2018 n a pcam kan r4r3 addgene 71275 pcam hyg r4r3 addgene 71274 software  (Addgene inc)

Journal: Frontiers in Immunology

Article Title: Identification of New Antibodies Targeting Malignant Plasma Cells for Immunotherapy by Next-Generation Sequencing-Assisted Phage Display

doi: 10.3389/fimmu.2022.908093

Figure Lengend Snippet: Binding analyses of monoclonal phage antibodies. (A) 1 x 10 6 L‐363 myeloma cells were incubated with 1 x 10 9 colony-forming-units (CFU) from the 41 different VH clonotypes and tested in ELISA experiments. Cell surface bound phages were detected using anti-M13-HRP antibody and absorbance was measured at 405 nm. 17 phages (green) showed a signal above the one obtained for CD7-specific phage (grey; dotted line), which was used as negative control. (B) Results of ELISA experiments performed equally with 1 x 10 6 PBMC from healthy donors are shown. 9 phage antibodies (green) bind low to moderate to PBMC (green) compared to cells alone (grey; dotted line). CD7-specific phage (grey) showed antigen-dependent binding to T cells and NK cells and served as positive control in these experiments. Mean values ± SEM of three independent experiments are shown for these ELISA experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 monoclonal phage vs. control. (C) The 9 most promising candidates were further tested in whole cell ELISA experiments with plasma cell lines (MM) L-363, U‐266, and INA-6, Raji Burkitt’s lymphoma (BL) cell line, T cell acute lymphoblastic leukemia cell line (T-ALL) CEM, human endothelial cells (endothelial) from healthy donors (HUVEC) and stable transfected CHO cells expressing either CD38, SLAMF7, BCMA, ICAM-1, CD40, CD56, CD70, CD138, FGFR3 or IL-6R. Color bar was set OD 405 nm = 0 (white) to OD 405 nm > 4 (dark green).

Article Snippet: The inhibition of the enzymatic activity of CD38 was measured using the CD38 Inhibitor Screening Assay Kit (Cyclase Activity) from BPS Bioscience (cat. no. 71275).

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Negative Control, Positive Control, Transfection, Expressing

Journal: Frontiers in Immunology

Article Title: Identification of New Antibodies Targeting Malignant Plasma Cells for Immunotherapy by Next-Generation Sequencing-Assisted Phage Display

doi: 10.3389/fimmu.2022.908093

Figure Lengend Snippet: Frequencies, V genes and CDRH3/CDRL3 aa sequences of the 9 myeloma binding antibodies.

Article Snippet: The inhibition of the enzymatic activity of CD38 was measured using the CD38 Inhibitor Screening Assay Kit (Cyclase Activity) from BPS Bioscience (cat. no. 71275).

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: Identification of New Antibodies Targeting Malignant Plasma Cells for Immunotherapy by Next-Generation Sequencing-Assisted Phage Display

doi: 10.3389/fimmu.2022.908093

Figure Lengend Snippet: Binding analysis of chimeric antibody #5-CD38-IgG1. (A) L-363 cells were incubated with increasing concentrations of #5-CD38-IgG1 (green), daratumumab (red), isatuximab (blue) and control IgG1 (grey). Antibody binding to cell surface CD38 was detected by goat anti-human Fc F(ab) 2 fragment-FITC and flow cytometry. Mean fluorescence intensity (MFI) ± SEM from three independent experiments are shown. ****p ≤ 0.0001 for CD38 antibodies vs. control IgG1. (B) Structures from co-crystallization of daratumumab Fab (red) and isatuximab Fab (blue) with the extracellular part of CD38 (grey, key residues of the catalytic active side indicated in black) merged by superposition of CD38 according references ( , ). (C) To check for overlapping epitopes of #5-CD38-IgG1 with daratumumab or isatuximab on cell surface expressed CD38, 0.2 x 10 6 L-363 cells were pre-incubated with 1 mg/ml #5-CD38-IgG1, daratumumab, isatuximab or control IgG1 prior incubation with 5 µg/ml DyLight755-labeled daratumumab (left graph), isatuximab (middle graph) or #5-CD38-IgG1 (right graph) and subsequently analyzed by flow cytometry. MFI ± SEM from three independent experiments are shown. ***p ≤ 0.001. (D) MNC from BM aspirates of three myeloma patients with 27% (left histogram), 4% (middle histogram) and 0.8% (right histogram) CD138-positive plasma cells, respectively, were checked for binding of FITC-labeled #5-CD38-IgG1 (#5, green) and corresponding control IgG1 antibody (black) by flow cytometry.

Article Snippet: The inhibition of the enzymatic activity of CD38 was measured using the CD38 Inhibitor Screening Assay Kit (Cyclase Activity) from BPS Bioscience (cat. no. 71275).

Techniques: Binding Assay, Incubation, Flow Cytometry, Fluorescence, Crystallization Assay, Labeling

Journal: Frontiers in Immunology

Article Title: Identification of New Antibodies Targeting Malignant Plasma Cells for Immunotherapy by Next-Generation Sequencing-Assisted Phage Display

doi: 10.3389/fimmu.2022.908093

Figure Lengend Snippet: Functional characterization of #5-CD38-IgG1. (A) To measure the induction of PCD, 0.5 x 10 6 Daudi cells were incubated with 1 µg/ml of the indicated antibodies for 20 h and afterwards stained with Annexin V-FITC and 7-AAD. Dead cells are shown as mean percentage of total Annexin V-positive cells ± SEM from three independent experiments. *p ≤ 0.05 CD38 antibodies vs. control IgG1. (B) ADCC was measured by standard 4 h chromium release assay using L-363 cells as target cells and PBMC of healthy donors as effectors cells in an effector-to-target cell (E:T) ratio of 80:1. Mean percentage of lysis ± SEM from six experiments with different PBMC donors are shown. ****p ≤ 0.0001 CD38 antibodies vs. control IgG1. (C) CDC was also measured by chromium release assay using 25% (v/v) serum of healthy donors and CD38-positive Daudi cells as targets. Mean percentage of lysis ± SEM from three independent experiments are shown. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 CD38 antibodies vs. control IgG1. (D) ADCP was measured by live cell imaging using pHrodo-labeled L-363 cells and macrophages generated from healthy donors in an E:T ratio of 1:1 with 10 µg/ml of the indicated antibodies. Phagocytosis was measured as red counts per image. Mean values ± SEM from four independent experiments are shown.

Article Snippet: The inhibition of the enzymatic activity of CD38 was measured using the CD38 Inhibitor Screening Assay Kit (Cyclase Activity) from BPS Bioscience (cat. no. 71275).

Techniques: Functional Assay, Incubation, Staining, Release Assay, Lysis, Live Cell Imaging, Labeling, Generated