Construction and application of chimeric cytokine receptor based on transforming growth factor TGF-beta

Abstract

The invention discloses construction and application of a chimeric cytokine receptor based on a transforming growth factor TGF-beta, the chimeric cytokine receptor 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, the intracellular fragment is an NKP46 intracellular fragment from human or other species, and the transmembrane fragment is a human TGFBR1 extracellular fragment or a human TGFBR2 extracellular fragment. The transmembrane segment is a transmembrane segment of transmembrane protein from human or other species, and the constructed chimeric cytokine receptor can be used for modifying immune cells such as T cells and NK cells, so that the effect of reversing a traditional inhibitory pathway on the immune cells is realized, and the inhibitory influence of TGF-beta on the immune cells is converted into activation.

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 a chimeric cytokine receptor based on transforming growth factor TGF-β. [Background technology]

[0002] In recent years, with the development of tumor immunotherapy, how to effectively overcome the complex tumor microenvironment of solid tumors has become a research focus; there are a variety of immunosuppressive factors in the tumor microenvironment, including immunosuppressive cells (such as regulatory T cells, myeloid-derived suppressor cells and tumor-associated macrophages), inhibitory factors (such as PGE2, TGF-β, IL-4 and IL-10) and immune checkpoint molecules (such as PD-1 / PD-L1 and CTLA-4); in addition, factors such as hypoxia and lack of metabolic resources further weaken the function of effector immune cells, resulting in the traditional cell immunotherapy in the treatment of solid tumors is far less effective than hematological tumors.

[0003] Chimeric cytokine receptor technology provides new possibilities for immunotherapy; the basic design concept of chimeric cytokine receptors is to utilize the high expression characteristics of specific immunosuppressive factors in the tumor microenvironment, combine the extracellular domain of the inhibitory factor receptor with the intracellular signal transduction domain of the activating receptor, and thus convert the inhibitory signal into an activating signal, 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 achimeric cytokine receptor.Mol Ther.2014Jun;22(6):1211-1220.doi:10.1038 / mt.2014.47.Epub 2014Mar20.PMID:24732709;PMCID:PMC4048899) The extracellular part of IL-4 receptor is combined with the intracellular part of IL-7 or IL-21 to form IL-4 / 7ICR or IL-4 / 21ICR, which significantly enhances the killing ability of CAR-T cells in the tumor microenvironment with high IL-4 expression after being transferred into CAR-T cells.

[0004] The transforming growth factor β (TGF-β) signaling pathway is an extremely important intracellular signal transduction pathway in the occurrence and development of tumors. 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 also can significantly inhibit the activity of CD8+T cells, NK cells, etc. through high expression in the tumor microenvironment, 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. In other words, how to use immune cells such as NK cells to reverse the inhibitory effect of TGF-β still needs to be solved. [Summary of the invention]

[0005] The purpose of the present invention is to solve the problems in the prior art and propose the construction and application of a chimeric cytokine receptor based on transforming growth factor TGF-β. The constructed chimeric cytokine receptor can be used to modify immune cells such as T cells and NK cells, thereby reversing the effects of traditional inhibitory pathways on immune cells and converting the inhibitory effect of TGF-β on immune cells into an activating effect.

[0006] To achieve the above objectives, the present invention proposes a chimeric cytokine receptor based on 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 NKP46 from humans or other species, and the transmembrane segment is a transmembrane segment of a transmembrane protein from humans or other species.

[0007] 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.

[0008] Preferably, the intracellular segment is a human NKP46 intracellular segment or a rodent NKP46 intracellular segment, and the transmembrane segment is a human TGFBR1 transmembrane segment, a human TGFBR2 transmembrane segment, a human NKP46 transmembrane segment or a rodent NKP46 transmembrane segment.

[0009] Furthermore, the nucleic acid sequence and polypeptide sequence of the human NKP46 intracellular fragment are shown in SEQ ID NO.11 and SEQ ID NO.12 in the sequence list, respectively, and the nucleic acid sequence and polypeptide sequence of the rodent NKP46 intracellular fragment are shown in SEQ ID NO.15 and SEQ ID NO.16 in the sequence list, respectively.

[0010] 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 NKP46 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 NKP46 transmembrane fragment are shown as SEQ ID NO.13 and SEQ ID NO.14 in the sequence listing, respectively.

[0011] The present invention also provides an expression vector, which is formed by connecting the above-mentioned chimeric cytokine receptor based on transforming growth factor TGF-β and a lentiviral vector.

[0012] Preferably, the lentiviral vector is a pCDH-EFS-Luc-T2A-EGFP vector.

[0013] 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 chimeric cytokine receptor based on transforming growth factor TGF-β on the cell membrane.

[0014] Preferably, the immune effector cells are T cells, NK cells or macrophages.

[0015] The present invention also provides a use of the above-mentioned chimeric cytokine receptor based on transforming growth factor TGF-β, the above-mentioned expression vector or the above-mentioned engineered cell in the preparation of solid tumor drugs.

[0016]

[0017]

[0018]

[0019] Table 1 Sequence Listing Beneficial effects of the present invention:

[0020] The present invention forms a new chimeric cytokine receptor (TGFBR / NKP46 CCR) by recombining the extracellular segment of the TGF-β receptor (TGFBR1 or TGFBR2) and the intracellular segment of the NK cell receptor NKP46. The extracellular segment of the TGF-β receptor can be used to bind to the TGF-β in the microenvironment to receive the signal, and the intracellular segment of NKP46 can be used to transduce the upstream signal to generate an activation signal in the cell. The present invention also introduces the chimeric cytokine receptor into immune effector cells (T cells, NK cells or macrophages, etc.) or stem cells (autologous, allogeneic, stem cell differentiation or specific cell lines) through gene transfection technology by constructing an expression vector, thereby using the chimeric cytokine receptor to reverse the inhibitory effect of TGF-β in the microenvironment on immune cells such as T cells and NK cells, so that the cells expressing the chimeric cytokine receptor still have high activity and function in an immunosuppressive environment rich in TGF-β, such as the ability to kill tumor cells, the ability to proliferate or differentiate, and the cytokine secretion function.

[0021] 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

[0022] Figure 1 is a schematic diagram of the structure of TGFRB2 / NKp46 CCR;

[0023] Figure 2 It is the restriction electrophoresis diagram of pUC57-TGFRB2 / NKP46 and pCDH-SFFV-Luc-T2A-EGFP vector;

[0024] Figure 3 is the vector map of pCDH-EFS-Luc-T2A-EGFP;

[0025] Figure 4 is the vector map of pCDH-TGFRB2 / NKP46;

[0026] Figure 5 This is the result of flow cytometry detection of the proportion of HEK293T cells infected with different gradient dilutions of lentivirus;

[0027] Figure 6 This is a flow cytometric analysis of peripheral blood mononuclear cells after purification and expansion of NK cells in vitro;

[0028] Figure 7 This is a flow cytometric analysis of the transfection efficiency of NK cells after TGFRB2 / NKP46 CCR lentivirus transfection;

[0029] Figure 8This is a comparison of the killing ability of Mock NK cells and TGFRB2 / NKP46 CCR-NK cells on K562 cells in a medium containing TGF-β50ng / ml;

[0030] Fig. 9 This is a comparison of the secretion levels of IFN-γ (left) and TNF-α (right) after co-culture of Mock NK cells and TGFRB2 / NKP46 CCR-NK cells with K562 cells in a medium containing 50 ng / ml of TGF-β;

[0031] Fig.10 This is a flow cytometric analysis of the transfection efficiency of NK cells transfected with TGFRB2 / NKP46 CCR and anti-Her2 CAR lentivirus;

[0032] Fig.11 It is a comparison of the killing ability of control NK cells, anti-Her-2 CAR-NK cells and TGFRB2 / NKP46-CCR / CAR-NK cells on gastric cancer cells N87 and AGS after co-culture in a medium containing TGF-β50ng / ml;

[0033] 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 / NKP46-CCR / CAR-NK cells with gastric cancer cells N87 and AGS in a culture medium containing TGF-β 50 ng / ml. [Specific implementation method]

[0034] Embodiment 1:

[0035] 1. Design and acquisition of TGFRB2 / NKp46 CCR:

[0036] The extracellular sequence of human TGFRB2, the transmembrane sequence of human NKP46, and the intracellular sequence of human NKP46 are sequentially combined and connected to form a structure such as Figure 1 The expression sequence (SEQ) of the chimeric cytokine receptor TGFRB2 / NKP46 CCR shown; subsequently, the sequence was synthesized and cloned into the pUC57 vector for preservation, and was named pUC57-TGFRB2 / NKP46; in addition, the nucleic acid sequence and polypeptide sequence of pUC57-TGFRB2 / NKP46 are shown in SEQ ID NO.17 and SEQ ID NO.18 in the sequence table, respectively.

[0037] 2. Construction of lentiviral vector:

[0038] First, the pUC57-TGFRB2 / NKP46 plasmid was extracted and digested with QuickCut restriction endonucleases BamHI and XbaI (Takara) (the specific digestion reaction system was to take 1 μg of pUC57-TGFRB2 / NKP46, 1 μl of BamHI and XbaI, 2 μl of 10X QuickCut Green Buffer, add water to 20 μl, and incubate in a 37°C water bath for 15 min); the digested products were separated by agarose gel electrophoresis, and DNA fragments were recovered using an agarose gel DNA fragment recovery kit (purchased from Takara) (the results are shown in Figure 2 From the electrophoresis diagram, it can be seen that the left figure shows the band of TGFRB2 / NKP46 CCR released after the pUC57-TGFRB2 / NKP46 vector was digested with BamHI and XbaI).

[0039] The same method was used to digest the pCDH-EFS-Luc-T2A-EGFP vector (see Figure 3 ), and the vector fragments after enzyme digestion were separated and recovered by agarose gel electrophoresis (results see Figure 2 From the electrophoresis diagram, it can be seen that the right side of the figure is the pCDH-SFFV-Luc-T2A-EGFP vector part after enzyme digestion).

[0040] The recovered TGFRB2 / NKP46 CCR fragment was connected to the vector after restriction digestion by T4 ligase (purchased from Takara) (the specific reaction system and conditions were as follows: first, the TGFRB2 / NKP46 CCR and pCDH-EFS-Luc-T2A-EGFP vector digested by BamHI and XbaI were quantified, and then added into the system at a molar ratio of 1:3 according to the vector and the target band; the reaction system also included 1 μl of 10X T4 DNA Ligase Buffer and 1 μl of ligase, which were made up to 10 μl with water, and connected at 16°C for 4 h or overnight); the ligation product was transformed into E. coli DH5a competent bacteria, cultured at 37°C overnight, and a single colony was picked. After amplification, the plasmid of the positive clone was extracted using a plasmid extraction kit (purchased from Axygene) according to the operating instructions of the kit. After restriction digestion and sequencing detection, the correct vector was named pCDH-TGFRB2 / NKP46 (see Figure 4 ).

[0041] 3. Lentivirus packaging and titer determination:

[0042] HEK293T cells with a confluence of 90% were passaged in 10 mm 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 h. When the cell confluence reached 30-40%, fresh serum-containing medium was replaced, and cultured for 2 h. Three packaging plasmids pMD2.G (3.24 μg), pMDL-G / P-RRE (4.5 μg), pRSV-REV (2.25 μg) and pCDH-TGFRB2 / NKP46 plasmid (11.52 μg) was co-transfected into HEK293T cells by calcium phosphate precipitation method, and fresh culture medium was replaced after 16 h of culture; GFP expression of HEK293T cells was observed under a fluorescence microscope after 24 h, and supernatant was collected after 48 h and filtered with a 0.45 μm filter to remove cell debris; the filtered supernatant was centrifuged in an ultrahigh-speed centrifuge at 20,000 r / min and 4°C for 3 h, the virus particle precipitate was collected, resuspended in serum-free 1640 culture medium, and stored in a -70°C refrigerator for later use.

[0043] HEK293T cells were cultured at 5 × 10 3 The cells were inoculated at a density of cells / well in a 96-well plate, each well containing 100 μL of DMEM high-glucose medium with 10% fetal bovine serum, and cultured overnight at 37°C and 5% CO2; 40 μL of pCDH-TGFRB2 / NKP46 lentiviral stock solution was taken separately, and 360 μL of DMEM high-glucose medium (containing 10% fetal bovine serum) with a final concentration of 8 μg / mL polybrene was added; and a 10-fold gradient dilution was performed; 90 μL of the culture supernatant was carefully aspirated from the 96-well plate inoculated with HEK293T cells, and then 90 μL of the above gradient diluted virus particle solution was added to each well, and each dilution was repeated for 3 wells; the culture was continued for 24 hours, the culture medium of each well was cleaned, and fresh culture medium without any virus particles was replaced, and the culture was continued for 24 hours. The proportion of fluorescent cells in each dose group was calculated by flow cytometry, and the original average virus titer was calculated. The results are shown in Figure 5 As shown, the titer of the pCDH-TGFRB2 / NKP46 lentiviral stock solution was calculated to be 7.31E7TU / ml.

[0044] Preparation of TGFRB2 / NKP46 CCR-NK cells:

[0045] 20 ml of fresh anticoagulated blood from healthy volunteers was collected and peripheral blood mononuclear cells (PBMCs) were separated using lymphocyte separation fluid (purchased from GE). After the separated cells were counted, 2.5×10 6 / well density in a 6-well plate coated with CD16 for 72 hours, and then changed to an ordinary 6-well plate for further culture and expansion for 72 hours; the cells were purified by NK magnetic bead sorting (purchased from Miltenyi Biotec), and 1640 (purchased from Thermo Scientific) culture medium containing 10% FBS + 200IU / ml IL-2 was added to continue induction culture to obtain highly active NK cells, and the CD3 and CD56 phenotype ratios were detected by flow cytometry; the test results are shown in Figure 6 , wherein the horizontal axis represents CD3, and the vertical axis represents CD56. The cell population represented by CD3 negative and CD56 positive is NK cells, indicating that the proportion of NK cells prepared by this method is greater than 70%.

[0046] The in vitro expanded NK cells were plated at a volume of approximately 2.5 × 10 6 NK cells were seeded into 24-well plates (BD Biosciences) and mixed with an appropriate amount of TGFRB2 / NKP46 CCR virus supernatant in the presence of a final concentration of Protamine sulfate 8ug / ml (Sigma-Aldrich) and BX7951.5uM (Sigma-Aldrich), with the final volume not exceeding 1ml; cytokines were supplemented, and the plate was centrifuged at 1000·g for 1h at room temperature; after centrifugation, the virus supernatant was not removed, and the plate was incubated at 37°C, 5% CO2 for 4-6h; after the incubation, a second centrifugation was performed at 1000·g for 1h at room temperature, and then 1ml of fresh NK cell growth medium was added from the wells; the cells were maintained in a medium supplemented with cytokines every day for 2 days to obtain NK cells expressing TGFRB2 / NKP46 CCR (hereinafter referred to as TGFRB2 / NKP46 CCR-NK) and further identified the transfection efficiency; the transfected NK cells were incubated with fluorescently labeled anti-TGFBR2 monoclonal antibody for 15 min at 4°C, washed and resuspended, and further flow cytometry was performed; the results are shown in Figure 7 As shown, the abscissa represents the expression of TGFRB2 / NKP46 CCR.

[0047] Detection of the killing ability of TGFRB2 / NKP46 CCR-NK cells on tumor cells:

[0048] K562 cells were adjusted to 1 × 10 6 / ml, add Calcein-AM with a final concentration of 5ug / ml for labeling, and incubate at 37℃ for 1h; then wash three times with PBS, resuspend the cells in complete medium containing TGF-β50ng / ml without phenol red 1640, and count; adjust the tumor cells to 10000 / well and add them to 96-well round-bottom plate; add 1×10 MockNK cells transfected with empty vector and TGFRB2 / NKP46 CCR-NK cells at E:T of 10:1, 5:1, 2.5:1, 1.25:1, 0.625:1 and 0.03125:1, respectively 5 , 5×10 4 , 2.5×10 4 , 1.25×10 4 , 0.625×10 4 and 0.03125×10 4 Tumor cells were added with 2% Triton X-100 and untreated groups, centrifuged at 100g for 5 min, co-cultured at 37°C for 3 h, and centrifuged at 300g for 5 min. 100ul of each well was transferred to a 96-well plate to detect the OD value. The test results are shown in Figure 8 As shown, the killing ability of TGFRB2 / NKP46 CCR-NK cells on K562 cells in the environment containing TGF-β was significantly higher than that of control NK cells.

[0049] Detection of cytokine secretion ability of TGFRB2 / NKP46 CCR-NK cells:

[0050] K562 cells were co-cultured with empty vector-transfected Mock NK cells and TGFRB2 / NKP46 CCR-NK cells in a medium containing 50 ng / ml TGF-β for 12 h at an E:T ratio of 2.5:1. The supernatant was then collected and the concentrations of IFN-γ and TNF-α in the supernatant were detected by ELISA. The test results are shown in Fig. 9 As shown, the secretion levels of IFN-γ and TNF-α of TGFRB2 / NKP46 CCR-NK cells co-cultured with K562 were significantly higher than those of the control group.

[0051] Embodiment 2:

[0052] Steps 1 to 3 are the same as those in Example 1, and the rest are as follows:

[0053] TGFRB2 / NKP46-CCR / CAR-NK cell preparation:

[0054] The in vitro expanded NK cells were plated at a volume of approximately 2.5 × 10 6NK cells were seeded into 24-well plates (BD Biosciences) and mixed with appropriate amounts of TGFRB2 / NKP46-CCR virus and anti-Her2CAR virus supernatant in the presence of final concentrations of Protamine sulfate 8ug / ml (Sigma-Aldrich) and BX7951.5uM (Sigma-Aldrich), with the final volume not exceeding 1ml; cytokines were supplemented and the plates were centrifuged at 1000·g for 1h at room temperature; after centrifugation, the viral supernatant was not removed and the plates were incubated at 37°C and 5% CO2 for 4-6h; after the incubation, a second centrifugation was performed at 1000·g for 1h at room temperature, and then 1ml of fresh NK cell growth medium was added from the wells; the cells were maintained in a medium supplemented with cytokines every day for 2 days to obtain cells expressing TGFRB2 / NKP46 CCR and anti-Her-2 CAR-expressing NK cells (hereinafter referred to as TGFRB2 / NKP46-CCR / CAR-NK) were further identified for transfection efficiency. The transfected NK cells were incubated with APC fluorescently labeled anti-TGFBR2 monoclonal antibody and PE fluorescently labeled recombinant human Her-2-Fc protein for 15 min at 4°C, washed and resuspended, and further flow cytometry was performed. The results are shown in Fig.10 As shown, the ordinate represents the expression of TGFRB2 / NKP46 CCR, and the abscissa represents the expression of anti-Her2 CAR.

[0055] Detection of the killing ability of TGFRB2 / NKP46-CCR / CAR-NK cells on tumor cells:

[0056] The Her-2 positive gastric cancer cells N87 and AGS were adjusted to 1×10 6 / ml, add Calcein-AM with a final concentration of 5ug / ml for labeling, incubate at 37℃ for 1h, wash three times with PBS, resuspend the cells in complete medium containing TGF-β50ng / ml without phenol red 1640, count; adjust the tumor cells to 10000 / well and add them to 96-well round bottom plate; add 1×10 empty vector transfected Mock NK cells, anti-Her-2CAR-NK cells and TGFRB2 / NKP46-CCR / CAR-NK cells according to E:T of 10:1, 5:1, 2.5:1, 1.25:1, 0.625:1 and 0.03125:1 respectively 5 , 5×10 4 , 2.5×10 4 , 1.25×10 4 , 0.625×10 4 and 0.03125×10 4Tumor cells were added with 2% Triton X-100 and untreated groups, centrifuged at 100g for 5 min, co-cultured at 37°C for 3 h, and centrifuged at 300g for 5 min. 100ul of each well was transferred to a 96-well plate to detect the OD value. The test results are shown in Fig.11 As shown, the killing ability of TGFRB2 / NKP46-CCR / 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.

[0057] Detection of cytokine secretion ability of TGFRB2 / NKP46-CCR / CAR-NK cells:

[0058] Her-2 positive gastric cancer cells N87 and AGS cells were co-cultured with empty vector transfected MockNK cells, anti-Her-2 CAR-NK cells and TGFRB2 / NKP46-CCR / CAR-NK cells in a medium containing TGF-β 50 ng / ml at an E:T ratio of 2.5:1 for 12 h, and the supernatant was collected for ELISA to detect the concentrations of IFN-γ and TNF-α in the supernatant; the test results are shown in Fig.12 As shown, the secretion levels of IFN-γ and TNF-α after co-culture of TGFRB2 / NKP46-CCR / CAR-NK cells with gastric cancer cells N87 and AGS were significantly higher than those in the control group.

[0059] 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. A chimeric cytokine receptor based on 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 the extracellular segment of human TGFBR1 or the extracellular segment of human TGFBR2, the intracellular segment is the intracellular segment of NKP46 from humans or other species, and the transmembrane segment is the transmembrane segment of a transmembrane protein from humans or other species.

2. The chimeric cytokine receptor based on 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 chimeric cytokine receptor based on transforming growth factor TGF-β according to claim 1, characterized in that: The intracellular segment is a human NKP46 intracellular segment or a rodent NKP46 intracellular segment, and the transmembrane segment is a human TGFBR1 transmembrane segment, a human TGFBR2 transmembrane segment, a human NKP46 transmembrane segment or a rodent NKP46 transmembrane segment.

4. The chimeric cytokine receptor based on transforming growth factor TGF-β according to claim 3, characterized in that: The nucleic acid sequence and polypeptide sequence of the human NKP46 intracellular fragment are shown in SEQ ID NO.11 and SEQ ID NO.12 in the sequence list, respectively, and the nucleic acid sequence and polypeptide sequence of the rodent NKP46 intracellular fragment are shown in SEQ ID NO.15 and SEQ ID NO.16 in the sequence list, respectively.

5. The chimeric cytokine receptor based on 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 list, 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 list, respectively; the nucleic acid sequence and polypeptide sequence of the human NKP46 transmembrane fragment are shown in SEQ ID NO.9 and SEQ ID NO.10 in the sequence list, respectively; the nucleic acid sequence and polypeptide sequence of the rodent NKP46 transmembrane fragment are shown in SEQ ID NO.13 and SEQ ID NO.14 in the sequence list, respectively.

6. An expression vector, characterized in that: The invention is formed by connecting the chimeric cytokine receptor based on transforming growth factor TGF-β according to any one of claims 1 to 5 with 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 according to claim 6 is introduced into immune effector cells or stem cells through gene transfection technology and can express the chimeric cytokine receptor based on transforming growth factor TGF-β according to 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 chimeric cytokine receptor based on 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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