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