Journal: Blood Advances

Article Title: Generation of a humanized afucosylated BAFF-R antibody with broad activity against human B-cell malignancies

doi: 10.1182/bloodadvances.2022008560

Figure Lengend Snippet: In vitro characterization of humanized anti-BAFF-R mAbs. (A) Binding affinities of 9 selected humanized Abs derived from the C90 sequence by ELISA, as described in Methods. (B) Specificity of 2 candidates, H90-4 and H90-5, analyzed by flow cytometry staining of human B-cell malignancy lines representative of the following diseases: Burkitt lymphoma (Raji), ALL (RS4;11), CLL (MEC-1), acute myeloid leukemia (HL-60), DLBCL (Ly 10), follicular lymphoma (RL), and MCL (JeKo-1, Z-138). (C) ADCC assays of H90-4 and H90-4 against B-cell lines as indicated. Anti-CD20 mAb rituximab was used as a positive control. Negative controls were human IgG and NK cells alone. Target cell lines were incubated with Abs at 5 μg/mL, and fresh NK cells at E:T ratio of 20:1 for 6 hours. Specific cytotoxicity was calculated as in Methods. Experiments were conducted in triplicate and data were analyzed by Student t test. (D) Flow cytometry of H90-4 and H90-5 (100 ng/10 6 cells) on human primary tumor samples. (E.) H90-4 and H-90 to 5 ADCC assays against primary patient tumor cells. Target cells were incubated with Abs at 5 μg/mL, and fresh NK cells at E:T ratio of 20:1 for 6 hours. (F) 293T cells were transfected with the BAFF-R wild-type or P21R/G64V variants as described in Methods. Cells were then stained with different amounts of H90-4/H90-5 with a secondary goat antihuman APC or antihuman BAFF-R mAb (11C1) as positive controls. Unstained cells and secondary Ab (antihuman IgG-APC) served as negative controls. Experiments were conducted in triplicate and analyzed by Student t test. Data shown are representative of 3 independent experiments. Ctrl, control.

Article Snippet: LakePharma (San Carlos, CA) was contracted to produce 3 light chain (VL) and 3 heavy-chain (VH) variants of our previously described BAFF-R chimeric C90 mAb (C90) to produce 9 humanized Ab variants.

Techniques: In Vitro, Binding Assay, Derivative Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Positive Control, Incubation, Transfection, Control

Journal: Blood Advances

Article Title: Generation of a humanized afucosylated BAFF-R antibody with broad activity against human B-cell malignancies

doi: 10.1182/bloodadvances.2022008560

Figure Lengend Snippet: Humanized C90 humanness score and binding affinity

Article Snippet: LakePharma (San Carlos, CA) was contracted to produce 3 light chain (VL) and 3 heavy-chain (VH) variants of our previously described BAFF-R chimeric C90 mAb (C90) to produce 9 humanized Ab variants.

Techniques: Binding Assay

Journal: Blood Advances

Article Title: Generation of a humanized afucosylated BAFF-R antibody with broad activity against human B-cell malignancies

doi: 10.1182/bloodadvances.2022008560

Figure Lengend Snippet: In vivo activity of humanized anti-BAFF-R mAbs. Z-138 MCL–luciferase cells (5 × 10 4 ) were injected IV into the tail veins of NSG mice on day 0. Mice were treated with 300 μg Abs, as indicated, together with 15 × 10 6 Nk92 176V cells or 2 × 10 6 NK cells enriched from a healthy donor and 5 × 10 4 IU IL-2 on days 3, 7, 11, and 15. Tumor growth was monitored by bioluminescent imaging on the days indicated. (A) Tumor growth after treatment with either humanized BAFF-R Ab (H90-5), chimeric BAFF-R Ab (C90), or control treatments of NK cells alone or PBS only. (B) Flux (photons/s) was determined by measuring bioluminescence on day 16, 23, 30, and 37 after tumor cell injection. (C) Kaplan-Meier survival curves for each group of mice. (D) Z138 tumor growth after treatment with either C90, H90-AF, and control groups of NK alone or PBS only. (E) Flux was determined by measuring bioluminescence of luciferase on day 21, 28, 35 and 42 after tumor cell injection. (F) Kaplan-Meier survival curves for each group of mice. PDX MCL9606 ( BIRC3 L548fs ) cells (3 × 10 6 ) were inoculated via tail vein into 8-week-old NSG mice on day 0. Mice were treated with 300 μg Abs (as indicated), IL-2, and NK cells, as described earlier for panel D, on days 3, 7, 11, and 15. Control groups received the same volume of injection with NK cells alone or PBS. (G) Flow cytometry for CD45 + /CD5 + /CD19 + cells representing MCL at the indicated times after tumor inoculation. Mice in the PBS and NK cells only control groups were all euthanized before week 7 because weight loss exceeded 20%, indicated by †. (H) Kaplan-Meier survival curves for each group of mice. Nalm6 ALL-luciferase cells (1 × 10 5 ) were injected IV into the tail veins of NSG mice on day 0. Mice were treated with 300 μg Abs, as indicated, together with 1.5 × 10 6 NK cells enriched from a healthy donor and 5 × 10 4 IU IL-2 on days 3, 7, 11, and 15. Tumor growth was monitored by bioluminescent imaging on the days indicated. (I) Tumor growth after treatment with either chimeric BAFF-R Ab (C90), humanized BAFF-R Ab (H90-5), afucosylated H90-5 (H90-AF), or control treatments of NK cells alone or PBS only. (J) Flux was determined by measuring bioluminescence of luciferase on days 16, 22, 29 and 36 after tumor cell injection. (K) Kaplan-Meier survival curves for each group of mice. ∗∗log-rank P values.

Article Snippet: LakePharma (San Carlos, CA) was contracted to produce 3 light chain (VL) and 3 heavy-chain (VH) variants of our previously described BAFF-R chimeric C90 mAb (C90) to produce 9 humanized Ab variants.

Techniques: In Vivo, Activity Assay, Luciferase, Injection, Imaging, Control, Flow Cytometry

Journal: Scientific Reports

Article Title: BAFF attenuates oxidative stress-induced cell death by the regulation of mitochondria membrane potential via Syk activation in WiL2-NS B lymphoblasts

doi: 10.1038/s41598-020-68628-5

Figure Lengend Snippet: Oxidative stress-induced cell death was inhibited by BAFF. ( A ) WiL2-NS cells were incubated with 10 μg/ml of a human BAFF-murine CD8 (BAFF-muCD8) biotinylated fusion protein on ice for 30 min. BAFF binding was visualized the incubation with phycoerythrin (PE)-conjugated streptavidin (SA) for 20 min and analyzed by flow cytometry. ( B ) WiL2-NS cells were incubated with biotinylated anti-human BAFF-R or TACI antibodies for 30 min followed by PE-conjugated SA for 20 min. At the same time, cells were incubated with FITC-labeled CD19. Expression of BAFF-R or TACI and CD19 on each cell was analyzed by flow cytometry as compared to control group incubated with isotype antibodies. ( C ) and ( D ) Cells were incubated in the RPMI 1640 medium with 10% or 1% FBS in the presence or absence of 20 ng/ml BAFF. Total number of cells ( C ) or dead cells ( D ) were counted with hemocytometer and estimated by trypan blue staining, respectively. Cell lysates were prepared and caspase 3 activity was measured by using Ac-DEVD-pNA, caspase 3 substrate. Caspase 3 activity was normalized with protein concentration ( E ). Data were the representative of four experiments. Data in the line ( C ) or bar ( D–E ) graph represent the means ± SD. * p < 0.05; ** p < 0.01; significant difference as compared to BAFF-untreated control with 10% FBS ( C , D and E ). # p < 0.05; significant difference as compared to BAFF-untreated control with 1% FBS ( D and E ).

Article Snippet: Recombinant human BAFF, biotinylated anti-human BAFF-R and TACI antibodies was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Incubation, Binding Assay, Flow Cytometry, Labeling, Expressing, Staining, Activity Assay, Protein Concentration