Journal: bioRxiv

Article Title: More than an attachment module: covalent inhibitor warheads influence BTK dynamics and function

doi: 10.64898/2026.05.07.723540

Figure Lengend Snippet: (a) Mapping the HDX-MS changes induced by inhibitor binding onto the structure of the BTK kinase domain. Differences > 1.0 Da are dark blue (decrease) or dark green (increase); differences 0.5 Da-1.0 Da are light blue (decrease) and light green (increase). Inhibitors are pink and C481 is yellow. Regions of increased deuterium uptake in the kinase domain C-lobe are labeled. (b) Covalent attachment of BTK with Tirabrutinib and Acalabrutinib is required to observe the dynamic changes in the C-lobe. Relative deuterium level of peptides in apo BTK is subtracted from the deuterium level of the corresponding peptide from each drug-bound form (D drug-bound -D apo ); scale shows magnitude of differences. Peptic peptides are shown from BTK N- to C-terminus, top to bottom, and sample time in deuterium is shown left to right. (c) Tirabrutinib or Acalabrutinib bound BTK linker-kinase domain (LKD) shows increased binding to PLCγ as compared to Ibrutinib and Zanubrutinib bound BTK LKD. Anti-His detects bound BTK, Ponceau stain shows total PLCγ cSH2. Bands were quantified and plotted as a histogram with the error bars representing standard deviation. Data shown is the average of three independent experiments. (d) Ibrutinib (red) is more effective than Tirabrutinib (blue) at inhibiting calcium flux in BCR stimulated Ramos B cells. Structures of both inhibitors are shown highlighting the 2-butynamide (blue) and acrylamide (red) warheads. Half maximal inhibitory concentration (IC 50 ) values and -log 10 IC 50 (pIC 50 ) values ± SD (n=6) of calcium flux inhibition are listed. (e) Data are as described in (d); IC 50 for acrylamide-Tirabrutinib (red) is lower than 2-butynamide-Ibrutinib (blue).

Article Snippet: Ramos B cells (ATCC, # CRL-1596) were maintained in RPMI 1640 medium (Gibco, #A1049101) supplemented with 10% FBS (Thermo Fisher Scientific) and Penicillin/Streptomycin (Thermo Fisher Scientific) at 37°C/5% CO 2 .

Techniques: Binding Assay, Labeling, Staining, Standard Deviation, Concentration Assay, Inhibition

Journal: bioRxiv

Article Title: SpaCir-VDJ: a broadly compatible circularization strategy for spatial immune repertoire profiling

doi: 10.64898/2026.04.26.720528

Figure Lengend Snippet: (A, B) Ranking of average SHM rates and spatial mapping of SHM groups within Tonsil 2. (C, D) AUC-based prioritization and expression patterns of genes associated with high- and low-SHM states. (E, F) Correlations between SHM rate and high- or low-SHM gene-expression scores. (G–I) Changes in B-cell, T-cell, and non-lymphoid cell abundance across SHM groups. (J) Experimental workflow for lentiviral shRNA perturbation and SHM validation in Ramos cells. (K) SHM-frequency distributions after knockdown of candidate genes and the AICDA positive control. (L–N) RT-qPCR-based validation of candidate downstream molecular changes, including AICDA, BCL6, and MSH6. (O–R) Relationships between SHM intensity and IGH diversity, sequence dispersion, TRB diversity, and IGH isotype composition.

Article Snippet: Ramos cells (Cat. No. CL-0483; Procell Life Science & Technology Co., Ltd.) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) in a humidified incubator at 37 °C with 5% CO2.

Techniques: Expressing, Gene Expression, shRNA, Biomarker Discovery, Knockdown, Positive Control, Quantitative RT-PCR, Sequencing, Dispersion

Journal: Frontiers in Oncology

Article Title: A novel anti-CD20, concabody, enhances immunotherapy efficacy by targeting MPZL1 and augmenting antibody-induced cell death

doi: 10.3389/fonc.2026.1748576

Figure Lengend Snippet: Concabodies, anti-CD20 antibodies fused with the MPZL1-binding lectin Con A, enhance direct cell death and antibody-dependent cellular cytotoxicity. (A) Schematic design of concabody and SDS-PAGE analysis of purified concabodies. These recombinant Con A–fused antibodies carry a 4×G4S linker(~2kDa) between the Fc region and Con A (~28 kDa). O, obinutuzumab; T-C, isotype control (trastuzumab)-Con A; R-C, rituximab-Con A; O-C, obinutuzumab-Con A. (B) Dose-dependent induction of direct cell death (DCD) by wild-type antibodies (TRA, RTX, OBI) and concabodies (T-C, R-C, O-C) in Raji cells (n=3). (C) Cell death induced by TRA-Con A or free Con A in Raji cells(n=3). (D) DCD induced by OBI and OBI-Con A in CD20 knockout Raji cells. (E) Comparison of DCD induced by wild-type anti-CD20 antibodies alone, free Con A plus antibody co-treatment, and concabodies in Raji cells (n=3). (F) MPZL1 dependency of R-C– and O-C–induced DCD assessed in doxycycline-inducible MPZL1 knockdown cells (DOX− vs . DOX+) in Raji cells(n=3). (G) Internalization efficiency of concabodies compared to wild-type antibodies in Raji cells (n=4). (H) DCD induced by concabodies in MPZL1 Y241F mutant cells, in which phosphorylation-dependent signaling is disrupted in Raji cells (n=3). EV, empty vector. (I) Lysosomal membrane permeabilization(LMP) (left) and direct cell death(DCD) (right) assays were conducted in Raji cells using wild-type antibodies (T, TRA; R, RTX; O, OBI) and concabodies (T-C, TRA-Con A; R-C, RTX-Con A; O-C, OBI-Con A)(n=3). (J) Lysosomal membrane permeabilization(LMP) (left) and Direct cell death(DCD) (right) assays were conducted in Ramos cells using wild-type antibodies (T, TRA; R, RTX; O, OBI) and concabodies (T-C, TRA-Con A; R-C, RTX-Con A; O-C, OBI-Con A)(n=3). (K) Increased antibody-dependent cellular cytotoxicity (ADCC) by concabodies (T-C, R-C, O-C) compared to wild-type antibodies(n=3). ADCC was measured by co-incubating 1×10 4 Raji cells with 5×10 4 healthy donor PBMCs. (L) Enhanced B-cell depletion by concabodies compared to wild-type antibodies in ex vivo assays using PBMCs from patients with chronic lymphocytic leukemia (CLL). Data are presented as mean ± SD. Statistical significance was determined using Welch’s t-test for comparisons between parental controls and specific groups (G, H) .For comparisons between multiple conjugate groups, One-way ANOVA followed by Tukey’s post-hoc test was employed. (F–J) Significance is indicated as follows: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001.

Article Snippet: CHO-K1, HEK293T and Ramos cells were purchased from the Korean Cell Line Bank.

Techniques: Binding Assay, SDS Page, Purification, Recombinant, Control, Knock-Out, Comparison, Knockdown, Mutagenesis, Phospho-proteomics, Plasmid Preparation, Membrane, Ex Vivo