran S, Watanabe N, Mohammed S, Keirnan J, Yanagisawa R, Anurathapan U, Rendon D, Heslop HE, Rooney CM, Brenner MK, Vera JF. Reversal of tumor immune inhibition using a chimeric cytokine receptor. Mol Ther. 2014 Jun; 22 (6): 1211-1220. doi: 10.1038 / mt.2014.47. Epub 2014 Mar 20. PMID: 24732709; PMCID: PMC4048899) forms IL-4 / 7ICR or IL-4 / 21ICR by combining the extracellular part of the IL-4 receptor with the intracellular part of IL-7 or IL-21, and after being transferred into CAR-T cells, it significantly enhances the killing ability of CAR-T cells in the tumor microenvironment with high IL-4 expression.

[0005] TGF-β can not only promote infiltration and metastasis by inducing changes in the matrix environment around the tumor, promoting tumor angiogenesis, and inducing epithelial-mesenchymal transition of tumor cells, but more importantly, its high expression in the tumor microenvironment significantly inhibits the activity of CD8+T cells and NK cells, becoming an important node in the immunosuppressive network; however, the design of the above two studies only targets the IL-4 cytokine in the tumor microenvironment, while the expression of inhibitory cytokines in different tumors is different, and different inhibitory cytokines have different inhibitory effects on different immune cells; therefore, how to combine TGF-β-related cytokine receptor technology to further optimize the anti-tumor activity of immune cells, while reducing their exhaustion in complex microenvironments, enhancing the efficacy of immunotherapy, and significantly improving the application potential of CAR-T and CAR-NK cell therapy in solid tumors needs to be solved urgently. [Summary of the invention]

[0006] The purpose of the present invention is to solve the problems in the prior art and propose the construction and application of a reverse cytokine receptor targeting transforming growth factor TGF-β. A reverse cytokine receptor targeting TGF-β can be constructed for immune cell modification, thereby reversing the negative regulation function of the TGF-β signaling pathway on immune cells and converting the inhibitory effect of TGF-β on immune cells into an activating effect.

[0007] To achieve the above objectives, the present invention provides a reverse cytokine receptor targeting transforming growth factor TGF-β, comprising an extracellular segment, a transmembrane segment and an intracellular segment connected in sequence, wherein the extracellular segment is an extracellular segment of human TGFBR1 or an extracellular segment of human TGFBR2, the intracellular segment is an intracellular segment of KIR3DS1 from humans or other species, and the transmembrane segment is a transmembrane segment of a transmembrane protein from humans or other species (preferably an intracellular segment of TGFBR1, TGFBR2 or KIR3DS1 from humans or other species).

[0008] Preferably, the nucleic acid sequence and polypeptide sequence of the human TGFBR1 extracellular fragment are shown as SEQ ID NO.1 and SEQ ID NO.2 in the sequence listing, respectively, and the nucleic acid sequence and polypeptide sequence of the human TGFBR2 extracellular fragment are shown as SEQ ID NO.5 and SEQ ID NO.6 in the sequence listing, respectively.

[0009] Preferably, the intracellular segment is a human KIR3DS1 intracellular segment or a rodent KIR3DS1 intracellular segment, and the transmembrane segment is a human TGFBR1 transmembrane segment, a human TGFBR2 transmembrane segment, a human KIR3DS1 transmembrane segment or a rodent KIR3DS1 transmembrane segment.

[0010] Furthermore, the nucleic acid sequence and polypeptide sequence of the human KIR3DS1 intracellular fragment are shown as SEQ ID NO.11 and SEQ ID NO.12 in the sequence listing, respectively, and the nucleic acid sequence and polypeptide sequence of the rodent KIR3DS1 intracellular fragment are shown as SEQ ID NO.15 and SEQ ID NO.16 in the sequence listing, respectively.

[0011] Furthermore, the nucleic acid sequence and polypeptide sequence of the human TGFBR1 transmembrane fragment are shown as SEQ ID NO.3 and SEQ ID NO.4 in the sequence listing, respectively, the nucleic acid sequence and polypeptide sequence of the human TGFBR2 transmembrane fragment are shown as SEQ ID NO.7 and SEQ ID NO.8 in the sequence listing, respectively, the nucleic acid sequence and polypeptide sequence of the human KIR3DS1 transmembrane fragment are shown as SEQ ID NO.9 and SEQ ID NO.10 in the sequence listing, respectively, and the nucleic acid sequence and polypeptide sequence of the rodent KIR3DS1 transmembrane fragment are shown as SEQ ID NO.13 and SEQ ID NO.14 in the sequence listing, respectively.

[0012] The present invention also provides an expression vector, which is formed by connecting the above-mentioned reverse cytokine receptor targeting transforming growth factor TGF-β with a lentiviral vector.

[0013] Preferably, the lentiviral vector is a pCDH-EFS-Luc-T2A-EGFP vector.

[0014] The present invention also provides an engineered cell, which is formed by introducing the above expression vector into immune effector cells or stem cells through gene transfection technology and can express the above reverse cytokine receptor targeting transforming growth factor TGF-β on the cell membrane.

[0015] Preferably, the immune effector cells are T cells, NK cells or macrophages.

[0016] The present invention also provides a use of the above reverse cytokine receptor targeting transforming growth factor TGF-β, the above expression vector or the above engineered cell in the preparation of solid tumor drugs.

[0017]

[0018]

[0019]

[0020] Table 1 Sequence Listing

[0021] Beneficial effects of the present invention:

[0022] The transforming growth factor β (TGF-β) signaling pathway plays a crucial role in the occurrence and progression of tumors, especially showing a significant effect in inhibiting the host immune system's attack on tumor cells. The present invention combines the extracellular segment of the TGF-β receptor (TGFBR1 or TGFBR2) with the intracellular segment of KIR3DS1 to construct a reverse cytokine receptor (TGFBR / KIR3DS1 ICR), and then uses the chimeric receptor to modify immune cells or stem cells through gene transduction technology, so that they express the artificially synthesized receptor on the cell membrane (i.e., obtain engineered cells expressing the reverse cytokine receptor), thereby enhancing their function in an immunosuppressive environment.

[0023] The reverse cytokine receptor targeting TGF-β can significantly enhance the anti-tumor function of immune cells through genetic modification. Specifically, the modified cells can convert TGF-β, an immunosuppressive signal source, into an activation signal (this process is achieved by fusing the extracellular domain of the TGF-β receptor with the intracellular domain of the activating cytokine receptor KIR3DS1); that is, after sensing TGF-β, the modified immune cells are no longer inhibited but activated, releasing more cytotoxic molecules (such as granzymes and perforins) and cytokines such as IFN-γ and TNF-α, thereby enhancing their ability to kill tumor cells; in addition, the modified cells can maintain a high proliferation rate in the tumor microenvironment while avoiding functional exhaustion caused by continuous inhibitory signals; this proliferation ability is particularly important in the complex immunosuppressive environment of solid tumors and can significantly improve the sustainability and effectiveness of treatment.

[0024] The features and advantages of the present invention will be described in detail through embodiments in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0025] Figure 1 is a schematic diagram of the structure of pUC57-TGFRB2 / KIR3DS1;

[0026] Figure 2 is the restriction electrophoresis diagram of pUC57-TGFRB2 / KIR3DS1 and pCDH-SFFV-Luc-T2A-EGFP vectors;

[0027] Figure 3 is the vector map of pCDH-EFS-Luc-T2A-EGFP;

[0028] Figure 4 is the vector map of pCDH-TGFRB2 / KIR3DS1;

[0029] Figure 5 This is the result of flow cytometry detection of the proportion of HEK293T cells infected with different gradient dilutions of lentivirus;

[0030] Figure 6 This is a flow cytometric analysis of peripheral blood mononuclear cells after purification and expansion of NK cells in vitro;

[0031] Figure 7 This is a flow cytometric analysis of the transfection efficiency of NK cells after TGFRB2 / KIR3DS1 ICR lentivirus transfection;

[0032] Figure 8 This is a comparison of the killing ability of Mock NK cells and TGFRB2 / KIR3DS1 ICR-NK cells against K562 cells in a medium containing TGF-β50ng / ml;

[0033] Fig. 9 This is a comparison of the secretion levels of IFN-γ (left) and TNF-α (right) after co-culture of MockNK cells and TGFRB2 / KIR3DS1 ICR-NK cells with K562 cells in a medium containing 50 ng / ml of TGF-β;

[0034] Fig.10 This is a flow cytometric analysis of the transfection efficiency of NK cells transfected with TGFRB2 / KIR3DS1 ICR and anti-Her2 CAR lentivirus;

[0035] Fig.11 This is a comparison of the killing abilities of control NK cells, anti-Her-2 CAR-NK cells, and TGFRB2 / KIR3DS1-ICR / CAR-NK cells against gastric cancer cells N87 and AGS after co-culture in a medium containing 50 ng / ml of TGF-β;

[0036] Fig.12 This is a comparison of the secretion levels of IFN-γ (left) and TNF-α (right) after co-culture of control NK cells, anti-Her-2 CAR-NK cells and TGFRB2 / KIR3DS1-ICR / CAR-NK cells with gastric cancer cells N87 and AGS in culture medium containing TGF-β 50 ng / ml. [Specific implementation method]

[0037] Embodiment 1:

[0038] 1. Design and acquisition of TGFRB2 / KIR3DS1 ICR:

[0039] The extracellular sequence of human TGFBR2, the transmembrane sequence of human KIR3DS1, and the intracellular sequence of human KIR3DS1 were sequentially connected to form a structure such as Figure 1The expression sequence (SEQ) of the reverse cytokine receptor TGFRB2 / KIR3DS1 ICR shown in the figure; subsequently, the sequence was synthesized and cloned into the pUC57 vector for storage, named pUC57-TGFRB2 / KIR3DS1; in addition, the nucleic acid sequence and polypeptide sequence of pUC57-TGFRB2 / KIR3DS1 are shown in SEQ ID NO.17 and SEQ ID NO.18 in the sequence table, respectively.

[0040] 2. Construction of expression vector:

[0041] First, DNA was extracted from the pUC57-TGFRB2 / KIR3DS1 plasmid and digested with QuickCut restriction endonucleases BamHI and XbaI (provided by Takara) (the specific digestion reaction system was to first take 1 μg of pUC57-TGFRB2 / KIR3DS1 plasmid, add 1 μL of BamHI and XbaI enzymes and 2 μL of 10X QuickCut Green Buffer, and then add water to a total of 20 μL and react in a water bath at 37°C for 15 minutes); after the digestion was completed, the products were separated by agarose gel electrophoresis, and then the DNA fragments were recovered using an agarose gel DNA fragment recovery kit (also purchased from Takara) (the results are shown in Figure 2 , the left side of the figure shows the band of TGFRB2 / KIR3DS1 ICR released after the pUC57-TGFRB2 / KIR3DS1 vector was digested with BamHI and XbaI).

[0042] Next, the same method was used to transform the pCDH-EFS-Luc-T2A-EGFP vector (see Figure 3 ) were digested with BamHI and XbaI, and the vector fragments after digestion were separated and recovered by agarose gel electrophoresis (results in Figure 2 , the right side of the figure shows a part of the pCDH-SFFV-Luc-T2A-EGFP vector after restriction digestion).

[0043] Subsequently, T4 ligase (provided by Takara) was used to connect the recovered TGFRB2 / KIR3DS1 ICR fragment with the pCDH-EFS-Luc-T2A-EGFP vector after restriction digestion (the specific operation steps of the ligation reaction are first quantifying the TGFRB2 / KIR3DS1 ICR fragment and the pCDH-EFS-Luc-T2A-EGFP vector digested with BamHI and XbaI, and then adding the two to the reaction system at a molar ratio of 1:3; the reaction system also includes 1 μL of 10X T4 DNA Ligase Buffer and 1 μL of ligase, and finally it is filled with water to 10 μL; the ligation reaction is carried out at 16°C for 4 hours or overnight); the ligation product is then transformed into E. coli DH5α competent cells and cultured overnight at 37°C; after selecting a single colony for expansion culture, the plasmid of the positive clone was extracted using a plasmid extraction kit (provided by Axygene) according to the instructions; after verification by restriction digestion and sequencing, the correct vector was named pCDH-TGFRB2 / KIR3DS1 (see Figure 4 ).

[0044] Lentiviral packaging and titer determination:

[0045] HEK293T cells with a confluence of 90% were passaged into 10 cm cell culture dishes, and DMEM high-glucose medium containing 10% fetal bovine serum was added, and cultured at 37°C and 5% CO2 for 24 hours; when the cell confluence reached 30-40%, fresh serum-containing medium was replaced and cultured for another 2 hours; then, HEK293T cells were co-transfected with three packaging plasmids (pMD2.G: 3.24 μg, pMDL-G / P-RRE: 4.5 μg, pRSV-REV: 2.25 μg) by calcium phosphate precipitation method. μg) and pCDH-TGFRB2 / KIR3DS1 plasmid (11.52 μg), cultured for 16 hours after transfection and replaced with fresh culture medium; 24 hours later, the expression of GFP in HEK293T cells was observed using a fluorescence microscope, and the supernatant was collected 48 hours later and filtered with a 0.45 μm filter to remove cell debris; the filtered supernatant was centrifuged at 20,000 rpm and 4°C in an ultracentrifuge for 3 hours, the precipitated virus particles were collected, resuspended with serum-free 1640 culture medium, and stored in a -70°C refrigerator for later use.

[0046] Next, HEK293T cells were seeded in a 96-well plate at a density of 5×10^3 cells / well, and 100 μL of DMEM high-glucose medium containing 10% fetal bovine serum was added to each well, and cultured overnight at 37°C and 5% CO2. Then, 40 μL of pCDH-TGFRB2 / KIR3DS1 lentiviral stock solution was taken, 360 μL of DMEM high-glucose medium containing a final concentration of 8 μg / mL polybrene was added, and 10-fold gradient dilution was performed. The HEK293T cells seeded in the 96-well plate were carefully removed. 90 μL of culture supernatant of 293T cells was added, and then 90 μL of the above-mentioned gradient diluted virus particle solution was added to each well, and each dilution concentration was repeated for 3 wells; after continuing to culture for 24 hours, the culture medium of each well was completely removed, and then replaced with fresh virus particle-free culture medium, and cultured for another 24 hours; the proportion of fluorescent cells in each dose group was calculated by flow cytometry, and the original average virus titer was calculated based on this; the results showed that the titer of the pCDH-TGFRB2 / KIR3DS1 lentiviral stock was 1.48E8 TU / ml (see Figure 5 ).

[0047] TGFRB2 / KIR3DS1 ICR-NK cell preparation:

[0048] 20 ml of fresh anticoagulated blood samples from healthy volunteers were used to separate peripheral blood mononuclear cells (PBMCs) using lymphocyte separation fluid purchased from GE; after counting the cells, the cells were seeded in a 6-well plate coated with CD16 at a density of 2.5×10^6 cells / well for stimulation culture for 72 hours; then, the cells were transferred to a common 6-well plate and continued to be cultured and expanded for 72 hours; then, the cells were sorted and purified using NK magnetic beads purchased from Miltenyi Biotec, and induced culture was performed by adding 1640 culture medium (provided by Thermo Scientific) containing 10% FBS and 200 IU / ml IL-2 to obtain highly active NK cells; the CD3 and CD56 phenotype ratios of the cells were detected by flow cytometry; the test results are shown in FIG. Figure 6 As shown, the horizontal axis represents CD3 and the vertical axis represents CD56; in the results, the cell population with CD3 negative and CD56 positive represents NK cells, indicating that the proportion of NK cells prepared by this method exceeds 90%.

[0049] The in vitro expanded NK cells were seeded into a 24-well plate (BD Biosciences) at a density of approximately 2.5×10^6 cells per well; an appropriate amount of TGFRB2 / KIR3DS1 ICR viral supernatant was mixed with the cells in the presence of 8 μg / ml Protamine sulfate (Sigma-Aldrich) and 1.5 μM BX795 (Sigma-Aldrich) at a final concentration of 1 ml; subsequently, cytokines were supplemented and centrifuged at 1000 g for 1 hour at room temperature; after centrifugation, the viral supernatant was not removed and the culture plate was incubated at 37°C and 5% CO2 for 4 to 6 hours; after the incubation, a second centrifugation was performed at 1000 g for 1 hour at room temperature, and then the supernatant in the well was replaced with 1 ml of fresh NK cell growth medium; over the next 2 days, cytokines were added to the culture medium every day to obtain NK cells expressing TGFRB2 / KIR3DS1 ICR (hereinafter referred to as TGFRB2 / KIR3DS1 ICR-NK).

[0050] On this basis, the transfection efficiency was further identified; in order to detect the transfected NK cells, fluorescently labeled anti-TGFBR2 monoclonal antibody was used for 15 minutes of incubation (4°C), washed and resuspended, and finally flow cytometry was performed; the test results are as follows Figure 7 As shown, the abscissa represents the expression of TGFRB2 / KIR3DS1 ICR.

[0051] Detection of the killing ability of TGFRB2 / KIR3DS1 ICR-NK cells on tumor cells in an inflammatory environment:

[0052] The concentration of K562 cells was adjusted to 1×10^6 / ml, and Calcein-AM with a final concentration of 5μg / ml was added for labeling, followed by incubation at 37°C for 1 hour; then, the cells were washed three times with PBS liquid, and then resuspended in phenol red-free 1640 complete medium containing 50ng / ml TGF-β for counting; the tumor cell density was adjusted to 10,000 / well and inoculated into 96-well round-bottom plates; according to different effector cell to target cell ratios (E:T), empty vector-transfected control NK cells and TGFRB2 / KIR3DS1 ICR-NK cells, and the number of cells in each group was set to 1×10^5, 5×10^4, 2.5×10^4, 1.25×10^4, 0.625×10^4 and 0.03125×10^4, and the ratios were 10:1, 5:1, 2.5:1, 1.25:1, 0.625:1 and 0.03125:1; in addition, a 2% Triton X-100 treatment group was set as a positive control, and an untreated control group was set; after co-culture for 3 hours, centrifugation was performed at 100g for 5 minutes and then at 300g for 5 minutes; 100 μL of culture medium was extracted from each well and entered into a 96 flat-bottom plate for OD value detection; the test results are shown in Figure 8 As shown, the killing ability of TGFRB2 / KIR3DS1 ICR-NK cells on K562 cells in an environment containing TGF-β was significantly better than that of control NK cells.

[0053] Detection of cytokine secretion ability of TGFRB2 / KIR3DS1 ICR-NK cells in inflammatory environment:

[0054] K562 cells were co-cultured with Mock NK cells transfected with an empty vector and TGFRB2 / KIR3DS1ICR-NK cells at a ratio of 2.5:1 in a medium containing 50 ng / ml TGF-β for 12 hours; then, the supernatant was collected for ELISA to determine the concentrations of IFN-γ and TNF-α therein; the test results are shown in Fig. 9 As shown, the secretion levels of IFN-γ and TNF-α of TGFRB2 / KIR3DS1 ICR-NK cells after co-culture with K562 cells were significantly higher than those of the control group.

[0055] Embodiment 2:

[0056] Steps 1 to 3 are the same as those in Example 1, and the rest are as follows:

[0057] 4. Preparation of TGFRB2 / KIR3DS1-ICR / CAR-NK cells:

[0058] The in vitro expanded NK cells were seeded into 24-well plates (BD Biosciences) at a density of approximately 2.5×10^6 cells per well. During this process, Protamine sulfate (Sigma-Aldrich) and 1.5μM BX795 (Sigma-Aldrich) were added at a final concentration of 8μg / ml and mixed with an appropriate amount of supernatant of TGFRB2 / KIR3DS1-ICR virus and anti-Her2 CAR virus, and the final volume was controlled within 1ml. Subsequently, cytokines were added and centrifuged at 1000g for 1 hour at room temperature. After centrifugation, the viral supernatant was not removed and the culture plate was incubated at 37°C and 5% CO2 for 4 to 6 hours. After the incubation, the plate was centrifuged again at 1000g for 1 hour at room temperature, and then the supernatant in the culture well was replaced with 1ml of fresh NK cell growth medium. In the next 2 days, cytokines were added every day to obtain cells expressing TGFRB2 / KIR3DS1 ICR and anti-Her-2 CAR-NK cells (hereinafter referred to as TGFRB2 / KIR3DS1-ICR / CAR-NK).

[0059] Afterwards, the transfection efficiency of the transfected NK cells was evaluated; the anti-TGFBR2 monoclonal antibody labeled with APC fluorescence and the recombinant human Her-2-Fc protein labeled with PE fluorescence were incubated at 4°C for 15 minutes, washed and resuspended, and then flow cytometry was performed; the test results were as follows Fig.10 As shown, the ordinate represents the expression level of TGFRB2 / KIR3DS1 ICR, and the abscissa represents the expression level of anti-Her2 CAR.

[0060] 5. Detection of the killing ability of TGFRB2 / KIR3DS1-ICR / CAR-NK cells on tumor cells in an inflammatory environment:

[0061] The concentration of Her-2 positive gastric cancer cells N87 and AGS was adjusted to 1×10^6 / ml, and Calcein-AM was added at a final concentration of 5μg / ml for labeling, and incubated at 37°C for 1 hour. After labeling, the cells were washed three times with PBS and resuspended in 50ng / ml The cells were counted in phenol red-free 1640 complete medium containing TGF-β; the number of tumor cells was adjusted to 10,000 / well and added to a 96-well round-bottom plate; according to the ratio of effector cells to target cells (E:T), empty vector-transfected control NK cells, anti-Her-2CAR-NK cells and TGFRB2 / KIR3DS1-ICR / CAR-NK cells were added at a ratio of 10:1, 5:1, 2.5:1, 1.25:1, 0.625:1 and 0.03125:1, respectively, and the cell numbers were 1×10^5, 5×10^4, 2.5×10^4, 1.25×10^4, 0.625×10^4 and 0.03125×10^4; in addition, a 2% Triton X-100 treated group and untreated group; co-cultured at 37°C for 3 hours, then centrifuged at 100g for 5 minutes, and then centrifuged at 300g for 5 minutes; 100 μL of culture medium was extracted from each well and transferred to a 96-well flat-bottom plate for OD value detection; the test results are shown in Fig.11 As shown, the killing efficacy of TGFRB2 / KIR3DS1-ICR / CAR-NK cells on gastric cancer cells N87 and AGS in an environment containing TGF-β was significantly higher than that of control NK cells and anti-Her-2 CAR-NK cells.

[0062] 6. Detection of cytokine secretion ability of TGFRB2 / KIR3DS1-ICR / CAR-NK cells in inflammatory environment:

[0063] Her-2 positive gastric cancer cells N87 and AGS cells were co-cultured with empty vector-transfected mock NK cells, anti-Her-2 CAR-NK cells, and TGFRB2 / KIR3DS1-ICR / CAR-NK cells in a medium containing 50 ng / ml TGF-β for 12 hours at an effector cell to target cell ratio of 2.5:1; then, the supernatant was collected and subjected to ELISA to measure the concentrations of IFN-γ and TNF-α therein; the test results are shown in FIG. Fig.12 As shown, the secretion levels of IFN-γ and TNF-α of TGFRB2 / KIR3DS1-ICR / CAR-NK cells were significantly higher than those of the control group after co-culture with gastric cancer cells N87 and AGS.

[0064] The above embodiments are intended to illustrate the present invention, not to limit the present invention. Any solution that is a simple transformation of the present invention belongs to the protection scope of the present invention.

Claims

1. Targeting the reverse cytokine receptor of transforming growth factor TGF-β, characterized in that: It includes an extracellular segment, a transmembrane segment and an intracellular segment connected in sequence, wherein the extracellular segment is an extracellular segment of human TGFBR1 or an extracellular segment of human TGFBR2, the intracellular segment is an intracellular segment of KIR3DS1 from humans or other species, and the transmembrane segment is a transmembrane segment of a transmembrane protein from humans or other species.

2. The reverse cytokine receptor targeting transforming growth factor TGF-β according to claim 1, characterized in that: The nucleic acid sequence and polypeptide sequence of the human TGFBR1 extracellular fragment are shown in SEQ ID NO.1 and SEQ ID NO.2 in the sequence list, respectively. The nucleic acid sequence and polypeptide sequence of the human TGFBR2 extracellular fragment are shown in SEQ ID NO.5 and SEQ ID NO.6 in the sequence list, respectively.

3. The reverse cytokine receptor targeting transforming growth factor TGF-β according to claim 1, characterized in that: The intracellular segment is a human KIR3DS1 intracellular segment or a rodent KIR3DS1 intracellular segment, and the transmembrane segment is a human TGFBR1 transmembrane segment, a human TGFBR2 transmembrane segment, a human KIR3DS1 transmembrane segment or a rodent KIR3DS1 transmembrane segment.

4. The reverse cytokine receptor targeting transforming growth factor TGF-β according to claim 3, characterized in that: The nucleic acid sequence and polypeptide sequence of the human KIR3DS1 intracellular fragment are shown in SEQ ID NO.11 and SEQ ID NO.12 in the sequence listing, respectively, and the nucleic acid sequence and polypeptide sequence of the rodent KIR3DS1 intracellular fragment are shown in SEQ ID NO.15 and SEQ ID NO.16 in the sequence listing, respectively.

5. The reverse cytokine receptor targeting transforming growth factor TGF-β according to claim 3, characterized in that: The nucleic acid sequence and polypeptide sequence of the human TGFBR1 transmembrane fragment are shown in SEQ ID NO.3 and SEQ ID NO.4 in the sequence listing, respectively; the nucleic acid sequence and polypeptide sequence of the human TGFBR2 transmembrane fragment are shown in SEQ ID NO.7 and SEQ ID NO.8 in the sequence listing, respectively; the nucleic acid sequence and polypeptide sequence of the human KIR3DS1 transmembrane fragment are shown in SEQ ID NO.9 and SEQ ID NO.10 in the sequence listing, respectively; the nucleic acid sequence and polypeptide sequence of the rodent KIR3DS1 transmembrane fragment are shown in SEQ ID NO.13 and SEQ ID NO.14 in the sequence listing, respectively.

6. An expression vector, characterized in that: The method is formed by connecting the reverse cytokine receptor targeting transforming growth factor TGF-β according to any one of claims 1 to 5 to a lentiviral vector.

7. The expression vector according to claim 6, characterized in that: The lentiviral vector is a pCDH-EFS-Luc-T2A-EGFP vector.

8. An engineered cell, characterized in that: The expression vector described in claim 6 is introduced into immune effector cells or stem cells through gene transfection technology and can express the reverse cytokine receptor targeting transforming growth factor TGF-β described in any one of claims 1 to 5 on the cell membrane.

9. The engineered cell according to claim 8, characterized in that: The immune effector cells are T cells, NK cells or macrophages.

10. Use of the reverse cytokine receptor targeting transforming growth factor TGF-β according to any one of claims 1 to 5, the expression vector according to claim 6 or the engineered cell according to claim 8 in the preparation of solid tumor drugs.

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