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: Scientific reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Figure 1. Unique fragments of the CDRs within the heavy and light chains of the new mouse monoclonal anti-HER2 antibody. The amino acid sequence corresponds to the characteristic nucleotide sequence of (A) anti-human HER2/70.27.58 mAb and (B) anti-human HER2/70.21.73.67 mAb.

Article Snippet: The concentration of HER2 protein in the analyzed sample was calculated based on the standard curve established with the recombinant HER2 protein Fc Tag (Acro BIOSYSTEMS, Newark, DE, USA) loaded on the plate at the concentration of 1.56–100 ng/ml.

Techniques: Sequencing

Journal: Scientific reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Figure 2. Anti-HER2 monoclonal antibodies production and characterization. (A) Morphology of the anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 hybridoma cells photographed at 20 × and 40 × magnification. (B) FPLC chromatograms were recorded during the purification of the anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 antibodies using affinity chromatography on the Protein A resin. (C) SDS-PAGE analysis of the purified anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 antibodies loaded at the amount of 1 µg/well on the 12% polyacrylamide gel under reducing conditions. (D) WB analysis of HER2 in whole cell lysates of the HER2 low expressing (MDA-MB-231) and HER2 high expressing (SK-BR-3, SK-OV-3) cells, probed with the home-made anti-human HER2/70.27.58 monoclonal antibody and detected with the secondary anti-mouse IgG-HRP (upper panel). The recombinant HER2 ECD protein was used as a reference. The loading control was performed with membrane probed with antibody binding β-actin (lower panel). (E) The formaldehyde-fixed SK-OV-3 cells were photographed in the bright field (BF) at the 40 × magnification. Immunofluorescence analysis was performed on cells stained with the commercial anti- HER2 ECD antibody followed by anti-mouse IgG-AlexaFluor594 (AF594) (red channel) and co-stained with the anti-HER2/70.27.58 or anti-HER2/70.21.73.67 antibodies detected with the AlexaFluor488-labeled (AF488) secondary antibody (green channel). Nuclei were stained with DAPI (blue channel) (F) Quantitative ELISA with the anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 antibodies loaded in a range of 0–5 µg/ ml on the plate coated with the recombinant chimera of the HER2 ECD-Fc protein. The signal generated from secondary antibody anti-mouse IgG-HRP was quantified by measuring absorbance at 450 nm and expressed after background subtraction (A450-A0).

Article Snippet: The concentration of HER2 protein in the analyzed sample was calculated based on the standard curve established with the recombinant HER2 protein Fc Tag (Acro BIOSYSTEMS, Newark, DE, USA) loaded on the plate at the concentration of 1.56–100 ng/ml.

Techniques: Bioprocessing, Purification, Affinity Chromatography, SDS Page, Expressing, Recombinant, Control, Membrane, Binding Assay, Immunofluorescence, Staining, Labeling, Enzyme-linked Immunosorbent Assay, Generated

Journal: Scientific reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Figure 3. Parameters of the sandwich ELISA for HER2 detection. (A) HER2 binding kinetics in the standard curve concentration range of 0.156–10 000 ng/well (1.56–100 ng/ml). Results are expressed as absorbance at 450 nm after background subtraction (A450-A0). (B) Assay accuracy was tested by comparison of HER2 level measured by ELISA in the samples of the known antigen concentration (mock samples). Data were collected for 2, 5, 10, 30, and 50 ng/ml of HER2 (given concentration; x-axis), covering both physiological and increased concentrations. Experimentally measured concentration [ng/ml] is shown on the y-axis. Error bars indicate SD.

Article Snippet: The concentration of HER2 protein in the analyzed sample was calculated based on the standard curve established with the recombinant HER2 protein Fc Tag (Acro BIOSYSTEMS, Newark, DE, USA) loaded on the plate at the concentration of 1.56–100 ng/ml.

Techniques: Sandwich ELISA, Binding Assay, Concentration Assay, Comparison, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Figure 4. HER2 expression in tumors from mice with xenografted human cancer cells. (A) Immunohistochemistry staining using anti-HER2/70.27.58 mAb of the mouse tumors induced with the human ovarian cancer cells (SK-OV-3) overexpressing HER2 and (B) human epithelial breast cancer cells (MDA-MB-231) with low expression of HER.

Article Snippet: The concentration of HER2 protein in the analyzed sample was calculated based on the standard curve established with the recombinant HER2 protein Fc Tag (Acro BIOSYSTEMS, Newark, DE, USA) loaded on the plate at the concentration of 1.56–100 ng/ml.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Pharmaceutics

Article Title: Biologic Evaluation of a Heterodimeric HER2-Albumin Targeted Affibody Molecule Produced by Chemo-Enzymatic Peptide Synthesis

doi: 10.3390/pharmaceutics14112519

Figure Lengend Snippet: ( A ) Overview of the Chemo-Enzymatic Peptide Synthesis (CEPS) method. Smaller peptide fragments are synthesized separately and subsequently joined by Omniligase. The detailed reaction scheme can be found in . ( B ) Schematic of PEP40233 produced by CEPS linking a variable fragment that is interchangeable for different binding specificities (here HER2) and a constant fragment comprising an ABD moiety for half-life extension. A chelating DOTA moiety may optionally be incorporated at the N-terminus during the chemical synthesis. ( C ) Schematic of the HER2 targeted Affibody molecule ABY-025 with a DOTA moiety conjugated at the C-terminus.

Article Snippet: The binding kinetics of PEP40233 to HER2 (rh-HER2-Fc, Sino Biological, Beijing, China), human serum albumin (HSA; Albumedix, Nottingham, UK) and mouse serum albumin (MSA; Sigma-Aldrich, Missouri, USA) were determined using Surface Plasmon Resonance (Biacore 8K, Cytiva, Uppsala, Sweden).

Techniques: Synthesized, Produced, Binding Assay

Journal: Pharmaceutics

Article Title: Biologic Evaluation of a Heterodimeric HER2-Albumin Targeted Affibody Molecule Produced by Chemo-Enzymatic Peptide Synthesis

doi: 10.3390/pharmaceutics14112519

Figure Lengend Snippet: ( A ) Single-cycle kinetics SPR sensorgrams showing PEP40233 concentration series (0.5, 1, 2, 4, 8 nM) interaction curve with, from left to right, immobilized Her2, HSA, and MSA. ( B ) A-B-A injection sensorgrams showing PEP40233 (8 nM) binding to HER2 in the presence of HSA/MSA. The data confirm binding to HER2 both in presence and absence of albumin.

Article Snippet: The binding kinetics of PEP40233 to HER2 (rh-HER2-Fc, Sino Biological, Beijing, China), human serum albumin (HSA; Albumedix, Nottingham, UK) and mouse serum albumin (MSA; Sigma-Aldrich, Missouri, USA) were determined using Surface Plasmon Resonance (Biacore 8K, Cytiva, Uppsala, Sweden).

Techniques: Concentration Assay, Injection, Binding Assay

Journal: Pharmaceutics

Article Title: Biologic Evaluation of a Heterodimeric HER2-Albumin Targeted Affibody Molecule Produced by Chemo-Enzymatic Peptide Synthesis

doi: 10.3390/pharmaceutics14112519

Figure Lengend Snippet: Kinetic parameters of PEP40233 interacting with HER2, HSA and MSA.

Article Snippet: The binding kinetics of PEP40233 to HER2 (rh-HER2-Fc, Sino Biological, Beijing, China), human serum albumin (HSA; Albumedix, Nottingham, UK) and mouse serum albumin (MSA; Sigma-Aldrich, Missouri, USA) were determined using Surface Plasmon Resonance (Biacore 8K, Cytiva, Uppsala, Sweden).

Techniques:

Journal: Pharmaceutics

Article Title: Biologic Evaluation of a Heterodimeric HER2-Albumin Targeted Affibody Molecule Produced by Chemo-Enzymatic Peptide Synthesis

doi: 10.3390/pharmaceutics14112519

Figure Lengend Snippet: In vitro specificity of [ 177 Lu]Lu-PEP40233 binding to ( A ) SKOV-3 and ( B ) BxPC3 cells in the presence and absence of HSA. Block refers to pre-saturation of HER2 receptors with unlabeled PEP40233. The data are presented as an average value from 3 samples ± SD. The data demonstrate specific binding to both cell lines, and both in the presence and absence of HSA.

Article Snippet: The binding kinetics of PEP40233 to HER2 (rh-HER2-Fc, Sino Biological, Beijing, China), human serum albumin (HSA; Albumedix, Nottingham, UK) and mouse serum albumin (MSA; Sigma-Aldrich, Missouri, USA) were determined using Surface Plasmon Resonance (Biacore 8K, Cytiva, Uppsala, Sweden).

Techniques: In Vitro, Binding Assay, Blocking Assay

Journal: Pharmaceutics

Article Title: Biologic Evaluation of a Heterodimeric HER2-Albumin Targeted Affibody Molecule Produced by Chemo-Enzymatic Peptide Synthesis

doi: 10.3390/pharmaceutics14112519

Figure Lengend Snippet: InteractionMap evaluation of the affinity of 177 Lu labeled PEP40233 binding to HER2-expressing SKOV-3 cells in the presence of HSA.

Article Snippet: The binding kinetics of PEP40233 to HER2 (rh-HER2-Fc, Sino Biological, Beijing, China), human serum albumin (HSA; Albumedix, Nottingham, UK) and mouse serum albumin (MSA; Sigma-Aldrich, Missouri, USA) were determined using Surface Plasmon Resonance (Biacore 8K, Cytiva, Uppsala, Sweden).

Techniques: Labeling, Binding Assay

Journal: Pharmaceutics

Article Title: Biologic Evaluation of a Heterodimeric HER2-Albumin Targeted Affibody Molecule Produced by Chemo-Enzymatic Peptide Synthesis

doi: 10.3390/pharmaceutics14112519

Figure Lengend Snippet: InteractionMap of [ 177 Lu]Lu-PEP40233 binding to HER2-expressing SKOV-3 cells in the presence of HSA. Binding was measured at concentrations of 0.25 nM, 0.75 nM, and 2.25 nM. Data are representatives from duplicates. The data show presence of two types of interactions with living HER2-expressing cells.

Article Snippet: The binding kinetics of PEP40233 to HER2 (rh-HER2-Fc, Sino Biological, Beijing, China), human serum albumin (HSA; Albumedix, Nottingham, UK) and mouse serum albumin (MSA; Sigma-Aldrich, Missouri, USA) were determined using Surface Plasmon Resonance (Biacore 8K, Cytiva, Uppsala, Sweden).

Techniques: Binding Assay, Expressing

Journal: Pharmaceutics

Article Title: Biologic Evaluation of a Heterodimeric HER2-Albumin Targeted Affibody Molecule Produced by Chemo-Enzymatic Peptide Synthesis

doi: 10.3390/pharmaceutics14112519

Figure Lengend Snippet: Biodistribution at 48 h post injection. ( A ) Biodistribution of [ 177 Lu]Lu-PEP40233 in BALB/C nu/nu mice bearing SKOV-3 and BxPC3 xenografts. Data are presented as an average value from four mice ± SD. The data show higher accumulation in xenografts with higher HER2 expression. ( B ) Comparison of biodistribution of [ 177 Lu]Lu-PEP40233 and [ 177 Lu]Lu-ABY-025 in SKOV-3 bearing xenografts. Data are presented as an average value from four mice ± SD. The data show clear reduction in the renal uptake and extension of residence in circulation for [ 177 Lu]Lu-PEP40233.

Article Snippet: The binding kinetics of PEP40233 to HER2 (rh-HER2-Fc, Sino Biological, Beijing, China), human serum albumin (HSA; Albumedix, Nottingham, UK) and mouse serum albumin (MSA; Sigma-Aldrich, Missouri, USA) were determined using Surface Plasmon Resonance (Biacore 8K, Cytiva, Uppsala, Sweden).

Techniques: Injection, Expressing, Comparison

Journal: Pharmaceutics

Article Title: Biologic Evaluation of a Heterodimeric HER2-Albumin Targeted Affibody Molecule Produced by Chemo-Enzymatic Peptide Synthesis

doi: 10.3390/pharmaceutics14112519

Figure Lengend Snippet: Imaging of mouse bearing SKOV-3 xenograft 48 h after injection of [ 177 Lu]Lu-PEP40233 using microSPECT/CT. Tumor (yellow arrow) and kidneys (white arrow) have elevated activity uptake, but tumor uptake exceeds the renal uptake. The scale is linear showing arbitrary units normalized to a maximum count rate.

Article Snippet: The binding kinetics of PEP40233 to HER2 (rh-HER2-Fc, Sino Biological, Beijing, China), human serum albumin (HSA; Albumedix, Nottingham, UK) and mouse serum albumin (MSA; Sigma-Aldrich, Missouri, USA) were determined using Surface Plasmon Resonance (Biacore 8K, Cytiva, Uppsala, Sweden).

Techniques: Imaging, Injection, Activity Assay

Journal: Scientific Reports

Article Title: A Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays

doi: 10.1038/s41598-017-07569-y

Figure Lengend Snippet: Molecular model of HER2 bound by two antibodies and two DARPins. The extracellular domain of HER2 (green) from four different crystal structures were superimposed to model the spatial proximity between the various binders. Trastuzumab is represented in red (PDB ID 1N8Z), Pertuzumab in blue (PDB ID 1S78), DARPin G3 in gray (PDB ID 4HRN), and DARPin 9.29 in brown (PDB ID 4HRL).

Article Snippet: Recombinant HER2 ECD with C-terminal His tag was purchased from Speed Biosystems and reconstituted in PBS at 100 μg/mL.

Techniques:

Journal: Scientific Reports

Article Title: A Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays

doi: 10.1038/s41598-017-07569-y

Figure Lengend Snippet: Validation of TEC using purified antibody fusions. TEC with the combination of β9-L73J and G3-β10 ( A ) or β9-9.29 and PertH-β10 ( B) . None of the fusions alone, nor the combination of synthetic β9 and β10 peptides added at the same concentration as the fusions produced significant luminescence. Assay was performed as in the screen of 100 pairs except 100 nM purified binders were incubated with the cells. ( C ) TEC signal (normalized to signal from Cell-titer Glo 2.0 to account for cell number) obtained by incubating with 100 nM β9-L73J/G3-β10 for 1 hr, washing 3 times, adding Δ11S/Nano-Glo substrate (1:20 diluted lysate and 10 μM, respectively) on various cell lines. Reported immunohistochemistry (IHC) scores for HER2 expression are indicated at top of graph. ( D ) Varying numbers of SKOV3 cells were incubated for 1 hr with a homogeneous TEC detection solution (12.5 nM β9-L73J, 2.5 nM G3-β10, 1:20 dilution of Δ11S lysate, and 10 μM Nano-Glo substrate). The limit of detection was determined to be 125 cells. All data are averages from three replicate wells with the error bars indicating the standard deviation, ***p < 0.001.

Article Snippet: Recombinant HER2 ECD with C-terminal His tag was purchased from Speed Biosystems and reconstituted in PBS at 100 μg/mL.

Techniques: Biomarker Discovery, Purification, Concentration Assay, Produced, Luminescence Assay, Incubation, Immunohistochemistry, Expressing, Standard Deviation

Journal: Scientific Reports

Article Title: A Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays

doi: 10.1038/s41598-017-07569-y

Figure Lengend Snippet: Signal Kinetics of TEC. ( A ) The signal kinetics with the combination of β9-L73J (12.5 nM)/G3-β10 (2.5 nM) or ( B ) β9-9.29 (2 nM)/PertH-β10 (20 nM). The kinetics of the single-step TEC assays are compared to two different conditions pre-equilibrated with HER2: antibody fusions (gray circles), or antibody fusions and Δ11S (blue squares). The difference between the pre-equilibrated solutions indicate the kinetics of binding between antibodies and HER2 and between the peptides and Δ11S. Δ11S lysate was diluted 1:20 and Nano-Glo substrate was 10 μM in all experiments.

Article Snippet: Recombinant HER2 ECD with C-terminal His tag was purchased from Speed Biosystems and reconstituted in PBS at 100 μg/mL.

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: A Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays

doi: 10.1038/s41598-017-07569-y

Figure Lengend Snippet: The linear range and limit of detection for homogeneous HER2 TEC assays. For these assays, a complete detection solution was used consisting of the β9- and β10-fused binders, Δ11S lysate diluted 1:20, and 10 μM Nano-Glo substrate. ( A ) For β9-L73J (12.5 nM)/G3-β10 (2.5 nM), the limit of detection was determined to be 137 pg/mL, and the signal was linear up to 11 ng/mL. ( B ) For β9-9.29 (2 nM)/PertH-β10 (20 nM), the limit of detection was determined to be 45 pg/mL, and the signal was linear up to approximately 100 ng/mL. ( C ) Demonstration that TEC can be used to detect HER2 in 100% human serum. The TEC signal was linear down to a change of sub-ng/mL concentrations (Δ = 900 pg/mL), p < 0.001 between response for 9.8, 10.7, 11.4, and 12.5 ng/mL HER2. In general, human serum contains a normal HER2 level of ~9.8 ng/mL, preventing the analysis of a “blank” sample. 15 ng/mL is the established threshold separating normal for elevated HER2 levels.

Article Snippet: Recombinant HER2 ECD with C-terminal His tag was purchased from Speed Biosystems and reconstituted in PBS at 100 μg/mL.

Techniques:

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Gating strategy for detection of hHER2-CAR-T cells. A median of > 90% of mouse CD8 T cells transduced with the hHER2-CAR retrovirus were positive for the transgene as judged by flow cytometry analysis. B. B16F10 cells were transfected with mammalian human HER2 construct with lipofectamine. Cells were grown in the presence of G418 and sorted twice to generate single cell clones. C. CAR-T-mediated B16-HER2 cell killing assay. Apoptotic cells were stained for Annexin V and analyzed by flow cytometry. D. Whole mouse lung before (left) and after (right) CUBIC clearing.

Article Snippet: Biotinylated goat anti-rat IgG (H+L) was purchased from Vector Lab. Recombinant human ErbB2/Her2 Fc chimera protein and human ErbB2/Her2-Alexa Fluor-594 were obtained from R&D systems.

Techniques: In Vivo, Transduction, Flow Cytometry, Transfection, Construct, Clone Assay, Staining

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Generation of GFP-ROSAβII-spectrin mouse model (Rosa26tm(CAG-LSL-Sptbn1-IRES-GFP)). Mouse Sptbn1 cDNA was inserted into the CAG-STOP-GFP-Rosa targeting vector, CTV, between a floxed Stop cassette and the internal ribosome entry site (IRES) followed by the enhanced Green Fluorescent Protein gene (eGFP). Transcription is under control of the CAG promoter. The targeting vector contained Rosa26 homology arms (1 kb 5′ and 3.8 kb 3′), so that the entire loxP-stop-loxP-Tmc2-IRES-GFP transcriptional cassette was inserted into the first intron of Rosa26 gene on chromosome 6. B & C. Flow cytometry analysis of IFNγ and TNFα expression in hHER2-CAR transfected T cells from GFP-ROSAβII-spectrin mouse (TβII-spectrin) after treated with PBS or Tat-Cre Recombinase. Cells were co-cultured with B16-HER2 cells. Data represent mean ± SEM. n = 9. D. CAR-mediated B16-HER2 cell killing assay with TβII-spectrin cells after treated with PBS or Tat-Cre Recombinase. Cell death was stained for NucSpot. Data represent mean ± SEM. n = 9. E. Activated CD4+ T cell (OT-II) migration on ICAM-1 coated plates ± CXCL12. Cells were treated with PTx (6 hr) or Gallein (30 min) where indicated. Data were collected from 2 independent experiments (n = 2, 17–34 individual cells per mouse). Data represent mean ± SEM Statistical analyses were performed using one-way ANOVA with Bonferroni post-test. *P = 0.007. F. The pie charts depict the proportion of CD4 T cells distributed in the tumour, blood, LN/spleen, or lung/liver 72 h post-injection. G. Expression levels of βII-spectrin and St3gal1 in human CD4 and CD8 T cells (before and after activation). Loading control: β-actin. Representative western blot images from three independent experiments are shown. H. Expression levels of βII-spectrin in human CD8 memory T cells (CD8+CD45RO+CD45RA–CD56–CD57–). Loading control: β-actin. Representative western blot images from three independent experiments are shown.

Article Snippet: Biotinylated goat anti-rat IgG (H+L) was purchased from Vector Lab. Recombinant human ErbB2/Her2 Fc chimera protein and human ErbB2/Her2-Alexa Fluor-594 were obtained from R&D systems.

Techniques: Expressing, Plasmid Preparation, Control, Flow Cytometry, Transfection, Cell Culture, Staining, Migration, Injection, Activation Assay, Western Blot