Journal: Antibody Therapeutics

Article Title: Biological activity validation of a computationally designed Rituximab/CD3 T cell engager targeting CD20+ cancers with multiple mechanisms of action

doi: 10.1093/abt/tbab024

Figure Lengend Snippet: Computer-aided design process of the “imbalanced” BsAb GB261. A) HCDRs in one arm of the parental CD20 (Rituximab analog) was replaced by the HCDRs of parental CD3 antibody to construct the preliminary CD20/CD3 BsAb. Several common VL sequences that share homology to VL (CD20) and VL (CD3) including Rituximab VL that may lead to loss of binding to CD3 but maintains binding to CD20 were designed and paired with the two VHs. These IgG-like BsAbs with different common VL candidates were constructed in the “knob into hole (KIH)” format comprising of mutations T366Y and Y407T (R). Then, T cell activation assays were performed to test the BsAb molecules and the version with Rituximab VL was selected as the lead BsAb due to its high safety/efficacy balance. Then, new mutations S354Y (Hydrophobic interaction) and Q347E (Ionic interaction) in the Fc domain were introduced to further improve heterodimer formation (ETYY format) . In addition, the pI of the two BsAb VHs was differentiated by introducing new mutations in the VH non-CDR framework regions. The deimmunization mutations were also introduced. B) The final lead molecule, GB261, was selected based on great safety/efficacy balance as well as great manufacturability represented by high expression level, high heterodimer formation percentage, easy purification process, and designed with great biochemical and biophysical characterization features. The 3D structural prediction of GB261 is shown with its CD20 binding arm, CD3 binding arm, and the Fc domain. This structure was generated with Schrodinger BioLuminate software. (Schrödinger Release 2020–3: BioLuminate, Schrödinger, LLC, New York, NY, 2020. https://www.schrodinger.com/products/bioluminate ).

Article Snippet: Recently, scientists from Genentech and Regeneron reported novel bispecific TEA with native human IgG format, which target CD20, a B cell marker, and the CD3 component of the T cell receptor, triggering redirected killing of B cells [ , ].

Techniques: Construct, Binding Assay, Activation Assay, Expressing, Purification, Generated, Software

Journal: Antibody Therapeutics

Article Title: Biological activity validation of a computationally designed Rituximab/CD3 T cell engager targeting CD20+ cancers with multiple mechanisms of action

doi: 10.1093/abt/tbab024

Figure Lengend Snippet: GB261 is weaker than BM in CD3 binding but is comparable in CD20 binding. A) Binding of designed CD20 (Rituximab analog with common VL) and CD3 antibodies (CD3 homodimer with common VL) to CD20+ Raji cells, compared with those of the parental CD20 (Rituximab analog) and CD3 antibodies. Cells were incubated with antibodies, labeled with Cy3-conjugated goat anti-human antibody, followed by FACS. The binding was presented as the percentage of cells positive for staining. B) Antibodies tested in were examined for binding to CD3+ Jurkat cells using the same method. C) Binding of GB261, BM and Rituximab analog to Raji cells. The cells were labeled with a Cy3-conjugated goat anti-human antibody and analyzed using FACS. The binding was quantified as the mean fluorescent intensity (MFI) of staining. D) Binding of GB261 and BM to Jurkat cells. E) Raji-GFP cells were mixed with Jurkat cells pre-stained with CellVue Claret Far Red fluorescent dye at 1:1 ratio and incubated overnight with 20 μg/ml antibodies, followed by the detection of cells by FACS. The events double positive for GFP and fluorescent dye were counted as bridged Raji-GFP and Jurkat cells. F) Raji-GFP cells were mixed with unstained Jurkat cells at 1:1 ratio, incubated with 20 μg/ml antibodies, and stained with DyLight 594-conjugated goat anti-human IgG. Two representative images showing the bridging of Raji-GFP cells and Jurkat cells in the presence of antibodies are shown. The display of the images has been adjusted non-uniformly for representation.

Article Snippet: Recently, scientists from Genentech and Regeneron reported novel bispecific TEA with native human IgG format, which target CD20, a B cell marker, and the CD3 component of the T cell receptor, triggering redirected killing of B cells [ , ].

Techniques: Binding Assay, Incubation, Labeling, Staining

Journal: Antibody Therapeutics

Article Title: Biological activity validation of a computationally designed Rituximab/CD3 T cell engager targeting CD20+ cancers with multiple mechanisms of action

doi: 10.1093/abt/tbab024

Figure Lengend Snippet: GB261 has balanced safety/efficacy. A) Jurkat (T cell) activation by GB261, BM, GB241, and an isotype IgG as performed by co-incubating with Raji (left), CHO cells (middle) and NK92-CD16 cells (right) at 1:1 ratio. The cells were labeled with anti-CD69 antibodies conjugated to PE and analyzed by flow cytometry. B) Upper panel, ADCC induced by either GB261, Designed CD20, Rituximab analog, or an isotype IgG. Raji cells labeled with Calcein AM were treated with antibodies and incubated with NK-92-CD16 cells. Lower panel examines whether the GB261-induced ADCC kills T cells that it binds to. Human PBMC were incubated with GB261, Rituximab analog, a CD3 mAb, and the isotype control antibody. T cell viability was analyzed by FACS. C) Upper panel, CDC induced by either GB261, Designed CD20, Rituximab analog, or an isotype IgG. Raji cells labeled with Calcein AM were treated with antibodies and incubated with complement-enriched human serum. Lower panel, Jurkat cells were incubated with antibodies as described in C Upper panel and the viability of the cells were analyzed by FACS.

Article Snippet: Recently, scientists from Genentech and Regeneron reported novel bispecific TEA with native human IgG format, which target CD20, a B cell marker, and the CD3 component of the T cell receptor, triggering redirected killing of B cells [ , ].

Techniques: Activation Assay, Labeling, Flow Cytometry, Incubation

Journal: Cell Stress & Chaperones

Article Title: Dynamics of heat shock protein 70 kDa in heat-shocked and hypoxic human endothelial cells

doi: 10.1016/j.cstres.2025.100085

Figure Lengend Snippet: Temporal modulation of human HSP high-molecular-weight complexes by heat shock in primary ECs. (a) Invitrogen’s NativeMark™ was subjected to Native PAGE, stained with Coomassie blue, and used as a standard to identify high-molecular-weight complexes in native PAGE. (b) Whole-cell lysates from HUVECs exposed to heat shock, recovery, or basal conditions were subjected to native PAGE and sequentially stained for HSP40 (CST, 4868S), HSC70 (Invitrogen PA5-27337), HSP70 (Invitrogen MA3-009), and HSP90 (CST, 4877S). Below, sodium dodecyl sulfate-PAGE for each protein is displayed. The data are representative of n = 3–6 independent experiments with cells from different lots (19TL127368, 19TL028325, 19TL023264, and 21TL195720). (c) Merged image of all the stained samples shown in (b). The data are representative of n = 3–6 independent experiments in cells from different lots (19TL127368, 19TL028325, 19TL023264, and 21TL195720). (d) Quantification of the high-molecular-weight complex intensities from specific proteins from (b) versus Basal n. The data are presented as one-way ANOVA, with Tukey’s post hoc test; ** P < 0.01, *** P < 0.001 versus the respective control; HSP40, n = 3; HSP70, n = 4; HSC70, n = 5; and HSP90, n = 6. (E) Schematic representation of the native PAGE procedure. Proteins in their native state from cell lysates were separated on a 6% polyacrylamide gel and transferred to a nitrocellulose membrane overnight. The membrane was blocked with nonfat milk, incubated with primary antibody overnight, and then incubated with secondary antibody. The membrane was scanned using the LI-COR Odyssey® 2-channel near-infrared fluorescence imaging system, and the fluorescence levels of the last HSP probed were quantified. The same membrane was then incubated overnight with a primary antibody against another chaperone, followed by secondary antibody incubation, scanning with the near-infrared fluorescence imager, and quantification. This process was repeated daily until all chaperones were stained on the same membrane, in this order: day 1 anti-HSP40; day 2 anti-HSC70; day 3 anti-HSP90; day 4 anti-HSP70. Abbreviations used: EC, endothelial cell; HSP, heat-shock protein; HUVEC, human vein.

Article Snippet: HUVEC , Lonza , 21TL195720 , Migration Tube formation Western blot Native PAGE Heat shock.

Techniques: High Molecular Weight, Clear Native PAGE, Staining, Control, Membrane, Incubation, Fluorescence, Imaging

Journal: Cell Stress & Chaperones

Article Title: Dynamics of heat shock protein 70 kDa in heat-shocked and hypoxic human endothelial cells

doi: 10.1016/j.cstres.2025.100085

Figure Lengend Snippet: Hypoxia induces decreases in multimeric HSPs in human coronary arteries (HCAECs) and vein endothelial cells (HUVECs). (a) Whole-cell lysates from HCAECs exposed to hypoxia, heat-shock recovery, or basal conditions were subjected to native PAGE and sequentially stained for HSC70 (Invitrogen PA5-27337), HSP70 (Invitrogen MA3-009), HSP90 (CST 4877S), and HSP40 (CST 4868S), as shown in the top panels. Below, sodium dodecyl sulfate-PAGE results for each protein are displayed. Data are representative of n = 3 independent experiments using two different lots of cells (20TL365545 and 20TL064651, Lonza). Both native PAGE and Western blot experiments were performed using two different lots of cells (20TL365545 and 20TL064651). (b) Quantification of HSC70, HSP70, and HSP90 high-molecular-weight complexes in (a). Data are presented as the means ± SEMs, one-way ANOVA, with Tukey’s post hoc n = 3, * P < 0.05, ** P < 0.01 versus basal conditions. Unpaired t test ( # P < 0.05) was used to compare only the HSP90 hypoxic and basal groups. (c) Whole-cell lysates from HUVECs exposed to hypoxia or basal conditions were subjected to native PAGE and sequentially stained for HSC70 (Invitrogen PA5-27337) and HSP90 (CST 4877S), as shown in the top panels. Below, sodium dodecyl sulfate-PAGE for each protein is displayed. The data are representative of n = 2 independent experiments using cells from lot 19TL028324. (d) Whole-cell lysates of HUVECs exposed to hypoxia or basal conditions were subjected to Western blot analysis for HSP40 (CST 4868S), total HSP70 (Invitrogen MA3-006), HSC70 (Invitrogen PA5-27337), and HSP90 (CST 4877S). The data are representative of n = 3 independent experiments using cells from lots 19TL028324 and 23TL086130. (e) Data from (d) are presented as the mean ± SEM, unpaired Student’s t test, n = 3 with * P < 0.05 and *** P < 0.001. β-actin, loading control. (f) HUVECs subjected to basal or hypoxic 1% O 2 (94% N 2 , 5% CO 2 ) for 24 h were subjected to Western blot analysis for HIF1α (CST, #48085) with β-actin (Sigma, A5441) loading control. The assay was performed three times using two different lots of cells (21TL195720 and 23TL086130).

Article Snippet: HUVEC , Lonza , 21TL195720 , Migration Tube formation Western blot Native PAGE Heat shock.

Techniques: Clear Native PAGE, Staining, Western Blot, High Molecular Weight, Control