Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Generation of multispecific DB-VHH constructs. Trastuzumab IgG1-K409R mAb (anti-HER2) C-termini are fused to VHHs directed against EGFR, IL6R or NKG2D via a GS-linker. CS06 IgG1-F405L mAb (anti-c-MET) is fused to the same VHH molecules in the same manner. After recombinant production and purification, parental IgG-VHHs are recombined pairwise by reduction and reoxidation. The matching K409R and F405L mutations drive the generation of heterodimeric multispecific DB-VHHs.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Construct, Recombinant, Purification

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Binding affinities measured for each paratope using recombinant antigens

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Recombinant, Construct

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Biolayer interferometry analysis of simultaneous antigen binding of tri- and tetraspecific DB-VHHs. (a), (b) and (c) show exemplary sensorgrams for trispecific molecules and (d) a tetraspecific DB-VHH. The first association step represents binding of the DB-VHH (200 nM) via its CS06 paratope to biotinylated c-MET immobilized to streptavidin biosensors. Second (and third for (D)) association step is performed using an IL6R, EGFR or NKG2D recombinant protein (200 nM). The last association step is performed using HER2 (200 nM). Kinetic buffer (KB) controls were applied as negative controls for each association step.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Recombinant

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Specific cell clustering due to simultaneous binding of the DB-VHHs to three different cancer cell lines. Flow cytometry cytograms represent the fluorescence signals of the different cell populations. (a) Cells without antibody construct. Upper left gate = HCC-1954 (HER2 +++ ) cells stained with DeepRed, lower left gate = MDA-MB-468 (EGFR +++ ) cells stained with CMRA, lower right gate = EBC-1 (c-MET ++ ) cells stained with CFSE, upper right gate = HCC-1954 + EBC-1 cell doublets. Events in all three fluorescence channels (cell triplets) were marked in red. Cells were incubated in the presence of 1 nM (b) bispecific DB, (c) and (d) trispecific DB-VHHs, (e) and (f) tetraspecific DB-VHHs.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Flow Cytometry, Fluorescence, Construct, Staining, Incubation

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Simultaneous interaction of the DB-VHHs with HCC-1954 and EBC-1 target cells and binding of recombinant IL6R. Flow cytometry cytograms represent the fluorescence signals of the two cell populations and bound recombinant IL6R, detected with an anti-His6 detection antibody. (a) Cells without antibody construct. Upper left gate = HCC-1954 (HER2+++) cells stained with DeepRed, lower right gate = EBC-1 (c-MET++) cells stained with CFSE, upper right gate = HCC-1954 + EBC-1 cell doublets. Events in all three fluorescence channels (HCC-1954 + EBC-1 + bound recombinant IL6R-His Tag) were marked in blue. Cells were incubated in the presence of 10 nM (b) bispecific DB, (c) and (d) trispecific DB-VHHs, (e) and (f) tetraspecific DB-VHHs.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Recombinant, Flow Cytometry, Fluorescence, Construct, Staining, Incubation

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: DB-VHHs elicit potent and specific NK cell-mediated target cell killing. Tumor cells were incubated with primary effector cells (NK cells) at a 1:5 ratio in the presence of antibody constructs in different concentrations. Error bars represent standard deviation of two biological replicates. Wildtype CS06 and trastuzumab with active Fc effector functioning were used as an ADCC reference (green). (a) and (b): c-MET-positive EBC-1 target cells, (c) and (d) HER2-overexpressing SK-BR-3 target cells. (a) and (c): NK cell cytotoxicity triggered by parental antibodies. (b) and (d): DB-VHHs serve as NK cell engager and mediate tumor cell killing.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Incubation, Construct, Standard Deviation

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet:

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Variant Assay, Hydrophobic Interaction Chromatography, Mutagenesis, Molecular Weight, High Performance Liquid Chromatography

Journal: Frontiers in Immunology

Article Title: Bispecific killer cell engagers employing species cross-reactive NKG2D binders redirect human and murine lymphocytes to ErbB2/HER2-positive malignancies

doi: 10.3389/fimmu.2024.1457887

Figure Lengend Snippet: Generation of bispecific killer cell engagers. (A) Schematic representation of prototypic hNKAB-ErbB2 (left) and species cross-reactive scNKAB-ErbB2 molecules (right) that consist of an N-terminal scFv antibody fragment binding to NKG2D, followed by hinge, CH2 and CH3 domains of human IgG4 (hIgG4), a (G 4 S) 2 linker, and an ErbB2-specific C-terminal scFv antibody fragment. mNKAB-ErbB2 (middle) carries the extracellular domain (ECD) of murine NKG2D ligand MULT-1 at the N-terminus, followed by hinge, CH2 and CH3 domains of murine IgG1 (mIgG1), a (G 4 S) 2 linker, and an ErbB2-specific C-terminal scFv antibody fragment. Disulfide bridges facilitating formation of homodimers are indicated by lines. (B) Analysis of purified NKAB antibodies by SDS-PAGE under reducing (R, left) and non-reducing (NR, right) conditions and Coomassie staining. NKAB monomers and dimers are indicated by arrowheads. (C) Binding of purified NKAB molecules at a concentration of 12.5 nM to NK-92 cells expressing human (hNKAR-NK-92) or murine (mNKAR-NK-92) NKG2D-CARs, and ErbB2-positive MDA-MB-453 and ErbB2-negative MDA-MB-468 breast carcinoma cells was investigated by flow cytometry as indicated. Unstained cells and cells only incubated with secondary antibody served as controls.

Article Snippet: Simultaneous binding of NKAB antibodies to ex vivo expanded primary effector cells and the target antigen ErbB2 was analyzed by incubation of the cells with NKAB molecules, followed by staining with PE-conjugated recombinant extracellular domain of ErbB2 (AcroBiosystems, Basel, Switzerland).

Techniques: Binding Assay, Purification, SDS Page, Staining, Concentration Assay, Expressing, Flow Cytometry, Incubation

Journal: Frontiers in Immunology

Article Title: Bispecific killer cell engagers employing species cross-reactive NKG2D binders redirect human and murine lymphocytes to ErbB2/HER2-positive malignancies

doi: 10.3389/fimmu.2024.1457887

Figure Lengend Snippet: Competition of MICA binding by bispecific killer cell engagers. (A) Binding of His-tagged soluble MICA (sMICA) (20 nM) to hNKAR-NK-92 cells in the absence of NKAB molecules (gray area) was determined by flow cytometry with APC-conjugated anti-His-tag antibody. Control cells were only stained with secondary antibody (dashed line). (B) Competition of sMICA binding by prototypic hNKAB-ErbB2 was confirmed by incubation of cells with 20 nM of sMICA in the absence (gray area) or presence of increasing concentrations of hNKAB-ErbB2 (left). mNKAB-ErbB2 which is unable to interact with human NKG2D was included as negative control (right). (C) The ability of species cross-reactive NKAB antibodies to compete binding of sMICA to NKAR-NK-92 cells was investigated in similar experiments with purified scNKAB-ErbB2 proteins as indicated.

Article Snippet: Simultaneous binding of NKAB antibodies to ex vivo expanded primary effector cells and the target antigen ErbB2 was analyzed by incubation of the cells with NKAB molecules, followed by staining with PE-conjugated recombinant extracellular domain of ErbB2 (AcroBiosystems, Basel, Switzerland).

Techniques: Binding Assay, Flow Cytometry, Control, Staining, Incubation, Negative Control, Purification

Journal: Frontiers in Immunology

Article Title: Bispecific killer cell engagers employing species cross-reactive NKG2D binders redirect human and murine lymphocytes to ErbB2/HER2-positive malignancies

doi: 10.3389/fimmu.2024.1457887

Figure Lengend Snippet: Bispecific binding of NKAB molecules to NKG2D-positive primary lymphocytes and ErbB2. Binding of purified scNKAB-ErbB2 proteins (orange), hNKAB-ErbB2 (red) and mNKAB-ErbB2 (blue) to gated NK, NKT-like and CD8 + T cell subpopulations of human PBMCs (A) , as well as ex vivo expanded murine NK cells (B) was analyzed by flow cytometry, detecting bound NKAB molecules with recombinant PE-conjugated ErbB2 extracellular domain to confirm simultaneous interaction with NKG2D and ErbB2. Cells stained with ErbB2-PE in the absence of an NKAB molecule (gray) served as controls. Middle panels show representative histograms of cells from one donor and one animal, respectively. Panels on the right display median fluorescence intensities (MFI). Mean values ± SD are shown; n=3 individual donors in (A) and n=2 individual animals in (B) . NKG2D surface expression by human NK, NKT-like and CD8 + T cell subpopulations and murine NK cells was confirmed by flow cytometry with NKG2D-specific antibodies (left panels).

Article Snippet: Simultaneous binding of NKAB antibodies to ex vivo expanded primary effector cells and the target antigen ErbB2 was analyzed by incubation of the cells with NKAB molecules, followed by staining with PE-conjugated recombinant extracellular domain of ErbB2 (AcroBiosystems, Basel, Switzerland).

Techniques: Binding Assay, Purification, Ex Vivo, Flow Cytometry, Recombinant, Staining, Fluorescence, Expressing

Journal: Frontiers in Immunology

Article Title: Bispecific killer cell engagers employing species cross-reactive NKG2D binders redirect human and murine lymphocytes to ErbB2/HER2-positive malignancies

doi: 10.3389/fimmu.2024.1457887

Figure Lengend Snippet: NKAB-mediated redirection of primary human and murine lymphocytes endogenously expressing NKG2D to ErbB2-expressing tumor cells. (A) Cytotoxicity of human PBMCs in the absence or presence of 0.64 nM of the indicated ErbB2-specific NKAB molecules against human K562 erythroleukemia, and CEM.NKR or CEM.NKR/ErbB2 T lymphoblastic cells was determined after 3 hours of co-incubation at an E/T ratio of 10:1. Samples kept in the absence of an antibody or incubated with 0.64 nM of trastuzumab or an ErbB2-specific scFv fusion protein containing a human IgG4 Fc domain (FRP5-Fc) were included as controls. (B) Cytotoxicity of murine splenocytes in the absence or presence of 0.64 nM of the indicated ErbB2-specific NKAB molecules against murine A20 B-cell lymphoma, and RMA/neo or RMA/neo/ErbB2 T lymphoblastic cells was determined after 4 hours of co-incubation at an E/T ratio of 20:1. Samples kept in the absence of an antibody or incubated with 0.64 nM of FRP5-Fc (IgG4) were included as controls. Mean values ± SD are shown; n=3 independent donors or animals. **, p < 0.01; *, p < 0.05. Statistical significance is indicated for differences in comparison to samples without antibody.

Article Snippet: Simultaneous binding of NKAB antibodies to ex vivo expanded primary effector cells and the target antigen ErbB2 was analyzed by incubation of the cells with NKAB molecules, followed by staining with PE-conjugated recombinant extracellular domain of ErbB2 (AcroBiosystems, Basel, Switzerland).

Techniques: Expressing, Incubation, Comparison

Journal: Frontiers in Immunology

Article Title: Bispecific killer cell engagers employing species cross-reactive NKG2D binders redirect human and murine lymphocytes to ErbB2/HER2-positive malignancies

doi: 10.3389/fimmu.2024.1457887

Figure Lengend Snippet: Redirection of NKG2D-CAR engineered NK-92 cells by ErbB2-targeted NKAB molecules. (A) NKAB-independent natural cytotoxicity of hNKAR-NK-92 (left panels) and mNKAR-NK-92 cells (right panels) against NK-sensitive K562 erythroleukemia cells (top) and NKAB-mediated killing of ErbB2-positive MDA-MB-453 breast carcinoma cells (middle) in the absence or presence of 0.64 nM of the indicated NKAB-ErbB2 molecules was determined after 3 hours of co-incubation at an E/T ratio of 5:1. ErbB2-negative MDA-MB-468 breast carcinoma cells (bottom) were included as control. Mean values ± SD are shown; n=3 independent experiments. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05. (B) Cytotoxic activity of hNKAR-NK-92 (top) or mNKAR-NK-92 cells (bottom) against MDA-MB-453 cells in the presence of increasing concentrations of the indicated NKAB-ErbB2 molecules was determined after 3 hours of co-incubation at an E/T ratio of 2:1. Data points represent mean values of the percentage of viable tumor cells normalized to values obtained after co-incubation of effector and target cells in the absence of NKAB antibodies. n=3 independent experiments.

Article Snippet: Simultaneous binding of NKAB antibodies to ex vivo expanded primary effector cells and the target antigen ErbB2 was analyzed by incubation of the cells with NKAB molecules, followed by staining with PE-conjugated recombinant extracellular domain of ErbB2 (AcroBiosystems, Basel, Switzerland).

Techniques: Incubation, Control, Activity Assay