Journal: Antibody Therapeutics

Article Title: Development of a T cell-redirecting bispecific antibody targeting B-cell maturation antigen for the suppression of multiple myeloma cell growth

doi: 10.1093/abt/tbac012

Figure Lengend Snippet: Generation of mouse anti-hBCMA monoclonal antibodies. (A) Detection of hBCMA on the surface of HEK 293T cells stably expressing the full-length human BCMA . HEK 293T cells were transduced with a lentiviral vector containing the human BCMA gene tagged with GFP. The GFP + HEK 293T cells (blue) were FACS-sorted, and the expression of BCMA on the cell surface was examined by flow cytometry using a mouse anti-human BCMA antibody. The parental HEK 293 T cells (red) were used as a negative control. (B) Validation of three clones of anti-human BCMA monoclonal antibodies by flow cytometry using hBCMA-expressing HEK 293T cells. The hBCMA + 293T cells were stained first with supernatants from subcloned three positive clones, followed by staining with a goat anti-mouse immunoglobulin Fc fragment antibody. (C) Validation of anti-hBCMA monoclonal antibody produced by clone F15F12G7 using MM cell lines RPMI 8226, KMS28, H929 and MM-1R by flow cytometry. MM cells were first stained with supernatants from clone F15F12G7, followed by staining with a goat anti-mouse immunoglobulin Fc fragment secondary antibody. Supernatants from a negative clone and a commercial mouse anti-human BCMA antibody (19F2 from Biolegend) were used as negative and positive controls, respectively. (D) Schematic diagram of production of chimeric anti-hBCMA antibodies. IgH and IgL variable region genes from the mouse monoclonal antibodies were cloned into pTarget-CH vector, which contains genes encoding for the human IgG1 heavy chain constant region gene. CHO cells were transfected with the plasmid DNA for the production of chimeric antibodies. (E) Validation of chimeric anti-hBCMA antibodies produced from CHO cell lines. The monoclonal antibodies of clone F15F12G7 produced by CHO cells were purified by protein A, and the binding to BCMA + HEK 293T cells was examined by flow cytometry. Antibody produced using a previously published sequence of anti-hBCMA monoclonal antibody C12A32 was included as the positive control, and the IgG1 isotype control antibody was used as a negative control. (F) Measurement of ADCC of anti-human BCMA antibody F15F12G7. MM cell line U266 labeled with Cell Trace Violet dye was cocultured with human PBMCs at different E:T ratios in the presence of antibody F15F12G7. After 24 h of culture, the cells were stained with fixed-viability dye, and the viability of U266 cells was examined by flow cytometry. The data shown are representative of more than three individual experiments for (A), (B), (C), (E) and (F).

Article Snippet: For measuring T cell proliferation, purified T cells were first labeled with Cell Trace Violet dye (BD Biosciences, USA) in the dark for 15 min. After washing with PBS, the labeled T cells were cocultured with KMS11 cells at an effector (E): target (T) cell ratio of 1:1 in the presence of the BCMA × CD3 bsAb at the indicated concentrations for 16 h at 37 °C, supplemented with 5% CO 2 in a humidified incubator.

Techniques: Stable Transfection, Expressing, Transduction, Plasmid Preparation, Flow Cytometry, Negative Control, Clone Assay, Staining, Produced, Transfection, Purification, Binding Assay, Sequencing, Positive Control, Control, Labeling

Journal: Antibody Therapeutics

Article Title: Development of a T cell-redirecting bispecific antibody targeting B-cell maturation antigen for the suppression of multiple myeloma cell growth

doi: 10.1093/abt/tbac012

Figure Lengend Snippet: BCMA × CD3 FabscFv bsAb induces efficient T cell-mediated MM cell killing. (A) Flow cytometric analysis of T cell-mediated MM cell killing in the presence of BCMA × CD3 bsAb. MM cells H929 and MM-1R were labeled with Cell Trace Violet dye and cocultured with purified human T cells at 1:1 ratio for 24 h in the presence of control and BCMA × CD3 bsAb (1 nM). The MM cells were stained with FVD, and their viabilities were determined by flow cytometry. (B) The antibody dose-dependent T cell-mediated killing of MM cells by BCMA × CD3 bispecific antibody. The luciferase-positive MM cells KMS11 were cocultured with T cells at 1:1 ratio in the presence of BCMA × CD3 bsAb at the various concentrations for 48 h. The viability of KMS11 cells was measured by luminescence. The percentage of MM cell killing was calculated by the following formula: % of killing = (OD T cell alone —OD T cell with BsAb )/OD T cell alone . The data shown are representative of more than three (A) and 5 (B) individual experiments.

Article Snippet: For measuring T cell proliferation, purified T cells were first labeled with Cell Trace Violet dye (BD Biosciences, USA) in the dark for 15 min. After washing with PBS, the labeled T cells were cocultured with KMS11 cells at an effector (E): target (T) cell ratio of 1:1 in the presence of the BCMA × CD3 bsAb at the indicated concentrations for 16 h at 37 °C, supplemented with 5% CO 2 in a humidified incubator.

Techniques: Labeling, Purification, Control, Staining, Flow Cytometry, Luciferase

Journal: Antibody Therapeutics

Article Title: Development of a T cell-redirecting bispecific antibody targeting B-cell maturation antigen for the suppression of multiple myeloma cell growth

doi: 10.1093/abt/tbac012

Figure Lengend Snippet: BCMA × CD3 FabscFv bsAb stimulates robust T cell proliferation and activation (A), (B) Flow cytometric analysis of T cell proliferation. Purified T cells were labeled with Cell Trace Violet dye and cocultured with KMS11 cells at a 1:1 ratio in the presence of 0.3 and 1 nM of BCMA × CD3 bsAb for 72 h followed by flow cytometric analysis (A). The division index of CD4 + and CD8 + T cells was calculated with Flow Jo software. (C) Flow cytometric analysis of Granzyme B in the CD4 + and CD8 + T cells cocultured with KMS11 cells in the presence of 1 nM of BCMA × CD3 bsAb for 16 h. (D) Antibody dose-dependent production of Granzyme B by CD4 + and CD8 + T cells. (E) Flow cytometric analysis of activation markers CD25 and CD69 in the CD4 + and CD8 + T cells cocultured with KMS11 cells in the absence or presence of 1 nM of BCMA × CD3 bsAb for 16 h. (F) Cytokine production by T cells. Purified T cells were cocultured with KMS11 cells in the presence of various concentrations of BCMA × CD3 bsAb. The supernatants were collected after 48 h and the production of IFNγ, IL-2, GM-CSF and TNFα were measured by ELISA. Shown are mean ± SD values for independent triplicates. The data shown are representative of more than three individual experiments.

Article Snippet: For measuring T cell proliferation, purified T cells were first labeled with Cell Trace Violet dye (BD Biosciences, USA) in the dark for 15 min. After washing with PBS, the labeled T cells were cocultured with KMS11 cells at an effector (E): target (T) cell ratio of 1:1 in the presence of the BCMA × CD3 bsAb at the indicated concentrations for 16 h at 37 °C, supplemented with 5% CO 2 in a humidified incubator.

Techniques: Activation Assay, Purification, Labeling, Software, Enzyme-linked Immunosorbent Assay