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: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity

doi: 10.1007/s00262-024-03642-4

Figure Lengend Snippet: Optimization of cell culture conditions for generating canine CAR T cells . The impact of cell culture conditions, including T cell activation and T cell expansion, were evaluated to identify the optimal conditions. A Diagram of human B7-H3 CAR construct used to generate canine B7-H3 CAR T cells. In B , flow cytometry was used to determine expression of canine CD5 + T cell CD4 and CD8 subsets by day 10 CAR T cell cultures. In C the relative CAR T cell transduction efficiency was determined by immunostaining with recombinant L-protein. Canine T cells were activated with PHA, CD3/CD28 beads, or aAPCs, and expanded in rhIL-2 (top row) or rcIL-2 (bottom row), and the percentage of L-protein positive cells determined by flow cytometry. In D , CAR T cells were generated from n = 6 dogs (3 healthy, 3 osteosarcoma), and the mean ± SEM cell numbers and percentages of CAR T cells (red) and non-transduced T cells (blue) determined for each of the culture activation and expansion conditions (x-axis) determined, illustrating greatest CAR T generation when rhIL-2 was used for expansion. E Representative CAR T cell cytotoxicity study, using the canine OS cell line Abrams as the target, as described in Methods. Target killing over time determined for B7-H3 CAR T cells cultured in IL-2 + IL-21 (red) or IL-2 + IL-21 + IL-7 and IL15 (green), demonstrating superior killing activity of CAR T cells expanded in the 4-cytokine cocktail. Assays were run in triplicate, and statistical significance was determined using Dunn’s multiple means comparison test; *P < 0.05, *****, P < 0.0001, ns not significant

Article Snippet: Expression of B7-H3 by osteosarcoma tumors and normal dog lymph node was assessed using anti-B7 H3 antibody (clone RBT-B7H3, BioSB) by immunohistochemistry using 4 um sections from formalin fixed tumor sections (FFPE).

Techniques: Cell Culture, Activation Assay, Construct, Flow Cytometry, Expressing, Transduction, Immunostaining, Recombinant, Generated, Activity Assay, Comparison

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity

doi: 10.1007/s00262-024-03642-4

Figure Lengend Snippet: Expression of B7-H3 by canine OS tumor tissues . Tumor biopsies from n = 8 different dogs with primary, appendicular OS tumors were assessed to determine the relative levels of B7-H3 expression In A , tissues were immunostained for B7-H3 expression (red) using a cross-reactive mAb, and expression compared to immunostaining with an irrelevant mAb. In B an image of normal dog lymph node showed negative staining of B7H3. In C , the relative levels of B7-H3 expression by each of these 8 tumor biopsies were determined by ImageJ software (using 5 random 20 × magnification areas) to calculate an H-score, and expression was compared to that of normal canine lymph node (LN) tissue

Article Snippet: Expression of B7-H3 by osteosarcoma tumors and normal dog lymph node was assessed using anti-B7 H3 antibody (clone RBT-B7H3, BioSB) by immunohistochemistry using 4 um sections from formalin fixed tumor sections (FFPE).

Techniques: Expressing, Immunostaining, Negative Staining, Software

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity

doi: 10.1007/s00262-024-03642-4

Figure Lengend Snippet: Variable B7-H3 expression by 7 different canine OS cell lines . The potential variability in OS cell line expression of B7-H3 was assessed using cell lines generated from 7 different canine OS tumors (as noted by either animal name, or a numeric designation). A – G depicts flow staining plots comparing B7H3 expression (blue histograms) to mouse IgG1 isotype control immunostaining (red histograms) for each cell line labeled in top right. H) Geometric mean MFI (mean florescence intensity) on log scale for each cell line B7H3 expression and corresponding isotype control staining in red

Article Snippet: Expression of B7-H3 by osteosarcoma tumors and normal dog lymph node was assessed using anti-B7 H3 antibody (clone RBT-B7H3, BioSB) by immunohistochemistry using 4 um sections from formalin fixed tumor sections (FFPE).

Techniques: Expressing, Generated, Staining, Control, Immunostaining, Labeling

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity

doi: 10.1007/s00262-024-03642-4

Figure Lengend Snippet: Functional assessment of canine B7-H3 CAR T cells in vitro . Canine B7-H3 CAR T cells were evaluated for their functional properties, including cytokine production and tumor cytotoxicity. In A , B7-H3 CAR T cells were cultured with 7 canine OS cell line targets, and production of IFN-γ was measured at 24 h. Cytokine production by non-transduced (NTD) T cells co-cultured with OS cell lines was included as a control. y-axis shows IFNg production in pg/mL. In B , killing of Abrams OS target cells by triplicate cultures of B7-H3 CAR T cells or NTD T cells was recorded over time using an Incucyte instrument, using an E:T ratio of 20:1 (effector CAR T to target cells) Y-axis shows total cell count (RFP red) with time elapsed on x-axis. Color legend shown in top right corner. In C , a different measure of cell killing (apoptosis induction by caspase 3 and 7 expression) was used to assess cytotoxicity of B7-H3 CAR T cells against either OSA8 target cells (B7-H3 high expressor), or D17 (B7-H3 low expressor) target cells. Y-axis shows total green (GFP- caspase) per image. In D), the impact of E:T ratios on target killing by B7-H3 CAR T cells was assessed, using Abrams OS cells as targets, at E:T ratios of 1:1, 5:1, and 20:1. Killing is depicted graphically as mean ± SEM . Statistical differences were determined by Dunn’s multiple means comparison, with **** = p < 0.0001 and ns = not significant

Article Snippet: Expression of B7-H3 by osteosarcoma tumors and normal dog lymph node was assessed using anti-B7 H3 antibody (clone RBT-B7H3, BioSB) by immunohistochemistry using 4 um sections from formalin fixed tumor sections (FFPE).

Techniques: Functional Assay, In Vitro, Cell Culture, Control, Cell Counting, Expressing, Comparison

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity

doi: 10.1007/s00262-024-03642-4

Figure Lengend Snippet: Functional in vitro activity of dual B7-H3-CXCR2 CAR T cells compared to single CAR B7-H3 CAR T cells . The ability of a chemokine receptor (CXCR2) to improve the activity of canine B7-H3 CAR T cells when the two molecules were co-expressed by transduced T cells was examined using in vitro studies. In A , the diagram of the dual B7-H3-CXCR2 construct is depicted. In B , the transduction efficiency of canine T cells with the dual CAR construct was evaluated by measuring L-protein binding (scFv detection) and with anti-huCXCR2 immunostaining, demonstrating expression of both molecules, along with the distribution of CD4 and CD8 CAR T cells. Flow cytometry dot plots showing percentage positive for each population. In C , to assess tumor target expression of a key chemokine (CXCL8) ligand for CXCR2, secretion of CXCL8 by 7 different canine OS tumor cell lines was measured, demonstrating that 6 of the 7 lines secreted large amounts of CXCL8 spontaneously. The ability of the huCXCR2 molecule to bind and become activated by canine CXCL8 was confirmed in D , using a transwell migration assay as described in Methods, with sustained migration of B7-H3-CXCR2 CAR T cells to a canine CXCL8 gradient. Serum containing medium was used as a positive control. In E , cytokine secretion following target Abrams cell engagement for 24 h was assessed, and revealed significantly greater production of both IFN-g and IL-2 with B7-H3-CXCR2 CAR T cells compared to B7-H3 CAR T cells, at equivalent E:T ratios (20:1)

Article Snippet: Expression of B7-H3 by osteosarcoma tumors and normal dog lymph node was assessed using anti-B7 H3 antibody (clone RBT-B7H3, BioSB) by immunohistochemistry using 4 um sections from formalin fixed tumor sections (FFPE).

Techniques: Functional Assay, In Vitro, Activity Assay, Construct, Transduction, Protein Binding, Immunostaining, Expressing, Flow Cytometry, Transwell Migration Assay, Migration, Positive Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity

doi: 10.1007/s00262-024-03642-4

Figure Lengend Snippet: Transcriptomic comparison of B7-H3 and B7-H3-CXCR2 CAR T cells . RNA sequencing was performed to help elucidate in greater detail the functional differences between the two CAR T cells. In A , principal component analysis (PCA) plots depict B7-H3 CAR T cells, B7-H3-CXCR2 CAR T cells, and non-transduced T cells from 3 different dogs with biological replicates shown connected by lines. In B , volcano plots depict significantly upregulated (red) and downregulated (blue) genes in B7-H3-CXCR2 vs B7-H3 CAR T cells. Gene set enrichment analysis (GSEA) in C revealed significant upregulation (right of central axis) of pathways related to RNA processing and myc targets, along with downregulation of certain G-protein coupled receptor activities. Constitutive secretion of CXCL8 by B7-H3-CXCR2 CAR T cells in the absence of target cells was observed in panel D

Article Snippet: Expression of B7-H3 by osteosarcoma tumors and normal dog lymph node was assessed using anti-B7 H3 antibody (clone RBT-B7H3, BioSB) by immunohistochemistry using 4 um sections from formalin fixed tumor sections (FFPE).

Techniques: Comparison, RNA Sequencing, Functional Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity

doi: 10.1007/s00262-024-03642-4

Figure Lengend Snippet: Activity of B7-H3 and B7-H3-CXCR2 CAR T cells compared in canine OS xenograft model . The relative ability of canine CAR T cells generated using the two different CAR constructs to control growth of a canine OS tumor (Abrams) in NSG mice was evaluated, with the study design illustrated in A . In B , the persistence of the two CAR T cells in blood of mice at day 14 was assessed using flow cytometric detection of canine CD5 T cells, revealing undetectable CAR T cells in B7-H3 CAR T injected mice; whereas, CAR T cells remained detectable in B7-H3-CXCR2 CAR T injected mice. Tumor responses to CAR T cell treatment were measured in C , using BLI, as noted in Methods, and revealed greater tumor control and elimination in mice treated with B7-H3-CXCR2 CAR T cells, compared to untreated mice or mice treated with B7-H3 CAR T cells. In D , tumor growth was quantitated using direct volume measurements (upper panel) or BLI measurements (lower panel) and revealed significantly greater tumor control in animals treated with B7-H3-CXCR2 CAR T cells. Data represent mean ± SEM of tumor area or photon flux, with * and ** reflecting statistical significance of p < 0.05 and p < 0.01 respectively, as determined by repeated measures ANOVA for tumor area

Article Snippet: Expression of B7-H3 by osteosarcoma tumors and normal dog lymph node was assessed using anti-B7 H3 antibody (clone RBT-B7H3, BioSB) by immunohistochemistry using 4 um sections from formalin fixed tumor sections (FFPE).

Techniques: Activity Assay, Generated, Construct, Control, Injection