Journal: Frontiers in Immunology

Article Title: LUBAC Suppresses IL-21-Induced Apoptosis in CD40-Activated Murine B Cells and Promotes Germinal Center B Cell Survival and the T-Dependent Antibody Response

doi: 10.3389/fimmu.2021.658048

Figure Lengend Snippet: SHARPIN deficiency exacerbates IL-21-induced death in CD154-stimulated B cells. (A) Flow cytometry analysis of plasma cell differentiation and B cells viability in C57 B cells stimulated with different doses of CD154, as indicated, in the absence or presence of IL-21 for 96 h (n=4, mean and s.e.m.; data at 1 unit of CD154 were analyzed for statistical differences between Sharpin +/+ and Sharpin cpdm B cells). (B, C) Flow cytometry analysis of proliferation, survival, and plasma cell differentiation of Sharpin +/+ and Sharpin cpdm B cells stimulated with CD154 or CD154 plus IL-21 for 96 (h) Division-linked B cell viability is depicted in (B) , representative of four independent experiments) and average divisions, live B cell proportions, and CD138 + plasma cell proportions were depicted in (C) . (D, E) Flow cytometry analysis of proliferation, survival of Sharpin +/+ and Sharpin cpdm B cells stimulated with CD154 (left panels) or αCD40 (right panels) at indicated doses in the presence of different doses of IL-21 for 96 h (n=3, mean and s.e.m.; data at 4 units of CD154 or 40 μg of αCD40 were analyzed for statistical differences between Sharpin +/+ and Sharpin cpdm B cells). (F) Immunoblotting of phosphorylated p65 and total p65 protein levels in Sharpin +/+ and Sharpin cpdm B cells after stimulation by αCD40 for the indicated time. Representative of two independent experiments. (G) Live cell proportions in Sharpin +/+ and Sharpin cpdm B cells stimulated with αCD40 plus IL-21 or IL-4 for 96 (h) * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; t-test.

Article Snippet: Other stimuli include an agonistic anti-CD40 Ab (clone FGK4.5; BioXcel; αCD40, 3 μg/ml or as indicated), TLR1/2 ligand Pam 3 CSK 4 (100 ng/ml, Invivogen), TLR4 ligand lipid A (1 μg/ml, Invivogen), TLR7 ligand R-848 (30 ng/ml, Invivogen), TLR9 ligand ODN1826 (sequence 5’-TCCATGACGTTCCTGACGTT-3’) with a phosphorothioate backbone (CpG, 1 μM; Eurofins), F(ab’)2 of a goat anti-mouse μ chain Ab (anti-μ F(ab’) 2, 1 μg/ml; αIgM; Southern Biotech), which crosslinks IgM BCR, or anti-Igδ mAb (clone 11-26c conjugated to dextran, αIgD/dex, 100 ng/ml; Fina Biosolutions), which crosslinks IgD BCR ( ).

Techniques: Flow Cytometry, Clinical Proteomics, Cell Differentiation, Western Blot

Journal: Frontiers in Immunology

Article Title: LUBAC Suppresses IL-21-Induced Apoptosis in CD40-Activated Murine B Cells and Promotes Germinal Center B Cell Survival and the T-Dependent Antibody Response

doi: 10.3389/fimmu.2021.658048

Figure Lengend Snippet: SHARPIN inhibits IL-21-induced apoptosis in CD154-stimulated B cells. (A) Flow cytometry analysis of apoptosis (Annexin V + 7–AAD lo ) and necrosis (Annexin V + 7–AAD hi ) in freshly isolated Sharpin +/+ and Sharpin cpdm B cells or after stimulation with CD154 (left) or αCD40 (right) plus nil or IL-21 for 96 h, with or without priming with CD154 or αCD40 for 4 h. (B, C) Flow cytometry analysis of apoptosis and necrosis in Sharpin +/+ and Sharpin cpdm B cells after priming with TLR ligands (as indicated, B ) or αIgD/dex (C) for 4 h and then stimulated with CD154 or αCD40 plus IL-21 for 92 h. (D) Flow cytometry analysis of the viability of Sharpin +/+ and Sharpin cpdm B cells stimulated with CD154 plus IL-21 in the presence of nil or pan-caspase inhibitor Z-VAD-FMK, caspase 9-specific inhibitor Z-LEHD-FMK or caspase 8-specific inhibitor Z-IETD-FMK (all 20 μM). (E) Mitochondrial membrane potential in B cells stimulated with CD154 and IL-21 at indicated doses for 48 h (n=4, mean and s.e.m.; data at 4 units of CD154 were analyzed for statistical differences between Sharpin +/+ and Sharpin cpdm B cells). * p < 0.05; ** p <0.01; *** p < 0.001; t-test.

Article Snippet: Other stimuli include an agonistic anti-CD40 Ab (clone FGK4.5; BioXcel; αCD40, 3 μg/ml or as indicated), TLR1/2 ligand Pam 3 CSK 4 (100 ng/ml, Invivogen), TLR4 ligand lipid A (1 μg/ml, Invivogen), TLR7 ligand R-848 (30 ng/ml, Invivogen), TLR9 ligand ODN1826 (sequence 5’-TCCATGACGTTCCTGACGTT-3’) with a phosphorothioate backbone (CpG, 1 μM; Eurofins), F(ab’)2 of a goat anti-mouse μ chain Ab (anti-μ F(ab’) 2, 1 μg/ml; αIgM; Southern Biotech), which crosslinks IgM BCR, or anti-Igδ mAb (clone 11-26c conjugated to dextran, αIgD/dex, 100 ng/ml; Fina Biosolutions), which crosslinks IgD BCR ( ).

Techniques: Flow Cytometry, Isolation, Membrane

Journal: Frontiers in Immunology

Article Title: LUBAC Suppresses IL-21-Induced Apoptosis in CD40-Activated Murine B Cells and Promotes Germinal Center B Cell Survival and the T-Dependent Antibody Response

doi: 10.3389/fimmu.2021.658048

Figure Lengend Snippet: Illustration of LUBAC-mediated suppression of IL-21-induced apoptosis in CD154-stimulated B cells. (A) CD40 activation in B cells upregulates LUBAC-catalyzed linear M1-Ub, including that of cFLIP, which leads to cFLIP stabilization and inhibition of caspase 8 activation. LUBAC plays a minor role to the major role of RAB7 in CD40-triggered NF-κB activation in B cells. (B) CD40 activation also induces the expression of anti-apoptotic factor BCL2 and BCL-XL. IL-21, however, dampens such induction and also upregulates expression of pro-apoptotic factor BIM. The combined effects of BCL2/BCL-XL downregulation and BIM upregulation would activate pro-apoptotic BAX/BAK complex to increase the mitochondria permeability and cytochrome C release into the cytoplasm to activate caspase 9. (C) In the absence of LUBAC, caspase 8 activation would amplify a putative caspase network, within which multiple caspases, including caspase 9 and caspase 3, would be activated in a synchronized manner due to positive-feedback loops, leading to the irreversible apoptosis process.

Article Snippet: Other stimuli include an agonistic anti-CD40 Ab (clone FGK4.5; BioXcel; αCD40, 3 μg/ml or as indicated), TLR1/2 ligand Pam 3 CSK 4 (100 ng/ml, Invivogen), TLR4 ligand lipid A (1 μg/ml, Invivogen), TLR7 ligand R-848 (30 ng/ml, Invivogen), TLR9 ligand ODN1826 (sequence 5’-TCCATGACGTTCCTGACGTT-3’) with a phosphorothioate backbone (CpG, 1 μM; Eurofins), F(ab’)2 of a goat anti-mouse μ chain Ab (anti-μ F(ab’) 2, 1 μg/ml; αIgM; Southern Biotech), which crosslinks IgM BCR, or anti-Igδ mAb (clone 11-26c conjugated to dextran, αIgD/dex, 100 ng/ml; Fina Biosolutions), which crosslinks IgD BCR ( ).

Techniques: Activation Assay, Inhibition, Expressing, Permeability

Journal: Bioengineering & Translational Medicine

Article Title: Viral vector‐based gene therapies in the clinic

doi: 10.1002/btm2.10258

Figure Lengend Snippet: Examples of current clinical trials for adenovirus‐based in vivo gene therapies, grouped by indication

Article Snippet: Ad , EnAd , Anti‐CD40 antibody , Metastatic cancer, epithelial tumor , NG‐350A (PsiOxus Therapeutics Ltd) , Intratumoral , NCT03852511 (Phase 1).

Techniques: In Vivo, Plasmid Preparation, Sequencing, Infection

Journal: Bioengineering & Translational Medicine

Article Title: Viral vector‐based gene therapies in the clinic

doi: 10.1002/btm2.10258

Figure Lengend Snippet: Examples of current clinical trials for herpes simplex virus (HSV)‐based in vivo gene therapies, grouped by indication

Article Snippet: Ad , EnAd , Anti‐CD40 antibody , Metastatic cancer, epithelial tumor , NG‐350A (PsiOxus Therapeutics Ltd) , Intratumoral , NCT03852511 (Phase 1).

Techniques: In Vivo, Plasmid Preparation

Journal: Nature Communications

Article Title: RAS/MEK/PI3K pathway inhibition augments response to CD40 agonism by targeting CD11b + Bregs thereby overcoming melanoma PD1-resistance

doi: 10.1038/s41467-025-67315-1

Figure Lengend Snippet: A Immunofluorescence staining in tumors of patients with metastatic melanoma resistant to αPD1 therapy. DNA (blue), CD8α (green), CD20 (red) and CD11b (yellow). Scale bars indicate 2 mm (left) and 50 μm (right). These results are representative of immunofluorescence staining performed on melanoma tumors from two patients. B – F Single-cell (sc) RNA-Seq analysis identifies CD20 + CD11b + PD-L1 + regulatory B cells in patient melanoma tumors. References: ( B ) Cell 2018, 175, 984–997. doi: j.cell.2018.09.006. C Cell 2018, 175, 998–1013.e1–e20. doi: j.cell.2018.12.034. Samples with total B-cell size <5 were excluded. D Frequency of CD20 + CD11b + PD-L1 + regulatory B cells in patient melanoma tumors at baseline and post αPD1 treatment. Data were derived from ( C ). E , F Patient melanoma scRNA-Seq analysis reveals a high CD40 mRNA expression in CD20 + CD11b + PD-L1 + Bregs. E n = 818 B cells from 26 patients, two-sided t -test; and ( F ) n = 1379 B cells from 30 patients. One-way analysis of variance (ANOVA) with post hoc test.

Article Snippet: For western blot analysis, mouse splenocytes were isolated from C57BL/6 mice and cultured in 10% FBS RPMI 1640 media with 10 μg/ml CD40 agonist antibody (Leinco Technologies, Cat#: C2825) for five days.

Techniques: Immunofluorescence, Staining, RNA Sequencing, Derivative Assay, Expressing

Journal: Nature Communications

Article Title: RAS/MEK/PI3K pathway inhibition augments response to CD40 agonism by targeting CD11b + Bregs thereby overcoming melanoma PD1-resistance

doi: 10.1038/s41467-025-67315-1

Figure Lengend Snippet: A 1014 cells were injected in C57BL/6 female mice. Treatment of C57BL/6 mice with aCD40 (30 μg/mouse every 3 days, intraperitoneal) started at Day 10 post tumor cell inoculation. Analysis of CD40 levels by flow cytometry of CD45 + CD19 + B cells prepared from tumor samples at day 22 post-treatment ( n = 5 per group) using a one-sided Mann–Whitney U test. B Splenocytes were isolated from C57BL/6 mice by magnetic separation, serum-free starved for 4hrs, and stimulated with or without 12.5 μg/ml aCD40. After 4 days of culture, splenic B cells were isolated by magnetic separation and stained for flow cytometric analysis ( n = 6 per group). C , D Tumor volume of 1014 melanoma in C57BL/6 female mice. Treatment with 200 μg/mouse αPD1 (every 3 days, intraperitoneal), 100 μg/mouse αCTLA4 (every 3 days, intraperitoneal), 30 μg/mouse agonist CD40 (aCD40, every 3 days, intraperitoneal), or IgG control antibody, starts at day 10 post tumor cell inoculation. E Frequency of CD19 + CD11b + regulatory B cells in 1014 melanoma tumors or tumor draining lymph node (TDLN) after 22 days of aCD40 treatment (every 3 days, intraperitoneal) TDLN IgG, n = 5; TDLN aCD40, n = 5; Tumor IgG, n = 4; Tumor aCD40, n = 5. F Flow cytometric analysis of CD45 + CD19 + B cells in the tumor microenvironment of 1014 melanoma after 3 weeks treatment of aCD40. Data were replicates from one experiment ( n = 5 mice per group). G C57BL/6 mice were intravenously injected with formalin-fixed 2 × 10 6 metastatic B16F10-Luc2 cells via the tail vein. Splenic CD8 + T and total B cells were isolated by magnetic separation after 2 days of tumor antigen priming. Total B cells were stimulated with 12.5 μg/ml aCD40 for 2 days and CD11b + B cells were isolated by magnetic separation. CD8 + cytotoxic T cell killing assay were performed via co-culturing CD11b + B cells, CD8 + T cells and B16F10-Luc2 cells in the conditioned RPMI complete medium containing 0.5 μg/ml of IL-7, 30U/ml of IL-2, and CD3/CD28 Dynabeads at a bead-to-cell ratio 2:1. Dead cells were removed and the luciferase activity of remaining live tumor cells was quantified to calculate % inhibition of CTL killing capacity. ( n = 3 independent experiments). Created in BioRender. Yan, C. (2026) https://BioRender.com/83jrya2 . Exact p values are provided as a Source Data file. H Splenic B cells were stimulated with 12.5 μg/ml aCD40 for 4 days. The CD11b - and CD11b + B cells were isolated by magnetic separation and restimulated with 12.5 μg/ml aCD40 for 30 min. Whole-cell extracts were harvested and immunoblotted. β-actin was used as a loading control for densitometry quantification. n = 3 independent experiments. One-way analysis of variance (ANOVA) with post hoc test. Source data are provided as a Source Data file.

Article Snippet: For western blot analysis, mouse splenocytes were isolated from C57BL/6 mice and cultured in 10% FBS RPMI 1640 media with 10 μg/ml CD40 agonist antibody (Leinco Technologies, Cat#: C2825) for five days.

Techniques: Injection, Flow Cytometry, MANN-WHITNEY, Isolation, Staining, Control, Luciferase, Activity Assay, Inhibition

Journal: Nature Communications

Article Title: RAS/MEK/PI3K pathway inhibition augments response to CD40 agonism by targeting CD11b + Bregs thereby overcoming melanoma PD1-resistance

doi: 10.1038/s41467-025-67315-1

Figure Lengend Snippet: A Tumor volume of 1014 melanoma in C57BL/6 female mice. Treatment with 300 mg/kg rigosertib (RGS, 5 days a week, oral gavage) or vehicle control (H 2 O) starts at day 10 post tumor cell inoculation. B Tumor samples were collected from each treatment group at 4 weeks of treatment and immune profiled by FACS analysis. All graphs show Mean ± SEM, n = 5 samples per group. C 1014 tumor cells were treated with 1 μM trametinib (T) and/or 1 μM RGS for 30–60 min. Whole-cell extracts were harvested and immunoblotted. β-actin was used as a loading control for densitometry quantification. n = 3 independent experiments. Source data are provided as a Source Data file. D RGS and T induce CD40 expression in 1014 cells. Cells were treated with RGS + /- T for 24 h. CD40 protein expression on the cell surface was detected by anti-CD40-FITC staining and flow cytometric analysis. n = 3 independent experiments. E Tumor volume of 1014 melanoma in C57BL/6 female mice. Treatment with 200 μg/mouse αPD1 (every 3 days, intraperitoneal), 300 mg/kg rigosertib (RGS, 5 days a week, oral gavage), and/or 1 mg/kg trametinib (T, 5 days a week, oral gavage), starts at day 8 post tumor cell inoculation, n = 7 ~ 10 mice per group. Created in BioRender. Yan, C. (2026) https://BioRender.com/83jrya2 . Exact p values are provided as a Source Data file. F Definition of time to resistance to drug: (1) the tumor is > 100 mm 3 and (2) has a > 30% increase of tumor volume compared to the previous measurement. Survival curves (resistance-free probabilities or survival probabilities) of treatment groups are estimated using the Kaplan–Meier Plotter and compared using the log-rank test, n = 7 ~ 9 mice per group. Exact p values are provided as a Source Data file. G Mouse body weight recorded for 20 days of treatment. Veh, n = 10; T, n = 8; RGS, n = 7, RGS + T, n = 9 mice. H Tumor and ( I ) Tumor-draining lymph node (TDLN) samples were collected from each treatment group at day 20 of treatment and immune profiled by FACS analysis, Veh, n = 5; T, n = 4; RGS, n = 4, RGS + T, n = 4 tumors. For tumor samples, we collected 200,000 live singlet cells from each tumor and recovered ~6000 CD45 + cells per tumor, regardless of treatment groups. Tc, CD8 + CD3 + cytotoxic T cells. Th, CD4 + CD3 + T helper cells. Exact p values are provided as a Source Data file.

Article Snippet: For western blot analysis, mouse splenocytes were isolated from C57BL/6 mice and cultured in 10% FBS RPMI 1640 media with 10 μg/ml CD40 agonist antibody (Leinco Technologies, Cat#: C2825) for five days.

Techniques: Control, Expressing, Staining

Journal: Nature Communications

Article Title: RAS/MEK/PI3K pathway inhibition augments response to CD40 agonism by targeting CD11b + Bregs thereby overcoming melanoma PD1-resistance

doi: 10.1038/s41467-025-67315-1

Figure Lengend Snippet: A , B Splenocytes were isolated from C57BL/6 mice, serum-free starved for 4 h, and stimulated with 12 μl/ml anti-CD3/CD28 Dynabeads plus 12.5 μg/ml agonist CD40 (aCD40). After 5 days of culture, cells were stained for FACS analysis. Exact p values are provided as a Source Data file. C Tumor volume of 1014 melanoma, created in BioRender. Yan, C. (2026) https://BioRender.com/83jrya2 , exact p values are provided as a Source Data file; and D Mouse body weight of tumor-bearing C57BL/6 female mice. Treatment with 200 μg/mouse αPD1 (2 lead-in doses, every 3 days, intraperitoneal), 30 μg/mouse aCD40 (every 3 days, intraperitoneal), plus 300 mg/kg rigosertib (RGS, 5 days a week, oral gavage) and/or 1 mg/kg trametinib (T, 5 days a week, oral gavage), starts at day 8 post tumor cell inoculation ( n = 10 per group). E Serum level of liver/kidney enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and blood urea nitrogen (BUN) at day 27 post-treatment ( n = 5 per group). Live CD45 + leukocytes in ( F ) Tumor and ( G ) Tumor-draining lymph node (TDLN) samples were concatenated after downsampling to, 25,000 and 10,000 events respectively, for t-SNE analysis through flow cytometry. All graphs show Mean ± SEM, n = 5 per group. H – K Frequency of B cells and effector cells in 1014 tumors and TDLN samples at day 27 post-treatment ( n = 5 per group). Exact p values are provided as a Source Data file. L L308 mouse protein array of 1014 tumor lysate samples at day 27 post-treatment. Data were pooled from replicates of one experiment ( n = 5 mice per group). Exact p values are provided as a Source Data file.

Article Snippet: For western blot analysis, mouse splenocytes were isolated from C57BL/6 mice and cultured in 10% FBS RPMI 1640 media with 10 μg/ml CD40 agonist antibody (Leinco Technologies, Cat#: C2825) for five days.

Techniques: Isolation, Staining, Flow Cytometry, Protein Array

Journal: Nature Communications

Article Title: RAS/MEK/PI3K pathway inhibition augments response to CD40 agonism by targeting CD11b + Bregs thereby overcoming melanoma PD1-resistance

doi: 10.1038/s41467-025-67315-1

Figure Lengend Snippet: A Correlation heatmap of top 25% differentially altered genes summarized across all treatment groups (15,903 genes plotted). Heatmap3 was used for cluster analysis and visualization. Significantly differential expressed genes with absolute fold change ≥ 2 and FDR adjusted p value ≤ 0.05 were detected by DESeq2. B CIBERSORT analysis of a leukocyte signature matrix (LM22) to deconvolution of 1014 tumors. Exact p values are provided as a Source Data file. C , D Hallmark Gene Set Enrichment Analysis (GSEA) of the transcriptomic profile of 1014 tumors at baseline or with agonist CD40 (aCD40) plus αPD1 treatment at 22 days using GSEA package. E – K TCR-β chain sequencing results were evaluated using the Archer Immunoverse analyzer. CDR3 sequences and frequency tables were extracted from the manufacturers’ analysis platform and imported for analysis using the Immunarch package ( https://immunarch.com ) in R. Comparisons were made within specific tumor types and across different tumor types (CD40-Ctrl vs CD40-OE) to evaluate drug effects, with the analysis conducted on a natural logarithmic scale. Number of ( E ) total and ( F ) unique TCR clonotypes. Exact p values are provided as a Source Data file. G Treatment effect (ratio of TCR clonotypes among treatment to clonotypes among Veh + IgG control) comparisons between CD40-Ctrl and CD40-OE tumors were performed using multiple comparisons test for generalized linear hypothesis test based on the negative Binomial generalized linear model. All graphs show Mean ± SD. H Estimated distribution of clonotype abundances. I Table summary of FDR (False Discovery Rate) adjustment. J Estimated relative abundance (also known as clonal space homeostasis), which characterizes the proportion of repertoire occupied by clonal groups with specific abundances indicated. K The top 20 most abundant TCRs of small- and medium-expanded clonotypes in TRBV26 are shown. Flow between samples indicates shared TCRs. Boxed CDR3s indicate most clonal TCRs in TRBV26. The Holm correction was used to adjust the p value for multiple comparisons. A – K Pooled values of n = 3 tumors per group.

Article Snippet: For western blot analysis, mouse splenocytes were isolated from C57BL/6 mice and cultured in 10% FBS RPMI 1640 media with 10 μg/ml CD40 agonist antibody (Leinco Technologies, Cat#: C2825) for five days.

Techniques: Sequencing, Control

Journal: Nature Communications

Article Title: RAS/MEK/PI3K pathway inhibition augments response to CD40 agonism by targeting CD11b + Bregs thereby overcoming melanoma PD1-resistance

doi: 10.1038/s41467-025-67315-1

Figure Lengend Snippet: A PDO cultures were developed using previously frozen patient melanoma tissue that was thawed and immediately subjected to fine needle aspiration. IL-2 was added to all cultures to ensure the survival and proliferation of T cells. PDOs were grown in organoid media containing 5% Matrigel. After allowing PDOs to establish for 3–4 days, treatments were initiated as follows: RGS, CD40 agonist (aCD40), αPD1, RGS + aCD40, RGS + αPD1, or RGS + aCD40 + αPD1. Cultures were allowed to grow for 10 days, and the organoid area was assessed and quantified at day 10, PDO-2624, n = 18 per group; PDO-3453A, n = 18 per group; PDO-3529 primary, n = 12 per group; PDO-3529LN, n = 12 per group; PDO-3101, n = 12 per group. All graphs show Min to Max with all points +/− SD. Exact p values are provided as a Source Data file. B , C , F Melanoma scRNA-Seq dataset . Analysis of malignant and immune cell gene expression of 6173 scRNA-seq profiles from 32 human melanoma tumors. D , E , G Melanoma scRNA-Seq dataset . Transcriptome analysis of 16,291 individual immune cells from 49 tumor samples of melanoma patients treated with checkpoint inhibitors. C , E Functional enrichment of Geneset and KEGG pathway over-representation analysis were performed on differentially expressed genes of CD20 + CD11b + PD-L1 + Bregs in patient melanoma tumors using WebGestaltR package (NULL). F , G Patient melanoma scRNA-Seq analysis reveals a distinct transcriptome profile of CD20 + CD11b + PD-L1 + Bregs. All graphs show Median ± SD. H The Breg gene signature (Sig. Bregs) is associated with poorer patient OS in multiple cancer types. Kaplan-Meier plots show OS for patients in the TCGA pan-cancer datasets . Patients were split into high and low expression of the Breg gene curated signature. Median OS survival is represented on the graph when available. P-values were calculated by log-rank test. GBM, glioblastoma; LGG, low-grade glioma; LUSC, lung squamous cell carcinoma; STAD, stomach adenocarcinoma; MESO, mesothelioma; OV, ovarian carcinoma.

Article Snippet: For western blot analysis, mouse splenocytes were isolated from C57BL/6 mice and cultured in 10% FBS RPMI 1640 media with 10 μg/ml CD40 agonist antibody (Leinco Technologies, Cat#: C2825) for five days.

Techniques: Gene Expression, Functional Assay, Expressing

Journal: Antibodies

Article Title: Immunogenicity Risk Assessment of Biotherapeutics Using an Ex Vivo B Cell Assay

doi: 10.3390/antib14030062

Figure Lengend Snippet: PBMCs were obtained as described in the methods. For B cell monoculture experiments, B cells were purified and cultured for seven days with 10 ng/mL of IL-4, 10 ng/mL of BAFF, and 1–10 µg/mL of anti-CD40 agonist antibody. After seven days B cell surface activation and maturity markers were measured by flow cytometry. ( A ) Representative histogram plots show a comparison between three bead-conjugated anti-CD40 agonist antibodies IBA 568, IBA569, and IBA 570. 5 µg of antibody was conjugated per 1 mg of beads and 0.5 mg of beads were used per 1 × 10 6 cells. ( B ) Representative histogram plots show a dose response of IBA570 ranging from 1–10 µg/mL. ( C ) B cells were treated with 1 µg/mL IBA 570 for seven days and flow cytometry was performed. * p < 0.05, ** p < 0.01, ns = not significant.

Article Snippet: Anti-CD40 agonist biosimilar antibodies were produced at Eli Lilly and Company, Indianapolis, IN.

Techniques: Purification, Cell Culture, Activation Assay, Flow Cytometry, Comparison

Journal: Antibodies

Article Title: Immunogenicity Risk Assessment of Biotherapeutics Using an Ex Vivo B Cell Assay

doi: 10.3390/antib14030062

Figure Lengend Snippet: The culture conditions support B cell proliferation and activation. CD8 + T cell-depleted PBMCs from two to four donors were seeded into six-well plates at a density of 3 × 10 6 cells/well with 10 ng/mL of IL-4 and 10 ng/mL of BAFF, and pre-coated with 1 µg/mL of anti-CD40 agonist mAb alone or plus 0.33 µM of mAb 1 and cultured for seven days. ( A ) After seven days, cells were harvested, and flow cytometry was performed to measure the positive cells for membrane IgD, IgM, IgG, and CD80/86 or ( B ) the median fluorescence intensity (MFI) of IgD, IgM, IgG, and CD80/86. ( C ) Prior to culture, cells were incubated with CellTrace Far Red Proliferation Dye, and proliferation was assessed on day seven, with representative plots shown. ( D ) Cell division index after seven days of culture. Next, CD8 + T cell-depleted PBMCs from three donors were seeded into six-well plates at a density of 4 × 10 6 cells/well or 12-well plates at a density of 3 × 10 6 cells/well and cultured for days with 10 ng/mL of IL-4 and 10 ng/mL of BAFF, and pre-coated with 0.1–1 µg/mL of anti-CD40. ( E ) After seven days, cells were harvested, and flow cytometry was performed to measure the positive cells for membrane IgD, IgM, IgG, and CD80/86 or ( F ) the median fluorescence intensity (MFI) of IgD, IgM, IgG, and CD80/86. ( G ) Prior to culture, cells were incubated with CellTrace Far Red Proliferation Dye, and proliferation was assessed on day seven, with representative plots shown. ( H ) Cell division index following seven days of culture. Next, PBMCs from three donors were seeded into six-well plates at a density of 4 × 10 6 cells/well and cultured with 10 ng/mL of IL-21, 10 ng/mL of IL-4, and 10 ng/mL of BAFF for seven days with mAb 1. ( I ) After seven days, cells were harvested, and flow cytometry was performed to measure the positive cells for membrane IgD, IgM, IgG, CD80/86, and CD138 or ( J ) the median fluorescence intensity (MFI) of IgD, IgM, IgG, and CD80/86. ( K ) Prior to culture, cells were incubated with CellTrace Far Red Proliferation Dye, and proliferation was assessed on day seven with representative plots shown. ( L ) Cell division index for control or mAb 1-treated cells after seven days of culture. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns = not significant.

Article Snippet: Anti-CD40 agonist biosimilar antibodies were produced at Eli Lilly and Company, Indianapolis, IN.

Techniques: Activation Assay, Cell Culture, Flow Cytometry, Membrane, Fluorescence, Incubation, Control, Standard Deviation

Journal: Antibodies

Article Title: Immunogenicity Risk Assessment of Biotherapeutics Using an Ex Vivo B Cell Assay

doi: 10.3390/antib14030062

Figure Lengend Snippet: IgG secretion is increased in response to treatment with the immunogenic mAb 1. CD8 + T cell-depleted PBMCs from four donors were seeded into 6-well plates at a density of 4 × 10 6 cells/well or 12-well plates at a density of 3 × 10 6 cells/well with 10 ng/mL of IL-21, 10 ng/mL of BAFF, and 10 ng/mL of IL-4, and pre-coated with 0.1 µg/mL of anti-CD40 agonist mAb alone or plus mAb 1 and cultured for seven days. ( A ) After seven days, cells were harvested, and flow cytometry was performed to measure the cells positive for membrane IgD, IgM, IgG, CD80/86, CD24, CD27, and CD138 or ( B ) the median fluorescence intensity (MFI) of IgD, IgM, IgG, and CD80/86. ( C ) Prior to culture, cells were incubated with CellTrace Far Red Proliferation Dye and proliferation was assessed on day seven, with representative plots shown. ( D ) Cell division index for control or mAb 1-treated cells following seven days of culture. ( E – I ) Additionally, culture supernatants were taken on day seven, and IgG and IgM secretion were measured. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns = not significant.

Article Snippet: Anti-CD40 agonist biosimilar antibodies were produced at Eli Lilly and Company, Indianapolis, IN.

Techniques: Cell Culture, Flow Cytometry, Membrane, Fluorescence, Incubation, Control, Standard Deviation

Journal: Antibodies

Article Title: Immunogenicity Risk Assessment of Biotherapeutics Using an Ex Vivo B Cell Assay

doi: 10.3390/antib14030062

Figure Lengend Snippet: mAbs with high clinical rates of ADAs (mAb 1 and Bococizumab) elicit a stronger CD27 + proliferation response compared to mAbs that have low clinical rates of ADAs (mAb 2 and mAb 3 CD8 + T cell-depleted PBMCs from 9 to 10 donors were seeded into 24-well plates at a density of 3 × 10 6 cells/well and cultured for seven days with 10 ng/mL of IL-21, 10 ng/mL of BAFF, and 10 ng/mL of IL-4, and pre-coated with 0.1 µg/mL of anti-CD40 agonist mAb alone or plus test article. After seven days, the culture supernatant was harvested, and flow cytometry was performed. ( A ) The fold change in CD27 + B cells following seven days of treatment in the 9–10 donors tested. ( B ) The fold change in CD27 − B cells following seven days of treatment in the 9–10 donors tested. ( C ) Representative plots showing proliferation of memory (CD27 + ) and non-memory B cells (CD27 − ). Data are presented as mean ± standard deviation. Dashed lines in ( A , B ) represent a two-fold increase. BS = biosimilar. ns = not significant, * p < 0.05. ns = not significant.

Article Snippet: Anti-CD40 agonist biosimilar antibodies were produced at Eli Lilly and Company, Indianapolis, IN.

Techniques: Cell Culture, Flow Cytometry, Standard Deviation

Journal: Antibodies

Article Title: Immunogenicity Risk Assessment of Biotherapeutics Using an Ex Vivo B Cell Assay

doi: 10.3390/antib14030062

Figure Lengend Snippet: T cell activation and IgG secretion can be obtained from the same donors. CD8 + T cell-depleted PBMCs from four donors were seeded into 24-well plates at a density of 3 × 10 6 cells/well and with 10 ng/mL of IL-21, 10 ng/mL of BAFF, and 10 ng/mL of IL-4, and pre-coated with 0.1 µg/mL of anti-CD40 agonist mAb alone or plus test article for 2 days to measure T cell activation or 7 days to measure IgG secretion. ( A ) After 2 days, cells were harvested, and flow cytometry was performed for CD134 and CD137 expression on CD4 + T helper cells. ( B ) After 7 days, culture supernatant was harvested, and IgG secretion was measured using the LEGENDPlex Human Immunoglobulin Isotyping Panel (8-Plex) from BioLegend. ( C ) Representative plots for CD134 and CD137 expression on CD4 + T helper cells. Dashed lines in ( A , B ) represent a two-fold increase. Data are presented as mean ± standard deviation. ns = not significant, * p < 0.05, ** p < 0.01.

Article Snippet: Anti-CD40 agonist biosimilar antibodies were produced at Eli Lilly and Company, Indianapolis, IN.

Techniques: Activation Assay, Flow Cytometry, Expressing, Standard Deviation

Journal: Antibodies

Article Title: Immunogenicity Risk Assessment of Biotherapeutics Using an Ex Vivo B Cell Assay

doi: 10.3390/antib14030062

Figure Lengend Snippet: IgG secretion in response to treatment with a panel of 51 mAbs comprising internal and external mAbs with varying clinical immunogenicity rates. CD8 + T cell-depleted PBMCs from 8 to 48 donors depending on the mAb (most mAbs were screened using 10 donors) were seeded into 24-well plates at a density of 3 × 10 6 cells/well with 10 ng/mL of IL-21, 10 ng/mL of BAFF, and 10 ng/mL of IL-4, and pre-coated with 0.1 µg/mL of anti-CD40 agonist alone or plus test article and cultured for seven days. After seven days, culture supernatant was harvested, and IgG secretion was analyzed for all 51 mAbs tested. ( A ) The fold change in IgG secretion compared to control cultures for internal mAbs. ( B ) The fold change in IgG secretion compared to control cultures for external mAbs. ( C ) Visualization of the fold change in IgG secretion compared to control cultures for low-, moderate-, and high-risk mAbs. ( D ) To test if formulation had an impact on the B cell IgG secretion response, we tested mAbs 13, 15, 19, and 20 in their original formulation compared to PBS formulation. Low-risk mAbs were defined as when <10% of patients developed ADA, moderate-risk mAbs were defined as when 10–25% of patients developed ADA, and high-risk mAbs were defined as when >25% of patients developed ADA. BS = biosimilar. Black bars represent the median fold change.

Article Snippet: Anti-CD40 agonist biosimilar antibodies were produced at Eli Lilly and Company, Indianapolis, IN.

Techniques: Immunopeptidomics, Cell Culture, Control, Formulation