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: Cancer cell

Article Title: Antitumor Responses in the Absence of Toxicity in Solid Tumors by Targeting B7-H3 via Chimeric Antigen Receptor T Cells

doi: 10.1016/j.ccell.2019.01.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 4Ig-B7-H3-His , Novus Biologicals , CAT# NBP2-59914-250ug.

Techniques: Generated, Polymer, Recombinant, Enzyme-linked Immunosorbent Assay, Isolation, Software, Imaging

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors

doi: 10.1158/1078-0432.CCR-18-0432

Figure Lengend Snippet: B7-H3 is highly expressed on pediatric solid tumors. A, Pediatric tumor microarrays were stained by IHC for the expression of B7-H3. Representative images of Ewing sarcoma (3+), glioblastoma multiforme (3+), medulloblastoma (2+), and alveolar rhabdomyosarcoma (3+, 2+, and 1+) samples are shown. H-scores were generated by multiplying the % cells positive × intensity seen for each core. H-scores are shown for pediatric sarcomas (B), neuroblastoma and Wilms tumor (C), and pediatric CNS tumors (D). RMS, rhabdomyosarcoma; EWS, Ewing sarcoma; DIPG, diffuse intrinsic pontine glioma.

Article Snippet: Lentiviral engineering of tumor cell lines NALM6-GL (GFP-Luciferase) cell lines stably overexpressing B7-H3 were produced by lentiviral transduction with supernatant containing the cDNA for B7-H3 (Origene, RC215064L1).

Techniques: Staining, Expressing, Generated, Wilms Tumor Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors

doi: 10.1158/1078-0432.CCR-18-0432

Figure Lengend Snippet: Expression of B7-H3 on pediatric tumors by IHC

Article Snippet: Lentiviral engineering of tumor cell lines NALM6-GL (GFP-Luciferase) cell lines stably overexpressing B7-H3 were produced by lentiviral transduction with supernatant containing the cDNA for B7-H3 (Origene, RC215064L1).

Techniques: Expressing, Staining, Wilms Tumor Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors

doi: 10.1158/1078-0432.CCR-18-0432

Figure Lengend Snippet: Systemically administered B7-H3 CAR T cells induce regression of osteosarcoma xenografts. A, B7-H3 CAR T cells produce IFNγ, TNFα, and IL2 following 24-hour in vitro coculture with MG63.3 osteosarcoma. Representative results of four experiments with 3 different PBMC donors are shown. B, Mouse model of orthotopic osteosarcoma: 1e6 MG63.3 tumor cells were injected into the periosteum of the tibia in NSG mice. Two to three weeks later, following onset of measurable tumors, 1e7 B7-H3 CAR+ T cells or irrelevant control CD19 CAR T cells were intravenously administered. C, Tumor growth was measured biweekly by digital caliper and tumor area was calculated. Values for individual mice, as well as mean values per treatment group (inset) are shown. D, Survival curves of mice treated as in B. Representative results of three experiments with 3 different PBMC donors are shown. E, Metastatic model of osteosarcoma: MG63.3-derived tumors were allowed to grow and metastasize before the mouse underwent amputation followed by administration of intravenous 1e7 B7-H3 CAR+ T cells. F, Survival curves of mice treated as in E. Representative results of four experiments with 3 different PBMC donors are shown. Error bars, SEM. P values were calculated as described in Materials and Methods.

Article Snippet: Lentiviral engineering of tumor cell lines NALM6-GL (GFP-Luciferase) cell lines stably overexpressing B7-H3 were produced by lentiviral transduction with supernatant containing the cDNA for B7-H3 (Origene, RC215064L1).

Techniques: In Vitro, Injection, Derivative Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors

doi: 10.1158/1078-0432.CCR-18-0432

Figure Lengend Snippet: Systemically administered B7-H3 CAR T cells induce regression of Ewing sarcoma xenografts. A, Mouse model of orthotopic Ewing sarcoma: 2e7 EW8 tumor cells were injected into the periosteum of the tibia in NSG mice. Two weeks later, 1e7 B7-H3 CAR+ T cells or irrelevant control CD19 CAR T cells were intravenously administered. B, Tumor growth was measured twice weekly by digital caliper and tumor area was calculated. Values for individual mice, as well as mean values per treatment group (inset) are shown. C, Survival curves of mice treated as in A. Representative results of two experiments with 2 different PBMC donors are shown. Error bars, SEM. P values were calculated as described in Materials and Methods.

Article Snippet: Lentiviral engineering of tumor cell lines NALM6-GL (GFP-Luciferase) cell lines stably overexpressing B7-H3 were produced by lentiviral transduction with supernatant containing the cDNA for B7-H3 (Origene, RC215064L1).

Techniques: Injection

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors

doi: 10.1158/1078-0432.CCR-18-0432

Figure Lengend Snippet: Systemically administered B7-H3 CAR T cells can clear medulloblastoma xenografts. A, B7-H3 CAR T cells were cocultured in vitro with medulloblastoma cell lines and patient-derived DIPG cell cultures and, 24 hours later, IFNγ, TNFα, and IL2 were measured in the supernatant. Representative results of three experiments with 3 different PBMC donors are shown. B, Orthotopic xenograft model of medulloblastoma: NSG mice were autochthonously injected with luciferase expressing DAOY medulloblastoma tumor cells. Following evidence of tumor engraftment by IVIS imaging, animals received 1e7 B7-H3 CAR+ T cells or CD19 CAR T cells intravenously. C, In vivo imaging of DAOY tumors treated with B7-H3 or CD19 CAR T cells. D, Tumor progression was measured by bioluminescence photometry and flux values (photons per second) were calculated using Living Image software. Values for individual mice, as well as mean values per treatment group (inset) are shown. Representative results of three experiments with three different PBMC donors are shown. E, Orthotopic xenograft model of c-myc–amplified medulloblastoma: D425 tumor cells expressing luciferase were autochthonously injected into NSG mice. Mice were treated with 1e7 B7-H3 CAR+ T cells or CD19 CAR T cells after 3–4 days, at which point tumor was detectable by IVIS imaging. F, Tumor progression was measured by bioluminescence photometry and flux values (photons per second) were calculated using Living Image software. Values for individual mice, as well as average values of living mice per treatment group (inset) are shown. G, In vivo imaging of D425 tumors treated with B7-H3 or CD19 CAR T cells. H, Survival curves of mice shown in G. Representative results of three experiments with three different T cell donors are shown. I, Confocal images of brains from D425-GFP+ tumor bearing mice treated with B7-H3 CAR-mCherry or CD19 CAR-mCherry T cells, harvested at two different time points after T-cell infusion. Representative image of two mice at two time points in one experiment. Error bars, SEM. P values were calculated as described in Materials and Methods.

Article Snippet: Lentiviral engineering of tumor cell lines NALM6-GL (GFP-Luciferase) cell lines stably overexpressing B7-H3 were produced by lentiviral transduction with supernatant containing the cDNA for B7-H3 (Origene, RC215064L1).

Techniques: In Vitro, Derivative Assay, Injection, Luciferase, Expressing, Imaging, In Vivo Imaging, Software, Amplification

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors

doi: 10.1158/1078-0432.CCR-18-0432

Figure Lengend Snippet: B7-H3 CAR T cells have limited activity against B7-H3 low expressing K562 xenografts. A, Mouse model of K562 leukemia: NSG mice were inoculated with K562, a myeloid leukemia that expresses low levels of B7-H3, and then treated with 1e7 B7-H3 CAR+ T cells or mock transduced control T cells 5 days later. B, Survival curves of mice treated as in A. Representative results of five experiments with three different PBMC donors are shown. C, Flow cytometric analysis of B7-H3 expression on the surface of K562 (leukemia), MG63.3 (osteosarcoma), EW8 (Ewing sarcoma), DAOY, and D425 (medulloblastoma) human cell lines. D, Number of B7-H3 surface molecules expressed by human tumor cell lines as estimated by Quantibrite kit.

Article Snippet: Lentiviral engineering of tumor cell lines NALM6-GL (GFP-Luciferase) cell lines stably overexpressing B7-H3 were produced by lentiviral transduction with supernatant containing the cDNA for B7-H3 (Origene, RC215064L1).

Techniques: Activity Assay, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors

doi: 10.1158/1078-0432.CCR-18-0432

Figure Lengend Snippet: B7-H3 CAR T cells require adequate antigen expression for in vitro and in vivo activity. A, Flow cytometry analysis of B7-H3 expression on single-cell clones derived from Nalm6 expressing different amounts of lentivirally expressed B7-H3. B, Number of B7-H3 surface molecules expressed by Nalm6-B7-H3 cell lines as estimated by Quantibrite kit. C, GFP+ Nalm6-B7H3 clones were cocultured with B7-H3 CAR T cells and tumor cell killing was measured in an Incucyte assay over 72 hours. Representative data of three experiments with three different PBMC donors is shown. D, Percentage of CAR T cells positive (left) and mean fluorescence index (right) for T-cell activation and degranulation markers CD69 and CD107a, as measured by flow cytometry 6 hours after coculture of B7-H3 CAR T cells with tumor cells expressing increasing amounts of B7-H3. Representative results of three experiments with three different PBMC donors are shown. E and F, Cytokine production by CAR T cells cocultured with tumor cells expressing increasing amounts of B7-H3. G, Mouse model for Nalm6-B7H3: 1e6 NALM6 cells expressing either low or medium amounts of B7-H3 were engrafted into mice by tail vein injection. Three days later, mice were injected with 1e7 B7-H3 CAR+ T cells or untransduced control T cells (MOCK). In vivo imaging of mice bearing (H) Nalm6-B7-H3-Medium leukemia or (I) Nalm6-B7-H3-Low leukemia. J and K, Tumor progression was measured by bioluminescence photometry and flux values (photons per second) were calculated using Living Image software. Values for individual mice are shown. Representative results of four (Nalm6-B7-H3-Med) and two (Nalm6-B7-H3-Low) experiments with two different PBMC donors are shown. N6, NALM6; GL, GFP-Luciferase.

Article Snippet: Lentiviral engineering of tumor cell lines NALM6-GL (GFP-Luciferase) cell lines stably overexpressing B7-H3 were produced by lentiviral transduction with supernatant containing the cDNA for B7-H3 (Origene, RC215064L1).

Techniques: Expressing, In Vitro, In Vivo, Activity Assay, Flow Cytometry, Clone Assay, Derivative Assay, Fluorescence, Activation Assay, Injection, In Vivo Imaging, Software, Luciferase