Journal: PLoS ONE

Article Title: Acetate Supplementation Induces Growth Arrest of NG2/PDGFRα-Positive Oligodendroglioma-Derived Tumor-Initiating Cells

doi: 10.1371/journal.pone.0080714

Figure Lengend Snippet: OG33 (A) and OG35 (B-D) cells were induced to differentiate over 6 days in vitro via activation of adenylyl cyclase (1 mM dibutyryl cAMP or 10 μM forskolin) or inhibition of MEK1/2 (1 μM PD035901), ErbB2 (1 μM PD174285), PI3K (5 μM and 10 μM LY294002), or mTOR (10 nM and 20 nM rapamycin [RAP]) signaling. DMSO (0.2% DMSO) was used as a control for PD174285, PD035901 and LY294002 treatments. Despite inhibition of PI3K-Akt-mTOR and ERK signaling pathways, both cells failed to differentiate into cells with oligodendroglial morphology (A, B) or increase immunoreactivity for GPAF or CNPase (not shown). MEK and ErbB2 inhibition actually increased OG35 proliferation in DM. While inhibition of PI3K-Akt-mTOR signaling induced morphological alterations in OG35 cells (B), this was not associated with increased CNPase (C) or ASPA (D) protein levels. In fact, ASPA expression decreased in the presence of 10 μM LY294002 and 20 nM rapamycin. Differentiation of Oli-Neu cells with cAMP for 4 days or inhibition of ErbB2 signaling for 2 days served as controls for oligodendrocyte cell morphology. DM - differentiation medium, SCM - stem cell medium. n = 3 independent cultures. *p < 0.05. Scale bar = 100 μm.

Article Snippet: For pharmacological induction of differentiation, OG33 and OG35 cells were plated in SCM or DM in the absence or presence of dibutyryl cAMP (1 mM in water, Sigma), forskolin (10 μM in ethanol, Sigma), the MEK1/2 inhibitor PD035901 (1 μM in dimethylsulfoxide [DMSO], Tocris Bioscience/R & D Systems; Minneapolis, MN), the ErbB2 inhibitor PD174285 (1 μM in DMSO, Santa Cruz Biotechnology), the PI3K inhibitor LY294002 (at 5 μM and 10 μM in DMSO, Tocris), the mTOR inhibitor rapamycin (at 10 nM and 20 nM in ethanol, Sigma) or 0.2% DMSO as a control (final DMSO concentration of 0.05% for PD174285, 0.1% for PD035901 and LY294002, and 0.2% for PD035901/LY294002/rapamycin treatment).

Techniques: In Vitro, Activation Assay, Inhibition, Control, Protein-Protein interactions, Expressing

Journal: Breast Cancer Research : BCR

Article Title: Anti-HER2 CD4 + T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer

doi: 10.1186/s13058-015-0584-1

Figure Lengend Snippet: Consolidated Standards of Reporting Trials (CONSORT) diagram of the study population. In this study, 87 patients with human epidermal growth factor receptor 2-positive ( HER2 pos ) breast cancer were enrolled; all tumors were histologically confirmed as invasive breast cancer ( IBC ) with HER2 overexpression (3+ or 2+/fluorescence in situ hybridization (FISH)-positive). Cohorts are labeled ( a – g ) for ease of comparison (of immune responses), and are referred to in Results . Time points at which blood was drawn are indicated ( red callout boxes ). Median follow up in the cohort treated with trastuzumab and chemotherapy ( T + C ) was 26 (IQR 16.5–31.0) months. pCR pathologic complete response, DCI type 1-polarized dendritic cell, Adj adjuvant, mo months

Article Snippet: After plates were blocked, cryopreserved PBMCs, isolated using density gradient centrifugation, were plated in triplicate (2 × 10 5 cells/well) and incubated at 37 °C for 24–36 h with either HER2 peptides (4 μg; Genscript, Piscataway, NJ, USA); media alone (unstimulated control); or positive control (anti-human CD3/CD28 antibodies (0.5 μg/mL; BD Pharmingen, San Jose, CA, USA)).

Techniques: Over Expression, Fluorescence, In Situ Hybridization, Labeling, Comparison, Adjuvant

Journal: Breast Cancer Research : BCR

Article Title: Anti-HER2 CD4 + T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer

doi: 10.1186/s13058-015-0584-1

Figure Lengend Snippet: Interferon (IFN)-γ + anti-human epidermal growth factor receptor 2 ( HER2 ) CD4 + T cell response variations in HER2 pos patients with invasive breast cancer (IBC). IFN-γ enzyme-linked immunosorbent spot analysis of HER2 peptide-pulsed peripheral blood mononuclear cells examined anti-HER2 CD4 + T cell responses in patients with HER2 pos IBC, stratified by anti-HER2 responsivity , response repertoire ( mean # of reactive peptides ), and cumulative response ( mean total SFC/10 6 cells ). Differences between ( a ) Treatment-naïve patients with HER2 pos IBC (n = 22) and those treated with trastuzumab and chemotherapy ( T + C ) (n = 65); and ( b ) patients treated with Neoadjuvant T + C (n = 40) and those treated with Adjuvant T + C (n = 25). SFC spot-forming cells

Article Snippet: After plates were blocked, cryopreserved PBMCs, isolated using density gradient centrifugation, were plated in triplicate (2 × 10 5 cells/well) and incubated at 37 °C for 24–36 h with either HER2 peptides (4 μg; Genscript, Piscataway, NJ, USA); media alone (unstimulated control); or positive control (anti-human CD3/CD28 antibodies (0.5 μg/mL; BD Pharmingen, San Jose, CA, USA)).

Techniques: ELISpot Assay, Adjuvant

Journal: Breast Cancer Research : BCR

Article Title: Anti-HER2 CD4 + T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer

doi: 10.1186/s13058-015-0584-1

Figure Lengend Snippet: Significant disparity in anti-human epidermal growth factor receptor 2 ( anti-HER 2) interferon (IFN)-γ + T cell immune responses between patients with pathologic complete response ( pCR ) and patients with non-pCR . a Significantly elevated anti-HER2 CD4 + T cell responses by IFN-γ enzyme-linked immunosorbent spot analysis (ELISPOT) are observed in patients with HER2 pos invasive breast cancer achieving pCR (n = 16) following neoadjuvant trastuzumab and chemotherapy (T + C), compared with patients with non-pCR (n = 24). Peripheral blood mononuclear cells (PBMC) from patients with pCR and non-pCR were stimulated ex vivo with six HER2-derived class II peptides and IFN-γ production via ELISPOT was compared. Responses are stratified by anti-HER2 responsivity, repertoire, and cumulative response. b Evaluable HLA-A2.1 pos PBMC from patients with pCR (n = 6; black bars ) and non-pCR (n = 4; white bars ) were stimulated ex vivo with two HER2-derived class I peptides, HER2 369 – 377 and HER2 689–697 , and IFN-γ production via ELISPOT was compared. Phorbol-12-myristate 13-acetate ( PMA ) and ionomycin served as positive control. Results are expressed as mean spot-forming cells ( SFC )/2 × 10 5 cells ± standard error of the mean

Article Snippet: After plates were blocked, cryopreserved PBMCs, isolated using density gradient centrifugation, were plated in triplicate (2 × 10 5 cells/well) and incubated at 37 °C for 24–36 h with either HER2 peptides (4 μg; Genscript, Piscataway, NJ, USA); media alone (unstimulated control); or positive control (anti-human CD3/CD28 antibodies (0.5 μg/mL; BD Pharmingen, San Jose, CA, USA)).

Techniques: ELISpot Assay, Enzyme-linked Immunospot, Ex Vivo, Derivative Assay, Positive Control

Journal: Breast Cancer Research : BCR

Article Title: Anti-HER2 CD4 + T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer

doi: 10.1186/s13058-015-0584-1

Figure Lengend Snippet: Anti-human epidermal growth factor receptor 2 ( anti-HER2 ) CD4 + T-helper type-1 (Th1) is the dominant phenotype contributing to interferon-γ + ( IFN-γ + ) CD4 + T cell deficit in patients without pathologic complete response ( non-pCR ). a Relative contributions of Th1 ( T-bet + IFN-γ + ) versus Th2 ( GATA-3 + IFN-γ + ) phenotypes to HER2 peptide-specific IFN-γ + cells in peripheral blood mononuclear cells ( PBMC ) from pCR and non-pCR patients. Representative stains within groups are shown after gating on CD4 + cells; results in adjoining histograms are expressed as mean proportions (%) ± standard error of the mean (SEM) as indicated. b Circulating HER2-specific IL-4 production does not vary between pCR and non-pCR patients, when assessed by responsivity , repertoire , and cumulative response . Results expressed as proportion or mean ± SEM ( top panel ). Donor-matched cumulative IFN-γ and IL-4 production (spot-forming cells ( SFC )/10 6 cells) across six HER2 class II peptides compared in pCR and non-pCR patients. Relative HER2-specific IFN-γ:IL-4 proportions (% depicted in graph) was significantly higher in pCR (IFN-γ/(IFN-γ + IL-4) = 73.9 %:IL-4/(IFN-γ + IL-4) = 26.1 %) compared with non-pCR patients (34.6 %:65.4 %). Absolute IFN-γ:IL-4 production ratio changed from 2.8:1 (pCR) to 0.5:1 (non-pCR) ( bottom left panel ). No relative shifts in IFN-γ:IL-4 production were observed to positive controls (anti-CD3/CD28 or anti-CD3, respectively) ( bottom right panel ). c HER2-specific IL-10 production is similar between pCR and non-pCR patients across all Th1 metrics. Results are expressed as proportion or mean ± SEM ( top panel ). Relative HER2-specific IFN-γ:IL-10 production was significantly higher in pCR (62.0:38.0 %) compared with non-pCR (23.8:76.2 %) patients. Absolute IFN-γ:IL-10 production ratio changed from 1.6:1 (pCR) to 0.3:1 (non-pCR) ( bottom left panel ). No relative shifts in IFN-γ:IL-4 production were observed to positive controls (anti-CD3/CD28 or anti-CD3, respectively) ( bottom right panel ); * p <0.05, ** p <0.01, *** p <0.001

Article Snippet: After plates were blocked, cryopreserved PBMCs, isolated using density gradient centrifugation, were plated in triplicate (2 × 10 5 cells/well) and incubated at 37 °C for 24–36 h with either HER2 peptides (4 μg; Genscript, Piscataway, NJ, USA); media alone (unstimulated control); or positive control (anti-human CD3/CD28 antibodies (0.5 μg/mL; BD Pharmingen, San Jose, CA, USA)).

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: Anti-HER2 CD4 + T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer

doi: 10.1186/s13058-015-0584-1

Figure Lengend Snippet: Anti-human epidermal growth factor receptor 2 (anti-HER2) T-helper type-1 (Th1) deficit in patients without pathologic complete response ( non-pC R) is not attributable to lack of immune competence, host-level T cell anergy, or increase in immunosuppressive phenotypes. Peripheral blood mononuclear cells ( PBMC ) from pCR and non-pCR patients did not differ significantly in ( a ) immune competence – measured by IFN-γ production to anti-CD3/anti-CD28 stimulus or recall stimuli tetanus toxoid and Candida albicans – by enzyme-linked immunosorbent spot. Results presented as median ± IQR interferon-γ (IFN-γ) spot-forming cells (SFC)/2 × 10 5 cells. b , c Relative proportions of CD3 + CD4 + ( b , top ) or CD3 + CD8 + T-cells ( b , bottom ), T reg (CD4 + CD25 + FoxP3 + ) ( c , top ) or myeloid-derived suppressor cells (MDSC) (CD11b + CD33 + HLA-DR − CD83 − ) ( c , bottom ) by flow cytometry. Representative stains within groups are shown; results in histograms are expressed as mean proportions (%) ± standard error of the mean as indicated

Article Snippet: After plates were blocked, cryopreserved PBMCs, isolated using density gradient centrifugation, were plated in triplicate (2 × 10 5 cells/well) and incubated at 37 °C for 24–36 h with either HER2 peptides (4 μg; Genscript, Piscataway, NJ, USA); media alone (unstimulated control); or positive control (anti-human CD3/CD28 antibodies (0.5 μg/mL; BD Pharmingen, San Jose, CA, USA)).

Techniques: ELISpot Assay, Derivative Assay, Flow Cytometry

Journal: Breast Cancer Research : BCR

Article Title: Anti-HER2 CD4 + T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer

doi: 10.1186/s13058-015-0584-1

Figure Lengend Snippet: Demographic and clinicopathologic characteristics of patients recruited to the ongoing phase I trial of HER2-pulsed DC1 vaccination in HER2 pos IBC patients with residual disease following neoadjuvant trastuzumab and chemotherapy (NCT02061423)

Article Snippet: After plates were blocked, cryopreserved PBMCs, isolated using density gradient centrifugation, were plated in triplicate (2 × 10 5 cells/well) and incubated at 37 °C for 24–36 h with either HER2 peptides (4 μg; Genscript, Piscataway, NJ, USA); media alone (unstimulated control); or positive control (anti-human CD3/CD28 antibodies (0.5 μg/mL; BD Pharmingen, San Jose, CA, USA)).

Techniques: Fluorescence In Situ Hybridization

Journal: Breast Cancer Research : BCR

Article Title: Anti-HER2 CD4 + T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer

doi: 10.1186/s13058-015-0584-1

Figure Lengend Snippet: Depressed anti-human epidermal growth factor receptor 2 (anti-HER2) T-helper type-1 (Th1) immunity is restored following HER2-pulsed type 1-polarized dendritic cell (DC1) immunization. a Anti-HER2 CD4 + T cell immune reactivity profiles in four patients with non-pCR ( UPCC # 26113–01 to −04 ) undergoing HER2-pulsed DC1 vaccination in the ongoing phase I trial NCT02061423 are demonstrated at three time points: pre-vaccination ( black ), 3 months post vaccination ( gray ), and 6 months post vaccination ( white ). For these time points, responses to individual HER2-derived class II peptides ( x-axis ; p42, p98, p328, p776, p927, p1166) are graphed, with anti-CD3/CD28 serving as positive control. Anti-HER2 Th1 repertoire and cumulative responses at these time points are listed in adjoining tables. b Anti-HER2 Th1 repertoire ( mean # of reactive peptides ) and cumulative response ( mean total SFC/10 6 cells ) increase progressively in patients with non-pCR at the 3-month and 6-month time points. SFC spot-forming cells

Article Snippet: After plates were blocked, cryopreserved PBMCs, isolated using density gradient centrifugation, were plated in triplicate (2 × 10 5 cells/well) and incubated at 37 °C for 24–36 h with either HER2 peptides (4 μg; Genscript, Piscataway, NJ, USA); media alone (unstimulated control); or positive control (anti-human CD3/CD28 antibodies (0.5 μg/mL; BD Pharmingen, San Jose, CA, USA)).

Techniques: Derivative Assay, Positive Control

Journal: Breast Cancer Research : BCR

Article Title: Anti-HER2 CD4 + T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer

doi: 10.1186/s13058-015-0584-1

Figure Lengend Snippet: Demographic and tumor-related characteristics of the study population: age, race, AJCC pathologic stage, hormone receptor status, and time from completion of trastuzumab (when applicable)

Article Snippet: After plates were blocked, cryopreserved PBMCs, isolated using density gradient centrifugation, were plated in triplicate (2 × 10 5 cells/well) and incubated at 37 °C for 24–36 h with either HER2 peptides (4 μg; Genscript, Piscataway, NJ, USA); media alone (unstimulated control); or positive control (anti-human CD3/CD28 antibodies (0.5 μg/mL; BD Pharmingen, San Jose, CA, USA)).

Techniques: Adjuvant, Biomarker Discovery

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