Application of B16 cell in preparation of cell model for specifically evaluating CAR-NK (Chimeric Antigen Receptor-Natural Killer) efficiency
By using the B16 cell model, it is verified that it does not express NK cell activation ligands and is killed by CAR-NK cells after expressing the target, which solves the problem that the prior art cannot specifically evaluate the efficacy of CAR molecules on CAR-NK cells, and achieves the goal of achieving The specific activation and efficient killing effect of CAR-NK cells was achieved.
Patent Information
- Application Number
- CN202411397634.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-08
- Publication Date
- 2025-01-07
AI Technical Summary
The existing target cell model cannot effectively distinguish the effects of CAR molecule-independent killing and potential activated receptors on target cells of CAR-NK cells, resulting in the inability to specifically evaluate the unique role of CAR molecules on CAR-NK cells.
B16 cells were used as a cell model to verify that they did not express the NK cell-activated ligand ICAM-1 to ensure that they cannot be killed by NK cells, but after expressing the corresponding target, it can be specifically killed by CAR-NK cells, which proves that B16 cells are It is an ideal model for evaluating the efficacy of CAR molecules on CAR-NK cells.
After expressing human target proteins, B16 cells can specifically activate CAR-NK cells, promoting their activation marker expression, cell coupling, granule polarization, degranulation and cytokine secretion, and are efficiently killed by CAR-NK cells only. , validate it as an effective cellular model for specifically evaluating CAR-NK efficacy.
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Figure CN119265138A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of cell models, and particularly relates to the application of B16 cells in preparing a cell model for specifically evaluating the efficacy of CAR-NK. Background Art
[0002] NK cells retain their killing effect on most tumors. CAR-NK refers to loading chimeric antigen receptors on NK cells. CAR-NK can kill target cells in a CAR molecule-dependent and -independent manner. In theory, this can better overcome target loss and tumor heterogeneity. However, it is precisely because of this characteristic that general target cells cannot specifically evaluate the unique effect of CAR molecules on CAR-NK. For example, although some studies have confirmed that the killing effect of CAR-NK with γ chain as the intracellular segment is better than that of ζ chain, some studies have reached the opposite conclusion. By comparison, the latter selected Raji cells as target cells. Raj cells are a cell line sensitive to NK cells. Therefore, it is impossible to distinguish the effects of CAR molecule-independent killing and the interaction between potential activating receptors and CAR molecules on CAR-NK killing of target cells. Therefore, the selection of appropriate target cells is crucial for the specific evaluation of the unique effect of CAR molecules on CAR-NK.
[0003] Eric O.Long from the National Institutes of Health (NIH) and Professor Yenan Bryceson from the Karolinska University School of Medicine in Sweden used Drosophila Schneider 2 cells, a cell model that is not derived from mammals and does not express NK cell activating or inhibitory ligands, making it possible to study the individual and synergistic effects of NK cell activation receptors. Considering the accessibility and ease of operation of cell models, it is believed that the ideal cell model for specifically evaluating the unique effects of CAR molecules on CAR-NK should have the following two characteristics: non-human, no expression of NK cell activating ligands, and cannot be killed by NK cells. According to these two conditions, the mouse melanoma B16 may be an ideal cell model. Current studies have shown that B16 hardly expresses NK cell activating ligands, and the only known mouse ligand that can be recognized by human NK cells is ICAM-1. Therefore, if in vitro experiments prove that B16 does not express ICAM-1 and is not killed by NK cells, but can be specifically killed by CAR-NK after expressing the corresponding targets, it can be proved that B16 is a cell model for specifically evaluating the unique effects of CAR molecules on CAR-NK. Summary of the invention
[0004] The present invention provides the use of B16 cells in preparing a cell model for specifically evaluating the efficacy of CAR-NK. B16 cells cannot be killed by NK cells, but can be killed by CAR-NK after expressing the corresponding target, proving that B16 is a cell model for specifically evaluating the unique effect of CAR molecules on CAR-NK.
[0005] The present invention provides the use of cells in preparing a cell model for specifically evaluating the efficacy of CAR-NK, wherein the cells are non-human and do not express NK cell activating ligands.
[0006] In a specific embodiment of the present invention, the NK cell activating ligand comprises ICAM-1.
[0007] In a specific embodiment of the present invention, the cells include murine melanoma B16 cells.
[0008] Beneficial effects: The present invention provides the use of cells in preparing a cell model for specifically evaluating the efficacy of CAR-NK, wherein the cells are non-human and do not express NK cell activating ligands; after the cells overexpress human target proteins, they can effectively promote CAR-NK to express activation markers, indicating that B16 cells overexpressing human target proteins can specifically activate CAR-NK. And the examples confirm that NK cells themselves or CAR-NK cannot kill B16 cells, while CAR-NK can efficiently kill B16 cells overexpressing human target proteins, indicating that B16 cells can be used as a cell model for specifically evaluating the efficacy of CAR-NK. BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Figure 1 Schematic diagram of co-culture for in vitro killing experiment;
[0010] Figure 2 Flow cytometry results to verify that B16 cells do not express ICAM-1;
[0011] Figure 3 Schematic diagram showing that B16 cells overexpressing CD19 can specifically promote the expression of CAR-NK activation marker CD69;
[0012] Figure 4 The figure shows the result of B16 cells overexpressing CD19 specifically coupling with CAR-NK cells;
[0013] Figure 5 This is a result diagram showing that B16 cells overexpressing CD19 can specifically promote the polarization of CAR-NK particles;
[0014] Figure 6 This is a result diagram showing that B16 cells overexpressing CD19 can specifically promote CAR-NK degranulation;
[0015] Figure 7 This is a result diagram showing that B16 cells overexpressing CD19 can be specifically killed by CAR-NK;
[0016] Figure 8 This is the result diagram showing that B16 cells overexpressing CD19 specifically promote CAR-NK to secrete cytokines;
[0017] Fig. 9 This is the result diagram showing that B16 can be specifically killed by CAR-NK after overexpressing B7H3 target protein. DETAILED DESCRIPTION
[0018] The present invention provides the use of cells in preparing a cell model for specifically evaluating the efficacy of CAR-NK, wherein the cells are non-human and do not express NK cell activating ligands.
[0019] In a specific embodiment of the present invention, the NK cell activating ligand includes ICAM-1, and the ICAM-1 is intercellular adhesion molecule-1. In a specific embodiment of the present invention, the cells include murine melanoma B16 cells. Currently known, the only murine ligand that can be recognized by human NK cells is ICAM-1. In one embodiment of the present invention, flow cytometry was used to verify that B16 cells do not express ICAM-1; compared with the negative control (Mock group), B16 cells overexpressing human CD19 protein can effectively promote CAR-NK to express the activation marker CD69, indicating that B16 cells overexpressing human CD19 protein can specifically activate CAR-NK; Mock group NK cells can hardly form cell coupling with B16 cells expressing human CD19 protein, while CAR-NK can efficiently bind to B16 cells expressing human CD19 protein. Form cell coupling; B16 cells overexpressing CD19 can specifically promote CAR-NK particle polarization, B16 cells overexpressing human CD19 protein cannot promote NK cell degranulation in the Mock group, but can significantly increase CAR-NK degranulation, indicating that B16 cells overexpressing CD19 can specifically promote CAR-NK degranulation; neither Mock group NK cells nor CAR-NK can kill B16, but only CAR-NK can efficiently kill B16 cells overexpressing human CD19 protein, indicating that CAR-NK can specifically kill B16 cells overexpressing CD19 target protein.
[0020] In another embodiment of the present invention, in order to illustrate that B16 cells as a cell model for specific evaluation of CAR-NK efficacy is independent of the overexpressed target protein, CAR-NK targeting B7H3 and a negative control (Mock) of the same vector were also constructed, and a B16 cell line overexpressing human B7H3 protein was constructed by lentiviral transfection. The killing experiment showed that neither NK-92MI cells themselves nor CAR-NK could kill B16, and only CAR-NK could efficiently kill B16 cells overexpressing human B7H3 protein, indicating that B16 can also be used as a cell model for specific evaluation of CAR-NK efficacy after overexpressing B7H3 target protein.
[0021] In summary, B16 cells can specifically activate CAR-NK after expressing human target protein, indicating that B16 cells can be used as a cell model to specifically evaluate the efficacy of CAR-NK.
[0022] In order to further illustrate the present invention, the application of B16 cells provided by the present invention in preparing a cell model for specifically evaluating the efficacy of CAR-NK is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.
[0023] Example 1
[0024] 1. Source and culture of B16 cells: B16 cells were purchased from Concord Cell Bank and cultured in 1640 complete medium (containing 10% serum and double antibody), adherent to the wall, and passaged by trypsin digestion.
[0025] Flow cytometry results Figure 2 As shown, B16 cells do not express ICAM-1, that is, B16 cells do not express NK cell activating ligands.
[0026] 2. Construction of B16 stable cells overexpressing a target protein: The stable cell line was constructed by lentiviral infection. The viral vector number was pHS-B-0085 (Beijing Hesheng Biotechnology Co., Ltd.), the element sequence was pLV-EF1a-hluc-P2A-Puro-CMV-MCS-3flag (Beijing Hesheng Biotechnology Co., Ltd.), and plasmid synthesis and virus packaging were completed by Beijing Hesheng Biotechnology Co., Ltd. The ratio of virus to cell number (MOI) during infection was 20. After 72 hours of transfection, the cells were selected with 2.0 μg / ml puromycin, and the expression of the target protein was verified by flow cytometry for two weeks.
[0027] 2.1 B16 cells overexpressing human CD19 protein can specifically activate CAR-NK
[0028] The NK-92MI cells were constructed as Figure 3The two CD19-targeting CAR-NKs and the negative control (Mock) of the same vector shown in a. To facilitate subsequent functional experiments, each CAR molecule carries green fluorescent protein GFP. Flow cytometry was used to verify the expression of CAR molecules ( Figure 3 b),
[0029] The specific construction process is as follows: Gene sequence codon optimization is performed using GenScript codon optimization software (https: / / www.genscript.com.cn / tools / gensmart-codon-optimization). After optimization, the gene sequence is sent to Anshengda for gene synthesis. The geneIDs involved are: CD8α (GenBank: NM_001145873.1), anti-CD19scFv (FMC63 monoclonal antibody), aCD28 (GenBank: AF222341.1), NKG2D (GenBank: AF461811.1), 2B4 (GenBank: AF107761.2), FCER1G (GenBank: NM_004106.2). MaxDNA Polymerase (Takara, Code No. R045Q) was used to obtain the relevant target gene fragment by PCR, and after NheI / XhoI double enzyme digestion, it was connected to the plasmid vector pIRES-EGFP-puro (Addgene, #45567). After the CAR molecular screening plasmid was constructed, the plasmid was extracted with a midi extraction kit (ZymoPUREIIPlasmidMidiprepKitCat#D4201) for use.
[0030] Take NK-92MI cells with good growth status, collect cells by centrifugation, resuspend cells with celetrix special electroporation solution 103buffer (partA: partB = 1:1), add CAR molecular screening plasmid to electroporate cells. Electroporation conditions: 100ul electroporation tube, 10M cells, CAR molecular screening plasmid 8ug, voltage cellline430V, electroporation solution 103buffer, electroporator model CeletrixCellElectroporatorModelCTX-1500ALE+. The electroporation operation was carried out according to the celetrix electroporator manual. After electroporation, the cells were transferred to 1 well of a 6-well plate for culture, and 3ml of NK-92MI cell special culture medium (Procell, CAT#CM-0533) was added to each well for culture. After electroporation, the cells were transferred to a 6-well plate, grown in a 37°C incubator for 3 days, and then cultured in separate wells. The cells in a single well of the 6-well plate were filled with about 80%, and 2.0μg / ml puromycin was added for selection. After two weeks, the flow cytometry was run to identify the positive rate.
[0031] At the same time, a B16 cell line overexpressing human CD19 protein was constructed by lentiviral transfection ( Figure 3 (c). Compared with Mock, B16 cells overexpressing human CD19 protein can effectively promote CAR-NK to express activation marker CD69 ( Figure 3 Middle d) indicates that B16 cells overexpressing human CD19 protein can specifically activate CAR-NK.
[0032] 2.2 B16 cells overexpressing human CD19 protein can couple with CAR-NK cells
[0033] NK cells kill target cells mainly through steps such as cell coupling formation, granule polarization, and degranulation, and then release perforin and granzymes. Cell coupling formation refers to the formation of a stable connection between NK cells and ligands on the surface of target cells through surface activation receptors. Cell coupling formation is the basis of granule polarization and degranulation. The most commonly used evaluation method for cell coupling formation is dual fluorescence flow cytometry detection. Mock and CAR-NK cells themselves carry green fluorescence, so this application uses live cell fluorescent dye (AAT Bioquest, 22622, PE channel) to dye B16 cells overexpressing human CD19 protein red. After co-culture, they are fixed with tissue fixative and flow cytometry. The double positivity of red and green represents cell coupling formation. The results are as follows: Figure 4 As shown, the NK cells in the Mock group could hardly form cell coupling with the B16 cells expressing the human CD19 protein, while the CAR-NK cells could efficiently form cell coupling with the B16 cells expressing the human CD19 protein.
[0034] 2.3 B16 cells overexpressing CD19 can specifically promote CAR-NK particle polarization
[0035] Granule polarization refers to the aggregation of perforin granzymes dispersed in the cytoplasm at the interface between NK cells and target cells after receiving activation signals. Granule polarization can mark the activation of NK cells and also prepare for efficient killing of target cells. After co-culture of Mock or CAR-NK with B16 cells overexpressing human CD19 protein, they were fixed with tissue fixative, membrane broken, blocked, and stained with fluorescent group-labeled perforin antibodies (Biolegend, 308124). Under a confocal microscope, it can be clearly seen that green is NK cells and perforin is red ( Figure 5 ), the perforin of NK cells in the Mock group was still scattered after co-culture, without particle polarization, while CAR-NK had different degrees of particle polarization, especially CAR10, where 126 out of 151 NK cells showed particle polarization, indicating that B16 cells overexpressing CD19 can specifically promote particle polarization of CAR-NK.
[0036] 2.4 B16 cells overexpressing CD19 can specifically promote CAR-NK degranulation
[0037] Degranulation refers to the process in which NK cells release perforin and granzyme in the cytoplasm after receiving activation signals. Degranulation more directly marks the activation of NK cells and is also the main means for NK cells to kill target cells. Currently, the most commonly used marker to evaluate degranulation is CD107a. After co-culture of Mock or CAR-NK with B16 cells overexpressing human CD19 protein, they were fixed with tissue fixative and stained with CD107a antibody labeled with fluorescent group (Biolegend, 328620). Degranulation was observed by flow cytometry. The results are as follows: Figure 6 As shown, B16 cells overexpressing human CD19 protein could not promote NK cell degranulation in the Mock group, but could significantly increase CAR-NK degranulation, indicating that B16 cells overexpressing CD19 could specifically promote CAR-NK degranulation.
[0038] 3. In vitro killing experiment: B16 cells and B16 stably transfected cells overexpressing a certain target protein were co-cultured with NK cells and CAR-NK, respectively. In vitro killing experiments were performed to verify that only B16 stably transfected cells expressing a certain target protein could be killed by CAR-NK. Figure 1 Co-cultivation was performed as described.
[0039] The results are as follows Figure 7 As shown, neither the Mock group NK cells nor the CAR-NK cells could kill B16, while only the CAR-NK cells could efficiently kill the B16 cells that overexpressed the human CD19 protein, indicating that the CAR-NK cells could specifically kill the B16 cells that overexpressed the CD19 target protein.
[0040] 4. In addition to directly killing target cells, cytokine secretion is also an important function of NK cells. After activation, NK cells can efficiently secrete cytokines to play an immunomodulatory role. B16 cells and B16 stable cells overexpressing CD19 target protein were co-cultured with NK cells and CAR-NK, respectively. The results showed that both Mock group NK cells and CAR-NK could not secrete cytokines after co-culture with B16 cells, while CAR-NK could secrete cytokine IFN-γ( Figure 8 ), indicating that B16 cells overexpressing CD19 can specifically promote CAR-NK to secrete cytokines.
[0041] 5. Using the same method as above, a CAR-NK targeting B7H3 and a negative control (Mock) of the same vector were constructed on NK-92MI cells ( Fig. 9In a), a B16 cell line overexpressing human B7H3 protein was constructed by lentiviral transfection ( Fig. 9 In middle b), the killing experiment showed that neither NK-92MI cells nor CAR-NK cells could kill B16 ( Fig. 9 c), while only CAR-NK can effectively kill B16 cells that overexpress human B7H3 protein ( Fig. 9 (d) in the middle shows that B16 can be used as a cell model to specifically evaluate the efficacy of CAR-NK after overexpressing the B7H3 target protein.
[0042] In summary, B16 cells can specifically activate CAR-NK after expressing human target protein, indicating that B16 cells can be used as a cell model to specifically evaluate the efficacy of CAR-NK.
[0043] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. Use of cells in preparing a cell model for specifically evaluating the efficacy of CAR-NK, characterized in that: The cells are non-human and do not express NK cell activating ligands.
2. The application according to claim 1, characterized in that: The NK cell activating ligand includes ICAM-1.
3. The application according to claim 1, characterized in that: The cells include murine melanoma B16 cells.