car specific antibody  (Jackson Immuno)

Journal: International Journal of Molecular Sciences

Article Title: Anti-Her2 CAR-NK92 Cells and Their Exosomes: Generation, Characterization, and Selective Cytotoxicity Against Her2-Positive Tumor Cells

doi: 10.3390/ijms26157648

Figure Lengend Snippet: Comparative flow cytometric analysis between untransduced and transduced NK92 cells. ( A , B ). Gating strategy for identifying live cells (red polygon). ( C , D ) GFP expression in transduced cells is significantly higher compared to untransduced cells, suggestive for successful transfection. ( E ) Fluorescence intensity histograms showing significant differences between the two groups (37.33 ± 4.13 vs. 3395.66 ± 111.065 MFUs, p < 0.001, df = 4, t = 55.5200). ( F , G ). CAR expression analysis using anti-G4S linker antibodies revealed a significantly higher expression of G4S linker (from the CAR molecule) in the transduced cell population. ( H ) Fluorescence intensity histogram showing a statistically significant difference in fluorescence emission in the PerCP spectrum in the transduced cell population (32.67 ± 28.41 MFUs vs. 486 ± 63.16 MFUs, p = 0.002, df = 4, t = 7.0600). ( I , J ). Anti-kappa light chain expression was significantly higher in the transduced cell population, with statistically significant differences as observed in the histogram (( K ), 13,355.5 ± 832.958 MFUs vs. 5473.5 ± 144.828 MFUs, p = 0.0045, df = 4, t = 14.9193).

Article Snippet: The nitrocellulose membranes were marked with exosome-specific antibodies (rabbit anti-CD63 primary antibody, cat. #25682-1-AP, rabbit anti-Alix, cat. #12422-1-AP, ProteinTech, Martinsried, Germany), anti-granzyme B antibodies (rabbit anti-granzyme B, cat. #13588-1-AP, ProteinTech, Martinsried, Germany) and CAR-specific antibodies (rabbit anti-CD3ζ, cat. #88083; rabbit anti-G4S linker, cat. #71645, Cell Signaling Technology, Leiden, The Netherlands), respectively, overnight, at 4 °C.

Techniques: Expressing, Transfection, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Anti-Her2 CAR-NK92 Cells and Their Exosomes: Generation, Characterization, and Selective Cytotoxicity Against Her2-Positive Tumor Cells

doi: 10.3390/ijms26157648

Figure Lengend Snippet: Comparison of exosome production and protein content between NK92 WT and CAR-NK92 cells. ( A ) Quantification of exosomes produced by NK92 WT and CAR-NK92 anti-Her2 cells after 72 h of incubation in serum-free media. Starting from 4 × 10 6 cells, NK92 WT produced 11.2 × 10 6 exosomes, while CAR-NK92 produced 10.4 × 10 6 exosomes, with no statistically significant difference between the two groups ( p = 0.50, n = 3). ( B ) Measurement of exosomal protein concentration showed 13.26 μg/μL for NK92 WT and 12.17 μg/μL for CAR-NK92 cells, also without significant difference ( p = 0.78, n = 3). Data represent mean ± SEM; ns = not significant.

Article Snippet: The nitrocellulose membranes were marked with exosome-specific antibodies (rabbit anti-CD63 primary antibody, cat. #25682-1-AP, rabbit anti-Alix, cat. #12422-1-AP, ProteinTech, Martinsried, Germany), anti-granzyme B antibodies (rabbit anti-granzyme B, cat. #13588-1-AP, ProteinTech, Martinsried, Germany) and CAR-specific antibodies (rabbit anti-CD3ζ, cat. #88083; rabbit anti-G4S linker, cat. #71645, Cell Signaling Technology, Leiden, The Netherlands), respectively, overnight, at 4 °C.

Techniques: Comparison, Produced, Incubation, Protein Concentration

Journal: International Journal of Molecular Sciences

Article Title: Anti-Her2 CAR-NK92 Cells and Their Exosomes: Generation, Characterization, and Selective Cytotoxicity Against Her2-Positive Tumor Cells

doi: 10.3390/ijms26157648

Figure Lengend Snippet: Western blot analysis of the whole cell lysate and the exosomal populations. ( A ) Both cell populations express CD3ζ, while CAR-NK92 cells express two bands containing CD3ζ, suggestive for CAR expression. Both cell populations express granzyme B, present both as monomers and multimers. Compared to whole cell lysate which stained positive for β-actin (loading control). ( B ) Exosomal lysate Western blot revealed that both exosomal populations were negative for β-actin, suggesting low contamination during isolation The exosomal specific markers CD63 and Alix were present in both exosomal populations, suggesting that exosomes are released via ESCRT-dependent and Alix-dependent pathways. Both exosomal populations express CD3ζ and granzyme B, while CAR expression, documented using the G4S antibody was present only in the exo-CAR-NK92 population; kDa—kilodaltons.

Article Snippet: The nitrocellulose membranes were marked with exosome-specific antibodies (rabbit anti-CD63 primary antibody, cat. #25682-1-AP, rabbit anti-Alix, cat. #12422-1-AP, ProteinTech, Martinsried, Germany), anti-granzyme B antibodies (rabbit anti-granzyme B, cat. #13588-1-AP, ProteinTech, Martinsried, Germany) and CAR-specific antibodies (rabbit anti-CD3ζ, cat. #88083; rabbit anti-G4S linker, cat. #71645, Cell Signaling Technology, Leiden, The Netherlands), respectively, overnight, at 4 °C.

Techniques: Western Blot, Expressing, Staining, Control, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Anti-Her2 CAR-NK92 Cells and Their Exosomes: Generation, Characterization, and Selective Cytotoxicity Against Her2-Positive Tumor Cells

doi: 10.3390/ijms26157648

Figure Lengend Snippet: Relative protein expression levels in NK92 WT, CAR-NK92, Exo-NK92, and Exo-CAR-NK92 cells. ( A ) Bar graph showing the relative signal intensity of CD3ζ, CAR, GzmB monomer, and GzmB dimer in NK92 WT (orange) and CAR-NK92 (green) cells, normalized to β-actin as a loading control. No significant differences were observed in expression of CD3ζ ( p = 0.1839, t = 3.365, df = 1), granzyme B monomers ( p = 0.1753, t = 3.540, df = 1) and dimers ( p = 0.12, t = 5.244, df = 1), while CAR expression was significantly increased in the transduced NK cell population ( p = 0.05, t = 12.791, df = 1). ( B ) Bar graph displaying the relative signal intensity of CD3ζ, GzmB, Alix, and CAR-G4S in Exo-NK92 (orange) and Exo-CAR-NK92 (blue) exosomes, normalized to CD63 as a loading control. No statistically significant differences were observed in terms of CD3ζ ( p = 0.1328, t = 4.725, df = 1), granzyme B ( p = 0.9187, t = 0.1285, df = 1) or Alix expression ( p = 0.4325, t = 1.238, df = 1), while the presence of CAR was significantly increased in the Exo-CARNK92 group ( p = 0.046, t = 13.81, df = 1). Data are presented as mean ± SEM; * p < 0.05, ns = not significant.

Article Snippet: The nitrocellulose membranes were marked with exosome-specific antibodies (rabbit anti-CD63 primary antibody, cat. #25682-1-AP, rabbit anti-Alix, cat. #12422-1-AP, ProteinTech, Martinsried, Germany), anti-granzyme B antibodies (rabbit anti-granzyme B, cat. #13588-1-AP, ProteinTech, Martinsried, Germany) and CAR-specific antibodies (rabbit anti-CD3ζ, cat. #88083; rabbit anti-G4S linker, cat. #71645, Cell Signaling Technology, Leiden, The Netherlands), respectively, overnight, at 4 °C.

Techniques: Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Anti-Her2 CAR-NK92 Cells and Their Exosomes: Generation, Characterization, and Selective Cytotoxicity Against Her2-Positive Tumor Cells

doi: 10.3390/ijms26157648

Figure Lengend Snippet: Analysis of the binding capacity of exo-NK92 and exo-CAR to SK-BR3 and MCF-7 cells using the In-cell ELISA technique. Cells were incubated with the described exosomes and subsequently stained with anti-CD63 antibodies to investigate the binding capacity of exosomes via surface markers. Absorbance intensity is directly proportional to the amount of CD63 present in the well. Control MCF-7 and SK-BR3 cells show no statistically significant differences in CD63 marker expression ( p = 0.8562, n = 3). Positivity for this marker is associated with continuous exosome production. When the cell lines were incubated with NK92-exosomes, an increase in optical density at 450 nm was observed. The difference was significant in the SK-BR3 cell population ( p = 0.013, n = 3) and close to statistical significance in the MCF-7 population ( p = 0.07, n = 3). In contrast, statistically significant differences were observed in the binding of Exo-CAR-NK92 to the Her2-positive SK-BR3 cell line compared to the constitutively Her2-negative MCF-7 cell line ( p = 0.0435, n = 3). Comparative analysis revealed that CAR incorporation did not significantly alter binding in MCF-7 cells ( p = 0.8481, n = 3), but significantly increased exosomal binding in Her2+ SK-BR3 cells ( p = 0.0369, n = 3); *— p < 0.05; ns—not significant.

Article Snippet: The nitrocellulose membranes were marked with exosome-specific antibodies (rabbit anti-CD63 primary antibody, cat. #25682-1-AP, rabbit anti-Alix, cat. #12422-1-AP, ProteinTech, Martinsried, Germany), anti-granzyme B antibodies (rabbit anti-granzyme B, cat. #13588-1-AP, ProteinTech, Martinsried, Germany) and CAR-specific antibodies (rabbit anti-CD3ζ, cat. #88083; rabbit anti-G4S linker, cat. #71645, Cell Signaling Technology, Leiden, The Netherlands), respectively, overnight, at 4 °C.

Techniques: Binding Assay, In-Cell ELISA, Incubation, Staining, Control, Marker, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Anti-Her2 CAR-NK92 Cells and Their Exosomes: Generation, Characterization, and Selective Cytotoxicity Against Her2-Positive Tumor Cells

doi: 10.3390/ijms26157648

Figure Lengend Snippet: Exosome-mediated cytotoxic effects of NK and CAR-NK cells on SK-BR3 and MCF-7 breast cancer cell lines. ( A ) Percentage of resazurin (AB) reduction indicating cytotoxicity of NK cell-derived and CAR-NK cell-derived exosomes at different concentrations (1, 5, and 10 μg) against SK-BR-3 and MCF-7 breast cancer cells. While both NK and CAR-NK-derived exosomes expressed significant cytotoxic effects, the most pronounced effect was observed in SK-BR3 cells treated with 10 μg of CAR-NK-derived exosomes ( p < 0.01). ( B ) Dose-dependent reduction in cell viability of SK-BR-3 and MCF-7 cells upon treatment with NK or CAR-NK cell-derived exosomes, as measured by viability assays. CAR-NK exosomes demonstrated greater cytotoxicity compared to NK exosomes across all tested doses. Data are presented as mean ± SEM; significance levels are denoted as * p < 0.05 and ** p < 0.01; AB—alamarBlue.

Article Snippet: The nitrocellulose membranes were marked with exosome-specific antibodies (rabbit anti-CD63 primary antibody, cat. #25682-1-AP, rabbit anti-Alix, cat. #12422-1-AP, ProteinTech, Martinsried, Germany), anti-granzyme B antibodies (rabbit anti-granzyme B, cat. #13588-1-AP, ProteinTech, Martinsried, Germany) and CAR-specific antibodies (rabbit anti-CD3ζ, cat. #88083; rabbit anti-G4S linker, cat. #71645, Cell Signaling Technology, Leiden, The Netherlands), respectively, overnight, at 4 °C.

Techniques: Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Anti-Her2 CAR-NK92 Cells and Their Exosomes: Generation, Characterization, and Selective Cytotoxicity Against Her2-Positive Tumor Cells

doi: 10.3390/ijms26157648

Figure Lengend Snippet: Schematic representation of the proposed mechanism by which exosomes derived from CAR-NK92 cells infiltrate the tumor microenvironment and mediate Her2-targeted cytotoxicity. Exo-CAR-NK exosomes specifically recognize and bind Her2 receptors on tumor cells, triggering downstream signaling pathways that induce tumor cell apoptosis either through direct Her2 receptor blockade or granzyme B release. The exosomal surface expression of CD44 facilitates binding to hyaluronic acid in the extracellular matrix, promoting enhanced infiltration into the tumor microenvironment. Additionally, CD2 on exosomes interacts with CD58 on T cells, NK cells, and CAR-NK cells, thereby amplifying the local immune response within the tumor microenvironment. Created in BioRender software version 4. Tirziu, A. (2025) https://BioRender.com/a1miim7 (accessed on 15 June 2025).

Article Snippet: The nitrocellulose membranes were marked with exosome-specific antibodies (rabbit anti-CD63 primary antibody, cat. #25682-1-AP, rabbit anti-Alix, cat. #12422-1-AP, ProteinTech, Martinsried, Germany), anti-granzyme B antibodies (rabbit anti-granzyme B, cat. #13588-1-AP, ProteinTech, Martinsried, Germany) and CAR-specific antibodies (rabbit anti-CD3ζ, cat. #88083; rabbit anti-G4S linker, cat. #71645, Cell Signaling Technology, Leiden, The Netherlands), respectively, overnight, at 4 °C.

Techniques: Derivative Assay, Protein-Protein interactions, Expressing, Binding Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Anti-Her2 CAR-NK92 Cells and Their Exosomes: Generation, Characterization, and Selective Cytotoxicity Against Her2-Positive Tumor Cells

doi: 10.3390/ijms26157648

Figure Lengend Snippet: Design and Structure of the Third-Generation Anti-Her2 CAR Vector. ( A ) Schematic representation of the pPB [Exp]-EF1A>CAR construct generated using the VectorBuilder platform. This bi-cistronic piggyBac transposon-based plasmid (9791 bp) contains the anti-Her2 CAR sequence driven by the EF1A promoter and flanked by 5′ and 3′ inverse terminal repeats (ITRs). The vector includes a Kozak consensus sequence, a CD8a leader, and the CAR transgene, followed by an internal ribosome entry site (IRES) and a dual selection cassette expressing EGFP and a puromycin resistance gene ( pac ) linked via a T2A sequence for bicistronic expression. This design facilitates both visual and antibiotic selection of successfully transduced cells. ( B ) Structural diagram of the anti-Her2 chimeric antigen receptor (CAR). The extracellular domain consists of a single-chain variable fragment (scFv) derived from trastuzumab (anti-Her2), composed of VH and VL regions linked by a flexible (G 4 S) 3 linker. This is fused to a CD8a hinge and transmembrane domain. The intracellular signaling domain contains the co-stimulatory molecules 4-1BB and CD28, followed by the CD3ζ activation domain.

Article Snippet: The nitrocellulose membranes were marked with exosome-specific antibodies (rabbit anti-CD63 primary antibody, cat. #25682-1-AP, rabbit anti-Alix, cat. #12422-1-AP, ProteinTech, Martinsried, Germany), anti-granzyme B antibodies (rabbit anti-granzyme B, cat. #13588-1-AP, ProteinTech, Martinsried, Germany) and CAR-specific antibodies (rabbit anti-CD3ζ, cat. #88083; rabbit anti-G4S linker, cat. #71645, Cell Signaling Technology, Leiden, The Netherlands), respectively, overnight, at 4 °C.

Techniques: Plasmid Preparation, Construct, Generated, Sequencing, Selection, Expressing, Derivative Assay, Activation Assay