mouse anti-human cd3ζ sc-1239  (Santa Cruz Biotechnology)

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Dynamics of CARζ/CPR41BB T-cell activation and CARIS formation in comparison with CAR41BBζ cells. A, CARζ/CPR41BB cells ( n = 3 donors) demonstrated significantly lower IL-2 and IFN-γ release compared with CAR41BBζ cells at 24 hours of coculture with LN229-GBM cells, with CAR28ζ cells consistently showing the higher Th1 cytokine production. Data are shown as the mean ± SD. **, P < 0.000; ****, P < 0.0001; two-way ANOVA with the Tukey multiple comparisons test. B, Western blot analysis for CAR-phosphoCD3ζ (pCD3) in T cells in a resting state (maintained in culture with IL-7/IL-15). The pCD3 to CD3 ratio is normalized to CARζ in each donor. *, P < 0.05; one-way ANOVA with the Tukey multiple comparisons test. C, Representative image capture showing the CARIS with tumor cell (LN229-GBM) and different CARζ/CPR41BB immune synapse parameters evaluated. Gating strategy is shown in the Supplementary Material. Spearman correlation ( D ) between the intensity of CAR and CPR at the CARIS and ( E ) between the intensity of actin and CPR at the CARIS, both assessed by imaging flow cytometry at 15, 30, and 60 minutes. F, CARζ/CPR41BB and CAR41BBζ cells show significantly higher percent (%) of F-actin at the CARIS compared with CARζ cells. ns, P > 0.5; **, P < 0.01; ***, P < 0.0001, two-way ANOVA with the Tukey post hoc test. Data are shown as the mean with 95% confidence interval (CI). G, CARζ/CPR41BB cells show significantly higher CPR intensity in the CARIS with WT LN229 GBM cells at 15, 30, and 60 minutes compared with conjugates with LN229-PD-L1 KO (Kruskal–Wallis and Wilcox pairwise). CPR intensity increased over time in both conditions. ns, not significant.

Article Snippet: The membranes were blocked with 5% milk and probed with mouse anti-human CD3ζ (Santa Cruz Biotechnology; sc-1239, RRID: AB_627020), mouse anti-human phosphoCD3ζ (BD Biosciences; 558402, RRID: AB_647307), and GAPDH (Cell Signaling Technology, 2118, RRID: AB_561053) mAbs followed by appropriate IRdye secondary antibodies (Li-Cor).

Techniques: Activation Assay, Comparison, Western Blot, Imaging, Flow Cytometry

Journal: Nature Communications

Article Title: Chimeric antigen receptor T cells targeting FcRH5 provide robust tumour-specific responses in murine xenograft models of multiple myeloma

doi: 10.1038/s41467-023-39395-4

Figure Lengend Snippet: a Schematic representation of FcRH5-specific CAR expression construct. b The cytolytic activity of mock T or FcRH5 CAR-T cells against indicated target cells at various effector: target cell (E/T) ratios was determined by a 6-h luciferase-based cytolytic assay. Data represent Mean ± SD from three independent experiments. c Mock T or FcRH5 CAR-T cells were co-cultured with target cells for 24 h, and cell-free supernatants were harvested for evaluating IFN-γ, IL-2 and TNF-α secretion. Mean ± SD, n = 3 independent co-cultures, unpaired two-tailed t -test. d Mock T or FcRH5 CAR-T cells were labeled with eFlour-670 and co-cultured with equal number of target cells for 5 days, and the dilution of eFlour-670 fluorescence signal was determined to reflect cell division. The plots were gated on CD3 + lymphocytes. The above experiments a-d were repeated with 2 different T cell donors. e Flow cytometric analysis of FcRH5 expression on CD138 + primary myeloma cells from three newly-diagnosed MM patients. Solid line represents staining with anti-FcRH5 mAb and dashed line represents staining with isotype control antibody. f The patient-derived myeloma cells were co-cultured with autologous mock T or FcRH5 CAR-T cells for 24 h, and cell-free supernatants were harvested for determination of IFN-γ secretion. Mean ± SD, n = 3 independent co-cultures, unpaired two-tailed Student t -test. g The patient-derived myeloma cells were labeled with eFlour-670 and co-cultured with autologous mock T or FcRH5 CAR-T cells at the E/T ratio of 5:1 for 6 h, and then cytolytic effect of T cells was determined by a flow cytometry-based assay. Mean ± SD, n = 3 independent co-cultures, two-tailed Student t test.

Article Snippet: The membrane was probed with mouse anti-human CD3ζ mAb (Santa Cruz, catalog number sc-166435, clone: E3, dilution:1:100) or GAPDH mAb (Biolegend, catalog number 649203, clone:FF26A/F9)) followed by incubation with a horseradish peroxidase–conjugated goat anti-mouse IgG antibody (Santa Cruz, catalog number sc-516102, dilution:1:1000) .

Techniques: Expressing, Construct, Activity Assay, Luciferase, Cell Culture, Two Tailed Test, Labeling, Fluorescence, Staining, Control, Derivative Assay, Flow Cytometry

Journal: Nature Communications

Article Title: Chimeric antigen receptor T cells targeting FcRH5 provide robust tumour-specific responses in murine xenograft models of multiple myeloma

doi: 10.1038/s41467-023-39395-4

Figure Lengend Snippet: a Experimental schematic: Male NOG mice aged 6–8 weeks were subcutaneously inoculated with 5 × 10 6 NCI-H929-CBR cells expressing click beetle red (CBR) luciferase on day −7, and were then intravenously infused with 5 × 10 6 mock T or FcRH5 CAR-T cells from two different donors ( n = 6 mice per group for each donor) when the tumors became palpable (day 0). b Representative tumor bioluminescence of NOG mice at different time points. c Graph showed the quantification of whole-body luminescence in NOG mice from each group at different time points with the lines connecting the mean values. Data shown are representative for results obtained in independent experiments with T-cell from 2 donors. Mean ± SD, n = 6 mice, unpaired two-tailed Student t test, d Kaplan–Meier curve for the overall survival of mice from different treatment group ( n = 12 mice per group). Log-rank (Mantel-Cox) test. e The percentage of T cells (gated on human CD45 + CD3 + ) in peripheral blood of NOG mice at different time points was determined by flow cytometric analysis. Data are representative of two independent experiments with T-cell from 2 donors ( n = 12 mice per group in total). Mean ± SD, unpaired two-tailed Student t test,.

Article Snippet: The membrane was probed with mouse anti-human CD3ζ mAb (Santa Cruz, catalog number sc-166435, clone: E3, dilution:1:100) or GAPDH mAb (Biolegend, catalog number 649203, clone:FF26A/F9)) followed by incubation with a horseradish peroxidase–conjugated goat anti-mouse IgG antibody (Santa Cruz, catalog number sc-516102, dilution:1:1000) .

Techniques: Expressing, Luciferase, Two Tailed Test

Journal: Nature Communications

Article Title: Chimeric antigen receptor T cells targeting FcRH5 provide robust tumour-specific responses in murine xenograft models of multiple myeloma

doi: 10.1038/s41467-023-39395-4

Figure Lengend Snippet: a Experimental schematic: Male NOG mice aged 6–8 weeks were intravenously inoculated with 5 × 10 6 MM.1s-FcRH5-CBR cells on day −7, and were then treated with 5 × 10 6 mock T, FcRH5 CAR-T cells or BCMA CAR-T cells from two different donors ( n = 6 mice per group for each donor) on day 0. b Representative tumor bioluminescence of NOG mice at different time points. c Graph showed the quantification of whole-body luminescence in NOG mice from each group at different time points with the lines connecting means. Data shown are representative for results obtained in independent experiments with T-cell from 2 donors. Mean ± SD, n = 6 mice, One-way ANOVA with Dunnett’s correction for multiple comparison. d Kaplan–Meier curve for the overall survival of mice from different treatment group ( n = 12 mice per group). Log-rank (Mantel–Cox) test. e The percentage of T cells (gated on human CD45 + CD3 + ) in peripheral blood of NOG mice was determined by flow cytometric analysis. Data are representative of two independent experiments with T-cell from 2 donors ( n = 12 mice per group in total). Mean ± SD, One-way ANOVA with Dunnett’s correction for multiple comparison.

Article Snippet: The membrane was probed with mouse anti-human CD3ζ mAb (Santa Cruz, catalog number sc-166435, clone: E3, dilution:1:100) or GAPDH mAb (Biolegend, catalog number 649203, clone:FF26A/F9)) followed by incubation with a horseradish peroxidase–conjugated goat anti-mouse IgG antibody (Santa Cruz, catalog number sc-516102, dilution:1:1000) .

Techniques: Comparison

Journal: Nature Communications

Article Title: Chimeric antigen receptor T cells targeting FcRH5 provide robust tumour-specific responses in murine xenograft models of multiple myeloma

doi: 10.1038/s41467-023-39395-4

Figure Lengend Snippet: a Cytolytic activity of bispecific CAR-T cells and mono-specific CAR-T cells against indicated target cells was determined by the 6-h luciferase-based cytolytic assay. Mean ± SD, n = 3 independent co-cultures. b Bispecific CAR-T cells or mono-specific CAR-T cells were co-incubated with indicated target cells for 24 h, and cell-free supernatants were harvested for evaluating IFN-γ, IL-2 and TNF-α secretion. Mean ± SD, n = 3 independent co-cultures. Experiments of ( a , b ) were repeated with two different T cell donors. c Experimental schematic: Male NOG mice aged 6–8 weeks mice were subcutaneously inoculated with 5 × 10 6 NCI-H929-CBR cells expressing click beetle red (CBR) luciferase on day −7, and were then intravenously infused with 5 × 10 6 mock T, FcRH5 CAR-T, BCMA CAR-T or FcRH5/BCMA CAR-T cells from two different donors ( n = 6 mice per group for each donor) on day 0 when the tumors became palpable. d Representative tumor bioluminescence of NOG mice at different time points. e Graph showed the quantification of whole-body luminescence in NOG mice from each group at different time points with the lines connecting means. Data shown are representative for results obtained in independent experiments with T-cell from 2 donors. Mean ± SD, n = 6 mice per group, One-way ANOVA with Dunnett’s correction for multiple comparison. f Kaplan–Meier curve for the overall survival of mice from different treatment group ( n = 12 mice per group). Log-rank (Mantel-Cox) test. g The percentage of T cells (gated on human CD45 + CD3 + ) in peripheral blood of NOG mice at different time points was determined by flow cytometric analysis. Data are representative of two independent experiments with T-cell from 2 donors ( n = 12 mice per group in total). Mean ± SD, One-way ANOVA with Dunnett’s correction for multiple comparison.

Article Snippet: The membrane was probed with mouse anti-human CD3ζ mAb (Santa Cruz, catalog number sc-166435, clone: E3, dilution:1:100) or GAPDH mAb (Biolegend, catalog number 649203, clone:FF26A/F9)) followed by incubation with a horseradish peroxidase–conjugated goat anti-mouse IgG antibody (Santa Cruz, catalog number sc-516102, dilution:1:1000) .

Techniques: Activity Assay, Luciferase, Incubation, Expressing, Comparison

Journal: Cancers

Article Title: CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells

doi: 10.3390/cancers15123081

Figure Lengend Snippet: Assessing the oligomerization state and surface organization of various chimeric antigen receptors in the unstimulated CAR T cell membrane. ( a ) Western blot analysis of HER2.z, HER2.CD28.z, HER2.41BB.z and HER2.CD28.41BB.z CAR T cells. The membranes were probed for anti-human CD3ζ. ( b ) Spot intensities were quantified using ImageJ/Fiji, and the proportion of dimer/oligomer states was determined. ( c ) Representative raw and segmented AiryScan images of fluorescently tagged CARs. Clusters were segregated by watershedding and quantified. The scale bar represents 2 µm. ( d ) Total intensity of the apical membrane slices. ( e ) Average number of clusters per 10 µm 2 apical membrane surface. ( f ) Distribution of individual cluster areas. ( g ) Integrated intensity of individual clusters. The charts represent mean ± SEM; nHER2.z = 54, 4 donors; nHER2.CD28.z = 44, 3 donors; nHER2.41BB.z = 47, 4 donors; nHER2.CD28.41BB.z = 54, 4 donors; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were probed with 1 µg/mL mouse anti-human CD3ζ (Cat.# 556366, BD Biosciences, San Jose, CA, USA) antibody overnight at 4 °C.

Techniques: Western Blot

Journal: Cancers

Article Title: CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells

doi: 10.3390/cancers15123081

Figure Lengend Snippet: Membrane diffusion analysis and tertiary structure modeling of HER2-specific chimeric antigen receptors. ( a ) Representative fluorescence fluctuation intensity traces of 10-s FCS runs. ( b ) Pooled autocorrelation curve for each CAR. Position of triplet lifetime (τ Triplet ), diffusion correlation times of the detached Alexa Fluor 647 conjugated monomer HER2 ECD (τ A647HER2 ) and the various CAR species (τ. z ; τ. CD28 . z ; τ. 41BB . z ; τ. CD28 . 41BB . z ) are indicated by arrows. ( c ) Diffusion coefficients of CAR constructs. The chart represents mean ± SEM, nHER2.z = 300, 4 donors; nHER2.CD28.z = 260, 3 donors; nHER2.41BB.z = 276, 4 donors; nHER2.CD28.41BB.z = 289, 4 donors; *** p < 0.001. ( d ) Schematic diagram illustrating the tertiary structure of the native TCR and the CAR constructs. TCR: T Cell Receptor complex extracellular domains; CD3z TM: CD3ζ transmembrane segment; CD3z IC: CD3ζ intracellular domain; FRP5 scFv: single chain variable fragment of FRP5 antibody targeting the HER2 ECD; IgG1 SH: short hinge linker derived from IgG1; CD28 TM: transmembrane segment derived from CD28; CD28 IC: costimulatory domain from CD28; 41BB IC: costimulatory domain from 41BB. Confidence scores of the predicted models and intramolecular disulfide bonds are indicated.

Article Snippet: The membranes were probed with 1 µg/mL mouse anti-human CD3ζ (Cat.# 556366, BD Biosciences, San Jose, CA, USA) antibody overnight at 4 °C.

Techniques: Diffusion-based Assay, Fluorescence, Construct, Derivative Assay

Journal: Cancers

Article Title: CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells

doi: 10.3390/cancers15123081

Figure Lengend Snippet: Early activation signaling events were determined by measuring CD3ζ and Lck phosphorylation. ( a ) Representative microscopic images following 15 min of effector–tumor co-culturing. ( b ) Quantitative image analysis of CD3ζ and Lck phosphorylation. The area of the immune synapse, the average and the integrated intensity of the phospho-specific label in the synapse are plotted showing the distribution of individual data points as well as their mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were probed with 1 µg/mL mouse anti-human CD3ζ (Cat.# 556366, BD Biosciences, San Jose, CA, USA) antibody overnight at 4 °C.

Techniques: Activation Assay

Journal: Frontiers in Oncology

Article Title: Use of phage display biopanning as a tool to design CAR-T cells against glioma stem cells

doi: 10.3389/fonc.2023.1124272

Figure Lengend Snippet: Dual peptide CAR-T cell design. (A) Diagram of the CAR construct designs. The E-28t28z and E-8t28z constructs use CD28 and CD8 as the hinge/transmembrane domains respectively, while both constructs employed CD28 for the co-stimulatory domain. The E-28t28z-tCD34 and E-8t28z-tCD34 constructs are identical to the E-28t28z and E-8t28z constructs but feature co-expression of truncated CD34 as an extracellular marker. (B) Diagram of a CAR-T cell with the different dual peptide-based constructs as the antigen-recognition domain. (C) CD34+ expression analysis by flow cytometry allows for a built-in quantification of transduction efficiency. (D) ELISA results displaying IFN-gamma secretion from CAR- or mock-transduced (GFP) T cells co-cultured overnight against T387 GSCs or cell-free media, **** = p < 0.0001. Values shown in the bar chart represent the average of two independent experiments. (E) CD3ζ western blotting with GAPDH as a housekeeping gene, used for additional support to confirm the presence or lack of the CAR following transduction. LTR, Long terminal repeat; S, signal peptide; P2A, Porcine teschovirus-1 2A self-cleaving peptide; tCD34, Truncated CD34.

Article Snippet: Membranes were incubated with 0.2 μg/mL mouse anti-human CD3ζ (Santa Cruz Biotechnology, SC-166275, Dallas, TX) and 0.04 μg/mL rabbit anti-human glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology, SC-25778, Dallas, TX).

Techniques: Construct, Expressing, Marker, Flow Cytometry, Transduction, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot