Journal: Advanced Science

Article Title: An Antibody‐Drug Conjugate for Multiple Myeloma Prepared by Multi‐Arm Linkers

doi: 10.1002/advs.202307852

Figure Lengend Snippet: Drug conjugation site and antigen‐binding of TE‐1146. a) TE‐1146 (red) and α‐CD38 (blue) underwent trypsin digestion, and the resulting peptides were analyzed by HPLC. An additional peak in the trypsin‐digested TE‐1146 profile is highlighted with an asterisk (*). b) The aa sequence of the peptide fragment containing the engineered Cys is shown above the spectrum. The y‐ion, y(4), and the b‐ions, b(3,5,6,7,8,9,11), are indicated above and below the sequence, respectively. c) The binding affinities of α‐CD38 and TE‐1146 to human CD38 were assessed via a cell‐based enzyme‐linked immunosorbent assay (ELISA) using a human CD38‐overexpressing 293T cell line. d) The binding abilities of TE‐1146 (solid curves) and daratumumab (dashed curves) to cell surface‐expressed CD38 were characterized by flow cytometry using U266‐CD38 + and H929 MM cell lines. The EC 50 , the concentration of each protein resulting in 50% of the maximal response of binding to target cells, is shown.

Article Snippet: About 2 × 10 5 of H929, MM.1S, U266, or Daudi cells were washed thrice with flow cytometry staining (FACS) buffer (PBS with 1% fetal bovine serum) and incubated with different concentrations of TE‐1146 and daratumumab (1 μM, 100, 10, and 1 nM, 100, 10 and 1 pM) for 20 min. After FACS buffer rinsing, cells were labeled with PE‐conjugated anti‐human IgG‐Fc antibody (Jackson ImmunoResearch) to detect the presence of TE‐1146 and daratumumab on the cell surface.

Techniques: Conjugation Assay, Binding Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Concentration Assay

Journal: Advanced Science

Article Title: An Antibody‐Drug Conjugate for Multiple Myeloma Prepared by Multi‐Arm Linkers

doi: 10.1002/advs.202307852

Figure Lengend Snippet: Internalization of TE‐1146 and subsequent lenalidomide release from drug bundles. a) The procedure to determine the ratio of TE‐1146 internalization into MM cells. Flow cytometry analysis of the degree of TE‐1146 internalization at indicated time points in b) live H929 cells and c) H929 cells fixed with 100% ethanol to abolish the internalization process. d) Confocal microscopy analysis of the internalization of TE‐1146 conjugated with VivoTag 645 at different incubated times (0, 1, 2, and 3 h). Upon binding to DNA AT regions, DAPI (4′,6‐diamidino‐2‐phenylindole) emits blue fluorescence and is used to detect cell nuclei. e) HPLC detection of lenalidomide release from TE‐1146 after incubating with MM cells. 1) lenalidomide in phosphate‐buffered saline (PBS); 2) RPMI medium; 3) lenalidomide in RPMI medium; 4) TE‐1146 in RPMI medium; 5) 5‐day cell culture supernatant of H929 cells; 6) 5‐day cell culture supernatant collected from 1 × 10 6 H929 cells incubated with TE‐1146.

Article Snippet: About 2 × 10 5 of H929, MM.1S, U266, or Daudi cells were washed thrice with flow cytometry staining (FACS) buffer (PBS with 1% fetal bovine serum) and incubated with different concentrations of TE‐1146 and daratumumab (1 μM, 100, 10, and 1 nM, 100, 10 and 1 pM) for 20 min. After FACS buffer rinsing, cells were labeled with PE‐conjugated anti‐human IgG‐Fc antibody (Jackson ImmunoResearch) to detect the presence of TE‐1146 and daratumumab on the cell surface.

Techniques: Flow Cytometry, Confocal Microscopy, Incubation, Binding Assay, Fluorescence, Saline, Cell Culture