Journal: Cancer science

Article Title: Immunotherapy based on bispecific T-cell engager with hIgG1 Fc sequence as a new therapeutic strategy in multiple myeloma.

doi: 10.1111/cas.12631

Figure Lengend Snippet: Fig. 1. Expression of aCD138-ScFv-hIgFc, aCD3- ScFv-hIgFc, and bispecific T-cell engager (BiTE)-hIgFc antibodies. (A) Twelve percent reducing SDS-PAGE (3 lg ⁄ lane) and Western blot analysis of the supernatant of the three expression products showing molecular weights close to those predicted (52, 52, and 79 kDa). (B) Amino acid sequence of BiTE-hIgFc (STL001). The orange letters represent the (G4S)3 or G4S linker. In addition, the signal peptide and hIgG1-Fc sequence are shown at the NH2-terminus and COOH-terminus, respectively.

Article Snippet: We cloned the mouse OKT3 ScFv gene into pFUSE-hIgFc vector (pfuse-hg1fc1; InvivoGen, San Diego, CA, USA) between the NcoI(579) and BglII(587) (New England Biolabs) cloning sites.

Techniques: Expressing, SDS Page, Western Blot, Sequencing

Journal: Cancer science

Article Title: Immunotherapy based on bispecific T-cell engager with hIgG1 Fc sequence as a new therapeutic strategy in multiple myeloma.

doi: 10.1111/cas.12631

Figure Lengend Snippet: Fig. 2. Flow cytometry analysis of bispecific T-cell engager (BiTE)-hIgFc (STL001), aCD138-ScFv-hIgFc, and aCD3-ScFv-hIgFc. BiTE-hIgFc (STL001), aCD138-ScFv-hIgFc, and aCD3-ScFv-hIgFc (all 10 lg ⁄ mL) stained RPMI-8226 (A, E, I), U266 (B, F, J), and Jurkat (C, G, K) cells and peripheral blood mononuclear cells (D, H, L) for 1 h. Finally, FITC-labeled anti-human IgG1-Fc antibody at 1:5000 dilution was used to label the harvested cells at room temperature for 30 min. After centrifugation and being washed twice, the cells were analyzed using a flow cytometer. Data shown are representative of three independent experiments.

Article Snippet: We cloned the mouse OKT3 ScFv gene into pFUSE-hIgFc vector (pfuse-hg1fc1; InvivoGen, San Diego, CA, USA) between the NcoI(579) and BglII(587) (New England Biolabs) cloning sites.

Techniques: Flow Cytometry, Staining, Labeling, Centrifugation, Cytometry

Journal: Cancer science

Article Title: Immunotherapy based on bispecific T-cell engager with hIgG1 Fc sequence as a new therapeutic strategy in multiple myeloma.

doi: 10.1111/cas.12631

Figure Lengend Snippet: Fig. 3. Determination of equilibrium dissociation constant (KD) of bispecific T-cell engager (BiTE)- hIgFc (STL001) and aCD138-ScFv-hIgFc. Biotin- labeled rCD138 (10 lg ⁄ mL) in Octet Kinetic buffer was loaded onto an streptavidin (SA) biosensor. For sensors A8–D8, 500.0, 250.0, 125.0, and 62.5 nM aCD138-ScFv-hIgFc (A) or 333.3, 166.7, 83.3, and 41.7 nM BiTE-hIgFc (STL001) (B) against the coated rCD138 protein were analyzed by the bio-layer interferometry approach in the KD determination experiment. The association and dissociation time was set to 300 and 1800 s, respectively. The KD of aCD138-ScFv-hIgFc and BiTE-hIgFc (STL001) against rCD138 protein were determined to be 13.5 and 29.5 nM, respectively. The blank kinetic buffer (Sensor E8 curve) or overloaded rBiTE solution (Sensor G8 curve) only was added to the rCD138- coated SA biosensor or blank SA biosensor, respectively, which was designed to verify that blank kinetic buffer or overloaded rBiTE solution cannot non-specifically bind the rCD138-coated SA biosensor or blank SA biosensor, respectively. Data shown are representative of three independent experiments.

Article Snippet: We cloned the mouse OKT3 ScFv gene into pFUSE-hIgFc vector (pfuse-hg1fc1; InvivoGen, San Diego, CA, USA) between the NcoI(579) and BglII(587) (New England Biolabs) cloning sites.

Techniques: Labeling