Chemotactic factor secreting type CAR-NK cell as well as preparation method and detection method thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a chemokine secreting type CAR-NK cell as well as a preparation method and a detection method thereof. The CAR-NK cell capable of expressing the T cell chemotactic factor CXCL16 is constructed, the T cell chemotactic factor CXCL16 can be activated and secreted after the chimeric antigen is combined with the ligand, so that the recruitment capacity on T cells is enhanced, and the effect of killing PD-L1 positive tumor cells is better improved. According to the method for detecting the chemotactic effectiveness of the T chemotactic factor secreting type CAR-NK cells in vitro, a Transwell migration experiment is combined with an immunofluorescent staining experiment and a flow cytometry experiment at the same time, and the influence of the chemotactic factors on the T cell chemotactic performance in vivo can be analyzed more comprehensively and accurately.

Description

Technical Field

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

[0002] Natural killer cells (NK) are a subpopulation of innate lymphocytes (ILCs), characterized by the ability to perform a series of effector functions on virus-infected or transformed cells without prior sensitization, mainly including cytotoxic effects and the release of pro-inflammatory cytokines. Natural killer cells (NK cells) are currently the most important immune cells known to kill tumor cells that have undergone tumor immunoediting, and are one of the important means of clinical tumor cell immunotherapy. Clinical studies have shown that allogeneic and autologous NK cell immunotherapy is safe. In 2009, the Ministry of Health issued the "Management Specifications for Autologous Immune Cell T Cell and NK Cell Therapy Technology (Draft for Comments)", which included NK cell immunotherapy technology as a third-category medical new technology in the scope of application of clinical treatment.

[0003] Currently, NK cell therapy (CAR-NK) based on chimeric antigen receptor (CAR) is in the early stages 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 have extracellular antigen recognition domains, transmembrane domains, and intracellular signal transduction domains. In addition, NK cells enhance their cytotoxicity and cytokine production through two co-stimulatory molecules, NKG2D and CD244 (2B4), thereby showing better tumor targeting and cytotoxicity than CAR-T cells. Studies have shown that allogeneic NK cells have a higher 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 cause cytokine release syndrome (CRS). In addition to recognizing tumor-associated antigens through single-chain antibodies, NK cells also recognize multiple 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, the inadequate persistence of infused NK cells in vivo without cytokine support is a significant limitation of adoptive NK cell therapy. Although this treatment may be safer, its efficacy may be limited. Secondly, rapid and effective homing to the tumor site is crucial to the effectiveness of adoptive cell therapy, a process that is regulated by the complex interaction between NK cells and chemokines released by tumor cells. Furthermore, immunosuppressive molecules, immunosuppressive cells, and an environment that is not conducive to immune cell function in the tumor microenvironment (TME) constitute the main obstacles to CAR-NK cell therapy. Finally, the lentiviral transduction system, as a common method for intracellular gene modification and delivery, also plays a key role in CAR-NK cell therapy.

[0005] Chemokines are cytokines that 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). 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 to help or hinder tumor growth. Currently, a variety of chemokine / chemokine receptor strategies have been used in preclinical studies of immunotherapy cells to promote immune cell targeting to tumors.

[0006] CXC motif ligand 16, or 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 seven transmembrane domains, and the CXCR6 / CXCL16 axis has been implicated in the development of a variety of autoimmune diseases and has been associated with clinical parameters of severity, activity, and prognosis in 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 function of Treg cells, a type of T lymphocyte that is primarily involved in cancer immune escape. CXCL16 is a chemoattractant 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 into the tumor microenvironment through CXCL16. CXCL16 acts as a chemoattractant for anti-tumor TILs, and its increased expression leads to tumor cell infiltration, resulting in an anti-cancer effect. In particular, CXCL16 promotes tumor infiltration through activated T cells, a process that is particularly important when CXCL16 expression is increased in tumor cells after radiotherapy. CXCL16 also increases the proliferation of CD4+ T cells, thereby fighting cancer.

[0007] Therefore, chemokine CXCL16 is of great significance in the treatment of tumor diseases. However, the current verification method for the effectiveness of chemokines is not comprehensive. Only through animal in vivo experiments, it is impossible to analyze the chemotactic effect of chemokine CXCL16 on T cells from multiple angles, which limits the application effect of CAR-NK to a certain extent. Summary of the invention

[0008] To this end, the object of the present invention is to provide a chemokine-secreting CAR-NK cell, and further disclose a method for preparing the same; The second object 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 effect of chemokine-secreting CAR-NK cells on T cell chemotaxis.

[0009] In order to solve the above technical problems, a method for preparing a chemokine-secreting CAR-NK cell according to the present invention comprises the following steps: (1) Full gene synthesis of a CAR sequence that can express CXCL16 and high-affinity PDL1-scFv; (2) inserting the CAR sequence into a lentiviral expression plasmid to obtain a shuttle plasmid; (3) taking stromal cells and the shuttle plasmid for lentiviral packaging to obtain lentivirus expressing CXCL16 and PDL1-scFv; (4) The separated NK cells are transfected and cultured with the lentivirus to obtain the desired CAR-NK cells expressing CXCL16 and PDL1-scFv.

[0010] Specifically, in 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, 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 the P2A cleavage peptide.

[0011] Specifically, in the preparation method of the chemokine-secreting CAR-NK cells, the lentiviral plasmid of the three-plasmid system is selected as the backbone for shuttle plasmid construction. The plasmid backbone is an improved lentiviral expression evaluation backbone, which has the advantage of being able to increase the expression amount of the lentivirus in 293FT cells, and has the special advantage of increasing the expression amount of the lentivirus.

[0012] Specifically, the preparation method of the chemokine-secreting CAR-NK cells is: 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.

[0013] Specifically, in the method for preparing the chemokine-secreting CAR-NK cells, the construction components of the 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, 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, 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 CD19scFv VL 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 SEQ ID NO.10; and / or, The amino acid sequence of 41BB is shown in SEQ ID NO.11, and the base sequence of 41BB is shown in SEQ ID NO.12; and / or, The amino acid sequence of DAP12 is shown in SEQ ID NO.13, and the base sequence of DAP12 is shown in SEQ ID NO.14; and / or, The amino acid sequence of P2A is shown in SEQ ID NO.15, and the base sequence of 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 base sequence of the CXCL16 is shown in SEQ ID NO.18.

[0014] Specifically, in the method for preparing the chemokine-secreting CAR-NK cells, in the step (2), the lentiviral expression plasmid includes pLV-IRES-PuroR; Preferably, the lentiviral expression plasmid further comprises auxiliary plasmids pSPAX2 and / or pMD2.G; Preferably, the CAR sequence is inserted between the NotI and XhoI restriction sites on the lentiviral expression plasmid.

[0015] Specifically, in the method for preparing the chemokine-secreting CAR-NK cells, in the step (3), the transfected cells include human embryonic kidney cell lines; Preferably, the matrix cells include HEK293FT cells, which can effectively cooperate with lentiviral expression plasmids, especially pLV-IRES-PuroR plasmids to improve application performance.

[0016] Specifically, in the method for preparing the chemokine-secreting CAR-NK cells, in the step (3), the lentiviral packaging system comprises: the matrix cells, the shuttle plasmid, the pSPAX2 plasmid, the pMD2.G plasmid, CaCl2 solution and BBS solution; Preferably, the culture medium in the lentivirus packaging step comprises DMEM culture medium containing 6-10% FBS; Preferably, the culture conditions of the lentiviral packaging step include 35-40°C, 3-8% CO 2 Perform cell culture.

[0017] Specifically, in the method for preparing chemokine-secreting CAR-NK cells, in step (4), the NK cells are derived from peripheral blood.

[0018] Specifically, in the method for preparing the chemokine-secreting CAR-NK cells, in step (4), the culture medium of the transfection culture step includes GT-T551 H3 culture medium. As an exemplary embodiment, a commercial culture medium of Takara Biotech of Japan can be selected, which is a special culture system for NK cells; Preferably, the conditions of the transfection culture step include 35-40°C, 3-8% CO 2 Perform cell culture.

[0019] The present invention also discloses a chemokine-secreting CAR-NK cell prepared by the method, which is denoted as PDL1-CXCL16-CAR-NK cell.

[0020] The present invention also discloses a method for detecting the chemotactic effectiveness of the chemokine-secreting CAR-NK cells, comprising the steps of respectively taking T cells and the CAR-NK cells for in vitro culture, and respectively using Transwell experiment, flow cytometry and immunofluorescence staining experiment for detection.

[0021] Specifically, the method for detecting the chemotactic effectiveness of the chemokine-secreting CAR-NK cells is: 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, and the upper chamber was T cells cultured without serum. Based on the Transwell cell migration experiment, 1-10% gelatin coating was additionally performed, and crystal violet was used to stain the T cells in the lower layer of the Transwell membrane; The flow cytometry and immunofluorescence staining experiments were used to detect the culture fluid in the lower chamber of the Transwell and the residual T cells at the bottom of the well.

[0022] The present invention constructs a CAR-NK cell that can express CXCL16, namely, PDL1-CXCL16-CAR-NK cell, which secretes a cytokine CXCL16 that regulates the migration and transport of immune cells, has a chemotactic effect on T lymphocytes, and enhances the recruitment ability of T cells through the expression of the chemokine CXCL16, thereby better improving the killing effect.

[0023] In order to verify the tropism effectiveness of the CAR-NK cells expressing CXCL16, the present invention cultured T cells and PDL1-CXCL16-CAR-NK cells in vitro, took the chemokine-containing culture medium and added it to the lower chamber of Transwell at the peak of CXCL16 secretion, and the upper chamber was serum-free cultured T cells. Based on the Transwell cell migration experiment, an additional 1-10% gelatin coating treatment was performed. Crystal violet was used to stain the T cells in the lower layer of the Transwell membrane. Flow cytometry and immunofluorescence staining were used to detect the culture fluid in the lower chamber and the residual T cells at the bottom of the well. The effect of chemokine CXCL16 secreting CAR-NK cells on the chemotaxis of T cells was more comprehensively analyzed, and the chemotaxis of chemokine secreting CAR-NK cells on T cells with CXCL16 chemokine performance could be more comprehensively characterized and verified.

[0024] The present invention enhances the recruitment of T cells by constructing CAR-NK cells expressing CXCL16, and better simulates the extracellular matrix environment through a modified Transwell migration experiment, while assisting in detecting the number of T cell migration by methods such as immunofluorescence staining experiments and flow cytometry experiments, thereby assisting in evaluating the migration ability of cells in vivo, and comprehensively verifying the chemotactic effect of CXCL16 on T cells.

[0025] The present invention improves the Transwell migration experiment and further coats the membrane with gelatin, which better simulates the in vivo environment and can more obviously reflect the invasive ability of T cells. The common transwell method only detects the number of cells under the membrane, and many cells will enter the culture fluid in the lower chamber and even adhere to the bottom of the culture well. The present invention adds the detection of the number of cells in the culture fluid in the lower chamber and the bottom of the well on the basic transwell method, which more truly reflects the changes of cxcl16 on the invasive ability of T cells, and its verification effect is better than that of the traditional method.

[0026] The method for detecting the chemotactic effectiveness of the chemokine-secreting CAR-NK cells described in the present invention is optimized in the Transwell cell migration experiment and covered with 1-10% gelatin, which can better mimic the extracellular matrix environment and detect the migration ability of cells. The migration of T cells requires the removal of the hindrance of gelatin to movement, so as to pass through the upper layer of gelatin in the chamber and then through the porous membrane of the chamber to achieve migration from the top chamber to the bottom.

[0027] The method for detecting the effectiveness of chemotactic factors secreting CAR-NK cells of the present invention is supplemented by flow cytometry to detect the residual T cells in the supernatant of the lower chamber due to the long migration time; at the same time, the experiment is supplemented by immunofluorescence staining to detect the residual T cells in the bottom of the lower chamber due to the 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 effect of chemokines on T cell chemotaxis in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein: Figure 1 (A) 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 PDL1-CXCL16-CAR design structure; (D) is a schematic diagram of the PDL1-CAR design 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 to the CAR structure through the P2A cleavage peptide; Figure 2 The plasmid construction process and verification results of the present invention; wherein (A) is a schematic diagram of the construction process, that is, the synthesized CAR sequence is inserted between the NotI and XhoI restriction sites on the pLV-IRES-PuroR plasmid; (B) is a diagram of double restriction digestion of the constructed plasmid using NotI and XhoI and agarose gel electrophoresis detection, the length of PDL1-CAR is 2838bp, the length of PDL1-CXCL16-CAR sequence is 3603bp, and the length of pLV-IRES-PuroR plasmid is 8081bp; Figure 3 The results of flow cytometry, where the horizontal axis represents the expression of G4S, the vertical axis represents the total number of cells detected, the red peaks are cells without antibody incubation, and the blue peaks are cells incubated with antibodies; Figure 4 It is the standard curve for CXCL16 content determination; Figure 5This is the crystal violet staining result; Figure 6 The results are measured by flow cytometry; Figure 7 Statistics for flow cytometry results; Figure 8 For immunofluorescence staining; Fig. 9 The positive area of ​​immunofluorescence staining was counted. DETAILED DESCRIPTION

[0029] In the following examples of the present invention, CAR-NK cells expressing CXCL16 are first constructed, and T cells and PDL1-CXCL16-CAR-NK cells are cultured in vitro, respectively. At the peak of CXCL16 secretion, the culture medium containing chemokines is added to the lower chamber of Transwell, and the upper chamber is T cells cultured without serum. Based on the Transwell cell migration experiment, 1-10% gelatin coating treatment is additionally performed. While crystal violet staining the T cells in the lower layer of the Transwell membrane, flow cytometry and immunofluorescence staining are used to detect the culture fluid in the lower chamber and the residual T cells at the bottom of the well, and the effect of chemokine CXCL16-secreting CAR-NK cells on T cell chemotaxis is more comprehensively analyzed.

[0030] Example 1 In this example, CAR-NK cells expressing CXCL16 and PDL1-scFv were constructed.

[0031] Plasmid construction In this example, PDL1-CAR and PDL1-CXCL16-CAR were constructed.

[0032] like Figure 1 (A) is a schematic diagram of the PDL1-CAR structure, (B) is a schematic diagram of the PDL1-CXCL16-CAR structure, and (C) is a schematic diagram of the PDL1-CAR design structure. 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 to the CAR structure through the P2A cleavage peptide; (D) is a schematic diagram of the PDL1-CXCL16-CAR design structure.

[0033] In this example, the PDL1 scFv-CD8α-41BB-DAP12-P2A-CXCL16 base sequence was synthesized based on the whole gene, the nucleotide sequence of which is shown in SEQ ID NO.2, and the amino acid sequence encoded by it is SEQ ID NO.1.

[0034] The PDL1 scFv-CD8α-41BB-DAP12-P2A-CXCL16 was further constructed into the lentiviral expression vector pLV-IRES-PuroR plasmid as a shuttle plasmid, and the lentivirus expressing CXCL16 and PDL1-scFv was 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 was constructed as a control, and its corresponding lentivirus was named P-LV.

[0035] The PDL1 scFv-CD8α-41BB-DAP12- P2A The amino acid sequence of -CXCL16 is as follows (SEQ ID NO.1): MKHLWFFLLLVAAPRWVLSEVKLQESGPSLVRPGASVKISCKASAYSFTSYWMHWVKQRPGQGLDWIG MIDPSDSEARLNQKFKDKATLTVDKSSSTTYIHLSSPTSEDSAVYYCARSYGYDGDYYLDVWGAGTTVTVSSGGGS GGGSGGGSGGGSDIVMTQSPSSSLSASLGGKVTITCKASQDINKYIAWYQHKPGKGPRLLIQYTSTLQPGIPSRFSG SGSGRDYSFSISNLEPEDIATYYCLQYDYLRTFGGGTKLEIKKL AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCEL RLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKSPGG ATNFSLLKQAGDVEENPGP MGRDLRPGSRVLLLLLLLLLVYLTQPGNGNEGSVTGSCYCGKRISSDSPPSVQFMNRLRKHLRAYHRCLYYTRFQL LSWSVCGGNKDPWVQELMSCLDLKECGHAYSGIVAHQKHLLPTSPPISQASEGASSDIHTPAQMLLSTLQSTQRPT LPVGSLSSDKELTRPNETTIHTAGHSLAVGPEAGENQKQPEKNAGPTARTSATVPVLCLLAIIFILTAAPSYVLCK RRRGQSPQSSPDLPVHYIPVAPDSNT .

[0036] The base sequence of PDL1 scFv-CD8α-41BB-DAP12-P2A-CXCL16 synthesized in this example is as follows (SEQ ID NO. 2):

[0037] In this example, the component information involved in the construction of PDL1 scFv-CD8α-41BB-DAP12-P2A-CXCL16 is as follows.

[0038] Anti-PD-L1-scFv VH amino acid sequence (SEQ ID NO.3): MKHLWFFLLLVAAPRWVLSEVKLQESGPSLVRPGASVKISCKASAYSFTSYWMHWVKQRPGQGLDWIGMIDPSDSEARLNQKFKDKATLTVDKSSSTTYIHLSSPTSEDSAVYYCARSYGYDGDYYLDVWGAGTTVTVSS.

[0039] Anti-PD-L1-scFv VH base sequence (SEQ ID NO.4):ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCCCCCAGATGGGTGCTGTCCGAGGTGAAGCTGCAGGAGTCAGGACCTAGCCTGGTTAGGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGCTTACTCATTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGTCTTGACTGGATTGGCATG ATTGATCCTTCCGACAGTGAAGCTAGTTAAATCAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGTACAACCTACATTCACCTCAGCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCTTATGGTTACGACGGGGACTACTACCTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA.

[0040] (G3S) 4 Linker amino acid sequence (SEQ ID NO.5): GGGSGGGSGGGSGGGS.

[0041] (G3S) 4 Linker base sequence (SEQ ID NO.6): GGCGGCGGCAGCGGCGGCGGCTCTGGAGGAGGATCTGGCGGCGGAAGC.

[0042] Anti CD19 scFv VL amino acid sequence (SEQ ID NO.7): DIVMTQSPSSLSASLGGKVTITCKASQDINKYIAWYQHKPGKGPRLLIQYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDYLRTFGGGTKLEIKKL.

[0043] Anti CD19 scFv VL base sequence (SEQ ID NO.8): GATATTGTGATGACCCAGTCTCCATCCTCACTGTCTGCATCTCTGGGAGGCAAAGTCACCATCACTTGCAAGGCAAGCCAAGACATTAACAAATATATAGCTTGGTACCAACACAAGCCTGGAAAAGGTCCTAGGCTGCTCATACAGTACACATCTACATTACAGCCAGGCATCCCATCAAGGTTCAGTGGAAGTGGGTCTGGGAGAGATTATTCCTTCAGCATCAGCAACCTGGAGCCTGAAGATATTGCAACTTATTATTGTCTACAGTATGATTATCTTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAAAGCTT.

[0044] CD8α Hinge amino acid sequence (SEQ ID NO.9): AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY.

[0045] CD8α Hinge base sequence (SEQ ID NO.10): GCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTAC.

[0046] 41BB amino acid sequence (SEQ ID NO.11): KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0047] 41BB base sequence (SEQ ID NO. 12): AAACGGGGCAGAAAGAAACTCCTGTATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG.

[0048] DAP12 amino acid sequence (SEQ ID NO. 13): RLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKSPGG.

[0049] DAP12 base sequence (SEQ ID NO. 14): CGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAATCCCCCGGGGGA.

[0050] P2A amino acid sequence (SEQ ID NO.15): ATNFSLLKQAGDVEENPGP.

[0051] P2A base sequence (SEQ ID NO. 16): GCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCG.

[0052] CXCL16 Amino Acid Sequence (SEQ ID NO.17): MGRDLRPGSRVLLLLLLLLLVYLTQPGNGNEGSVTGSCYCGKRISSDSPPSVQFMNRLRKHLRAYHRCLYYTRFQLLSWSVCGGNKDPWVQELMSCLDLKECGHAYSGIVAHQKHLLPTSPPISQASEGASSDIHTPAQMLLSTLQSTQRPTLPVGSLSSDKELTRPNETTIHTAGHSLAVGPEAGENQKQPEKNAGPTARTSATVPVLCLLAIIFILTAAPSYVLCKRRRGQSPQSSPDLPVHYIPVAPDSNT。

[0053] CXCL16 base sequence (SEQ ID NO.18): ATGGGACGGGACTTGCGGCCCGGGTCCCGCGTGCTCCTGCTCCTGCTTCTGCTCCTGCTGGTGTACCTGACTCAGCCAGGCAATGGCAACGAGGGCAGCGTCACTGGAAGTTGTTATTGTGGTAAAAGAATTTCTTCCGACTCCCCGCCATCGGTTCAGTTCATGAATCGTCTCCGGAAACACCTGAGAGCTTACCATCGGTGTCTATACTACACGAGGTTCCAGCTCCTTTCCTGGAGCGTGTGTGGAGGCAACAAGGACCCATGGGTTCAGGAATTGATGAGCTGTCTTGATCTCAAAGAATGTGGACATGCTTACTCGGGGATTGTGGCCCACCAGAAGCATTTACTTCCTACCAGCCCCCCAATTTCTCAGGCCTCAGAGGGGGCATCTTCAGATATCCACACCCCTGCCCAGATGCTCCTGTCCACCTTGCAGTCCACTCAGCGCCCCACCCTCCCAGTAGGATCACTGTCCTCGGACAAAGAGCTCACTCGTCCCAATGAAACCACCATTCACACTGCGGGCCACAGTCTGGCAGTTGGGCCTGAGGCTGGGGAGAACCAGAAGCAGCCGGAAAAAAATGCTGGTCCCACAGCCAGGACATCAGCCACAGTGCCGGTCCTGTGCCTCCTGGCCATCATCTTCATCCTCACCGCAGCCCCTTCCTATGTGCTGTGCAAGAGGAGGAGGGGGCAGTCACCGCAGTCCTCTCCAGATCTGCCGGTTCATTATATACCTGTGGCACCTGACTCTAATACCTGA。

[0054] Plasmid construction verification In this example, the plasmid constructed above was verified, and the results are shown in the appendix Figure 2 as follows.

[0055] As shown in the appendix Figure 2(A) is a schematic diagram of the construction process, in which the synthesized CAR sequence is inserted into the middle of the NotI and XhoI restriction sites on the lentiviral expression plasmid (a three-plasmid system is used in this example, in which the backbone plasmid is pLV-IRES-PuroR and the auxiliary plasmids are pSPAX2 and pMD2.G); (B) is a double restriction digestion of the constructed plasmid using NotI and XhoI and agarose gel electrophoresis detection diagram, PDL1-CAR is 2838bp in length, PDL1-CXCL16-CAR sequence is 3603bp in length, and pLV-IRES-PuroR plasmid is 8081bp in length. It can be seen that the plasmid required in this example was successfully constructed.

[0056] Example 2 Lentiviral packaging The stored HEK293FT cells were taken out from liquid nitrogen and quickly placed in a 37°C water bath for rapid thawing.

[0057] The cells in the cryopreservation solution were added to 10 mL of DMEM complete medium containing 10% FBS and centrifuged at 300 g for 5 min at room temperature.

[0058] Discard the supernatant, resuspend the pellet in 2 mL of DMEM complete medium containing 10% FBS, and add it to a 10 cm 2 The cell culture dish was placed in a 37°C, 5% CO 2 Cell culture incubator.

[0059] When the cell confluence reached 80% under a microscope, the culture medium in the dish was discarded and replaced with DMEM culture medium containing 6% FBS without antibiotics, and the dish was placed in a 37°C, 5% CO2 cell culture incubator for 1 hour.

[0060] Take a clean sterile 1.5 mL EP tube and add 781 μL of Cacl 2 Solution, 10 μg shuttle plasmid, 7.5 μg pSPAX2 plasmid, 2.5 μg pMD2.G plasmid were added to Cacl 2 solution, mix well.

[0061] Take a clean sterile 1.5 mL EP tube, add 781 μL of BBS solution into it, mix the mixture from the previous step with the BBS solution, and let it stand at room temperature for 20 minutes.

[0062] Cacl 2- The plasmid-BBS mixed solution was evenly added to the 10cm 2 The cell culture dish was placed in a 37°C, 5% CO2 cell culture incubator for 12 h.

[0063] After 12 h, the culture medium was replaced with 30 mL of DMEM complete culture medium containing 10% FBS and placed in a 37°C, 5% CO2 cell culture incubator for 48 h.

[0064] The culture supernatant was harvested and centrifuged at 4000 g for 10 min at 4°C.

[0065] The supernatant was filtered with a 0.45 μm filter membrane, placed in an ultra-high speed centrifuge, and centrifuged at 4°C, 25,000 rpm for 120 min. The supernatant was discarded and the precipitate was resuspended with 200 μL PBS to obtain the lentivirus concentrate.

[0066] Lentiviral titer detection 293FT cells were inoculated in 24-well plates and cultured overnight. 1, 2, and 4 μL of lentiviral solution were added for infection, and 1 mg / mL polybrene was added to promote infection. After 48 hours, 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: lentiviral titer (TU / mL) = 293FT cell number × positive cell percentage × 10 3 / Virus liquid volume (μL), the P / S-LV titer harvested this time was 1.8x10 8 TU / mL, P-LV titer was 1.2×10 8 TU / mL.

[0067] Example 3 Isolation of peripheral blood NK cells 4 mL of peripheral whole blood was collected from the subjects using EDTA anticoagulant tubes and NK cells were isolated from the peripheral blood using the MACSxpress Whole Blood NK Cell Isolation Kit from Miltenyi, Germany, within 4 hours. The specific steps are as follows: 1) Separation buffer preparation: dilute MACS BSA stock solution: auto MACS Rinsing solution at 1:20 and precool at 4°C; 2) Take a 15 mL centrifuge tube, add 1.5 mL of peripheral blood sample to the bottom of the tube, take 75 μL of Miltenyi Whole Blood CD56 magnetic beads into the blood, vortex for 2-3 seconds to mix, and incubate at 4°C for 15 minutes; 3) Add 8 mL of Separation buffer, mix well, and centrifuge at 445g for 10 min at room temperature; 4) Discard the supernatant and add 3 mL of Separation buffer; 5) Balance the Whole Blood Column: Fix the column on the magnetic stand and add 3 mL of Separation buffer; 6) After the liquid in the column stops dripping, quickly add the labeled cell suspension and discard the column liquid; 7) After the cell suspension stops dripping, quickly add 3 mL of Separation buffer and discard the column fluid; 8) Repeat step 7) twice. When 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 propeller to push out the cell suspension in the column at a uniform speed. 9) Centrifuge at room temperature and 2000 rpm for 10 min, discard the supernatant, and resuspend the cells to 3x10 5 cells / mL.

[0068] Example 4 This example prepares CAR-NK based on the scheme in Example 1.

[0069] Take a new sterile 24-well plate and inoculate NK cells at a density of 3 × 10 cells per well. 5 cells / mL, add polybrene to each well to a final concentration of 8 μg / mL, mix by pipetting, add 100 μL of lentivirus concentrate, and place in a 37°C, 5% CO 2 Incubate in a cell culture incubator for 12 h.

[0070] After culturing for 12 h, add 2 mL of GT-T551 H3 medium to the wells and place in a 37 °C, 5% CO2 cell culture incubator for 24 h.

[0071] After culturing for 24 h, discard 2 mL of the culture medium, add 2 mL of fresh GT-T551 H3 culture medium, place in a 37°C, 5% CO2 cell culture incubator, and culture for 24 h.

[0072] After culturing for 24 hours, discard 2 mL of the culture medium, add 2 mL of fresh GT-T551 H3 culture medium, place in a 37°C, 5% CO2 cell culture incubator, and culture for 24 hours.

[0073] Cells were taken for flow cytometry detection, and the detection antibody was 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 flow cytometry test results are shown in the attached Figure 3, where the horizontal axis represents the expression of G4S, the vertical axis represents the total number of cells detected, the red peak represents cells without antibody incubation, and the blue peak represents cells incubated with antibody.

[0074] It can be seen that the CAR-NK cells in this example were successfully prepared.

[0075] Example 5 This example is used to examine the chemotactic effect of CXCL16 on T cells.

[0076] Experimental Grouping The experiment in this embodiment was divided into four groups: Group 1 was treated with 1-10% gelatin coating on Transwell, and PDL1-CXCL16-CAR-NK was selected for chemokine-secreting cells; Group 2 was treated with 1-10% gelatin coating on Transwell, and PDL1-CAR-NK was selected for chemokine-secreting cells; Group 3 was untreated Transwell, and PDL1-CXCL16-CAR-NK was selected for chemokine-secreting cells; Group 4 was untreated Transwell, and PDL1-CAR-NK was selected for chemokine-secreting cells; and Groups 3 and 4 were not subjected to flow cytometry detection and immunofluorescence staining data statistics, and all other conditions were the same.

[0077] T cell culture in vitro This experimental example protocol was performed under sterile conditions.

[0078] On day 0 of the experiment, each group received 2.5 × 10 5 PBMCs were inoculated into a single well of a 48-well plate; T cells were cultured until the 6th day and divided into wells at appropriate density; on the 7th day of the experiment, T cells in each group were cultured in serum-free medium.

[0079] Transwell Membrane Pretreatment This experimental example protocol was performed under sterile conditions.

[0080] On the 7th day of the experiment, the Transwell membrane was coated with 1-10% gelatin in group 1 and group 2, and no pretreatment was done in groups 3 and 4. All four groups were kept at 4°C overnight.

[0081] CXCL16 expression detection This experimental example protocol was performed under sterile conditions.

[0082] On the 8th day of the experiment, the secretion of chemokine CXCL16 in each group was detected by ELISA.

[0083] In this example, the standard curve for CXCL16 content determination is shown in the attached Figure 4 The expression of CXCL16, i.e., the concentration of CXCL16, is shown in Table 1 below.

[0084] Table 1 CXCL16 concentration table

[0085] As shown in the results in Table 1 , the secretion of chemokine CXCL16 by PDL1-CAR-NK in groups 2 and 4 was lower than that by PDL1-CXCL16-CAR-NK in groups 1 and 3.

[0086] Improved Transwell cell migration assay This experimental example protocol was performed under sterile conditions.

[0087] On the eighth day of the experiment, the adhesive slide was placed in a single well of a 24-well plate, 1 mL of PDL1-CXCL16-CAR-NK cell suspension was added to well 1 of group 1, 1 mL of PDL1-CAR-NK cell suspension was added to well 2 of group 3, 1 mL of PDL1-CXCL16-CAR-NK cell suspension was added to well 4 of group 5, and 1 mL of PDL1-CAR-NK cell suspension was added to well 6 of group 7. The treated four groups of Transwell chambers were placed in the corresponding wells with tweezers. 100 μL of 5 × 10 6 T cell suspension of 100 cells / mL was added to the upper chamber and incubated at 37°C for 4 h.

[0088] Crystal violet staining The culture medium in the upper chamber was discarded, and four sets of Transwell chambers were taken out. The upper and lower layers of the membrane were washed with PBS for 3 times each, and the non-migrated cells in the upper chamber were gently wiped off with a moistened cotton swab.

[0089] Place the Transwell chamber into a new well, add 500 μL of paraformaldehyde to the lower chamber, place the chamber in a fixed position for 30 minutes, discard the paraformaldehyde, and air dry the chamber.

[0090] Add 500 μL of crystal violet solution to the lower chamber, place the chamber in the staining chamber for 10 minutes, discard the crystal violet solution, wash with PBS, and dry the chamber.

[0091] The chamber membrane was cut off with a blade, placed on a glass slide, sealed with neutral gum, and photographed under an upright microscope.

[0092] In this embodiment, the crystal violet staining results are shown in the attached Figure 5As shown. It can be seen that coating Transwell with 1-10% gelatin can better mimic the extracellular matrix environment and detect the migration ability of cells. The migration of T cells needs to remove the obstacles to movement caused by gelatin, pass through the upper gelatin of the chamber, and then pass through the porous membrane of the chamber to achieve migration from the top chamber to the bottom. Group 1 and Group 2 are obviously easier to count the differences in the amount of T cell migration, and can also more realistically evaluate the effect in vivo.

[0093] Flow cytometry All the culture medium in the lower chamber of Group 1 and Group 2 was aspirated for flow cytometry. The flow cytometry test results and statistical results are shown in the attached Figure 6-7 shown.

[0094] The results showed that more T cells remained in the supernatant of the lower chamber in the PDL1-CXCL16-CAR-NK group than in the PDL1-CAR-NK group.

[0095] Immunofluorescence staining The slides in the wells of Groups 1 and 2 were fixed with 4% paraformaldehyde and subjected to Dapi immunofluorescence staining. Photos were taken with an inverted fluorescence microscope, and the percentage of the fluorescent area was calculated.

[0096] In this experimental example, the immunofluorescence staining diagram and the statistical results of the immunofluorescence staining positive area are shown in the attached Figure 8-9 shown.

[0097] The results showed that more T cells remained at the bottom of the lower chamber in the PDL1-CXCL16-CAR-NK group than in the PDL1-CAR-NK group.

[0098] In summary, this study successfully established a method for preparing and detecting secretory chimeric antigen receptor NK cells that can chemotactic T cells.

[0099] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing chemokine-secreting CAR-NK cells, characterized in that: The steps include: (1) Full gene synthesis of CAR sequences that can express CXCL16 and PDL1-scFv; (2) inserting the CAR sequence into a lentiviral expression plasmid to obtain a shuttle plasmid; (3) taking stromal cells and the shuttle plasmid for lentiviral packaging to obtain lentivirus expressing CXCL16 and PDL1-scFv; (4) The separated NK cells are transfected and cultured with the lentivirus to obtain the desired CAR-NK cells expressing CXCL16 and PDL1-scFv.

2. The method for preparing chemokine-secreting CAR-NK cells according to claim 1, characterized in that: In step (1), the CAR sequence includes PDL1 scFv-CD8α-41BB-DAP12-P2A-CXCL16; wherein, 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 the P2A cleavage peptide.

3. The method for preparing chemokine-secreting CAR-NK cells according to claim 2, characterized in that: 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.

4. The method for preparing chemokine-secreting CAR-NK cells according to claim 3, 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; wherein, 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 CD19 scFv VL 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 SEQ ID NO.10; and / or, The amino acid sequence of 41BB is shown in SEQ ID NO.11, and the base sequence of 41BB is shown in SEQ ID NO.12; and / or, The amino acid sequence of DAP12 is shown in SEQ ID NO.13, and the base sequence of DAP12 is shown in SEQ ID NO.14; and / or, The amino acid sequence of P2A is shown in SEQ ID NO.15, and the base sequence of 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 base sequence of the CXCL16 is shown in SEQ ID NO.

18.

5. The method for preparing the chemokine-secreting CAR-NK cell according to any one of claims 1 to 4, characterized in that: In the step (2), the lentiviral expression plasmid includes pLV-IRES-PuroR; The lentiviral expression plasmid further comprises auxiliary plasmids pSPAX2 and / or pMD2.G; The CAR sequence is inserted between the NotI and XhoI restriction sites on the lentiviral expression plasmid.

6. The method for preparing chemokine-secreting CAR-NK cells according to claim 5, characterized in that: In step (3): The transfected cells include human embryonic kidney cell line; and / or, The lentiviral packaging system comprises: the matrix cells, the shuttle plasmid, the pSPAX2 plasmid, the pMD2.G plasmid, a CaCl2 solution and a BBS solution.

7. The method for preparing chemokine-secreting CAR-NK cells according to claim 6, characterized in that: In step (4): The NK cells are from peripheral blood; The culture medium in the transfection culture step includes GT-T551 H3 culture medium.

8. A chemokine-secreting CAR-NK cell prepared by the method according to any one of claims 1 to 7.

9. A method for detecting the chemotactic effectiveness of the chemokine-secreting CAR-NK cells according to claim 8, characterized in that: The method comprises the steps of respectively taking T cells and the CAR-NK cells according to claim 8 for in vitro culture, and respectively detecting them by using Transwell assay, flow cytometry and immunofluorescence staining assay.

10. The method for detecting the chemotactic effectiveness of the chemokine-secreting CAR-NK cells according to claim 9, characterized in that: 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, and the upper chamber was T cells cultured without serum. Based on the Transwell cell migration experiment, 1-10% gelatin coating was performed, and crystal violet was used to stain the T cells in the lower layer of the Transwell membrane; The flow cytometry and immunofluorescence staining experiments were used to detect the culture fluid in the lower chamber of the Transwell and the residual T cells at the bottom of the well.

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