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: Frontiers in Oncology

Article Title: BAFF/APRIL System Is Functional in B-Cell Acute Lymphoblastic Leukemia in a Disease Subtype Manner

doi: 10.3389/fonc.2019.00594

Figure Lengend Snippet: Pattern of mRNA expression of BAFF/APRIL system and E2A-PBX1 binding to BAFFR promoter. For (A–F) the vertical axis represents the normalized relative gene expression values. (A) Receptors and (B) ligands mRNA levels in patients with B- ( n = 63) and T- ( n = 7) lineage ALL and in mononuclear cells from BM ( n = 4), PB ( n = 5), pure mature B-cells ( n = 3) and pure monocytes ( n = 3) from healthy donors, and EBV cell lines ( n = 4). The bars represent mean values and the lines the standard error of mean. The discontinuous lines represent the relative mRNA expression of receptors in monocytes that serve as a negative control group. (C) BAFFR , (D) BCMA , and (E) APRIL mRNA levels are correlated with the maturation stage of B-lymphoblasts. The lines in the dot plots represent the median value for each B-ALL subtype; p-value in each plot refers to Kruskal-Wallis H test. (F) Increased BAFFR transcript levels in patients with pre-B-ALL carrying the E2A-PBX1 translocation; p-value refers to Mann-Whitney U test. (G) E2A-PBX1 binding to the promoter of BAFFR gene in the pre-B-ALL cell line 697. Cross-linked chromatin from 697 cells was used in ChIPs with anti-E2A-PBX1 antibody. IgG was utilized in parallel with anti-E2A-PBX1 antibody, as control. Two sets of primers were used: One set specific for the amplification of the BAFFR gene promoter and one set of negative control primers for amplification of a region lacking PBX motifs. Bars represent mean and standard error values of specific enrichments (fold differences) vs. input obtained in at least three independent experiments. The statistical significance (Student's t -tests) of the difference of enrichment for promoter sequences vs. the IgG control ( p = 0.019) and negative control region (ns, not significant) are indicated.

Article Snippet: Western blot analysis was performed as described before ( , ), using anti-NF-κB2 (Millipore), anti-BAFFR (CT; Enzo, Lausen, Switzerland) or anti-actin (Santa Cruz, CA, USA) antibodies.

Techniques: Expressing, Binding Assay, Negative Control, Translocation Assay, MANN-WHITNEY, Amplification

Journal: Frontiers in Oncology

Article Title: BAFF/APRIL System Is Functional in B-Cell Acute Lymphoblastic Leukemia in a Disease Subtype Manner

doi: 10.3389/fonc.2019.00594

Figure Lengend Snippet: BAFF activates the alternative pathway of NF-κB on BAFFR expressing blasts. (A) Western blot analysis of NF-κB2 processing in mononuclear cells from B-ALL patients and in 697 cells, incubated overnight with increasing concentrations of 60-mer BAFF. (B) Western blot analysis with the antibody that recognizes also the C-terminal fragment of BAFFR (~22 kDa) showed BAFF-mediated degradation of BAFFR in B-lymphoblasts expressing the receptor. Six hundred ninety-seven cells display an abnormal migration pattern of BAFFR protein (of lower molecular weight) compared to primary B-lymphoblasts (intact BAFFR: ~50 & 36 kDa); ns, non-specific.

Article Snippet: Western blot analysis was performed as described before ( , ), using anti-NF-κB2 (Millipore), anti-BAFFR (CT; Enzo, Lausen, Switzerland) or anti-actin (Santa Cruz, CA, USA) antibodies.

Techniques: Expressing, Western Blot, Incubation, Migration, Molecular Weight

Journal: Frontiers in Oncology

Article Title: BAFF/APRIL System Is Functional in B-Cell Acute Lymphoblastic Leukemia in a Disease Subtype Manner

doi: 10.3389/fonc.2019.00594

Figure Lengend Snippet: BAFF enhances dexamethasone-induced apoptosis of 697 cells and cell death of BAFFR expressing BCR-ABL + pre-B-lymphoblasts. (A) Malignant B-lymphoblasts were incubated with 60-mer BAFF (0.0488–12.5 ng/mL) for 5 and 17 h; the effect of BAFF-treatment in pure normal B-cells that served as a positive control, was evaluated after 3 days. Survival index represents the ratio of 7-AAD − CD19 + cells at each concentration of the ligand to the untreated 7-AAD − CD19 + cells. The graph shows the mean and standard error of mean of the survival index at each group (BAFFR −ve BCR-ABL −ve : common & pro-B-ALL, BAFFR +ve BCR-ABL −ve : common B-ALL, BAFFR +ve BCR-ABL +ve : pre-B-ALL, control B-cells: pure normal peripheral B-cells). (B) Flow cytometry analysis for Annexin V in 697 cells treated with/without BAFF and with/without dexamethasone at day 5. (C) Percentage of viable cell counts (Annexin V − 7-AAD − cells) of 697 cells incubated with/without 3-mer or 60-mer BAFF and with/without 20 ng/mL dexamethasone (DEX) for 5 days (DEX vs. DEX+BAFF p = 0.004); p-value refers to Wilcoxon signed-rank test between cells treated only with dexamethasone and those treated with dexamethasone and BAFF. (D) Western blot analysis of NF-κB2 processing in 697 cells treated with 5 ng/mL 60-mer BAFF and 10–20 ng/mL dexamethasone for 3 sequential days; NA: not applicable. (E) Normalization (fold difference) of p52/p100 degradation for each culture condition to that of the untreated cells per each day. (F) Western blot analysis of BAFFR (CT) in 697 cells treated with 5 ng/mL 60-mer BAFF and 10–20 ng/mL dexamethasone for 3 sequential days. (G) Normalization (fold difference) of intact BAFFR/actin for each culture condition to that of untreated cells per each day.

Article Snippet: Western blot analysis was performed as described before ( , ), using anti-NF-κB2 (Millipore), anti-BAFFR (CT; Enzo, Lausen, Switzerland) or anti-actin (Santa Cruz, CA, USA) antibodies.

Techniques: Expressing, Incubation, Positive Control, Concentration Assay, Flow Cytometry, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Requirement for BAFF and APRIL during B Cell Development in GALT

doi: 10.4049/jimmunol.1000146

Figure Lengend Snippet: Flow cytometric detection of BR3 and BAFF on appendix, PB, and splenic B cells. A, Cells stained with anti-IgM and goat anti-BR3 followed by biotinylated rabbit (Fab) anti-goat Ig and streptavidin PE. B, Gated IgM+ cells stained with goat anti-BAFF followed by biotinylated rabbit anti-goat Ig and streptavidin PE. C, Anti-Ig– and CD40L-activated B cells stained with anti-IgM and rBAFF.

Article Snippet: Flow cytometry and immunohistochemistry For flow cytometry, Abs and indirect reagents were as follows: anti-IgM (clone 367; BD Biosciences, San Jose, CA), anti-CD4 (clone Ken-4; BD Biosciences), anti-rabbit L chain (KLK stock), anti-CD79a (clone HM47; BD Biosciences), anti-CD9 (clone MM2; Antigenix America, Huntington Station, NY), anti-CD43 (clone L11/43; Antigenix America), anti-CD14 (clone K4; Antigenix America), anti-CD11b (clone 198; Antigenix America), anti-MHC class II (MHC-II; clone 2C4; BD Biosciences), anti-CD44 (clone W4/86; Chemicon International, Temecula, CA), goat anti-human BAFF (Antigenix America) ( 38 ), goat anti-human BR3 (R&D Systems, Minneapolis, MN) ( 38 ), FITC goat Fab anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), and streptavidin-PE/APC (BD Biosciences).

Techniques: Staining