Journal: Cell

Article Title: Enhanced safety and efficacy of protease-regulated CAR-T cell receptors.

doi: 10.1016/j.cell.2022.03.041

Figure Lengend Snippet: Figure 1. The optimized SNIP design, trans SNIP BBz, confers robust control over CAR-T cytotoxicity (A) Schematic illustration of the cis SNIP BBz system (left). CAR-CD3z immunoblots of protein lysates from anti-B7H3 cis SNIP BBz CAR-T cells ± 3 mM GPV (middle). Cytotoxicity of day 10 anti-B7H3 cis SNIP BBz CAR-T cells (right). (B) Schematic illustration of the cis SNIP scFv system (left), flow cytometry of surface B7H3 CAR (middle), and tumor killing (right). (C) Schematic illustration of the trans SNIP BBz system (left) and tumor killing (right). (D) Schematic of various combinations of CD8a and CD28 Tm for the CAR and protease modules (left). Tumor killing in the absence (SNIP OFF, middle) or presence (SNIP ON, right) of GPV. (E) CD3z immunoblots of the 8/8 Tm configuration.

Article Snippet: Recombinant B7H3-, HER2-, and human ROR1-Fc (R&D systems) were used to detection B7H3, HER2, and ROR1 surface CAR, respectively.

Techniques: Control, Western Blot, Cytometry

Journal: Cell

Article Title: Enhanced safety and efficacy of protease-regulated CAR-T cell receptors.

doi: 10.1016/j.cell.2022.03.041

Figure Lengend Snippet: Figure 2. Leaky cytotoxic activity is driven by high antigen density on target lines and ameliorated by substituting cleavage sites with high catalytic activity (A and B) Tumor BLI of mice inoculated with Nalm6-B7H3 leukemia cells and were then treated with B7H3.BBz CAR or mock T cells 4 days later. SNIP-treated mice were given 50 mg/kg GPV and 25 mg/kg ritonavir (RTV) (SNIP ON) or vehicle control (SNIP OFF) 3 times per day by oral gavage. RTV is a pharmacokinetic enhancer of GPV. (C and D) Tumor BLI of mice inoculated with MED8A cells in the right flank and cerebellum and were then treated with B7H3.BBz CAR or mock T cells 5 days later. (E) Cytotoxic activity of SNIP B7H3.BBz bearing different cleavage sites (CSs) against GFP-labeled tumor cells in the absence of GPV (SNIP OFF). Data are mean ± SEM of triplicate wells.

Article Snippet: Recombinant B7H3-, HER2-, and human ROR1-Fc (R&D systems) were used to detection B7H3, HER2, and ROR1 surface CAR, respectively.

Techniques: Activity Assay, Control, Labeling

Journal: Cell

Article Title: Enhanced safety and efficacy of protease-regulated CAR-T cell receptors.

doi: 10.1016/j.cell.2022.03.041

Figure Lengend Snippet: Figure 3. SNIP CAR-T cells display enhanced antitumor efficacy in orthotopic solid tumor models (A) Schematic of the CHLA255 neuroblastoma kidney capsule tumor model using GD2-specific CARs. (B) Tumor progression of CHLA255 by BLI. (C) Quantification of BLI (left) and survival curves of mice in the CHLA255 neuroblastoma model (right). (D) Schematic of the 143B osteosarcoma tumor model using HER2-specific CARs. (E) Tumor progression in the 143B osteosarcoma was monitored by tumor volume measurements (left) and survival curves in the 143B model (right). (F) Schematic of the MED8A medulloblastoma brain tumor model using B7H3-specific CARs. (G) Tumor progression of MED8A BLI.

Article Snippet: Recombinant B7H3-, HER2-, and human ROR1-Fc (R&D systems) were used to detection B7H3, HER2, and ROR1 surface CAR, respectively.

Techniques:

Journal: Molecular Cancer Therapeutics

Article Title: DS-7300a, a DNA Topoisomerase I Inhibitor, DXd-Based Antibody–Drug Conjugate Targeting B7-H3, Exerts Potent Antitumor Activities in Preclinical Models

doi: 10.1158/1535-7163.mct-21-0554

Figure Lengend Snippet: Figure 2. Characteristics of DS-7300a. A, Schematic structure of DS-7300a. B and C, Binding activity of DS-7300a against human B7 family proteins. Recombinant proteins of human B7-H1, B7-H2, B7-H3 (4Ig), B7-H4, B7-H5, B7-H6, B7–1, B7–2, and PD-L2 were incubated with DS-7300a or isotype control ADC and binding activities were measured by ELISA. CHO-K1 cells expressing human B7-H3 (4Ig), HHLA2, and BTNL2 were incubated with DS-7300a and binding activities were measured by cell- based ELISA. Each value represents the mean and SD (N ¼ 3). D, Species cross-reactivity of DS-7300a. CHO-K1 cells expressing human (4Ig), cynomolgus monkey (4Ig), rat, and mouse B7-H3 were incubated with DS-7300a and binding activities were evaluated by cell-based ELISA. Each value represents the mean and SD (N ¼ 3). E, In vitro stability of DS-7300a in plasma. The release rate of DXd from 100 mg/mL DS-7300a in human, monkey, rat, and mouse plasma was calculated using the mean concentration of the released DXd (N ¼ 3).

Article Snippet: Immunoplates were coated with recombinant C-terminal Histagged human B7 family proteins, 0.05 mg/mL hB7-H1 (PD-L1), hB7-H2, hB7-H3 (4Ig), hB7-H5, hB7-H6, hB7–1, hB7–2, and hPDL2 or 0.2 mg/mL hB7-H4 overnight at 4 C, washed, blocked with 1% BSA, and then incubated with 40 mg/mL DS-7300a or isotype control ADC for 1 hour at 22 C. All of the human B7 family proteins were purchased from R&D Systems.

Techniques: Binding Assay, Activity Assay, Recombinant, Incubation, Control, Enzyme-linked Immunosorbent Assay, Expressing, In-Cell ELISA, In Vitro, Clinical Proteomics, Concentration Assay

Journal: Molecular Cancer Therapeutics

Article Title: DS-7300a, a DNA Topoisomerase I Inhibitor, DXd-Based Antibody–Drug Conjugate Targeting B7-H3, Exerts Potent Antitumor Activities in Preclinical Models

doi: 10.1158/1535-7163.mct-21-0554

Figure Lengend Snippet: Figure 3. Invitro cytotoxic effects of DS-7300a. A and B, B7-H3 expression on human cancer cells and in vitro cell growth inhibition by DS-7300a. RH-41 (rhabdomyosarcoma), MFE-280 (endometrial adenocarcinoma), and CCRF-CEM (acute lymphocytic leukemia) cells were stained with PE-conjugated isotype control (mIgG2b, gray open histogram) or antihuman B7-H3 antibody (gray closed histogram) and then analyzed by flow cytometry. Cells were cultured with DS-7300a, parental anti–B7- H3 Ab, and isotype control ADC for 6 days, and then cell viability was examined. Each value represents the mean and SD (N ¼ 3). C, DNA damage and apoptosis induced by DS-7300a. RH-41 cells were treated with 10 mg/mL DS-7300a, parental anti–B7-H3 Ab, isotype control ADC, or 10 nmol/L DXd for 72 hours. Then, the cells were harvested and analyzed by Western blotting for phosphorylated Chk1 (pChk1) as a DNA damage marker and for cleaved PARP as an apoptosis marker.

Article Snippet: Immunoplates were coated with recombinant C-terminal Histagged human B7 family proteins, 0.05 mg/mL hB7-H1 (PD-L1), hB7-H2, hB7-H3 (4Ig), hB7-H5, hB7-H6, hB7–1, hB7–2, and hPDL2 or 0.2 mg/mL hB7-H4 overnight at 4 C, washed, blocked with 1% BSA, and then incubated with 40 mg/mL DS-7300a or isotype control ADC for 1 hour at 22 C. All of the human B7 family proteins were purchased from R&D Systems.

Techniques: Expressing, In Vitro, Inhibition, Staining, Control, Cytometry, Cell Culture, Western Blot, Marker

Journal: Molecular Cancer Therapeutics

Article Title: DS-7300a, a DNA Topoisomerase I Inhibitor, DXd-Based Antibody–Drug Conjugate Targeting B7-H3, Exerts Potent Antitumor Activities in Preclinical Models

doi: 10.1158/1535-7163.mct-21-0554

Figure Lengend Snippet: Figure 4. Antitumor activities of DS-7300a in xenograft mouse models. Representative B7-H3 IHC images of tumors and H-scores were also shown for each model. Scale bar, 100 mm. The indicated H-score represents the mean of H-scores in three tumor samples. The arrowhead indicates the time point of treatment. Each value represents the mean and SE of tumor volume. A, Antitumor activities of DS-7300a in cell line–derived xenograft models. Mice inoculated with human cancer cells, RH-41, MFE- 280, or Calu-6 (NSCLC) were treated with DS-7300a, vehicle control, or isotype control ADC intravenously on days 0 and 14 (N ¼ 6 mice/group). B, Antitumor activities of DS-7300a invarious tumor types of PDX models. PDX mice were treated with 10 mg/kg DS-7300a or vehicle control intravenously on days0 and 14 (N ¼ 5 mice/group). CTG-2093, SCLC PDX; CTG-0166, NSCLC PDX; CTG-0820, head and neck cancer PDX; CTG-1061, bladder cancer PDX.

Article Snippet: Immunoplates were coated with recombinant C-terminal Histagged human B7 family proteins, 0.05 mg/mL hB7-H1 (PD-L1), hB7-H2, hB7-H3 (4Ig), hB7-H5, hB7-H6, hB7–1, hB7–2, and hPDL2 or 0.2 mg/mL hB7-H4 overnight at 4 C, washed, blocked with 1% BSA, and then incubated with 40 mg/mL DS-7300a or isotype control ADC for 1 hour at 22 C. All of the human B7 family proteins were purchased from R&D Systems.

Techniques: Derivative Assay, Control

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