Journal: iScience

Article Title: Development of chimeric antigen receptor (CAR)-T cells targeting A56 viral protein implanted by oncolytic virus

doi: 10.1016/j.isci.2024.109256

Figure Lengend Snippet: Essential domain of A56 for its localization on cell plasma membrane (A) Confocal immunofluorescence (IF) microscopy of OVV-infected A549 tumor cells showing A56 expression; nuclei (blue), OVV (green), A56 (red); scale bar, 10 μm. (B) Full A56 construct design compared with those of various A56 mutant constructs that do not have one or more domains; Sg, signaling domain; IgV, IgV-like domain; TDR, tandem repeat domain; S, stalk region; TM, transmembrane domain; CT, cytoplasmic tail. (C) Merged confocal images of A56 localization on the plasma membranes of U2-OS tumor cells upon full A56-expressing plasmid transduction compared to proteins enclosed within the Golgi or ER from transduction with mutant A56 vectors; nuclei (blue), A56 (green), Golgi apparatus or endoplasmic reticulum (red); scale bar, 10 μm.

Article Snippet: For A56 detection on the plasma membrane, the cells were labeled with 1μg of anti-A56 antibody (Mouse IgG1 w/o kappa light chain) (LakePharma, Variant 3) diluted in PBS supplemented with 2% FBS for 30 min at 4°C.

Techniques: Clinical Proteomics, Membrane, Immunofluorescence, Microscopy, Infection, Expressing, Construct, Mutagenesis, Plasmid Preparation, Transduction

Journal: iScience

Article Title: Development of chimeric antigen receptor (CAR)-T cells targeting A56 viral protein implanted by oncolytic virus

doi: 10.1016/j.isci.2024.109256

Figure Lengend Snippet: Localization of A56 on solid tumor plasma membrane and distribution in tumor tissue (A) OTS-412 were treated in A549, HCT-116, HeLa cancer cells at 0.05 and 0.1 MOI for 24 h followed by detection of A56 expression via flow cytometry. Dead cells were excluded by Zombie Aqua (ZA) staining. For intracellular detection of A56, fixation/permeabilization was performed before anti-A56 staining. (B) Analysis of A56 expression compared to A27L (a vaccinia virus cytosolic protein) expression on tumor tissue collected 4 days post-OV injection of HT-29 tumor-bearing mice. Scale bar, 100 px (=438 μm). (C) IF analysis of tumor-specific A56 expression and non-expression on other tissues from VX2 tumor-bearing New Zealand White rabbits collected on day (D) 5, 14, and 28 after intravenous injection of OTS-412/HU; The data shown are representative of six and two individual rabbits from the treatment and control groups, respectively. Scale bar, 200 μm. (D) Quantitation of A56 expression detected from each tissue of Figure 2 C presented in mean intensity of fluorescence. Data are represented as mean ± SEM. (E) Distribution of OTS-412 in the OTS-412/HU combination group presented in mean viral copies per 25 mg of each tissue and 1 mL of PBMC analyzed until D28 post virus injection. Data are represented as mean ± SEM. BM; bone marrow, AG; adrenal gland.

Article Snippet: For A56 detection on the plasma membrane, the cells were labeled with 1μg of anti-A56 antibody (Mouse IgG1 w/o kappa light chain) (LakePharma, Variant 3) diluted in PBS supplemented with 2% FBS for 30 min at 4°C.

Techniques: Clinical Proteomics, Membrane, Expressing, Flow Cytometry, Staining, Virus, Injection, Control, Quantitation Assay, Fluorescence

Journal: iScience

Article Title: Development of chimeric antigen receptor (CAR)-T cells targeting A56 viral protein implanted by oncolytic virus

doi: 10.1016/j.isci.2024.109256

Figure Lengend Snippet: In vitro and in vivo functionality of A56-specific A56 CAR-T cells (A) Real-time cytotoxicity of A56 CAR-T cells against HCT-116 cells infected with 0.05 MOI of OTS-412. (B) Quantification of A56 CAR-T cell cytotoxicity in various human cancer cell lines infected with OTS-412. (∗∗∗ p < 0.001, comparison of T cell groups; n = 2 for A549, HCT-116, HeLa, SK-MEL-5 and n = 3 for MCF7 and PC-3 by two-tailed unpaired t test). Data are represented as the mean value ± SEM. (C) Aggregation of A56 CAR-T cells targeting tumor cells observed every 12 h until day 4 in real-time. (D) Antigen-dependent anti-tumor efficacy of A56 CAR-T cells in HCT-116 tumor-bearing mice after intratumoral T cell injection following OTS-412 injection with or without HU (∗ p < 0.05, ∗∗ p < 0.01, comparison of T cell groups; saline, n = 5; treatment groups, n = 4, two-way ANOVA with multiple comparison). Data are represented as the mean value ± SD. (E) IHC analysis of A56 expression and infiltrated hCD3 + T cells in tumors isolated from HCT-116 tumor-bearing mice 21 days post-intravenous T cell treatment. Scale bar, 20 μm (left panel) and quantitation of tumor-infiltrating A56 CAR-T cells positive for hCD3 (right panel) (∗ p = 0.01, n = 3, two-tailed unpaired t test). Data are presented as the mean ± SD. MOI, multiplicity of infection; IHC, immunohistochemistry.

Article Snippet: For A56 detection on the plasma membrane, the cells were labeled with 1μg of anti-A56 antibody (Mouse IgG1 w/o kappa light chain) (LakePharma, Variant 3) diluted in PBS supplemented with 2% FBS for 30 min at 4°C.

Techniques: In Vitro, In Vivo, Infection, Comparison, Two Tailed Test, Injection, Saline, Expressing, Isolation, Quantitation Assay, Immunohistochemistry

Journal: iScience

Article Title: Development of chimeric antigen receptor (CAR)-T cells targeting A56 viral protein implanted by oncolytic virus

doi: 10.1016/j.isci.2024.109256

Figure Lengend Snippet:

Article Snippet: For A56 detection on the plasma membrane, the cells were labeled with 1μg of anti-A56 antibody (Mouse IgG1 w/o kappa light chain) (LakePharma, Variant 3) diluted in PBS supplemented with 2% FBS for 30 min at 4°C.

Techniques: Staining, Virus, Recombinant, Isolation, Saline, Plasmid Preparation, Cell Isolation, Expressing, Software

Journal: American Journal of Cancer Research

Article Title: Antibody and fragment-based PET imaging of CTLA-4+ T-cells in humanized mouse models

doi:

Figure Lengend Snippet: In vitro studies. (A) Salivary gland tissues contain infiltrating human T-cells, as evidenced through co-staining of human CD3 and CTLA-4. (B) Intact ipilimumab and ipilimumab-F(ab’)2 retain their binding specificity to CTLA-4+ tissues. Scale bar (A) 20 µm, (B) 10 µm. (C) Results of flow cytometry binding assay with human PBMCs indicate higher binding of the targeted over nontargeted agent. (D) Significantly higher binding of ipilimumab and ipilimumab-F(ab’)2 to PBMCs is verified through quantitative mean fluorescence intensity analysis of flow results. **P<0.05.

Article Snippet: Ipilimumab was digested using the Promega IdeS protease.

Techniques: In Vitro, Staining, Binding Assay, Flow Cytometry, Fluorescence

Journal: American Journal of Cancer Research

Article Title: Antibody and fragment-based PET imaging of CTLA-4+ T-cells in humanized mouse models

doi:

Figure Lengend Snippet: 64Cu-NOTA-Ipilimumab PET. PET imaging of 64Cu-NOTA-ipilimumab in humanized (PBL) and control (NBSGW) mice. Salivary glands (sites of T-cell infiltration) are outlined by red dashed circles. Maximum intensity projection (MIP) images are shown, representative of 3-5 mice per group.

Article Snippet: Ipilimumab was digested using the Promega IdeS protease.

Techniques: Imaging

Journal: American Journal of Cancer Research

Article Title: Antibody and fragment-based PET imaging of CTLA-4+ T-cells in humanized mouse models

doi:

Figure Lengend Snippet: 64Cu-NOTA-Ipilimumab-F(ab’)2 PET. PET imaging of 64Cu-NOTA-ipilimumab-F(ab’)2 in humanized (PBL) and control (NBSGW) mice. Salivary glands (sites of T-cell infiltration) are outlined by red dashed circles. Maximum intensity projection (MIP) images are shown, representative of 3-5 mice per group.

Article Snippet: Ipilimumab was digested using the Promega IdeS protease.

Techniques: Imaging

Journal: Cancers

Article Title: Current and Future Therapies for Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers14102417

Figure Lengend Snippet: Summary table of discussed clinical trials investigating novel therapeutics for PDAC.

Article Snippet: The Cancer Centre at Johns Hopkins is currently in phase 1 of a trial utilising a pooled KRAS peptide vaccine with polyinosinic–polycytidylic acid adjuvant in combination with two monoclonal antibodies: ipilimumab and nivolumab [ ].

Techniques: Clinical Proteomics, Mutagenesis