A chemokine-secreting CAR-NK cell, a preparation method thereof, and a detection method thereof

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to a chemokine-secreting CAR-NK cell and its preparation method and detection method. The present invention constructs a CAR-NK cell that can express the T cell chemokine CXCL16. After the chimeric antigen binds to the ligand, it can activate and secrete the T chemokine CXCL16 to enhance the recruitment ability of T cells, and better improve the effect of killing PD-L1 positive tumor cells. The method for detecting the chemotactic effectiveness of T chemokine-secreting CAR-NK cells in vitro according to the present invention combines three detection methods, namely, the Transwell migration experiment, the immunofluorescence staining experiment, and the flow cytometry experiment, to more comprehensively and accurately analyze the influence of chemokines on the chemotaxis of T cells in vivo.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a chemokine-secreting CAR-NK cell, a preparation method thereof, and a detection method thereof. Background Art

[0002] Natural killer cells (NK) are a subset of innate lymphoid cells (ILC), characterized by the ability to perform a series of effector functions on virus-infected or transformed cells without prior sensitization, mainly including cytotoxicity and the release of pro-inflammatory cytokines. Natural killer cells (NK cells) are currently the main immune cells known to kill tumor cells that have undergone tumor immune editing, and are one of the important means of tumor cell immunotherapy in clinical practice. Clinical studies have shown that allogeneic and autologous NK cell immunotherapy is safe. In 2009, the Ministry of Health issued the "Administrative Specification for Autologous Immune Cell T Cell and NK Cell Therapy Technologies (Draft for Comment)", including NK cell immunotherapy technology as a new type of medical technology of the third category in the scope of clinical treatment applications.

[0003] Currently, chimeric antigen receptor (CAR)-based NK cell therapy (CAR-NK) is in the initial stage of research, and the number of related preclinical and clinical trials is increasing year by year. Similar to the widely used CAR-T cells, CAR-NK cells also possess an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain. In addition, NK cells enhance their cytotoxic ability and cytokine production through two co-stimulatory molecules, NKG2D and CD244 (2B4), thus showing better tumor targeting and cytotoxicity compared to CAR-T cells. Research shows that allogeneic NK cells have high safety in adoptive cell therapy (ACT) because they usually do not cause graft-versus-host disease (GVHD). Moreover, NK cells only secrete a small amount of interferon-γ (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF), and do not produce IL-1 and IL-6 that trigger cytokine release syndrome (CRS). In addition, in addition to recognizing tumor-associated antigens through single-chain antibodies, NK cells also recognize a variety of ligands through multiple receptors, such as natural cytotoxicity receptors (NKp46, NKp44, NKp30), NKG2D, and DNAM-1 (CD226), to exert anti-tumor effects. In clinical samples, NK cells are abundant and can be expanded from peripheral blood (PB), umbilical cord blood (UCB), human embryonic stem cells (HESC), induced pluripotent stem cells (IPSC), and NK-92 cell lines.

[0004] However, CAR-NK therapy also faces several challenges. First, without cytokine support, the persistence of infused NK cells in the body is insufficient, which is a significant limitation of adoptive NK cell therapy. Although this treatment may be safer, its efficacy may be limited as a result. Second, rapid and efficient homing to the tumor site is crucial for the success of adoptive cell therapy, and this process is regulated by the complex interaction between chemokines released by NK cells and tumor cells. Moreover, immunosuppressive molecules, immunosuppressive cells, and an environment that is unfavorable for immune cell function in the tumor microenvironment (TME) constitute the main obstacles to CAR-NK cell therapy. Finally, the lentiviral transduction system, as a commonly used method for intracellular gene modification and delivery, also plays a key role in CAR-NK cell therapy.

[0005] Chemokines are a type of cytokine that can regulate the migration and trafficking of immune cells. Moreover, chemotactic gradients are crucial for recruiting effector cells to sites of inflammation, including the tumor microenvironment (TME). The chemokine expression in solid tumors is secreted by stromal cells, tumor cells, and tumor-associated immune cells to determine which immune cells are recruited to the TME, thereby helping or hindering tumor growth. Currently, multiple chemokine / chemokine receptor strategies have been used in preclinical studies of immunotherapeutic cells to promote the targeting of immune cells to tumors.

[0006] C-X-C motif ligand 16, namely CXCL16, is a chemokine belonging to the ELR-CXC subfamily, and its function is to bind to the chemokine receptor CXCR6. CXCR6 is a G protein-coupled receptor with 7 transmembrane domains. The CXCR6 / CXCL16 axis is related to the development of various autoimmune diseases and is associated with clinical parameters such as the severity, activity, and prognosis of diseases such as multiple sclerosis, autoimmune hepatitis, rheumatoid arthritis, Crohn's disease, and psoriasis. CXCL16 is expressed in various immune cells, such as dendritic cells, monocytes, macrophages, and B cells. During autoimmune diseases, CXCL16 can promote the adhesion of immune cells such as monocytes, T cells, and NKT cells to endothelial cells and dendritic cells. Studies have shown that CXCL16 is involved in the recruitment and pro-tumorigenic functions of Treg cells, which are a type of T lymphocyte mainly involved in cancer immune escape. CXCL16 is a chemotactic agent for T cells. In tumors such as nasopharyngeal carcinoma and renal cell carcinoma, Treg cells show higher CXCR6 expression compared to Treg cells in the blood, indicating that these cells can be recruited to the tumor microenvironment through CXCL16. As a chemotactic agent for anti-tumor TILs, the increased expression of CXCL16 leads to tumor cell infiltration, thereby producing an anti-cancer effect. In particular, CXCL16 promotes tumor infiltration through activated T cells, and this process is particularly important when the expression of CXCL16 increases in tumor cells after radiotherapy. CXCL16 also increases the proliferation of CD4+ T cells, thereby counteracting cancer.

[0007] Therefore, the chemokine CXCL16 has very important significance in the treatment of tumor diseases. However, the current verification methods for the effectiveness of chemokines are not comprehensive. Only through in vivo animal experiments, the chemotactic effect of chemokine CXCL16 on T cells cannot be analyzed from multiple perspectives, which to a certain extent limits the application effect of CAR-NK. Summary of the Invention

[0008] For this reason, the purpose of the present invention is to provide a chemokine-secreting CAR-NK cell and further disclose its preparation method;

[0009] The second purpose of the present invention is to provide a method for detecting the chemotactic effectiveness of the above-mentioned chemokine-secreting CAR-NK cells, which can more comprehensively analyze the influence of chemokine-secreting CAR-NK cells on the chemotaxis of T cells.

[0010] To solve the above technical problems, a preparation method of a chemokine-secreting CAR-NK cell according to the present invention includes the following steps:

[0011] (1) Synthesize the CAR sequence that can express CXCL16 and high-affinity PDL1-scFv entirely by gene;

[0012] (2) Insert the CAR sequence into a lentiviral expression plasmid to obtain a shuttle plasmid;

[0013] (3) Take stromal cells and the shuttle plasmid for lentiviral packaging to obtain a lentivirus expressing CXCL16 and PDL1-scFv;

[0014] (4) Take the isolated NK cells and transfect and culture them with the lentivirus to obtain the desired CAR-NK cells expressing CXCL16 and PDL1-scFv.

[0015] Specifically, for the method for preparing the chemokine-secreting CAR-NK cells, in step (1), the CAR sequence includes PDL1 scFv-CD8α-41BB-DAP12-P2A-CXCL16; wherein,

[0016] The hinge region is the human CD8α hinge region, the transmembrane segment is the human 41BB transmembrane segment, the intracellular segment is the human DAP12 intracellular segment, and CXCL16 is fused to the CAR structure through a P2A cleavage peptide.

[0017] Specifically, in the method for preparing the chemokine-secreting CAR-NK cells, a lentiviral plasmid of the three-plasmid system is selected as the backbone for constructing the shuttle plasmid. This plasmid backbone is an improved lentiviral expression plasmid backbone, and its advantage is that it can increase the expression level of lentivirus in 293FT cells and has a special advantage of improving the lentivirus expression level.

[0018] Specifically, the method for preparing the chemokine-secreting CAR-NK cells:

[0019] The base sequence of PDL1 scFv-CD8α-41BB-DAP12-P2A-CXCL16 is shown in SEQ ID NO.2;

[0020] The amino acid sequence encoded by PDL1 scFv-CD8α-41BB-DAP12-P2A-CXCL16 is shown in SEQ ID NO.1.

[0021] Specifically, for the method for preparing the chemokine-secreting CAR-NK cells, the construction components of PDL1 scFv-CD8α-41BB-DAP12-P2A-CXCL16 include Anti-PD-L1-scFv VH, (G3S)4 Linker, Anti-PD-L1-scFv VL, CD8α Hinge, 41BB, DAP12, P2A, and CXCL16; wherein,

[0022] The amino acid sequence of the Anti-PD-L1-scFv VH is shown in SEQ ID NO.3, and the base sequence of the Anti CD19scFv VH is shown in SEQ ID NO.4; and / or,

[0023] The amino acid sequence of the (G3S)4 Linker is shown in SEQ ID NO.5, and the base sequence of the (G3S)4 Linker is shown in SEQ ID NO.6; and / or,

[0024] The amino acid sequence of the Anti-PD-L1-scFv VL is shown in SEQ ID NO.7, and the base sequence of the Anti CD19scFv VL is shown in SEQ ID NO.8; and / or,

[0025] The amino acid sequence of the CD8α Hinge is shown in SEQ ID NO.9, and the base sequence of the CD8α Hinge is shown in SEQ ID NO.10; and / or,

[0026] The amino acid sequence of the 41BB is shown in SEQ ID NO.11, and the base sequence of the 41BB is shown in SEQ IDNO.12; and / or,

[0027] The amino acid sequence of the DAP12 is shown in SEQ ID NO.13, and the base sequence of the DAP12 is shown in SEQ IDNO.14; and / or,

[0028] The amino acid sequence of the P2A is shown in SEQ ID NO.15, and the base sequence of the P2A is shown in SEQ ID NO.16; and / or,

[0029] The amino acid sequence of the CXCL16 is shown in SEQ ID NO.17, and the base sequence of the CXCL16 is shown in SEQ IDNO.18.

[0030] Specifically, for the method for preparing the chemokine-secreting CAR-NK cells, in step (2), the lentiviral expression plasmid includes pLV-IRES-PuroR;

[0031] Preferably, the lentiviral expression plasmid further includes the helper plasmid pSPAX2 and / or pMD2.G;

[0032] Preferably, the CAR sequence is inserted between the NotI and XhoI restriction enzyme sites on the lentiviral expression plasmid.

[0033] Specifically, in the method for preparing the chemokine-secreting CAR-NK cells, in step (3), the transfected cells include human embryonic kidney cell lines;

[0034] Preferably, the stromal cells include HEK293FT cells, which can effectively cooperate with lentiviral expression plasmids, especially the pLV-IRES-PuroR plasmid, to improve the application performance.

[0035] Specifically, in the method for preparing the chemokine-secreting CAR-NK cells, in step (3), the lentiviral packaging system includes: the stromal cells, the shuttle plasmid, the pSPAX2 plasmid, the pMD2.G plasmid, the CaCl2 solution, and the BBS solution;

[0036] Preferably, the culture medium for the lentiviral packaging step includes DMEM medium containing 6-10% FBS;

[0037] Preferably, the culture conditions for the lentiviral packaging step include culturing the cells at 35-40 °C and 3-8% CO2.

[0038] Specifically, in the method for preparing the chemokine-secreting CAR-NK cells, in step (4), the NK cells are derived from peripheral blood.

[0039] Specifically, in the method for preparing the chemokine-secreting CAR-NK cells, in step (4), the culture medium for the transfection and culture step includes GT-T551 H3 medium. As an exemplary embodiment, a commercially available medium from Takara Bio, Japan, can be selected, which is a special culture system for NK cells;

[0040] Preferably, the conditions for the transfection and culture step include culturing the cells at 35-40 °C and 3-8% CO2.

[0041] The present invention also discloses a chemokine-secreting CAR-NK cell prepared by the method, denoted as PDL1-CXCL16-CAR-NK cell.

[0042] The present invention also discloses a method for detecting the chemotactic effectiveness of the chemokine-secreting CAR-NK cells, including the steps of separately culturing T cells and the CAR-NK cells in vitro, and the steps of detecting by Transwell experiment, flow cytometry, and immunofluorescence staining experiment respectively.

[0043] Specifically, the method for detecting the chemotactic effectiveness of the chemokine-secreting CAR-NK cells:

[0044] In the Transwell experiment, at the peak of CXCL16 secretion, the culture medium containing the CAR-NK cells was added to the lower chamber of the Transwell. The upper chamber contained T cells cultured in serum-free medium. Based on the Transwell cell migration experiment, an additional 1-10% gelatin coating treatment was performed, and the T cells in the lower layer of the Transwell membrane were stained with crystal violet.

[0045] The flow cytometry and immunofluorescence staining experiments detected the culture medium in the lower chamber of the Transwell and the residual T cells at the bottom of the well.

[0046] The present invention constructs a CAR-NK cell capable of expressing CXCL16, namely PDL1-CXCL16-CAR-NK cell. CXCL16, a cytokine secreted by it that regulates the migration and transportation of immune cells, has a chemotactic effect on T lymphocytes, and enhances the recruitment ability of T cells through the expression of chemokine CXCL16, thereby better improving the killing effect.

[0047] To verify the chemotactic effectiveness of the CXCL16-expressing CAR-NK cells of the present invention, after culturing T cells and PDL1-CXCL16-CAR-NK cells separately in vitro, at the peak of CXCL16 secretion, the culture medium containing the chemokine was added to the lower chamber of the Transwell. The upper chamber contained T cells cultured in serum-free medium. Based on the Transwell cell migration experiment, an additional 1-10% gelatin coating treatment was performed. While staining the T cells in the lower layer of the Transwell membrane with crystal violet, flow cytometry and immunofluorescence staining were used to detect the culture medium in the lower chamber and the residual T cells at the bottom of the well, more comprehensively analyzing the effect of the chemokine CXCL16-secreting CAR-NK cells on the chemotaxis of T cells, and being able to more comprehensively characterize and verify the chemotaxis of the chemotactic secretory CAR-NK cells expressing CXCL16 on T cells.

[0048] The present invention enhances the recruitment of T cells by constructing CXCL16-expressing CAR-NK cells, better mimics the extracellular matrix environment through the improved Transwell migration experiment, and at the same time uses methods such as immunofluorescence staining experiment and flow cytometry experiment to detect the number of migrated T cells, thereby assisting in evaluating the migration ability of cells in vivo and comprehensively verifying the chemotactic effect of CXCL16 on T cells.

[0049] By improving the Transwell migration assay, the present invention further coated the membrane with gelatin to better simulate the in vivo environment and more significantly reflect the invasion ability of T cells. In the ordinary Transwell method, only the number of cells under the membrane is detected. There are still many cells that enter the culture medium in the lower chamber and even attach to the bottom of the culture well. The present invention adds the detection of the number of cells in the lower chamber culture medium and at the bottom of the well on the basis of the basic Transwell method, which can more truly reflect the change of CXCL16 on the invasion ability of T cells, and its verification effect is better than the traditional method.

[0050] The method for detecting the chemotactic effectiveness of the chemokine-secreting CAR-NK cells according to the present invention optimizes the Transwell cell migration assay, covering 1-10% gelatin, which can better mimic the extracellular matrix environment and detect the migration ability of cells. The migration of T cells requires removing the hindrance of gelatin to movement, passing through the gelatin on the upper layer of the chamber, and then passing through the porous membrane of the chamber to achieve migration from the upper chamber to the bottom.

[0051] The method for detecting the chemotactic effectiveness of the chemokine-secreting CAR-NK cells according to the present invention is supplemented with flow cytometry to detect the residual T cells in the supernatant of the lower chamber due to too long migration time; at the same time, the experiment is supplemented with immunofluorescence staining to detect the residual T cells at the bottom of the lower chamber due to too long migration time. The detection method of the present invention combines three detection methods and then performs data statistics to more comprehensively and accurately analyze the influence of chemokines on the chemotaxis of T cells in vivo. Brief Description of the Drawings

[0052] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention in combination with the drawings, wherein,

[0053] Figure 1 In (A), it is a schematic diagram of the PDL1-CXCL16-CAR structure; (B) is a schematic diagram of the PDL1-CAR structure; (C) is a schematic diagram of the designed PDL1-CXCL16-CAR structure; (D) is a schematic diagram of the designed PDL1-CAR structure; wherein, the hinge region uses the human CD8α hinge region, the transmembrane segment is the human 41BB transmembrane segment, the intracellular segment is the human DAP12 intracellular segment, and CXCL16 is fused with the CAR structure through the P2A cleavage peptide;

[0054] Figure 2For the plasmid construction process and verification results of the present invention; among them, (A) is a schematic diagram of the construction process, that is, the synthesized CAR sequence is inserted between the NotI and XhoI restriction enzyme sites on the pLV-IRES-PuroR plasmid; (B) is a diagram of double digestion of the constructed plasmid with NotI and XhoI and agarose gel electrophoresis detection. The length of PDL1-CAR is 2838bp, the length of the PDL1-CXCL16-CAR sequence is 3603bp, and the length of the pLV-IRES-PuroR plasmid is 8081bp;

[0055] Figure 3 For the results detected by flow cytometry; among them, the abscissa represents the expression of G4S, the ordinate represents the total number of detected cells, the red peak is the cells without incubated antibody, and the blue peak is the cells incubated with antibody;

[0056] Figure 4 For the standard curve of CXCL16 content determination;

[0057] Figure 5 For the results of crystal violet staining map;

[0058] Figure 6 For the results detected by flow cytometer;

[0059] Figure 7 For the statistics of flow cytometry results;

[0060] Figure 8 For the immunofluorescence staining map;

[0061] Figure 9 For the statistics of the positive area of immunofluorescence staining. Detailed implementation manners

[0062] In the following examples of the present invention, first, CAR-NK cells expressing CXCL16 were constructed, and T cells and PDL1-CXCL16-CAR-NK cells were cultured in vitro respectively. At the peak of CXCL16 secretion, the culture medium containing chemokine was taken and added to the lower chamber of Transwell. The upper chamber was T cells cultured without serum. Based on the Transwell cell migration experiment, an additional 1-10% gelatin coating treatment was carried out. While crystal violet staining the T cells in the lower layer of the Transwell membrane, flow cytometry and immunofluorescence staining were supplemented to detect the culture solution in the lower chamber and the residual T cells at the bottom of the well, and the influence of chemokine CXCL16-secreting CAR-NK cells on the chemotaxis of T cells was analyzed more comprehensively.

[0063] Example 1

[0064] In this example, CAR-NK cells expressing CXCL16 and PDL1-scFv were constructed.

[0065] Plasmid construction

[0066] In this embodiment, PDL1-CAR and PDL1-CXCL16-CAR are constructed.

[0067] As Figure 1 shown in (A) is the schematic diagram of the PDL1-CAR structure, (B) is the schematic diagram of the PDL1-CXCL16-CAR structure, (C) is the schematic diagram of the designed PDL1-CAR structure. The hinge region uses the human CD8α hinge region, the transmembrane segment is the human 41BB transmembrane segment, and the intracellular segment is the human DAP12 intracellular segment. CXCL16 is fused to the CAR structure through the P2A cleavage peptide; (D) is the schematic diagram of the designed PDL1-CXCL16-CAR structure.

[0068] In this embodiment, based on the full gene synthesis of the PDL1 scFv-CD8α-41BB-DAP12-P2A-CXCL16 base sequence, its nucleotide sequence is as shown in SEQ ID NO.2, and the encoded amino acid sequence is SEQ ID NO.1.

[0069] Furthermore, the PDL1 scFv-CD8α-41BB-DAP12-P2A-CXCL16 is constructed onto the lentiviral expression vector pLV-IRES-PuroR plasmid as a shuttle plasmid, and lentivirus expressing CXCL16 and PDL1-scFv is packaged and harvested, named P / C-LV, which can express CXCL16 and PDL1 scFv after infecting NK cells. At the same time, a plasmid without CXCL16 is constructed as a control, and its corresponding lentivirus is named P-LV.

[0070] The amino acid sequence of the PDL1 scFv-CD8α-41BB-DAP12- P2A -CXCL16 constructed in this embodiment is as follows (SEQ ID NO.1):

[0071] MKHLWFFLLLVAAPRWVLSEVKLQESGPSLVRPGASVKISCKASAYSFTSYWMHWVKQRPGQGLDWIG MIDPSDSEARLNQKFKDKATLTVDKSSSTTYIHLSSPTSEDSAVYYCARSYGYDGDYYLDVWGAGTTVTVSSGGGS GGGSGGGSGGGSDIVMTQSPSSLSASLGGKVTITCKASQDINKYIAWYQHKPGKGPRLLIQYTSTLQPGIPSRFSG SGSGRDYSFSISNLEPEDIATYYCLQYDYLRTFGGGTKLEIKKL AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCEL RLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKSPGG ATNFSLLKQAGDVEENPGP MGRDLRPGSRVLLLLLLLLLVYLTQPGNGNEGSVTGSCYCGKRISSDSPPSVQFMNRLRKHLRAYHRCLYYTRFQL LSWSVCGGNKDPWVQELMSCLDLKECGHAYSGIVAHQKHLLPTSPPISQASEGASSDIHTPAQMLLSTLQSTQRPT LPVGSLSSDKELTRPNETTIHTAGHSLAVGPEAGENQKQPEKNAGPTARTSATVPVLCLLAIIFILTAAPSYVLCK RRRGQSPQSSPDLPVHYIPVAPDSNT 。

[0072] The base sequence of PDL1 scFv-CD8α-41BB-DAP12-P2A-CXCL16 synthesized in this example is as follows (SEQ ID NO.2):

[0073]

[0074] In this embodiment, the component information involved in constructing PDL1 scFv-CD8α-41BB-DAP12-P2A-CXCL16 is as follows.

[0075] Anti-PD-L1-scFv VH amino acid sequence (SEQ ID NO.3): MKHLWFFLLLVAAPRWVLSEVKLQESGPSLVRPGASVKISCKASAYSFTSYWMHWVKQRPGQGLDWIGMIDPSDSEARLNQKFKDKATLTVDKSSSTTYIHLSSPTSEDSAVYYCARSYGYDGDYYLDVWGAGTTVTVSS.

[0076] Anti-PD-L1-scFv VH base sequence (SEQ ID NO.4): ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCCCCCAGATGGGTGCTGTCCGAGGTGAAGCTGCAGGAGTCAGGACCTAGCCTGGTTAGGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGCTTACTCATTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGTCTTGACTGGATTGGCATGATTGATCCTTCCGACAGTGAAGCTAGGTTAAATCAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGTACAACCTACATTCACCTCAGCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCTTATGGTTACGACGGGGACTACTACCTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA.

[0077] (G3S)4Linker amino acid sequence (SEQ ID NO.5): GGGSGGGSGGGSGGGS.

[0078] (G3S)4Linker base sequence (SEQ ID NO.6): GGCGGCGGCAGCGGCGGCGGCTCTGGAGGAGGATCTGGCGGCGGAAGC.

[0079] Anti-PD-L1-scFv VL amino acid sequence (SEQ ID NO.7): DIVMTQSPSSLSASLGGKVTITCKASQDINKYIAWYQHKPGKGPRLLIQYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDYLRTFGGGTKLEIKKL.

[0080] Anti-PD-L1-scFv VL base sequence (SEQ ID NO.8):

[0081] GATATTGTGATGACCCAGTCTCCATCCTCACTGTCTGCATCTCTGGGAGGCAAAGTCACCATCACTTGCAAGGCAAGCCAAGACATTAACAAATATATAGCTTGGTACCAACACAAGCCTGGAAAAGGTCCTAGGCTGCTCATACAGTACACATCTACATTACAGCCAGGCATCCCATCAAGGTTCAGTGGAAGTGGGTCTGGGAGAGATTATTCCTTCAGCATCAGCAACCTGGAGCCTGAAGATATTGCAACTTATTATTGTCTACAGTATGATTATCTTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAAAGCTT.

[0082] CD8α Hinge amino acid sequence (SEQ ID NO.9): AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY.

[0083] CD8α Hinge base sequence (SEQ ID NO.10): GCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTAC.

[0084] 41BB amino acid sequence (SEQ ID NO.11): KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0085] 41BB nucleotide sequence (SEQ ID NO.12): AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG.

[0086] DAP12 amino acid sequence (SEQ ID NO.13): RLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKSPGG.

[0087] DAP12 nucleotide sequence (SEQ ID NO.14): CGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAATCCCCCGGGGGA.

[0088] P2A amino acid sequence (SEQ ID NO.15): ATNFSLLKQAGDVEENPGP.

[0089] P2A nucleotide sequence (SEQ ID NO.16): GCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCG.

[0090] CXCL16 Amino Acid Sequence (SEQ ID NO.17): MGRDLRPGSRVLLLLLLLLLVYLTQPGNGNEGSVTGSCYCGKRISSDSPPSVQFMNRLRKHLRAYHRCLYYTRFQLLSWSVCGGNKDPWVQELMSCLDLKECGHAYSGIVAHQKHLLPTSPPISQASEGASSDIHTPAQMLLSTLQSTQRPTLPVGSLSSDKELTRPNETTIHTAGHSLAVGPEAGENQKQPEKNAGPTARTSATVPVLCLLAIIFILTAAPSYVLCKRRRGQSPQSSPDLPVHYIPVAPDSNT。

[0091] CXCL16 base sequence (SEQ ID NO.18): ATGGGACGGGACTTGCGGCCCGGGTCCCGCGTGCTCCTGCTCCTGCTTCTGCTCCTGCTGGTGTACCTGACTCAGCCAGGCAATGGCAACGAGGGCAGCGTCACTGGAAGTTGTTATTGTGGTAAAAGAATTTCTTCCGACTCCCCGCCATCGGTTCAGTTCATGAATCGTCTCCGGAAACACCTGAGAGCTTACCATCGGTGTCTATACTACACGAGGTTCCAGCTCCTTTCCTGGAGCGTGTGTGGAGGCAACAAGGACCCATGGGTTCAGGAATTGATGAGCTGTCTTGATCTCAAAGAATGTGGACATGCTTACTCGGGGATTGTGGCCCACCAGAAGCATTTACTTCCTACCAGCCCCCCAATTTCTCAGGCCTCAGAGGGGGCATCTTCAGATATCCACACCCCTGCCCAGATGCTCCTGTCCACCTTGCAGTCCACTCAGCGCCCCACCCTCCCAGTAGGATCACTGTCCTCGGACAAAGAGCTCACTCGTCCCAATGAAACCACCATTCACACTGCGGGCCACAGTCTGGCAGTTGGGCCTGAGGCTGGGGAGAACCAGAAGCAGCCGGAAAAAAATGCTGGTCCCACAGCCAGGACATCAGCCACAGTGCCGGTCCTGTGCCTCCTGGCCATCATCTTCATCCTCACCGCAGCCCCTTCCTATGTGCTGTGCAAGAGGAGGAGGGGGCAGTCACCGCAGTCCTCTCCAGATCTGCCGGTTCATTATATACCTGTGGCACCTGACTCTAATACCTGA。

[0092] Verification of plasmid construction

[0093] In this example, the plasmid constructed above was verified, and the results are as shown in the appendix Figure 2 as follows.

[0094] As shown in the appendix Figure 2In (A) is a schematic diagram of the construction process. The synthesized CAR sequence is inserted into the middle of the NotI and XhoI restriction enzyme sites on the lentiviral expression plasmid (in this example, a three-plasmid system is used, where the backbone plasmid is pLV-IRES-PuroR, and the helper plasmids are pSPAX2 and pMD2.G); (B) is a diagram of double digestion of the constructed plasmid with NotI and XhoI and agarose gel electrophoresis detection. The length of PDL1-CAR is 2838 bp, the length of the PDL1-CXCL16-CAR sequence is 3603 bp, and the length of the pLV-IRES-PuroR plasmid is 8081 bp. It can be seen that the plasmid required in this example was successfully constructed.

[0095] Example 2

[0096] Lentivirus packaging

[0097] Take out the stored HEK293FT cells from liquid nitrogen and quickly put them into a 37°C water bath for rapid thawing.

[0098] Add the cells in the cryopreservation solution to 10 mL of DMEM complete medium containing 10% FBS, and centrifuge at 300 g for 5 min at room temperature.

[0099] Discard the supernatant, resuspend the precipitate with 2 mL of DMEM complete medium containing 10% FBS, and add it to a 10 cm 2 cell culture dish, shake it crosswise, and place it in a 37°C, 5% CO2 cell culture incubator.

[0100] When observing the cell confluence under the microscope at 80%, discard the medium in the dish, replace it with DMEM medium containing 6% FBS without antibiotics, and place it in a 37°C, 5% CO2 cell culture incubator for static incubation for 1 h.

[0101] Take a clean sterile 1.5 mL EP tube, add 781 μL of Cacl2 solution to it, add 10 μg of shuttle plasmid, 7.5 μg of pSPAX2 plasmid, and 2.5 μg of pMD2.G plasmid to the Cacl2 solution, and mix well.

[0102] Take a clean sterile 1.5 mL EP tube, add 781 μL of BBS solution to it, mix the above mixture with the BBS solution, and let it stand at room temperature for 20 min.

[0103] The Cacl 2- plasmid-BBS mixed solution is evenly added to a 10 cm 2 cell culture dish, and placed in a 37°C, 5% CO2 cell culture incubator for 12 h.

[0104] After 12 h, the culture medium was replaced with 30 mL of complete DMEM medium containing 10% FBS, and placed in a cell culture incubator at 37 °C and 5% CO2 for 48 h.

[0105] The culture medium supernatant was harvested and centrifuged at 4000 g for 10 min at 4 °C.

[0106] After filtering the supernatant with a 0.45 μm filter membrane, it was placed in an ultra-high-speed centrifuge and centrifuged at 25000 rpm for 120 min at 4 °C. The supernatant was discarded, and the precipitate was resuspended with 200 μL of PBS to obtain the lentivirus concentrate.

[0107] Lentivirus titer detection

[0108] 293FT cells were seeded in a 24-well plate and cultured overnight. 1, 2, and 4 μL of lentivirus solution were added respectively for infection, and 1 mg / mL of Polybrene was added simultaneously to promote infection. After 48 h, recombinant protein L was used to bind to the CAR Sc Fv sequence expressed on the cell surface, and the percentage of positive cells was detected by FCM. According to the calculation formula: Lentivirus titer (TU / mL) = number of 293FT cells × percentage of positive cells × 10 3 / volume of virus solution (μL). The titer of P / S-LV harvested this time was 1.8x10 8 TU / mL, and the titer of P-LV was 1.2×10 8 TU / mL.

[0109] Example 3

[0110] Isolation of peripheral blood NK

[0111] 4 mL of peripheral whole blood of the research subjects was collected using an EDTA anticoagulant tube, and NK cells were sorted from the peripheral blood using the MACSxpress Whole Blood NK Cell Isolation Kit of Miltenyi Company, Germany within 4 hours. The specific operation steps are as follows:

[0112] 1) Preparation of Separation buffer: Dilute MACS BSA stock solution: auto MACS Rinsing solution at a ratio of 1:20 and pre-cool at 4 °C;

[0113] 2) Take a 15 mL centrifuge tube, add 1.5 mL of peripheral blood sample to the bottom of the tube, add 75 μL of Miltenyi Whole Blood CD56 magnetic beads into the blood, vortex for 2 - 3 s to mix evenly, and incubate at 4 °C for 15 min;

[0114] 3) Add 8 mL of Separation buffer, mix well, centrifuge at 445 g for 10 min at room temperature;

[0115] 4) Discard the supernatant and add 3 mL of Separation buffer;

[0116] 5) Equilibrate the Whole Blood Column: Fix the column on the magnetic stand and add 3 mL of Separation buffer;

[0117] 6) After the liquid in the column stops dripping, quickly add the labeled cell suspension and discard the flow-through;

[0118] 7) After the cell suspension stops dripping, quickly add 3 mL of Separation buffer and discard the flow-through;

[0119] 8) Repeat step 7) twice. After the liquid stops dripping, remove the column and place it on a new 15 mL centrifuge tube. Add 4 mL of liquid to the column and quickly insert the plunger to push out the cell suspension in the column at a uniform speed;

[0120] 9) Centrifuge at 2000 rpm for 10 min at room temperature, discard the supernatant, and resuspend the cells in GT-T551 H3 to 3x10 5 cells / mL.

[0121] Example 4

[0122] This example is based on the protocol in Example 1 for the preparation of CAR-NK.

[0123] Take a new sterile 24-well plate and seed NK cells at a density of 3×10 5 cells / mL. Add polybrene to each well to a final concentration of 8 μg / mL. After pipetting and mixing, add 100 μL of lentivirus concentrate and place it in a 37°C, 5% CO2 cell culture incubator for 12 h.

[0124] After 12 h of culture, add 2 mL of GT-T551 H3 medium to the wells and place it in a 37°C, 5% CO2 cell culture incubator for 24 h.

[0125] After 24 h of culture, discard the 2 mL of medium, add fresh 2 mL of GT-T551 H3 medium, and place it in a 37°C, 5% CO2 cell culture incubator for 24 h.

[0126] After continuing to culture for 24 h, discard the 2 mL of medium, add fresh 2 mL of GT-T551 H3 medium, and place it in a 37°C, 5% CO2 cell culture incubator for 24 h.

[0127] Take cells for flow cytometry detection. The detection antibody is Anti-(G4S)n (B02H1)mAb(APC). Flow cytometry was used to detect the CAR positive rate of PDL1-CAR-NK cells and PDL1-CXCL16-CAR-NK cells. The results of flow cytometry detection are shown in the appendix Figure 3 , where the abscissa represents the expression of G4S, the ordinate represents the total number of detected cells, the red peak is the cells without antibody incubation, and the blue peak is the cells incubated with antibody.

[0128] It can be seen that the CAR-NK cells were successfully prepared in this example.

[0129] Example 5

[0130] This example is used to test the chemotactic effect of CXCL16 on T cells.

[0131] Experimental grouping

[0132] This experimental example was divided into four groups: Group 1 was treated with 1-10% gelatin-coated Transwell, and the chemokine-secreting cells were PDL1-CXCL16-CAR-NK; Group 2 was treated with 1-10% gelatin-coated Transwell, and the chemokine-secreting cells were PDL1-CAR-NK; Group 3 was untreated Transwell, and the chemokine-secreting cells were PDL1-CXCL16-CAR-NK; Group 4 was untreated Transwell, and the chemokine-secreting cells were PDL1-CAR-NK; and in Group 3 and Group 4, flow cytometry detection and immunofluorescence staining data statistics were not performed, and the remaining conditions were all the same.

[0133] In vitro culture of T cells

[0134] This experimental example was carried out under sterile conditions.

[0135] On day 0 of the experiment, each group seeded 2.5 × 10 5 PBMCs into a single well of a 48-well plate; until day 6 for T cell culture and sub-culturing at an appropriate density; on day 7 of the experiment, each group replaced the T cells with serum-free medium for culture.

[0136] Pretreatment of Transwell membrane

[0137] This experimental example was carried out under sterile conditions.

[0138] On day 7 of the experiment, Group 1 coated the Transwell membrane with 1-10% gelatin, Group 2 coated the Transwell membrane with 1-10% gelatin, Group 3 and Group 4 were not pretreated, and all four groups were incubated overnight at 4°C.

[0139] Detection of CXCL16 expression

[0140] This experimental example protocol was carried out under aseptic conditions.

[0141] On the 8th day of the experiment, the secretion amount of chemokine CXCL16 in each group was detected by ELISA.

[0142] In this example, the standard curve for the determination of CXCL16 content is as attached Figure 4 , and the expression of CXCL16, that is, the CXCL16 concentration, is shown in Table 1 below.

[0143] Table 1 CXCL16 Concentration Table

[0144]

[0145] As shown in the results of Table 1, the secretion amount of the PDL1-CAR-NK chemokine CXCL16 in Group 2 and Group 4 was lower than that of the PDL1-CXCL16-CAR-NK in Group 1 and Group 3.

[0146] Modified Transwell cell migration assay

[0147] This experimental example protocol was carried out under aseptic conditions.

[0148] On the 8th day of the experiment, the adherent glass slides were placed in a single well of a 24-well plate. 1 mL of the PDL1-CXCL16-CAR-NK cell suspension was added to the well of Group 1, 1 mL of the PDL1-CAR-NK cell suspension was added to the well of Group 2, 1 mL of the PDL1-CXCL16-CAR-NK cell suspension was added to the well of Group 3, and 1 mL of the PDL1-CAR-NK cell suspension was added to the well of Group 4. The four processed Transwell chambers were respectively placed in the corresponding wells with forceps. 100 μL of the T cell suspension with a density of 5 × 10 6 cells / mL was added to the upper chamber, and incubated at 37 °C for 4 h.

[0149] Crystal violet staining

[0150] The medium in the upper chamber was discarded, the four Transwell chambers were taken out, and the upper and lower layers of the membrane were washed 3 times each with PBS. The non-migrated cells in the upper chamber were gently wiped off with a moist cotton swab.

[0151] The Transwell chamber was placed in a new well, 500 μL of paraformaldehyde was added to the lower chamber, and the chamber was fixed for 30 minutes. The paraformaldehyde was discarded, and the chamber was air-dried.

[0152] 500 μL of crystal violet solution was added to the lower chamber, and the chamber was stained for 10 minutes. The crystal violet solution was discarded, and it was washed clean with PBS and air-dried.

[0153] Use a blade to cut off the chamber membrane, place it on a glass slide, seal it with neutral balsam, and take pictures with an upright microscope.

[0154] In this example, the crystal violet staining results are as shown in the appendix. Figure 5 As shown, coating the Transwell with 1-10% gelatin can better mimic the extracellular matrix environment and detect the migration ability of cells. The migration of T cells requires removing the hindrance of gelatin to movement, passing through the gelatin on the upper layer of the chamber, and then through the porous membrane of the chamber to achieve migration from the top chamber to the bottom. Group 1 and Group 2 are significantly easier to statistically analyze the difference in the amount of T cell migration and can more realistically evaluate the effect in vivo.

[0155] Flow cytometry detection

[0156] Absorb all the lower chamber culture media of Group 1 and Group 2 for flow cytometry detection. The flow cytometry detection results and statistical results are respectively as shown in the appendix. Figure 6 - 7 As shown.

[0157] As shown in the results, there are more T cell residues in the lower chamber supernatant of the PDL1-CXCL16-CAR-NK group than in the PDL1-CAR-NK group.

[0158] Immunofluorescence staining

[0159] Fix the glass slides in the wells of Group 1 and Group 2 with 4% paraformaldehyde and perform Dapi immunofluorescence staining. Take pictures with a fluorescence inverted microscope and statistically analyze the proportion of the fluorescence area.

[0160] In this experimental example, the immunofluorescence staining pictures and the statistical results of the positive area of immunofluorescence staining are respectively shown in the appendix. Figure 8 - 9 As shown.

[0161] As shown in the results, there are more T cell residues in the bottom of the lower chamber wells of the PDL1-CXCL16-CAR-NK group than in the PDL1-CAR-NK group.

[0162] In summary, this study successfully established a method for the preparation and detection of a secretory chimeric antigen receptor NK cell that can chemotax T cells.

[0163] Obviously, the above embodiments are only examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or alterations derived therefrom are still within the protection scope of this invention.

Claims

1. A method for preparing chemokine-secreting CAR-NK cells, characterized in that, It includes the following steps: (1) Synthesize the CAR sequence that can express CXCL16 and PDL1-scFv entirely by gene synthesis; The CAR sequence includes PDL1 scFv-CD8α-41BB-DAP12-P2A-CXCL16; where The hinge region is the human CD8α hinge region, the transmembrane segment is the human 41BB transmembrane segment, the intracellular segment is the human DAP12 intracellular segment, and CXCL16 is fused with the CAR structure through the P2A cleavage peptide; The base sequence of the PDL1 scFv-CD8α-41BB-DAP12-P2A-CXCL16 is shown in SEQ ID NO.2; The amino acid sequence encoded by the PDL1 scFv-CD8α-41BB-DAP12-P2A-CXCL16 is shown in SEQ ID NO.1; (2) Insert the CAR sequence into the lentiviral expression plasmid to obtain a shuttle plasmid; (3) Take transfected cells and package lentivirus with the shuttle plasmid to obtain lentivirus expressing CXCL16 and PDL1-scFv; (4) Take isolated NK cells and transfect and culture them with the lentivirus to obtain the required CAR-NK cells expressing CXCL16 and PDL1-scFv.

2. The preparation method of the chemokine-secreting CAR-NK cells according to claim 1, characterized in that, The construction components of the PDL1scFv-CD8α-41BB-DAP12-P2A-CXCL16 include Anti-PD-L1-scFv VH, (G3S)4Linker, Anti-PD-L1-scFv VL, CD8α Hinge, 41BB, DAP12, P2A, and CXCL16; where The amino acid sequence of the Anti-PD-L1-scFv VH is shown in SEQ ID NO.3, and the base sequence of the Anti -PD-L1scFvVH is shown in SEQ ID NO.4; and / or The amino acid sequence of the (G3S)4 Linker is shown in SEQ ID NO.5, and the base sequence of the (G3S)4 Linker is shown in SEQ ID NO.6; and / or The amino acid sequence of the Anti-PD-L1-scFv VL is shown in SEQ ID NO.7, and the base sequence of the Anti PD-L1 scFvVL is shown in SEQ ID NO.8; and / or The amino acid sequence of the CD8α Hinge is shown in SEQ ID NO.9, and the base sequence of the CD8α Hinge is shown in SEQID NO.10; and / or The amino acid sequence of the 41BB is shown in SEQ ID NO.11, and the base sequence of the 41BB is shown in SEQ ID NO.12; and / or The amino acid sequence of the DAP12 is shown in SEQ ID NO.13, and the base sequence of the DAP12 is shown in SEQ ID NO.14; and / or The amino acid sequence of the P2A is shown in SEQ ID NO.15, and the nucleotide sequence of the P2A is shown in SEQ ID NO.16; and / or, The amino acid sequence of the CXCL16 is shown in SEQ ID NO.17, and the nucleotide sequence of the CXCL16 is shown in SEQ ID NO.

18.

3. The method for preparing the chemokine-secreting CAR-NK cells according to claim 1 or 2, characterized in that, In the step (2), the lentiviral expression plasmid includes pLV-IRES-PuroR; The lentiviral expression plasmid further includes the helper plasmid pSPAX2 and / or pMD2.G; The CAR sequence is inserted between the NotI and XhoI restriction enzyme sites on the lentiviral expression plasmid.

4. The preparation method of the chemokine-secreting CAR-NK cells according to claim 3, wherein In the step (3): The transfected cells include the human embryonic kidney cell line; and / or, The lentiviral packaging system includes: the transfected cells, the shuttle plasmid, the pSPAX2 plasmid, the pMD2.G plasmid, the CaCl2 solution, and the BBS solution.

5. The preparation method of the chemokine-secreting CAR-NK cells according to claim 4, wherein In the step (4): The NK cells are derived from peripheral blood; The medium for the transfection and culture step includes the GT-T551 H3 medium.

6. A chemokine-secreting CAR-NK cell prepared by the method according to any one of claims 1-5.

7. A method for detecting the chemotactic effectiveness of the chemokine-secreting CAR-NK cells according to claim 6, characterized in that, It includes the steps of separately culturing T cells and the CAR-NK cells according to claim 6 in vitro, and the steps of detecting by using Transwell experiment, flow cytometry, and immunofluorescence staining experiment respectively.

8. The method for detecting the chemotactic effectiveness of the chemokine-secreting CAR-NK cell according to claim 7, wherein: In the Transwell experiment, at the peak of CXCL16 secretion, the medium containing the CAR-NK cells is added to the lower chamber of the Transwell, and the upper chamber contains T cells cultured in serum-free medium. The Transwell cell migration experiment is used as the basis for 1-10% gelatin coating treatment, and the T cells in the lower layer of the Transwell membrane are stained with crystal violet; The flow cytometry and the immunofluorescence staining experiment detect the culture solution in the lower chamber of the Transwell and the residual T cells at the bottom of the well.

Images