goat anti human nectin  (R&D Systems)

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 1 High expression of Nectin4 and FAP in a variety of cancers. (A) Expression of Nectin4 and FAP in glioma, liposarcoma, and leiomyosarcoma was assessed by IHC. (B) Expression of Nectin4 on lung-metastasized esophageal cancer, lung-metastasized liver cancer, and bone-metastasized triple-negative breast cancer (TNBC). Also see Supplementary Figure 3. Nectin4 is mainly located in the membrane (strongly positive) and cytoplasm (weakly positive) of cancer cells; FAP is mainly located in the membrane (strongly positive) and cytoplasm (weakly positive) of stromal cells, shown in brown.

Article Snippet: The sections were rewarmed at 37°C for at least 45 min and incubated in the secondary antibody enhancement solution at room temperature for 20 min and then the secondary goat F (ab) anti-mouse IgG H&L (HRP) antibody (Abcam) at 37°C for 30 min. For Nectin4 staining, sections were stained with 10 mg/ ml primary goat anti-human Nectin4, affinity-purified polyclonal antibody (Catalog # AF2659, R&D Systems) at 4°C overnight.

Techniques: Expressing, Membrane

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 2 CAR structure and characterization of Nectin4-7.19 CAR-T cells. (A) Schematic illustration of Nectin4 CAR and Nectin4-7.19 CAR lentiviral vector. LTR: long terminal repeats; SP: CD8 signal peptide; TM: transmembrane region; P2A: 2A polypeptide element. (B) CAR expression in Nectin4 CAR-T and Nectin4-7.19 CAR-T cells was measured by flow cytometry. UTD indicates the untransduced T cells as a negative control. (C) Relative CAR expression in CD4+ and CD8+ T subsets. (D) Representative CAR-T cell phenotyping plot based on CD45RA and CCR7 in CD4+

Article Snippet: The sections were rewarmed at 37°C for at least 45 min and incubated in the secondary antibody enhancement solution at room temperature for 20 min and then the secondary goat F (ab) anti-mouse IgG H&L (HRP) antibody (Abcam) at 37°C for 30 min. For Nectin4 staining, sections were stained with 10 mg/ ml primary goat anti-human Nectin4, affinity-purified polyclonal antibody (Catalog # AF2659, R&D Systems) at 4°C overnight.

Techniques: Plasmid Preparation, Expressing, Cytometry, Negative Control

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 3 Efficient cytotoxicity of Nectin4-7.19 CAR-T cells in vitro. (A) Expression of Nectin4 on a panel of cancer cell lines. (B) Cytotoxicity of Nectin4 CAR-T cells against ABC-1, HT1376, and MDA-MB-453 cells was detected by xCELLigence RTCA label-free technology. The left panel compares the cytotoxicity between Nectin4 CAR-T and CD19 CAR-T cells against target cells at an Effect/Target ratio of 10:1; the right panel shows the killing efficacy of Nectin4 CAR-T cells at different Effect/Target ratios. Arrows refer to the addition of CAR-T cells. The y-axis is the normalized cell index generated by the RTCA software and displayed in real time to reflect the vitality of tumor cells. The x-axis is the time of cell culture in hours. (C) Nectin4 and GFP expression in Luc. ABC-1 cells transfected with lentivirus encoding the Luciferase-T2A-GFP gene. (D) Quantified data on the specific lytic levels of Nectin4 CAR-T and Nectin4-7.19 CAR-T cells against Luc. ABC-1 cells were assessed by luciferase bio-luminescence technique at different Effect/Target ratios in vitro. UTD served as a negative control. (E) Expression level of immune checkpoints was detected by flow cytometry after co-culture of Nectin4 CAR-T or Nectin4-7.19 CAR-T cells with ABC-1 cells at an Effect/Target ratio of 1:1 for 5 days. Data represent the mean ± SD of three independent experiments; ns, no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001, t-test.

Article Snippet: The sections were rewarmed at 37°C for at least 45 min and incubated in the secondary antibody enhancement solution at room temperature for 20 min and then the secondary goat F (ab) anti-mouse IgG H&L (HRP) antibody (Abcam) at 37°C for 30 min. For Nectin4 staining, sections were stained with 10 mg/ ml primary goat anti-human Nectin4, affinity-purified polyclonal antibody (Catalog # AF2659, R&D Systems) at 4°C overnight.

Techniques: In Vitro, Expressing, Generated, Software, Cell Culture, Transfection, Luciferase, Negative Control, Cytometry, Co-Culture Assay

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 4 Therapeutic effect of Nectin4 mCAR-T cells on metastatic colorectal cancer in fully immune-competent mice. (A) The murine CAR construct was inserted upstream of an IRES-GFP marker in the MSCV retroviral plasmid pMIGR1. (B) mCAR expression of Nectin4 mCAR-T cells transfected with pMIGR1-mCAR-IRES-GFP retroviral particles. mUTD indicates the untransduced mouse T cells. (C) Nectin4 and GFP expression of Luc. MC38 cells and hNectin4-Luc. MC38 cells. (D) Quantified data on the specific lytic levels of Nectin4 mCAR-T cells against Luc. MC38 or hNectin4-Luc. MC38 cells were assessed by luciferase bio-luminescence technique at different Effect/Target ratios in vitro. ***p < 0.001, t-test. (E) Secretion of IFN-g in CD4+ and CD8+ T subsets was assessed by flow cytometry after co-culture of Nectin4 mCAR-T cells or mUTD with hNectin4-Luc. MC38 cells for 12 h. ***p < 0.001, t-test. (F, G) C57BL/6 mice were s.c. inoculated with 1 × 106 hNectin4-Luc. MC38 cells on Day 0 and injected i.v. with 5.0 × 105 to 5.0 × 106 Nectin4 mCAR-T cells on Day 10. A total of 5.0 × 106 mUTD served as a negative control (N = 6 mice per group). Solid lines represent each individual mouse (F). Kaplan–Meier survival curve is shown in (G). p-values of log-rank tests were as follows: p = 0.35 (mUTD vs. 0.5 × 106 Nectin4 mCAR-T); p = 0.09 (mUTD vs. 1.5 × 106 Nectin4 mCAR-T); p = 0.0012 (mUTD vs. 5.0 × 106

Article Snippet: The sections were rewarmed at 37°C for at least 45 min and incubated in the secondary antibody enhancement solution at room temperature for 20 min and then the secondary goat F (ab) anti-mouse IgG H&L (HRP) antibody (Abcam) at 37°C for 30 min. For Nectin4 staining, sections were stained with 10 mg/ ml primary goat anti-human Nectin4, affinity-purified polyclonal antibody (Catalog # AF2659, R&D Systems) at 4°C overnight.

Techniques: Construct, Marker, Retroviral, Plasmid Preparation, Expressing, Transfection, Luciferase, In Vitro, Cytometry, Co-Culture Assay, Injection, Negative Control

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 5 Significant anti-tumor effect of Nectin4-7.19 CAR-T therapy on metastatic lung cancer without on-target off-tumor toxicity. (A) NSG mice were i.v. inoculated with 1.0 × 106 Luc. ABC-1 cells on Day 0 and received an administration of 3 × 106 Nectin4-7.19 CAR-T cells on Day 7 (N = 3 mice per group). Mice treated with the same dosage of UTD cells served as a negative control. (B–D) Tumor xenografts were monitored via bioluminescence imaging. Representative bioluminescence images of three independent experiments are shown in (B); bioluminescence kinetics are shown in (C); solid lines represent each individual mouse. Kaplan–Meier survival curve is shown in (D), p = 0.0246 (Nectin4-7.19 CAR-T vs. UTD), N = 3, log-rank test. (E) Body weight of mice since the tumor inoculation.

Article Snippet: The sections were rewarmed at 37°C for at least 45 min and incubated in the secondary antibody enhancement solution at room temperature for 20 min and then the secondary goat F (ab) anti-mouse IgG H&L (HRP) antibody (Abcam) at 37°C for 30 min. For Nectin4 staining, sections were stained with 10 mg/ ml primary goat anti-human Nectin4, affinity-purified polyclonal antibody (Catalog # AF2659, R&D Systems) at 4°C overnight.

Techniques: Negative Control, Imaging

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 6 Synergistic effect of Nectin4-7.19 CAR-T with FAP-12 CAR-T therapy on metastatic lung cancer mouse model. (A) Schematic illustration of FAP CAR and FAP-12 CAR lentiviral vector. LS: leader signal. (B) CAR expression on FAP CAR-T and FAP-12 CAR-T cells. (C) Expression of CD45RA and CD45RO in CD4+ or CD8+ T subset to assess the subtypes of T cells. (D) 293T cells were transduced with lentivirus encoding the human FAP-Firefly-Luciferase-GFP gene or the murine FAP-Firefly-Luciferase-GFP gene to generate hFAP-Luc. 293T and mFAP-Luc. 293T cells, respectively. Expression of GFP was measured by flow cytometry. 293T cells served as negative controls. (E) Quantified data on the specific lytic levels of FAP CAR-T cells against hFAP-Luc. 293T and mFAP-Luc. 293T cells were assessed by luciferase bio-luminescence technique at different Effect/Target ratios in vitro. UTD served as a negative control. (F) Cytotoxicity of FAP CAR-T and FAP-12 CAR-T cells was detected at an Effect/Target ratio of 10:1 by xCELLigence RTCA label-free technology. (G) Specific lysis of FAP CAR-T and FAP-12 CAR-T cells against hFAP- Luc. 293T was detected by luciferase bio-luminescence technique at different Effect/Target ratios in vitro. (H) NSG mice were inoculated with 1.0 × 106 Luc. ABC-1 cells i.v. on Day 0 and received an administration of 2 × 106 FAP-12 CAR-T cells, 2 × 106 Nectin4-7.19 CAR-T cells, or an admixture of 1 × 106 Nectin4-7.19 CAR-T cells and 1 × 106 FAP-12 CAR-T cells on Day 3 (N = 3 mice per group). A total of 2.0 × 106 UTD served as a negative control. (I) Tumor xenografts were monitored via bioluminescence imaging. Representative bioluminescence images of three independent experiments in each group of mice were shown. (J) Bioluminescence kinetics of the tumor growth in the model. (K) Kaplan– Meier survival curve. p-values of log-rank tests were as follows: p = 0.0246 (Nectin4-7.19+FAP-12 vs. UTD); p = 0.0246 (Nectin4-7.19+FAP-12 vs. FAP-12); p = 0.1161 (Nectin4-7.19+FAP-12 vs. Nectin4-7.19), N = 3. (L) Body weight of mice since the tumor inoculation. Data represent the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, t-test.

Article Snippet: The sections were rewarmed at 37°C for at least 45 min and incubated in the secondary antibody enhancement solution at room temperature for 20 min and then the secondary goat F (ab) anti-mouse IgG H&L (HRP) antibody (Abcam) at 37°C for 30 min. For Nectin4 staining, sections were stained with 10 mg/ ml primary goat anti-human Nectin4, affinity-purified polyclonal antibody (Catalog # AF2659, R&D Systems) at 4°C overnight.

Techniques: Plasmid Preparation, Expressing, Transduction, Luciferase, Cytometry, In Vitro, Negative Control, Lysis, Imaging