Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Expression, cytotoxic activity, and cytokine secretion of T cells expressing altered version of FcγRI. A, Illustration of the modified FcγRI structure with fused signaling domains: A2G (left), FcγRI composed of α-chain and homodimer of FcRγ chain; AG2G (middle), FcγRI α-chain with fused domain of FcRγ chain and homodimer of FcRγ; AGO2GO (right), FcγRI α-chain with fused FcRγ and OX40 domains and homodimer of FcRγ with fused OX40 domain. B, Flow cytometry analysis of A2G, AG2G, and AGO2GO expression following retroviral transduction of healthy donor T cells. C, Mean numbers of HT29 target cells expressing HER2 calculated by incuCyte software through 48-hour incubation with the three FcγRI-based receptor expressing T cells, with 1:1 E:T ratios with or without 6 μg/mL trastuzumab (data shows one of two experiments repeats, n = 4). Each sample is normalized to its cell number at time zero. Statistical significance was calculated using two-way ANOVA with Sidak correction for multiple comparisons. D, Representative incuCyte images showing HER2-expressing HT29 cells, imaged and counted in live imaging system after 48 hours of incubation with different construct-expressing T cells with 1:1 E:T ratio. Scale bar is 100 μm. E, Human Luminex Discovery Assay measurement of cytokine levels in supernatants from 48 hours of culture of T cells 4:1 E:T ratio with HT29 cells expressing HER2 ( n = 4). Graphs show mean ±SD. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Expressing, Activity Assay, Modification, Flow Cytometry, Retroviral, Transduction, Software, Incubation, Imaging, Construct, Luminex

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Characterization of AG2G-expressing T cells, activation, and cytotoxicity. A, ELISA measurement of cytokine levels of AG2G-expressing T cells after 48-hour incubation with HT29 cells expressing HER2. B, IFNγ and TNFα levels measured by ELISA in supernatants from 48-hour incubation of AG2G-expressing T cells and HT29 cells expressing HER2 in different E:T ratios. C, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-expressing T cells in combination with 0, 2.5, 5, 10 mg/mL IVIG, with or without 30 μg/mL trastuzumab ( n = 4). D, ELISA measurement of IFNγ levels of AG2G-expressing cells cocultured with HT29 cells expressing HER2 described in B . E, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-expressing T cells in combination with different concentrations of trastuzumab over 96 hours ( n = 2). F, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with different E:T ratios of AG2G-expressing T cells that were isolated for their CD4 (left) or CD8 (middle) population or with no isolation (right). G, ELISA measurement of cytokine levels from supernatants of 48-hour culture of 4:1 E:T ratios of isolated CD4, isolated CD8, or AG2G-expressing T cells with HT29 cells expressing HER2 and 30 μg/mL trastuzumab. Graphs show mean ±SD. Statistical significance was calculated using Student t test for a, two-way ANOVA with Sidak correction for multiple comparisons for B and C , two-way ANOVA with Tukey's correction for multiple comparisons for E and F . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Software, Isolation

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: AG2G-expressing T cells differentiate between cells expressing high and low antigen levels, are more specific, and less exhausted, compared with classic CAR T cells. A, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with transduced T cells in combination with antibodies (60 μg/mL each, n = 4). B, ELISA measurement of IFNγ levels in 48-hour supernatants of AG2G-transduced T cells incubated with HER2-expressing HT29 target cells, and with tumor-binding trastuzumab, or irrelevant antibodies cetuximab and rituximab (60 μg/mL). C, Mean counts of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-transduced T cells and 30 μg/mL trastuzumab, compared with trastuzumab-derived CAR (4:1 E:T ratio, n = 4). Each sample is normalized to its cell number at time zero. D, Normalized confluence of kidney epithelial cells, calculated by incuCyte software, following incubation with AG2G-transduced T cells and 30 μg/mL trastuzumab, compared with trastuzumab-derived CAR (4:1 E:T ratio, n = 4). Each sample is normalized to its cell confluence at time zero. E, ELISA measurement of IFNγ levels in 48-hour supernatant obtained from transduced T cells and kidney epithel described in D ( n = 4). F, ELISA measurement of IFNγ, granzyme B and TNFα levels in 24-hour supernatants of 10 5 AG2G-expressing T cells or CAR T cells incubated on immobilized trastuzumab or rHER2, in rising concentrations (mmol/mL) respectively. G, Confocal microscope imaging of internalization kinetics of PE-labeled IgG by AG2G-expressing cells (top), or PE-labeled HER2 by CAR T cells (bottom). H, PD-1, LAG-3, and TIM3 flow cytometry analysis of AG2G-expressing cells or CAR T cells after 48-hour incubation with or without 12 mmol/mL immobilized trastuzumab or HER2, respectively. Graphs show mean ± SD. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons for A , C , D , and H . Two-way ANOVA with Sidak correction for multiple comparisons for E and F . One-way ANOVA with Dunnett correction for multiple comparisons for B . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Expressing, Software, Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Derivative Assay, Microscopy, Imaging, Labeling, Flow Cytometry

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Human T cells expressing AG2G exert specific tumor cytotoxicity while sparing normal cells. A, Flow cytometry expression analysis of HER2 on different tumor cell lines and primary human normal cells. B, ELISA measurement of IFNγ levels in 48-hour supernatants of AG2G-expressing T cells with different tumor cells or primary normal cells in 4:1 E:T ratio and 60 μg/mL trastuzumab (results shown are from 3 different donors combined, n = 12). C, Quantification of HER2 receptor numbers using DAKO QIFIKIT (Agilent) beads by flow cytometer. Individual populations of the calibration are gated, a linear regression is calculated from the MFI and the ABC values of the five calibration bead populations using MS Excel. Using the linear regression, the ABCs of the beads coated with αHER2 is calculated from their MFI values. SDs are also retrieved from gated populations and transformed using linear regression. D, Mean counts of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-transduced T cells or ACTR707 and 30 μg/mL trastuzumab (4:1 E:T ratio, n = 4). Each sample is normalized to its cell number at time zero. E, Normalized confluence of kidney epithelial (epithel) cells, calculated by incuCyte software, following incubation with AG2G-transduced T cells or ACTR707 and 30 μg/mL trastuzumab. Each sample is normalized to its cell confluence at time zero. Graphs are identical and described in . and but with the comparison to ACTR707 instead of CAR T. F, ELISA measurement of IFNγ levels in 48-hour supernatant obtained from transduced T cells and kidney epithel described in D and E ( n = 4). G, ELISA measurement of IFNγ levels in 24 hours supernatants of AG2G-expressing T cells or ACTR707 incubated with medium containing different concentration of IVIG ( n = 3). Graphs show mean ±SD. Statistical significance was calculated using two-way ANOVA with Sidak correction for multiple comparisons. ****, P < 0.0001. Error bars represent standard error. Endothel, endothelial cells.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Transformation Assay, Software, Incubation, Comparison, Concentration Assay

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Systemic administration of AG2G-expressing T cells in combination with trastuzumab eradicates HER2-expressing tumor cells in vivo . A, NCI-N87 tumor volume measured by caliper over 42 days following treatment initiation. NSG mice were injected with 2.5×10 6 NCI-N87 tumor cells, after tumor reached 70 mm 3 mice were randomized and treated once a week for 3 times (treatments are marked with arrows) with either intraperitoneal injection of saline or 250-μg trastuzumab and intravenous injection of saline or 5×10 6 AG2G-expressing T cells ( n = 6). B, Endpoint analysis of tumor volume measured by caliper, 42 days following treatment initiation. C, Endpoint analysis of tumor weight measured using scales, 42 days following treatment initiation. D, Histology images taken by x4 objective light microscopy, showing whole sections of tumors stained with H&E ( n = 6; N.D., not detected - 2 mice from AG2G+trastuzumab group). Scale bar is 5 mm. E, Representative image taken by x4 and x40 objectives using light microscopy, of one tumor from AG2G+trastuzumab group stained with anti-human CD3 antibody by IHC. F, Tumor volume measured by caliper in NSG mice treated with a single injection of T cells (10 7 ) 13 days after tumor inoculation (2.5×10 6 NCI-N87). Antibodies were injected once a week for a total of 3 times (250 μg/mouse), n = 5. G, NSG mice were injected with 1.7×10 6 NCI-N87 tumor cells, after tumor reached 100 to 200 mm 3 mice were randomized and treated once with intravenous injection of 5×10 6 AG2G-expressing T cells and with intraperitoneal injection of saline or 250-μg trastuzumab. On days 1, 7, 14, 28 following treatment, 3 mice from each group were sacrificed and RNA was extracted from blood and tumors for real-time PCR analysis of AG2G-expressing cells ( n = 12). Graphs show mean ± SD. Graphs A – C show one of two independent experiments performed. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons for A and B , one-way ANOVA with Holm-Sidak correction for multiple comparisons for C . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Expressing, In Vivo, Injection, Saline, Light Microscopy, Staining, Real-time Polymerase Chain Reaction

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Retroviral transduction of γδ-T cells with AG2G endows them with antitumor ADCC. A, Flow cytometry analysis of sham or AG2G-transduced αβ-T cells (two left panels, activated with IL2 and anti-CD3); sham or AG2G-transduced γδ-T cells (two right panels, activated with IL2 and zoledronic acid). B, Live cell numbers of HT29-HER2 expressing cells, calculated by incuCyte software, cocultured with AG2G-expressing γδ-T cells or AG2G-expressing αβ-T cells, with or without trastuzumab ( n = 3). C, Live cell numbers of HT29-HER2 expressing cells, calculated by incuCyte software, cocultured with sham untransduced or AG2G-expressing γδ-T cells with or without trastuzumab, in E:T ratios of 1:1, 2:1 or 4:1 ( n = 4). D, ELISA measurement of IFNγ levels in supernatants of 48-hour sham or AG2G-expressing γδ-T cells cocultured with HT29-HER2 expressing cells, with or without trastuzumab (E:T ratio 4:1, n = 3). Graphs show mean ±SD. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons for B and C . Two-way ANOVA with Sidak correction for multiple comparisons for D . *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Retroviral, Transduction, Flow Cytometry, Expressing, Software, Enzyme-linked Immunosorbent Assay

Journal: Cancer Research

Article Title: A Wnt-Independent LGR4–EGFR Signaling Axis in Cancer Metastasis

doi: 10.1158/0008-5472.can-21-1112

Figure Lengend Snippet: Figure 4. LGR4 increases phospho-EGFR and total EGFR protein levels. A, Antibody microarray and in silico analysis identify potential targets of LGR4. B, LGR4 KO decreases phospho-EGFR and total EGFR levels in MDA231 cells upon EGF stimulation. C, Inducible expression of LGR4 enhances phospho-EGFR and total EGFR levels in MDA231 cells in the absence of recombinant EGF. D, LGR4 KO reduces levels of phospho-Src and phospho-FAK in MDA468 cells in the absence of recombinant EGF. E, Overexpression of LGR4 enhances both EGFR and HER2 levels in HCC1954 cells in the absence of recombinant EGF. Means SEMs are shown. P values were calculated by two-sided unpaired t test (B–E). n.s., not significant.

Article Snippet: Primary antibodies used in the study included LGR4 (7E7) rat antibody (37), EGFR (D38B1) XP Rabbit antibody (Cell Signaling Technology, 4276), EGFR (A-10) mouse antibody (Santa Cruz, sc373746), P-EGFR (Y1068) XP rabbit antibody (Cell Signaling Technology, 3777), MET mouse antibody (Cell Signaling Technology, 3127), HER2 (44E7) mouse antibody (Cell Signaling Technology, 2248), HA-tag mouse antibody (BioLegend 901501), b-catenin mouse antibody (BD Transduction Laboratories, 610153), Ub (P4D1) mouse antibody (Santa Cruz, sc-8017), CBL mouse antibody (Santa Cruz, sc1651), andGAPDHrabbit antibody (SantaCruz, sc-25778).

Techniques: Microarray, In Silico, Expressing, Recombinant, Over Expression

Journal: Analytical chemistry

Article Title: Rapid Enrichment and Detection of Extracellular Vesicles Enabled by CuS-Enclosed Microgels

doi: 10.1021/acs.analchem.9b04485

Figure Lengend Snippet: Quantification of EV concentrations with the 300-nm CuS-MG in 1× PBS or Serum. Calibration curves in 1×PBS using a) anti-CD63, b) anti-HER2; and in serum using c) anti-CD63 or d) anti-HER2. CL0 = chemiluminescence without EVs. [ABEI] = 0.5 mM, [H2O2] =1 mM, pH=11.

Article Snippet: The biotinylated mouse anti-human ErbB2/HER2 (Recombinant Monoclonal Human IgG 1 Clone Hu5), mouse anti-human CD63 (Clone mem-259), and biotinylated mouse anti-human CD63 (Clone NVG-2) was obtained from R&D systems, Sino Biological, and BioLegend, respectively.

Techniques:

Journal: Analytical chemistry

Article Title: Rapid Enrichment and Detection of Extracellular Vesicles Enabled by CuS-Enclosed Microgels

doi: 10.1021/acs.analchem.9b04485

Figure Lengend Snippet: Detection of cell released EVs using CuS-MG. The cell culture medium was collected from three cell lines: MCF-10A, MDA-MB-231 and SK-BR-3. a) Chemiluminescence resulted from EV detection in the culture media of three cell lines targeting CD63 and anti-HER2; b) PCA plot using the chemiluminescence data shown in a); c) Linear correlation between CD63 quantification results obtained by ELISA and the chemiluminescent signals from CuS-MG in our assay targeting CD63. [ABEI] = 0.5 mM, [H2O2] = 1 mM, pH=11.

Article Snippet: The biotinylated mouse anti-human ErbB2/HER2 (Recombinant Monoclonal Human IgG 1 Clone Hu5), mouse anti-human CD63 (Clone mem-259), and biotinylated mouse anti-human CD63 (Clone NVG-2) was obtained from R&D systems, Sino Biological, and BioLegend, respectively.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cancers

Article Title: Retargeting of NK-92 Cells against High-Risk Rhabdomyosarcomas by Means of an ERBB2 (HER2/Neu)-Specific Chimeric Antigen Receptor

doi: 10.3390/cancers13061443

Figure Lengend Snippet: ERBB2 expression on alveolar rhabdomyosarcoma (aRMS) cells. RH30, RH41, and ( A ) primary tumor cell suspensions showed different variability in size (forward (FSC) vs. side scatter (SSC)) and displayed low but homogenous ERBB2 expression compared to isotype controls. The MDA-MB-453 and MDA-MB-468 breast cancer cell lines served as positive and negative controls, respectively ( B , D ). Compared to tumor cell suspensions stable ERBB2 expression was detectable on tumor spheroids during tumor growth ( C , E ).

Article Snippet: The effector cell line NK-92/5.28.z was analyzed for ERBB2-CAR expression using an ERBB2-IgG-Fc chimera (Sino Biological Inc., Beijing, China) after nonspecific Fc receptor blocking (Human TruStain FcX™, BioLegend, London, UK) and incubation on ice for 20 min. To visualize the chimera, a secondary anti-IgG-Fc monoclonal antibody conjugated with APC (BioLegend, London, UK) was used.

Techniques: Expressing

Journal: Cancers

Article Title: Retargeting of NK-92 Cells against High-Risk Rhabdomyosarcomas by Means of an ERBB2 (HER2/Neu)-Specific Chimeric Antigen Receptor

doi: 10.3390/cancers13061443

Figure Lengend Snippet: Specific cytotoxicity of NK-92/5.28.z cells against aRMS cell lines growing in suspension or as tumor cell monolayers. NK-92/5.28.z cells were compared with unmodified parental NK-92 cells in a 3-h cytotoxicity assay against cell suspensions of ( A ) RH30 cells, ( B ) RH41 cells, ( E ) primary aRMS cells, ( C ) MDA-MB-453 cells (which served as a positive control), and ( D ) MDA-MB-468 cells (which were used as a negative control). The effector-to-target (E:T) ratios ranged from 40:1 to 5:1 or from 40:1 to 1:1 (in primary RMS cells). Tumor cell lysis was ERBB2-specific and significantly increased when NK-92/5.28.z cells were used, even at low E:T ratios. In a 16-h cytotoxicity assay, the killing capacity of NK-92/5.28.z cells against monolayers of ( F ) RH30, ( G ) RH41, ( H ) MDA-MB-453, and ( I ) MDA-MB-468 tumor cells was assessed in comparison to that of parental NK-92 cells by a Celigo cell cytometer. ARMS monolayers were lysed to a significantly greater extent by NK-92/5.28.z cells than by unmodified parental NK-92 cells. Differences were considered significant for p < 0.05 (*), p < 0.01 (**), p < 0.005 (***), and p < 0.0001 (****), or not significant (ns).

Article Snippet: The effector cell line NK-92/5.28.z was analyzed for ERBB2-CAR expression using an ERBB2-IgG-Fc chimera (Sino Biological Inc., Beijing, China) after nonspecific Fc receptor blocking (Human TruStain FcX™, BioLegend, London, UK) and incubation on ice for 20 min. To visualize the chimera, a secondary anti-IgG-Fc monoclonal antibody conjugated with APC (BioLegend, London, UK) was used.

Techniques: Cytotoxicity Assay, Positive Control, Negative Control, Lysis, Cytometry