Journal: International Journal of Molecular Sciences

Article Title: A Novel MICB-Targeting CAR-NK Cells for the Treatment of Pancreatic Cancer

doi: 10.3390/ijms27010500

Figure Lengend Snippet: Rational design and construction of the Anti-MICB-CAR. ( A ) Schematic of the Anti-MICB chimeric antigen receptor (Anti-MICB-CAR). ( B ) The plasmid structure of the Anti-MICB chimeric antigen receptor lentiviral vector. ( C ) The expression of Anti-MICB-CAR in NK cells was analyzed via flow cytometry. ( D ) The secretion of IL-15 by Anti-MICB-CAR-NK cells was detected by ELISA. NC: supernatant of 1 × 10 6 NK cells, Anti-MICB–CAR–NK: supernatant of 1 × 10 6 Anti-MICB-CAR-NK cells. Supernatants of Anti-MICB-CAR-NK cells were significantly elevated up to 48.88 pg/mL compared to the control NK cell supernatants. p = 0.0024. ( E ) The supernatant of 1 × 10 6 Anti-MICB-CAR-NK cells was filtered and incubated with PANC-1 for 1 h. MICB Protein hFc and IgG1 Fc FITC antibody staining were performed and free Anti-MICB-scFv was detected by flow cytometry. Statistical analysis was performed using a two-tailed Student’s t -test; ** p < 0.01.

Article Snippet: 10 6 NK cells centrifugation, resuspend in 1 mL PBS, 20 μL of serum for 45 min, 20 μL of anti-human CD56 Antibody APC (Biolegend, Cat: 362503, San Diego, CA, USA), and 20 μL of FITC anti-human CD3 antibody (Biolegend, Cat: 300305, San Diego, CA, USA), and incubate in the dark for 1.5 h. Anti-MICB-CAR expression on gene-modified NK cells using flow cytometry analyzed with an Anti-MICB-CAR Detection Reagent, containing a recombinantly expressed fusion protein consisting of the human MICB extracellular domains and a specifically mutated human lgG1 Fc region (Sino Biological, Recombinant Human MICB Protein-His & hFc Tag, Cat:10759-H03H, Beijing, China) and secondary addition of anti-Human IgG1 Fc-FITC-antibody (Invitrogen, anti-Human IgG1 Fc Secondary Antibody, Cat: A-10631, Carlsbad, CA, USA).

Techniques: Plasmid Preparation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control, Incubation, Staining, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: A Novel MICB-Targeting CAR-NK Cells for the Treatment of Pancreatic Cancer

doi: 10.3390/ijms27010500

Figure Lengend Snippet: Anti-MICB-CAR-NK cells demonstrate potent in vitro activity against tumors with high expression of MICB. ( A ) Western blot detection of MICB expression in PANC-1, A549, HepG2, BxPC-3, and AsPC-1 human tumor cell lines. ( B ) The qPCR results showed in the MICB gene expression compared to the five tumor cell lines. ( C ) The Effect-to-Target Ratio (E/T Ratio) of Anti-MICB-CAR-NK cells on tumor cell PANC-1. ( D ) The mortality rate of five types of tumor cells treated with Anti-MICB-CAR-NK cells after 24 h with CCK-8 Assay, PANC-1(71.37%), A549(31.78%), HepG2(53.83%), BxPC-3(62.26%), and AsPC-1(39.86%), E/T Ratio 1:1. ( E ) Anti-MICB-CAR-NK treatment of AsPC-1 and PANC–1 tumor cells with differential MICB expression for 24 h by flow cytometry, E/T Ratio 1:1. The results showed that the viability of AsPC-1 was 68.9% and the viability of PANC-1 was 54.6%. ( F – H ) PANC-1, HepG2, and A549 tumor cells treated with NK cells, Anti-MICB-CAR-NK cells, and Anti-MICB-CAR-NK supernatant + NK cells for 24 h, E/T Ratio 1:1; CCK8 detects mortality rate. Anti-MICB-CAR-NK cells significantly enhanced the anti-tumor ability compared to NK cells. PANC-1: 68.37%, p = 0.0005. HepG2: 57.3%, p = 0.0027. A549: 33.18%, p = 0.0342. Supernatant of Anti-MICB-CAR-NK cells co-treated tumor cells with NK cells also promoted tumor cell killing compared to NK. PANC-1: 57.66%, p = 0.0006. HepG2: 48.21%, p = 0.0037. A549: 31.55%, p = 0.0446. ( I ) NC-NK cells, non-transduced NK(NT-NK), NT-NK supernatant + NK cells, Anti-MICB-CAR-NK supernatant + NK cells, Anti-MICB-CAR-NK cells treated with PANC-1 tumor cells for 24 h, E/T Ratio 1:1. The viability of each group was detected by flow cytometry and the results showed that NK cells: 89.4%, non-transduced NK(NT-NK): 86.6%, NT-NK supernatant + NK cells: 88.7%, Anti-MICB-CAR-NK supernatant + NK cells: 62.6%, and Anti-MICB-CAR-NK cells: 55.5%. Statistical analysis was performed using a two-tailed Student’s t -test; * p < 0.05, *** p < 0.001.

Article Snippet: 10 6 NK cells centrifugation, resuspend in 1 mL PBS, 20 μL of serum for 45 min, 20 μL of anti-human CD56 Antibody APC (Biolegend, Cat: 362503, San Diego, CA, USA), and 20 μL of FITC anti-human CD3 antibody (Biolegend, Cat: 300305, San Diego, CA, USA), and incubate in the dark for 1.5 h. Anti-MICB-CAR expression on gene-modified NK cells using flow cytometry analyzed with an Anti-MICB-CAR Detection Reagent, containing a recombinantly expressed fusion protein consisting of the human MICB extracellular domains and a specifically mutated human lgG1 Fc region (Sino Biological, Recombinant Human MICB Protein-His & hFc Tag, Cat:10759-H03H, Beijing, China) and secondary addition of anti-Human IgG1 Fc-FITC-antibody (Invitrogen, anti-Human IgG1 Fc Secondary Antibody, Cat: A-10631, Carlsbad, CA, USA).

Techniques: In Vitro, Activity Assay, Expressing, Western Blot, Gene Expression, CCK-8 Assay, Flow Cytometry, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: A Novel MICB-Targeting CAR-NK Cells for the Treatment of Pancreatic Cancer

doi: 10.3390/ijms27010500

Figure Lengend Snippet: The anti-tumor mechanism induced by Anti-MICB-CAR-NK cells. ( A – D ) The release of perforin, granzyme B, TNF-α, and IFN-γ from PANC-1, HepG2, and A549 cells. NK cells and Anti-MICB-CAR-NK cells were treated with tumor cells for 24 h, with an E/T ratio of 1:1. Control represents complete medium cultivation. The supernatant was collected and detected by ELISA. Compared to the NK group, the Anti-MICB-CAR-NK group exhibited significantly higher levels of TNF-α expression (PANC-1, p = 0.0072; AsPC-1, p = 0.035; HepG2, p = 0.0076; A549, p = 0.0066) and IFN-γ expression (PANC-1, p = 0.0072; AsPC-1, p = 0.0162; HepG2, p = 0.002; A549, p = 0.0006) following treatment with PANC-1, AsPC-1, HepG2, and A549 tumor cells. Similarly, Granzyme B expression levels were significantly elevated in the Anti-MICB-CAR-NK group (PANC-1, p = 0.0007; HepG2, p = 0.0023; A549, p = 0.0004), as were Perforin expression levels (PANC-1, p = 0.0021; HepG2, p = 0.042; A549, p = 0.0022) after treatment with PANC-1, HepG2, and A549 tumor cells. Additionally, the Anti-MICB-CAR-NK group demonstrated significantly increased IL-15 expression following treatment with PANC-1 and A549 tumor cells (PANC-1, p = 0.0012; A549, p = 0.0025) compared to the NK group. ( E , F ) Elisa and flow cytometry were used to detect the levels of IL-15 and Anti-MICB-scFv in the tumor cells’ supernatants treated with Anti-MICB-CAR-NK cells for 24 h. ( G ) Study on MICB expression in PANC-1 treated with Anti-MICB-CAR-NK cells for 24 h using Western blot technique. Statistical analysis was performed using a two-tailed Student’s t -test; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: 10 6 NK cells centrifugation, resuspend in 1 mL PBS, 20 μL of serum for 45 min, 20 μL of anti-human CD56 Antibody APC (Biolegend, Cat: 362503, San Diego, CA, USA), and 20 μL of FITC anti-human CD3 antibody (Biolegend, Cat: 300305, San Diego, CA, USA), and incubate in the dark for 1.5 h. Anti-MICB-CAR expression on gene-modified NK cells using flow cytometry analyzed with an Anti-MICB-CAR Detection Reagent, containing a recombinantly expressed fusion protein consisting of the human MICB extracellular domains and a specifically mutated human lgG1 Fc region (Sino Biological, Recombinant Human MICB Protein-His & hFc Tag, Cat:10759-H03H, Beijing, China) and secondary addition of anti-Human IgG1 Fc-FITC-antibody (Invitrogen, anti-Human IgG1 Fc Secondary Antibody, Cat: A-10631, Carlsbad, CA, USA).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Western Blot, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: A Novel MICB-Targeting CAR-NK Cells for the Treatment of Pancreatic Cancer

doi: 10.3390/ijms27010500

Figure Lengend Snippet: Anti-MICB-CAR-NK cells exhibit tumor regression in the PANC-1 Xenograft Model. ( A ) Flow chart of Anti-MICB-CAR-NK cells treatment for PANC-1 Transplanted Tumors. On the 0th day, PANC-1 cells were inoculated and on the 13th day, PBS, NK cells, and Anti-MICB-CAR-NK cells were injected into mice via tail vein at a dose of 5 × 10 6 cells per mouse. Record tumor growth observed every other day. ( B ) Growth curve of mouse transplanted tumor. ( C , D ) Resected tumors from each group were imaged and Weighed at the end of the experiment. NC: 834.31 ± 197.26 (mg), NK: 606.69 ± 141.37 (mg), Anti-MICB-CAR-NK: 447.32 ± 136.13 (mg). ( E ) Anti-tumor rate of Anti-MICB-CAR-NK cells against PANC-1 transplanted tumors. The tumor inhibition rate of NK compared to NC was 27.28%, p = 0.07. The tumor inhibition rate of Anti-MICB-CAR-NK compared to NC was 46.38%, p = 0.007. ( F ) Mouse serum was collected at the end of the experiment and the amounts of IL-15, IFN-γ, and TNF-α were measured by ELISA. The IL-15, IFN—γ, and TNF—α in the Anti-MICB-CAR-NK group were significantly increased compared to the NK group, with a p -value of 0.0013 for IL-15, 0.013 for IFN—γ, and 0.003 for TNF—α. ( G , H ) Mouse tumor tissues were analyzed by immunohistochemistry for MICB expression and NK cell infiltration within the tumor tissue. Statistical analysis was performed using a two-tailed Student’s t -test; * p < 0.05, ** p < 0.01.

Article Snippet: 10 6 NK cells centrifugation, resuspend in 1 mL PBS, 20 μL of serum for 45 min, 20 μL of anti-human CD56 Antibody APC (Biolegend, Cat: 362503, San Diego, CA, USA), and 20 μL of FITC anti-human CD3 antibody (Biolegend, Cat: 300305, San Diego, CA, USA), and incubate in the dark for 1.5 h. Anti-MICB-CAR expression on gene-modified NK cells using flow cytometry analyzed with an Anti-MICB-CAR Detection Reagent, containing a recombinantly expressed fusion protein consisting of the human MICB extracellular domains and a specifically mutated human lgG1 Fc region (Sino Biological, Recombinant Human MICB Protein-His & hFc Tag, Cat:10759-H03H, Beijing, China) and secondary addition of anti-Human IgG1 Fc-FITC-antibody (Invitrogen, anti-Human IgG1 Fc Secondary Antibody, Cat: A-10631, Carlsbad, CA, USA).

Techniques: Injection, Inhibition, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Expressing, Two Tailed Test

Journal: JCI Insight

Article Title: Balanced engagement of activating and inhibitory receptors mitigates human NK cell exhaustion

doi: 10.1172/jci.insight.150079

Figure Lengend Snippet: ( A ) Schematic representing the in vitro model of exhaustion. Agonists of NKp46 (anti-NKp46) and NKG2D (MICA and MICB) were adsorbed onto tissue culture plates and used to stimulate NK cells for 7 days. Plate-bound isotype IgG served as a control. Both groups received 1 ng/mL IL-15. ( B ) NK cells harvested from isotype-coated and exhaustion plates (day 7) were incubated with K-562 targets for 4 hours (E/T: 2:1). Cytokine production (IFN-γ and TNF-α) and degranulation (CD107a) were measured via flow cytometry. Isotype NK cells (top row) in blue, exhausted NK cells (bottom row) in red. E/T, effector/target. ( C – E ) Quantification of cytokine production and degranulation as percentage of parent population (live, CD3 – CD56 + cells) and mean fluorescence intensity (MFI) ( n = 4). Paired t tests were used for comparisons. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: A total of 300 μL of PBS containing 5 μg/mL anti-NKp46 (R&D Systems; catalog MAB1850), 1.25 μg/mL recombinant MICA Fc-chimera (R&D Systems; catalog 1300-MA), and 1.25 μg/mL recombinant MICB Fc-chimera (R&D Systems; catalog 1599-MB) was added to 24-well plates and incubated at 4°C overnight.

Techniques: In Vitro, Control, Incubation, Flow Cytometry, Fluorescence