Journal: Journal of Cellular and Molecular Medicine

Article Title: NR1D1 ‐transactivated lncRNA NUTM2A‐AS1 promotes chemoresistance and immune evasion in neuroblastoma via inhibiting B7‐H3 degradation

doi: 10.1111/jcmm.18360

Figure Lengend Snippet: NUTM2A‐AS1 is associated with B7‐H3 and inhibits B7‐H3 ubiquitination level. (A,B) The graphs depict the protein scores of the 11 proteins identified through RNA pull‐down and LC–MS analysis. (C,D) The graphs illustrate the exponentially modified protein abundance index (emPAI) of the 11 proteins identified by RNA pull‐down assays and LC–MS. (E) B7‐H3 was specifically pulled down by biotin‐labelled sense NUTM2A‐AS1 (S) but not NUTM2A‐AS1 anti‐sense (AS) RNA in the indicated cells. (F) RNA immunoprecipitation (RIP) assays were conducted using anti‐B7‐H3 antibodies with extracts from neuroblastoma (NB) cells. Relative enrichment represents the RNA levels associated with B7‐H3 relative to an input control, comparing anti‐B7‐H3 antibody immunoprecipitation with IgG antibody. GAPDH mRNA served as the negative control. (G) B7‐H3 mRNA expression in NUTM2A‐AS1 knockdown cells was assessed by qRT‐PCR assay. (H,I) B7‐H3 protein expression in NUTM2A‐AS1 knockdown cells was examined by western blot assay. (J) NB cells with NUTM2A‐AS1 knockdown and control cells were treated with or without MG132 (5 μM) for 12 h, and cell lysates were analysed by western blotting. (K) NB cells with or without siRNAs specific for NUTM2A‐AS1 were treated with 20 μg/mL cycloheximide (CHX) or a vehicle for the indicated periods, and B7‐H3 levels were analysed by western blotting. (L) Western blot analysis of the ubiquitination of B7‐H3 in NUTM2A‐AS1 knockdown SK‐N‐SH cells. ***p < 0.001.

Article Snippet: Antibodies for western blot analysis are as follows: B7‐H3 (R&D Systems, #AF1027, 1:250) and GAPDH (Abcam, #EPR16891, 1:10000).

Techniques: Ubiquitin Proteomics, Liquid Chromatography with Mass Spectroscopy, Modification, Quantitative Proteomics, RNA Immunoprecipitation, Control, Immunoprecipitation, Negative Control, Expressing, Knockdown, Quantitative RT-PCR, Western Blot

Journal: Clinical Cancer Research

Article Title: B7-H3 CAR T Cells Are Effective against Ependymomas but Limited by Tumor Size and Immune Response

doi: 10.1158/1078-0432.CCR-24-3083

Figure Lengend Snippet: Expression of key target antigens in pediatric ependymoma. A, B7-H3, HER2, GD2, CD70, EphA2, GPC2, GPC3, and IL13Ra2 gene expression in primary pediatric ST ( n = 43), PF ( n = 88), and spinal (SP; n = 4) ependymomas. B, Representative IHC images of primary ST, PF, and SP ependymomas stained for B7-H3. Scale bar, 50 μm. C, H scores of pediatric ependymoma samples ( n = 44). The red line indicates matched primary and relapsed samples from the same patient. D, Murine Cd276 ( B7-h3 ), Her2 , B4galnt1 , Cd70 , Epha2 , Gpc2 , Gpc3 , and IL13Ra2 expression in mouse IUE–derived ZR fus1 tumors and matched normal brain. E, Binding profiles of ZR fus1 , H3K27ac CUT&RUN, and ATAC sequencing shown at the B7-h3 loci of ZR fus1 cells. F, Differential B7-h3 gene expression between nontargeting control and ZFTA-RELA KO tumors across three different cell passages of dissociated tumor cells.

Article Snippet: B7-H3 and HER2 were detected with Alexa Fluor 647 anti-human CD276 (R&D Systems, cat. # FAB1027R-100UG, RRID: AB_3644987) and Alexa Fluor 647 anti-human CD340 (ERBB2/HER2; BioLegend, cat. # 324412, RRID: AB_2262300), respectively.

Techniques: Expressing, Gene Expression, Staining, Derivative Assay, Binding Assay, Sequencing, Control

Journal: Cancer Research

Article Title: B7-H3–Targeted CAR-Vδ1T Cells Exhibit Potent Broad-Spectrum Activity against Solid Tumors

doi: 10.1158/0008-5472.CAN-24-0195

Figure Lengend Snippet: In vitro expansion and transduction of Vδ1T cells. A, Schematic diagram of manufacturing “off-the shelf” allogeneic B7-H3-CAR-Vδ1T cells in vitro . B, Fold expansion of Vδ1T cells in vitro at the end of the expansion protocol. C, The purity of Vδ1T cells in the final products by flow cytometry. D, The B7-H3-CAR–positive rate in the Vδ1T cells and one of the donor’s detection data by flow cytometry. E, NCR (NKG2D/DNAM1/NKp30/NKp44/NKp46) expression of the final cell products detected by flow cytometry. The above data were derived from six different healthy donors and are presented as the mean ± SD. NCR, natural cytotoxic receptor.

Article Snippet: The B7-H3 expression in tumor cells was detected using the anti-human B7-H3 antibody (Miltenyi Biotec, Clone: REA1094).

Techniques: In Vitro, Transduction, Flow Cytometry, Expressing, Derivative Assay

Journal: Cancer Research

Article Title: B7-H3–Targeted CAR-Vδ1T Cells Exhibit Potent Broad-Spectrum Activity against Solid Tumors

doi: 10.1158/0008-5472.CAN-24-0195

Figure Lengend Snippet: In vitro cytotoxicity and functional persistence of B7-H3-CAR-Vδ1T cells. A, Construct diagram of B7-H3-CAR (named AG-CAR) containing antigen-binding region (B7-H3 scFv), hinge region (IgG4-Fc), transmembrane domain (CD28), costimulatory domain (CD28), and intracellular domain (CD3ζ). B, The detection of CAR-positive rate of Vδ1T cells by flow cytometry. C, Cytotoxicity of AG-CAR-Vδ1T against overexpressed B7-H3–positive tumor cell line Raji-B7-H3 in vitro (E:T = 1:1 and 5:1, T = 24 hours). Three independent experiments. D, Cytokine production (IFNγ, TNFα, and IL2) in the supernatant of AG-CAR-Vδ1T cells after coincubation with Raji-B7-H3 cells (E:T = 5:1). E, The growth inhibition effect of AG-CAR-Vδ1T against solid tumor cells in vitro using the real-time analysis system (xCELLigence), including SK-N-AS, A549, HCT-15, and SW1990 (E:T = 1:2). F, Cytokine production (IFNγ, TNFα, and IL2) in the coincubated supernatant of AG-CAR-Vδ1T cells with the above solid tumor cells. G, Antigen-repeated stimulation assay to evaluate antitumor persistence of CAR-Vδ1T and Vδ1T against Raji-B7-H3 cells in vitro . The number of antigen stimulation was four times. Three independent experiments. H, Adding exogenous cytokine IL2 in the coincubation medium enhanced the antitumor durability of CAR-Vδ1T cells against Raji-B7-H3 cells, five times. Three independent experiments. I, Scheme of the in vivo evaluation of a single dose, with CAR-Vδ1T cells intravenously (1 × 10 7 cells/mouse) combined with intraperitoneal IL2 treatment (at a dose of 2,000 IU/per mouse every other day) in SK-N-AS tumor-bearing NPG mice. J, Tumor growth curve of SK-N-AS. n = 5 mice/group. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant, two-way ANOVA.

Article Snippet: The B7-H3 expression in tumor cells was detected using the anti-human B7-H3 antibody (Miltenyi Biotec, Clone: REA1094).

Techniques: In Vitro, Functional Assay, Construct, Binding Assay, Flow Cytometry, Inhibition, In Vivo

Journal: Cancer Research

Article Title: B7-H3–Targeted CAR-Vδ1T Cells Exhibit Potent Broad-Spectrum Activity against Solid Tumors

doi: 10.1158/0008-5472.CAN-24-0195

Figure Lengend Snippet: CAR-Vδ1T cells coexpressing IL2 display enhanced functional persistence in vitro . A, Construct of B7-H3-CAR without IL2 (AG-CAR) and coexpressing IL2 (AQ-CAR-IL2). B, IL2 could be detected in the culture supernatant of AQ-CAR-IL2 group, data derived from three different donors. C, Compared the antitumor durability of AQ-CAR-IL2 group with AG-CAR group in the antigen-repeated stimulation assay, five times. Three independent experiments. D, The absolute number of CAR-Vδ1T cells after each antigen-repeated stimulation. E, Cytokine production (IFNγ, TNFα, and IL2) in the coincubated supernatant after each antigen-repeated stimulation. F, The growth inhibition effect of AQ-CAR-IL2-Vδ1T against solid tumor cells in vitro using real-time cell analysis system (xCELLigence), including SK-N-AS, A549, HCT-15, and SW1990. G, Cytokine production (IFNγ, TNFα, and IL2) in the coincubated supernatant of AQ-CAR-IL2-Vδ1T cells with the above solid tumor cells. The above data are all presented as the mean ± SD. **, P < 0.01; ****, P < 0.0001, two-way ANOVA.

Article Snippet: The B7-H3 expression in tumor cells was detected using the anti-human B7-H3 antibody (Miltenyi Biotec, Clone: REA1094).

Techniques: Functional Assay, In Vitro, Construct, Derivative Assay, Inhibition, Cell Analysis

Journal: Cancers

Article Title: Adapter Chimeric Antigen Receptor (AdCAR)-Engineered NK-92 Cells for the Multiplex Targeting of Bone Metastases

doi: 10.3390/cancers13051124

Figure Lengend Snippet: Cell line immunophentoyping. Tumor cells were co-incubated with primary biotinylated antibodies. Antigen expression was detected using a secondary PE-coupled anti-biotin antibody. Percentage of stained cells and median fluorescence index (MFI) values were calculated using staining with the secondary antibody alone as negative control.

Article Snippet: Antigen Clone Antibody Order # Lot Supplier CD146 541-10B2 n/a 130-092-852 5190627154 Miltenyi Biotec, Bergisch Gladbach, Germany CD171 REA163 n/a 130-100-702 5190607129 Miltenyi Biotec, Bergisch Gladbach, Germany CD200 OX-104 n/a 130-106-064 5191021606 Miltenyi Biotec, Bergisch Gladbach, Germany CD221 REA271 n/a 130-103-973 5190627184 Miltenyi Biotec, Bergisch Gladbach, Germany CD271 REA844 n/a 130-112-608 5190627191 Miltenyi Biotec, Bergisch Gladbach, Germany CD274 n/a Atezolizumab n/a n/a Hoffmann-La Roche, Basel, Switzerland CD276 FM276 n/a 130-095-514 5190627174 Miltenyi Biotec, Bergisch Gladbach, Germany CD340 n/a Trastuzumab n/a n/a Hoffmann-La Roche, Basel, Switzerland EGFR n/a Cetuximab n/a n/a Merck KgaA, Darmstadt, Germany GD2 n/a Dinutuximab beta n/a n/a Eusa Pharma, Hertfordshire, Great Britain

Techniques: Expressing, Staining, Fluorescence, Control

Journal: Nature medicine

Article Title: Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors

doi: 10.1038/s41591-020-0821-8

Figure Lengend Snippet: a, Flow cytometry analysis of B7-H3 expression on the ATRT cell lines BT16 (ATRT-TYR), CHLA-2 (ATRT-SHH) and VU-397 (ATRT-MYC). b,c, Representative IHC images showing B7-H3 expression on ATRT tumors (b) and two infant ATRT cases with normal adjacent cerebellum and cerebrum (top, close up; bottom, overview) (c). d,e, H-score values of B7-H3 IHC analyses calculated by multiplying the percentage of cells positive by 1, 2 or 3 based on expression level (d). Ordinary one-way ANOVA TYR versus SHH, P = 0.9955; TYR versus MYC, P = 0.9491; SHH versus MYC, P = 0.9870 (statistics were calculated by comparing only the ATRT subgroups). ATRT, n = 47; TYR n = 13; SHH n = 15; MYC n = 11; subgroup unknown, n = 8. Liver, n = 3; cortex, n = 2; cerebellum, n = 2. BT12 and BT16 ATRT cell lines were parental (UNTRT) or transduced with viral vector expressing SMARCB1. Five days later, B7-H3 and SMARCB1 protein expression levels were analyzed by western blotting (e). f, Flow cytometry analysis of B7-H3 expression from cells treated as in e. g, Western blot analysis of B7-H3 and BRG1/SMARCA4 protein in SMARCB1-deficient ATRT cell lines (BT12 and BT16) 5 d after shRNA SMARCA4 knockdown with two different short hairpins. Densitometry quantification of the fold change of B7-H3 and SMARCA4 signals was normalized to the loading control (β-actin). h, Flow cytometry analysis of B7-H3 expression on the surface of cells treated as in g. i,j, mRNA expression levels of B7-H3 in normal tissue from developing and mature cerebrum (i) (prenatal, n = 32; pediatric, n = 12; adult, n = 9) (i) and cerebellum (j) (prenatal, n = 33; pediatric, n = 17; adult, n = 8). wpc, weeks postconception. RPKM, reads per kilobase of transcript per million mapped reads. k, Representative IHC images of B7-H3 staining on normal prenatal (left), infant (middle) and pediatric brain (right). l, Summary of H-score values of B7-H3 on ATRTs (n = 47), normal prenatal brain (n = 10), infant brain (n = 7) and pediatric brain (n = 11). Ordinary one-way ANOVA ATRT versus prenatal brain, P = 0.104; ATRT versus infant brain, ****P < 10−15; ATRT versus pediatric brain, ****P < 10−15; prenatal brain versus infant brain, ****P = 8 × 107; prenatal brain versus pediatric brain, ****P = 5 × 10−9; infant brain versus pediatric brain, P = 0.9741. a,e,f,g,h, Representative of two independent experiments. b,k, Experiments were performed once. All data shown are the mean ± s.d.

Article Snippet: Staining for B7-H3 expression on tumor lines was performed with phycoerythrin (PE)- or allophycocyanin (APC)-conjugated mouse anti-human B7-H3 antibody (clone 185504; R&D Systems).

Techniques: Flow Cytometry, Expressing, Transduction, Plasmid Preparation, Western Blot, shRNA, Staining

Journal: Nature medicine

Article Title: Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors

doi: 10.1038/s41591-020-0821-8

Figure Lengend Snippet: a, Cytometric Bead Array of interferon γ release 24 hours after co-culture of B7-H3 CAR T cells or CD19 CAR T cells (control) with ATRT tumor cells (B7-H3 CAR T cells vs. CD19 CAR T cells: BT12 ****p = 5×10−7; BT16 p = 0.086; ATRT-CHB-1 ****p = 5×10−6; VU397 ****p = 5×10−8; CHLA-2 ****p = 9×10−6; CHLA-4 ****p = 5×10−7; CHLA-5 ***p = 9×10−4; ATRT13808 ****p = 7×10−5) (unpaired t-test, two-tailed). b, Killing assay of BT16 ATRT tumor cells when co-cultured with B7-H3 CAR T cells or CD19 CAR T cells (control) at different E:T ratios (B7-H3 CAR T cells vs. CD19 CAR T cells: 1:1 ****p = 5×10−5; 1:4 ***p = 3×10−4; 1:8 ****p = 2×10−6; 1:16: **p = 0.003) (Two-Way Anova). c, Representative images obtained 72 hours after co-culture of BT16 ATRT tumor cells (red) with B7-H3 or CD19 CAR T cells in a 1:4 ratio. d, Killing assay of BT12 ATRT tumor cells when co-cultured with B7-H3 CAR T cells or CD19 CAR T cells (control) at different E:T ratios (B7-H3 CAR T cells vs. CD19 CAR T cells: 5:1 ****p = 3×10−6; 1:1 ****p = 8×10−5; 1:5 ****p = 5×10−6) (Two-Way Anova). All data are means ± s.e.m. (a-d) n = 3 independent samples, experiments have been conducted three times.

Article Snippet: Staining for B7-H3 expression on tumor lines was performed with phycoerythrin (PE)- or allophycocyanin (APC)-conjugated mouse anti-human B7-H3 antibody (clone 185504; R&D Systems).

Techniques: In Vitro, Co-Culture Assay, Two Tailed Test, Cell Culture

Journal: Nature medicine

Article Title: Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors

doi: 10.1038/s41591-020-0821-8

Figure Lengend Snippet: a, Flow cytometric analysis of B7-H3 expression on ATRT cell lines. Representative of two independent experiments is shown b, Quantification of B7-H3 molecules per cell on ATRT cell lines using flow cytometry (TYR n = 3, SHH n = 3, MYC n = 3; TYR vs. SHH p = 0.700; TYR vs. MYC p = 0.400; SHH vs MYC p = 0.200) (Mann-Whitney test, two-tailed). c, B7-H3 mRNA upon SMARCB1 re-expression from datamining of Chauvin et al (GSE98277 Cell Rep1017) published inducible SMARCB1 system in a SMARCB1-deficient rhabdoid cell line (n = 3 for all time points) 0 days vs. 2 days **p = 0.007; 0 days vs. 4 days *p = 0.011; 0 days vs. 7 days *p = 0.012; 0 days vs. 14 days **p = 0.007) (unpaired t-test, two-tailed). d, ChipSeq H3K27Ac data of the promoter region of B7-H3 in primary ATRT tumors. e, ChipSeq SMARCA4 data at the promoter region of B7-H3 in primary ATRT tumors f, RT-qPCR analysis of B7-H3 and SMARCA4 mRNA expression levels in SMARCB1-deficient ATRT cell lines (BT12 and BT16) five days after shRNA SMARCA4 knock down (k.d.) with two different short hairpins. Graph represents ΔΔCt relative to cells transduced with empty vector. Triplicates were run in each experiment. Representative results of two independent experiments is shown g, Correlation of normalized SMARCA4 and normalized B7-H3 expression from primary ATRT tumors (GSE70678) (n = 49) (r = 0.32, p = 0.026) (Pearson correlation, two-sided) h, Correlation of mRNA expression of SMARCA4 and B7-H3 during normal brain development (prenatal n = 237; pediatric n = 178; adult n = 109) (r = 0.86; p = <1×10−15) (Pearson correlation, two-sided). (i) mRNA expression levels of miR29c during and after normal brain development (prenatal n = 237; pediatric n = 178; adult n = 109). j, miR29 nanostring counts in ATRT cells lines and mature and immature neurons. k, ChipSeq H3K27Ac data around miR29 locus in primary ATRT tumors. All data are means ± s.d.

Article Snippet: Staining for B7-H3 expression on tumor lines was performed with phycoerythrin (PE)- or allophycocyanin (APC)-conjugated mouse anti-human B7-H3 antibody (clone 185504; R&D Systems).

Techniques: Expressing, Flow Cytometry, MANN-WHITNEY, Two Tailed Test, Quantitative RT-PCR, shRNA, Transduction, Plasmid Preparation

Journal: Nature medicine

Article Title: Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors

doi: 10.1038/s41591-020-0821-8

Figure Lengend Snippet: a, Experimental setup (IT, ICV: 1 × 106 CAR T cells; IV: 10 × 106 CAR T cells). LTR, long terminal repeat. b, Bicistronic vector expressing NanoLuc luciferase and the CAR T cell fused to a fluorescent protein. c, BLI signal emanating from the CAR T cells was measured at the indicated time points. d, Quantification of BLI signal in the brain of mice on day 5 after treatment (n = 7 IT, n = 8 ICV and n = 5 IV B7-H3 CAR T cell-treated mice). IT versus ICV, P = 0.613; IT versus IV, *P = 0.048; ICV versus IV, *P = 0.011 (two-tailed Mann–Whitney U-test). Experiments were conducted in two independent cohorts of mice. e, Immunofluorescence microscopy imaging of brains from mice on day 2 after treatment. B7-H3 or control CD19 CAR T cells are shown in green in mice that were previously engrafted with BT16 ATRT cells (red). Tiled images across engrafted regions (top), representative confocal microscopy of B7-H3 CAR T cell-treated groups (middle) and CD19 - CAR T cell-treated groups (bottom) are shown. Scanning of 1:12 series from three independent mice per group. f,g, Quantification (f) and representative flow cytometry (g) showing persisting T cells in the brain following administration of 1 × 106 (IT and ICV) or 10 × 106 (IV) B7-H3 CAR T cells (n = 6 IT, n = 6 ICV and n = 6 IV B7-H3 CAR T cell-treated mice). Pooled data from two independent experiments (IT versus ICV, P = 0.394; IT versus IV, P = 0.732; ICV versus IV, P = 0.394). Two-tailed Mann–Whitney U-test. h, BLI 13 and 27 d after B7-H3 CAR T cell treatment. i, Quantification of BLI signal in the trunk of mice on day 27 after treatment (IT versus ICV, *P = 0.026; IT versus IV, P = 0.178; ICV versus IV, P = 0.329; n = 6 IT, n = 6 ICV and n = 5 IV B7-H3 CAR T cell-treated mice). Two-tailed Mann–Whitney U-test. All data shown are the mean ± s.e.m.

Article Snippet: Staining for B7-H3 expression on tumor lines was performed with phycoerythrin (PE)- or allophycocyanin (APC)-conjugated mouse anti-human B7-H3 antibody (clone 185504; R&D Systems).

Techniques: Plasmid Preparation, Expressing, Luciferase, Two Tailed Test, MANN-WHITNEY, Immunofluorescence, Microscopy, Imaging, Confocal Microscopy, Flow Cytometry

Journal: Nature medicine

Article Title: Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors

doi: 10.1038/s41591-020-0821-8

Figure Lengend Snippet: a, Representative IHC image of B7-H3 staining of IC-pPDX-69 and IC-pPDX-159 ATRT PDX after engraftment (IC-pPDX-69: H Score 210 and IC-pPDX-159: H score 210). Staining was performed one time. b, Experimental overview for the evaluation of in vivo efficacy of B7-H3 CAR T cells against IC-pPDX-69 c,d, and IC-pPDX-159 e,f, Summary (c,e) and individual (d,f) tumor measurements after 10×106 B7-H3 CAR T cells or CD19 CAR T cells (control) (c) p = 2×10−6; (e) p = 6×10−5 (Two-way Anova). All data are means ± s.e.m. Experiment has been performed one time (n = 10 mice per group).

Article Snippet: Staining for B7-H3 expression on tumor lines was performed with phycoerythrin (PE)- or allophycocyanin (APC)-conjugated mouse anti-human B7-H3 antibody (clone 185504; R&D Systems).

Techniques: In Vivo, Staining

Journal: Nature medicine

Article Title: Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors

doi: 10.1038/s41591-020-0821-8

Figure Lengend Snippet: a, Representative IHC image of B7-H3 staining of the BT16 ATRT xenograft (H-score = 285). Staining was performed once. b, Experimental overview of the evaluation of in vivo efficacy of B7-H3 CAR T cells comparing different routes of CAR T cell administration. Mouse cartoon adapted from Gholamin et al.44. c, Intratumoral (IT) group 1 × 106 CAR T cells. d, Intracerebroventricular (ICV) group 1 × 106 CAR T cells. e, IV group 1 × 106 CAR T cells. BLI was obtained serially; the flux curve and corresponding bioluminescent images at designated time points are shown. Kaplan-Meier analysis of B7-H3 versus CD19 (control) CAR T cell-treated mice (IT, P = 0.0003; ICV, P = 0.0007; IV, P = 0.74). Two-tailed, log-rank Mantel–Cox test. Experiments were conducted in two independent cohorts of mice. f,g, Heatmap of cytokines detected in the serum (f) and CSF (g) from mice 7 d after CAR T cell treatment (at curative doses: IT, ICV 1 × 106 CAR T cells; IV 10 × 106 CAR T cells). Z-score values were calculated for each cytokine; for better visualization purposes, the z-score was limited to 2 s.d.; three mice per group, except for the control group in serum, n = 4. Statistics were calculated using box plots. Overall cytokine secretion in serum: CD19 IT versus B7-H3 IT, P = 0.638; CD19 ICV versus B7-H3 ICV, P = 0.025; CD19 IV versus B7-H3 IV, P = 1 × 10−7; B7-H3 IT versus B7-H3 ICV, P = 0.039; B7-H3 IT versus B7-H3 IV, P = 8 × 10−10; B7-H3 ICV versus B7-H3 IV, P = 6 × 10−11. Overall cytokine secretion in the CSF: CD19 IT versus B7-H3 IT, P = 0.039; CD19 ICV versus B7-H3 ICV, P = 7 × 10−7; CD19 IV versus B7-H3 IV, P = 0.0004; B7-H3 IT versus B7-H3 ICV, P = 0.024; B7-H3 IT versus B7-H3 IV, P = 0.133; B7-H3 ICV versus B7-H3 IV, P = 0.094. Two-tailed Mann–Whitney U-test.

Article Snippet: Staining for B7-H3 expression on tumor lines was performed with phycoerythrin (PE)- or allophycocyanin (APC)-conjugated mouse anti-human B7-H3 antibody (clone 185504; R&D Systems).

Techniques: Staining, In Vivo, Two Tailed Test, MANN-WHITNEY

Journal: Nature medicine

Article Title: Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors

doi: 10.1038/s41591-020-0821-8

Figure Lengend Snippet: a, Representative IHC image of B7-H3 staining of BT12 ATRT xenograft (H Score: 270). Staining was performed one time. b, IT Group (B7-H3 (1×106) and CD19 (1×106) CAR T cells), c, ICV Group (B7-H3 (1×106) and CD19 (1×106) CAR T cells), d, IV Group (1×106 or 10×106) B7-H3 and CD19 (10×106) CAR T cells). (c-e) Bioluminescence (BLI) was obtained serially and flux curve for individual mice are shown. Kaplan-Meier analysis (n = 1) of B7-H3 or CD19 (control) CAR T cell treated mice (IT p = 0.0023; ICV p = 0.0031); IV (1×106) p = 0.6015; IV (10×106) p = 0.0038). (Log-rank (Mantel-Cox) test, two-tailed). One mouse in the B7-H3 (10×106) IV group died while imaging and was censored from analysis. Mio=million.

Article Snippet: Staining for B7-H3 expression on tumor lines was performed with phycoerythrin (PE)- or allophycocyanin (APC)-conjugated mouse anti-human B7-H3 antibody (clone 185504; R&D Systems).

Techniques: In Vivo, Staining, Two Tailed Test, Imaging

Journal: Nature medicine

Article Title: Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors

doi: 10.1038/s41591-020-0821-8

Figure Lengend Snippet: In vivo dose response testing of B7H3 CAR T cells administered ICV (a,b) or IV (c,d). Bioluminescence (BLI) was obtained serially and flux curve for individual mice are shown for ICV (a) or IV (c) treatment. Kaplan-Meier analysis of B7-H3 or CD19 (control) CAR T cell treated mice in ICV (b) or IV (d) treatment. Stated p values are compared to CD19 control. (Log-rank (Mantel-Cox) test, two-tailed) n = 5 biologically independent animals. Experiment has been performed one time. Mio=million.

Article Snippet: Staining for B7-H3 expression on tumor lines was performed with phycoerythrin (PE)- or allophycocyanin (APC)-conjugated mouse anti-human B7-H3 antibody (clone 185504; R&D Systems).

Techniques: In Vivo, Two Tailed Test

Journal: Nature medicine

Article Title: Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors

doi: 10.1038/s41591-020-0821-8

Figure Lengend Snippet: a, INFγ, IL-4, IL-10 levels in serum and CSF from mice 7 days after CAR T cell treatment (For IT and ICV: 1×106 CAR T cells, IV: 10×106 CAR T cells). Significantly higher INFγ, IL-4 and IL-10 concentrations in the serum of the B7-H3 IV group compared to all other groups (Serum INFγ: B7-H3 IV vs. IT **p = 0.0033; B7-H3 IV vs. ICV **p = 0.0033; Serum IL-4: B7-H3 IV vs. IT ***p = 0.0008; B7-H3 IV vs. ICV ***p = 0.0008; Serum IL-10: B7-H3 IV vs. IT *p = 0.01; B7-H3 IV vs. ICV *p = 0.01). No significant differences in the CSF between B7-H3 CAR IV and locoregional treated mice (IT, ICV). (Ordinary one-way Anova). All data are means ± s.e.m. 3 mice per group. Serum from mice 7 days b, and 13 days c, after CAR T cell treatment (at indicated dose levels and CD19 IV 10×106 and ICV 1×106 CAR T cells) demonstrates significantly higher inflammatory cytokines on day7 after CAR T cell administration in B7-H3 IV group compared to B7H3 ICV. Strong reduction of cytokine levels on day 13 after CAR T cell administration. Z score values were calculated for each cytokine across the timepoints day 7 and day 13/all samples; for better visualization purposes z score was limited to 2 SD; 3 mice per group. Statistics were calculated using box plots. (Overall cytokine secretion in serum: Day7: Least curative CAR T cell dose: B7-H3 2.5×106 IV vs B7-H3 0.5 ×106 ICV p = 3×10−13; B7-H3 2.5×106 IV vs B7-H3 0.25 ×106 ICV p = 2×10−13; Day 7 vs. Day13: B7-H3 2.5 ×106 p = 3×10−11; B7-H3 0.5 ×106 ICV p = 0.9921, B7-H3 0.25 ×106 ICV p = 0.9957) (Mann-Whitney Test, two-tailed).

Article Snippet: Staining for B7-H3 expression on tumor lines was performed with phycoerythrin (PE)- or allophycocyanin (APC)-conjugated mouse anti-human B7-H3 antibody (clone 185504; R&D Systems).

Techniques: MANN-WHITNEY, Two Tailed Test

Journal: Nature medicine

Article Title: Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors

doi: 10.1038/s41591-020-0821-8

Figure Lengend Snippet: a, Flow cytometry demonstrating that fluorescent protein fused to CAR construct correlated with CAR surface expression. Experiment was performed two times. b, Representative immunofluorescence confocal microscopy of Ki67 staining of B7-H3 or CD19 CAR T cells in the brain. B7-H3 CAR T cells are positive for Ki 67, CD19 CAR T cells are negative. Scanning of 1:12 series from 3 independent mice/group. c, Quantification (n = 3 per group) and d, representative flow cytometry histogram of CAR T cells detected in single cell suspensions from mouse spleens 47 days after one time treatment (IT vs. ICV p = 0.200, IT vs. IV p = 0.800, ICV vs. IV p = 0.200) (Mann-Whitney test, two-tailed). e, Quantification of CAR T cell /T cell ratio in spleen after one time CAR T cell treatment (n = 3 per group) (ICV vs. IV *p = 0.041) (IT vs. IV p = 0.288) (unpaired t-test, two-tailed) All data are means ± s.e.m.

Article Snippet: Staining for B7-H3 expression on tumor lines was performed with phycoerythrin (PE)- or allophycocyanin (APC)-conjugated mouse anti-human B7-H3 antibody (clone 185504; R&D Systems).

Techniques: Flow Cytometry, Construct, Expressing, Immunofluorescence, Confocal Microscopy, Staining, MANN-WHITNEY, Two Tailed Test

Journal: Nature medicine

Article Title: Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors

doi: 10.1038/s41591-020-0821-8

Figure Lengend Snippet: a, Treatment scheme for in vivo validation of functional CAR T cell persistence (IT, ICV, 1 × 106 CAR T cells; IV, 10 × 106 CAR T cells). b,c, Flow cytometry (b) and western blot analysis (c) of B7-H3 expression on a CRSIPR–cas9-edited (knockout) ATRT cell line. Representative of two independent experiments. d, Confocal microscopy analysis of persisting B7-H3 CAR T cells in the brain 40 d after a single administration in mice treated IT, ICV or IV. Scanning of individual sections with persisting CAR T cells from three independent mice per group was performed and a representative immunofluorescence image is shown. e, Pooled flux values of two independent experiments (one rechallenge with parental line and one parental versus knockout line; IT, n = 10 parental versus 3 knockout; ICV, n = 9 parental versus 3 knockout; IV, n = 9 parental versus 3 knockout). f, BLI images of one representative experiment 6 d after orthotopic rechallenge with BT16 or BT16/B7-H3 knockout tumor cells (IT group: BT16 versus B7-H3 knockout, **P = 0.007; ICV group: BT16 versus B7-H3 knockout, **P = 0.009; IV group: BT16 versus B7-H3 knockout, **P = 0.009). Two-tailed Mann–Whitney U-test. g, Pooled flux values of two independent experiments (ICV: n = 5 parental versus 5 knockout; IV: n = 8 parental versus 8 knockout). h, BLI images after flank rechallenge with BT16 or BT16/B7-H3 knockout tumor cells (ICV group: BT16 versus B7-H3 knockout, **P = 0.008; IV group: BT16 versus B7-H3 knockout, ***P = 0.0002; BT16 ICV versus BT16 IV, P = 0.9433). Two-tailed Mann–Whitney U-test. All data shown are the mean ± s.e.m.

Article Snippet: Staining for B7-H3 expression on tumor lines was performed with phycoerythrin (PE)- or allophycocyanin (APC)-conjugated mouse anti-human B7-H3 antibody (clone 185504; R&D Systems).

Techniques: In Vivo, Functional Assay, Flow Cytometry, Western Blot, Expressing, Knock-Out, Confocal Microscopy, Immunofluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Nature medicine

Article Title: Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors

doi: 10.1038/s41591-020-0821-8

Figure Lengend Snippet: a, Proliferation assay of BT16 vs. B7-H3 KO BT16 tumor cells (p = 0.05) (Two-way Anova) n = 3 independent samples, experiment has been conducted three times. b, Representative images of BT16 and BT16 B7-H3 KO ATRT tumor cells. Experiment has been conducted two times c, Flux curve and survival d, of BT16 vs BT16 B7-H3 KO flank xenografts (p = 0.0031) (n = 5 independent animals) (Log-rank (Mantel-Cox) test, two-tailed). Experiment has been performed one time. All data are means ± s.e.m.

Article Snippet: Staining for B7-H3 expression on tumor lines was performed with phycoerythrin (PE)- or allophycocyanin (APC)-conjugated mouse anti-human B7-H3 antibody (clone 185504; R&D Systems).

Techniques: CRISPR, Knock-Out, In Vitro, In Vivo, Proliferation Assay, Two Tailed Test

Journal: Molecular cancer therapeutics

Article Title: Comprehensive surfaceome profiling to identify and validate novel cell-surface targets in osteosarcoma

doi: 10.1158/1535-7163.MCT-21-0836

Figure Lengend Snippet: (A) The workflow of the integrative proteomic and transcriptomic approach used to identify immunotherapeutic targets in osteosarcomas. (B) Expression profile of the cell-surface proteins identified by mass spectrometry in osteosarcoma cell lines and PDX models. (C) Expression profile of the 209 overexpressed surface protein-encoding genes in 98 osteosarcoma patients from the TARGET database (TARGET OS) and 17 osteosarcoma cell lines that we analyzed (OSC). The 11 candidate surface proteins and the 4 candidates that overlapped with existing drug targets are marked. The 4 candidate targets (MT1-MMP, MRC2, CD276, and LRRC15) were highly expressed in most of the patient samples and cell lines.

Article Snippet: Antibodies for MT1-MMP (R&D Systems, FAB9181A, 1:40), MRC2 (kindly provided by Dr. Niels Behrendt, University of Copenhagen, Copenhagen, Denmark, clone 2h9, 1:500) ( 27 ), and CD276 (R&D Systems, FAB1027P, 1:40) were added.

Techniques: Expressing, Mass Spectrometry

Journal: Molecular cancer therapeutics

Article Title: Comprehensive surfaceome profiling to identify and validate novel cell-surface targets in osteosarcoma

doi: 10.1158/1535-7163.MCT-21-0836

Figure Lengend Snippet: (A-D) RNA-seq data showed MT1-MMP (A), MRC2 (B), CD276 (C), and LRRC15 (D) were overexpressed in osteosarcoma compared to a range of normal tissues. The boxes represent the Q1 and Q3 of the data. The bars represent the median. (E-G) MT1-MMP (E), MRC2 (F), and CD276 (G) had higher expression in osteosarcoma compared to other pediatric cancers (OS osteosarcoma, MEL melanoma, RHB rhabdomyosarcoma, CPC choroid plexus carcinoma, HGG high-grade glioma, EPD ependymoma, ACT adrenocortical carcinoma, WLM Wilms’ tumor, NBL neuroblastoma, LGG low-grade glioma, RB retinoblastoma, AML acute myeloid leukemia, MLL mixed-lineage leukemia, MB medulloblastoma, BALL B-cell acute lymphoblastic leukemia, TALL T-cell acute lymphoblastic leukemia; TPM Transcripts Per Million, FPKM Fragments Per Kilobase Million).

Article Snippet: Antibodies for MT1-MMP (R&D Systems, FAB9181A, 1:40), MRC2 (kindly provided by Dr. Niels Behrendt, University of Copenhagen, Copenhagen, Denmark, clone 2h9, 1:500) ( 27 ), and CD276 (R&D Systems, FAB1027P, 1:40) were added.

Techniques: RNA Sequencing Assay, Expressing, Wilms Tumor Assay

Journal: Molecular cancer therapeutics

Article Title: Comprehensive surfaceome profiling to identify and validate novel cell-surface targets in osteosarcoma

doi: 10.1158/1535-7163.MCT-21-0836

Figure Lengend Snippet: (A, B) Western blots of MT1-MMP, MRC2, and CD276 in a panel of osteosarcoma cell lines (n=8; A) and PDXs (n=8; B). (C) Flow cytometry analysis of 7 osteosarcoma cell lines. Gray plots represent unstained controls and colored plots represent staining with MT1-MMP, MRC2, and CD276 antibodies.

Article Snippet: Antibodies for MT1-MMP (R&D Systems, FAB9181A, 1:40), MRC2 (kindly provided by Dr. Niels Behrendt, University of Copenhagen, Copenhagen, Denmark, clone 2h9, 1:500) ( 27 ), and CD276 (R&D Systems, FAB1027P, 1:40) were added.

Techniques: Western Blot, Flow Cytometry, Staining

Journal: Molecular cancer therapeutics

Article Title: Comprehensive surfaceome profiling to identify and validate novel cell-surface targets in osteosarcoma

doi: 10.1158/1535-7163.MCT-21-0836

Figure Lengend Snippet: (A-C) Representative membrane-staining examples of MT1-MMP in a patient sample (A), PDX (B), and testes (negative control; C). (D-F) Representative membrane-staining examples of MRC2 in a patient sample (D), PDX (E), and placenta (negative control; F). (G-I) Representative membrane-staining examples of CD276 in a patient sample (G), PDX (H), and placenta (mild positive; I). (J, K) Summary of IHC staining H-score of MT1-MMP, MRC2, and CD276 in the tissue microarray for 37 osteosarcoma patients (J) and 19 PDX models (K). Boxes indicate standard deviation and error bars represent data range.

Article Snippet: Antibodies for MT1-MMP (R&D Systems, FAB9181A, 1:40), MRC2 (kindly provided by Dr. Niels Behrendt, University of Copenhagen, Copenhagen, Denmark, clone 2h9, 1:500) ( 27 ), and CD276 (R&D Systems, FAB1027P, 1:40) were added.

Techniques: Staining, Negative Control, Immunohistochemistry, Microarray, Standard Deviation

Journal: BMC Cancer

Article Title: Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer

doi: 10.1186/1471-2407-9-463

Figure Lengend Snippet: Immunohistochemical characterization of B7-H3 expression in 4 representative human pancreatic cancer tissue sections . Positive B7-H3 immunoreactivity was detectable in pancreatic cancer cells (brown staining). Representative tissue sections of strong intensity (A), moderate intensity (B), and weak intensity (C). (D) Negative control.

Article Snippet: Cells were harvested and stained with anti-human B7-H3-FITC mAb (R&D Systems) or control anti-human IgG-FITC (BD Pharmingen, Heidelberg, Germany).

Techniques: Immunohistochemical staining, Expressing, Staining, Negative Control

Journal: BMC Cancer

Article Title: Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer

doi: 10.1186/1471-2407-9-463

Figure Lengend Snippet: Clinicopathological characteristics according to tumor B7-H3 expression.

Article Snippet: Cells were harvested and stained with anti-human B7-H3-FITC mAb (R&D Systems) or control anti-human IgG-FITC (BD Pharmingen, Heidelberg, Germany).

Techniques: Expressing

Journal: BMC Cancer

Article Title: Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer

doi: 10.1186/1471-2407-9-463

Figure Lengend Snippet: Overall survival of 68 pancreatic cancer patients in relation with B7-H3 protein expression . Patients with high tumor B7-H3 protein expression had a significantly better prognosis than patients without or with low tumor B7-H3 protein expression (p = 0.0067). The median survival of patients with high tumor B7-H3 protein expression was 17.8 months in contrast to 11.0 months in patients without or with low tumor B7-H3 protein expression.

Article Snippet: Cells were harvested and stained with anti-human B7-H3-FITC mAb (R&D Systems) or control anti-human IgG-FITC (BD Pharmingen, Heidelberg, Germany).

Techniques: Expressing

Journal: BMC Cancer

Article Title: Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer

doi: 10.1186/1471-2407-9-463

Figure Lengend Snippet: IFN-γ mRNA expression in 28 pancreatic cancer tissues and 10 normal pancreatic tissues . (A) IFN-γ mRNA expression was significantly upregulated in pancreatic cancer tissues compared to normal pancreas (p < 0.05). (B) Correlation analysis of B7-H3 mRNA and IFN-γ mRNA expression in pancreatic cancer (n = 28) revealed a positive correlation between B7-H3 and IFN-γ mRNA expression (p = 0.0225; Spearman rho 0.4297).

Article Snippet: Cells were harvested and stained with anti-human B7-H3-FITC mAb (R&D Systems) or control anti-human IgG-FITC (BD Pharmingen, Heidelberg, Germany).

Techniques: Expressing

Journal: BMC Cancer

Article Title: Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer

doi: 10.1186/1471-2407-9-463

Figure Lengend Snippet: Effects of treatment with IFN-γ or IL-4 on Panc-1 and SU86.86 pancreatic cancer cells . Cells were treated with either 2000 IU/ml of recombinant IFN-γ or 40 ng/ml of recombinant IL-4 for 24 or 48 hours and analyzed by FACS. (B) IFN-γ treatment resulted in a marked increase of B7-H3 expression on SU86.86 cells after 24 hours (green line) and 48 hours (red line). (D) IL-4 treatment resulted in a marked increase of B7-H3 expression on SU86.86 cells after 48 hours (red line), but not after 24 hours (green line). In contrast, (A) IFN-γ treatment led to a slight decrease of B7-H3 expression on Panc-1 cells 24 hours after stimulation (green line) with normalization 48 hours after stimulation (red line). (C) IL-4 treatment led to a slight decrease of B7-H3 expression on Panc-1 cells 24 hours after stimulation (green line) and 48 hours after stimulation (red line). The blue lines display B7-H3 expression without stimulation by IFN-γ and IL-4. The grey lines display the (isotope) control group.

Article Snippet: Cells were harvested and stained with anti-human B7-H3-FITC mAb (R&D Systems) or control anti-human IgG-FITC (BD Pharmingen, Heidelberg, Germany).

Techniques: Recombinant, Expressing, Control

Journal: BMC Cancer

Article Title: Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer

doi: 10.1186/1471-2407-9-463

Figure Lengend Snippet: Representative immunohistochemical stainings for B7-H3 (A, C) and CD8 (B, D) in pancreatic cancer tissues . (A) Pancreatic cancer tissue section with strong B7-H3 immunoreactivity. (B) Consecutive section with immunostaining for CD8 shows the infiltration of numerous CD8+ T cells (arrows). (C) Pancreatic cancer tissue section with weak tumor B7-H3 immunoreactivity. (D) Consecutive section with immunostaining for CD8 shows no CD8+ tumor-infiltrating T cells.

Article Snippet: Cells were harvested and stained with anti-human B7-H3-FITC mAb (R&D Systems) or control anti-human IgG-FITC (BD Pharmingen, Heidelberg, Germany).

Techniques: Immunohistochemical staining, Immunostaining

Journal: BMC Cancer

Article Title: Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer

doi: 10.1186/1471-2407-9-463

Figure Lengend Snippet: Semi-quantitative analysis of CD8+ T cells in pancreatic cancer . In areas with high tumor B7-H3 expression, the prevalence of CD8+ T cells was significantly increased compared to areas with low tumor B7-H3 expression (p = 0.018).

Article Snippet: Cells were harvested and stained with anti-human B7-H3-FITC mAb (R&D Systems) or control anti-human IgG-FITC (BD Pharmingen, Heidelberg, Germany).

Techniques: Expressing