Journal: bioRxiv

Article Title: Treatment with anti-HER2 chimeric antigen receptor tumor-infiltrating lymphocytes (CAR-TILs) is safe and associated with antitumor efficacy in mice and companion dogs

doi: 10.1101/2022.09.11.507449

Figure Lengend Snippet: mRNA encoding anti-HER2 CAR was electroporated into TILs from five cutaneous melanomas (MM1, MM3, MM4, MM5, and MM6) and one uveal melanoma (UM22) to produce CAR-TILs. CAR expression was detected by HER2-biotin binding at 5-16 hours post-transfection (a). MM1 TILs and CAR-TILs were co-cultured with the parental or HER2 KO 92-1 uveal melanoma cell line for 4-6 hours, followed by flow cytometry to measure degranulation (CD107a expression) (b) or IFN-γ-secreting cells (c). TILs and CAR-TILs not co-cultured with 92-1 cells (TILs alone; TA) were used as controls in b-c. Alternatively, cells were co-cultured for 48 h, and the supernatant was collected for analysis of secreted IFN-γ by ELISA (d). 92-1 cells not co-cultured with TILs (no TILs) were used as a control. Data are presented as mean ± standard deviation (SD) of duplicates. The experiments were performed twice, and representative results are shown.

Article Snippet: For analysis of HER2 protein binding capacity of CAR-TILs, 100.000 cells were incubated with 1 μg biotinylated HER2 protein (Abcam) for 30 min at 4 °C, followed by incubation with an allophycocyanin-conjugated streptavidin antibody (Jackson Immuno Research) for 25 min at 4 °C, and flow cytometry analysis was performed using an Accuri C6 flow cytometer (BD) equipped with the BD Accuri C6 software.

Techniques: Expressing, Binding Assay, Transfection, Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: bioRxiv

Article Title: Treatment with anti-HER2 chimeric antigen receptor tumor-infiltrating lymphocytes (CAR-TILs) is safe and associated with antitumor efficacy in mice and companion dogs

doi: 10.1101/2022.09.11.507449

Figure Lengend Snippet: Flow cytometry data showing HER2 CAR expression after HER2 CAR mRNA electroporation by detecting bound biotinylated HER2 protein. Representative plots for samples MM1, MM3, MM4, MM5, MM6 and UM22.

Article Snippet: For analysis of HER2 protein binding capacity of CAR-TILs, 100.000 cells were incubated with 1 μg biotinylated HER2 protein (Abcam) for 30 min at 4 °C, followed by incubation with an allophycocyanin-conjugated streptavidin antibody (Jackson Immuno Research) for 25 min at 4 °C, and flow cytometry analysis was performed using an Accuri C6 flow cytometer (BD) equipped with the BD Accuri C6 software.

Techniques: Flow Cytometry, Expressing, Electroporation

Journal: bioRxiv

Article Title: Treatment with anti-HER2 chimeric antigen receptor tumor-infiltrating lymphocytes (CAR-TILs) is safe and associated with antitumor efficacy in mice and companion dogs

doi: 10.1101/2022.09.11.507449

Figure Lengend Snippet: (a-b) TILs and CAR-TILs from MM1 were co-cultured with the parental or HER2 KO 92-1 uveal melanoma cell line for 24 hours, followed by cleaved caspase 3 (CC3) detection in the tumor cells using flow cytometry (a). Additionally, TILs and CAR-TILs from MM5 were co-cultured with autologous cancer cells for 24 hours, followed by cleaved CC3 detection using flow cytometry (b). Cancer cells not treated with TILs were used as a control (no TILs). (c-g) Viability of autologous cancer cells was measured by luciferase signal detected after 48 hours co-culture with increasing doses of TILs and CAR-TILs from UM22 (c), MM3 (d), MM4 (e), MM5 (f) and MM6 (g) in the indicated ratios (TILs:cancer cells). Data is presented as mean with SD of triplicates. Asterisks represent p-values of difference between similar doses of TILs and CAR-TILs. The experiments were performed twice, and representative data from one experiment is shown.

Article Snippet: For analysis of HER2 protein binding capacity of CAR-TILs, 100.000 cells were incubated with 1 μg biotinylated HER2 protein (Abcam) for 30 min at 4 °C, followed by incubation with an allophycocyanin-conjugated streptavidin antibody (Jackson Immuno Research) for 25 min at 4 °C, and flow cytometry analysis was performed using an Accuri C6 flow cytometer (BD) equipped with the BD Accuri C6 software.

Techniques: Cell Culture, Flow Cytometry, Luciferase, Co-Culture Assay

Journal: bioRxiv

Article Title: Treatment with anti-HER2 chimeric antigen receptor tumor-infiltrating lymphocytes (CAR-TILs) is safe and associated with antitumor efficacy in mice and companion dogs

doi: 10.1101/2022.09.11.507449

Figure Lengend Snippet: HER2 expression detected by qPCR (a) and immunohistochemistry (b) in tumors from four companion dogs (dogs 1-4). Canine cell lines D17.os and CF41.mg and canine PBMC were used as controls in a. (c) Expression of human melanoma marker Melan-a in tumors from dogs 3 and 4 with melanoma. (d) Classification of canine tumor biopsies from dogs 3 and 4 based on gene expression relative to TCGA cohort of > 10000 human tumor samples from 32 cancer types. The similarities between canine melanoma and samples in TCGA were visualized using tSNE dimensionality reduction. SKCM: melanoma cases. (e-f) TILs and mRNA electroporated CAR-TILs from dog 3 were co-cultured with the canine cell lines CF41.mg (e) and D17.os (f) for 24 h. The experiment was performed twice and representative data (n=3, data ± SD) from one experiment are shown. (g) Photograph of Dog 3 with metastatic oral malignant melanoma enrolled in the FIDO trial evaluating the safety of CAR-TIL treatment in companion dogs.

Article Snippet: For analysis of HER2 protein binding capacity of CAR-TILs, 100.000 cells were incubated with 1 μg biotinylated HER2 protein (Abcam) for 30 min at 4 °C, followed by incubation with an allophycocyanin-conjugated streptavidin antibody (Jackson Immuno Research) for 25 min at 4 °C, and flow cytometry analysis was performed using an Accuri C6 flow cytometer (BD) equipped with the BD Accuri C6 software.

Techniques: Expressing, Immunohistochemistry, Marker, Cell Culture

Journal: bioRxiv

Article Title: Noncovalent antibody catenation on a target surface drastically increases the antigen-binding avidity

doi: 10.1101/2022.07.12.499671

Figure Lengend Snippet: The binding kinetics were measured with the indicated targets immobilized on a sensor tip. The concentration of the antibodies was varied as shown. The experimental signals and fitted curves are shown in red and black, respectively. For curve fitting, 1:1 binding was assumed. The kinetic parameters are shown in the insets. k a , association rate constant; k d , dissociation rate constant. (A) High-affinity antibody. Trastuzumab(N30A/H91A) exhibited the K D of 2.1 nM for the immobilized ectodomain of HER2. (B) Low-affinity antibody. glCV30 exhibits the K D of 51.2 nM for the immobilized RBD of SARS-CoV-2. For both Trastuzumab(N30A/H91A)-SDF-1α and glCV30-SDF-1α, the K D values could not be accurately determined due to the instrumental insensitivity ( K D < 10 pM). The experiments were performed in triplicates, and representative sensorgrams are shown.

Article Snippet: Biotinylated SARS-CoV-2 RBD (Acrobio system) or biotinylated Her2/ERBB2 (Sino Biological) was loaded to a streptavidin biosensor tip (Sartorius) for 120 s. A baseline was determined by incubating the sensor with Kinetics Buffer (Sartorius) for 60 s. Antibody samples at different concentrations went through the association phase for 240 s and the dissociation phase for 720 s. All reactions were carried out in the Kinetics Buffer (Satorius).

Techniques: Binding Assay, Concentration Assay

Journal: Protein Engineering, Design and Selection

Article Title: Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process

doi: 10.1093/protein/gzv029

Figure Lengend Snippet: Primary screen of DARPins selected against HER2 using SNAP display. Four rounds of affinity selections were performed and 90 DARPins from the initial library, second and fourth round were tested for binding in a crude extract ELISA. Binding of DARPins to HER2 was detected using an HRP-conjugated antibody. The orange line indicates the ELISA signal of DARPin G3-HAVD in each screen. Over 90% of the clones screened gave a signal >5-fold over background while ∼30% of the selected DARPins showed greater binding signals compared with the parent DARPin G3-HAVD.

Article Snippet: The displayed protein library was incubated with biotinylated HER2 (purchased from R&D Systems, cat. no. 1129-ER-050) and captured on streptavidin-coated beads (Dynal).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Clone Assay

Journal: Protein Engineering, Design and Selection

Article Title: Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process

doi: 10.1093/protein/gzv029

Figure Lengend Snippet: ( A ) The frequency of mutations measured in the sequenced DARPin mutants is plotted onto the structure of DARPin H10-2-G3 (shown as putty cartoon): frequently mutated positions are colour-coded and rendered with backbone thickness proportional to mutation rate. Fully conserved positions are shown in dark blue. The position that accumulated ∼42% of sequence diversity, H52, is shown in red. The positions most frequently mutated are in proximity of the binding interface. Overall, positions mutated in greater than 10% of the sequenced population are all located in framework positions in the designed N2C DARPin library. Mutations do, however, map also on framework positions away from the binding interface. The interacting domain of HER2 is represented as a semi-transparent sand-coloured surface. ( B ) The published X-ray structure of DARPin H10-2-G3 (green) in complex with HER2 (blue) ( Jost et al. , 2013 ) was used to visualise the likely locations of the mutations obtained in DARPin F1. DARPin H10-2-G3 was affinity matured from the same parent clone (DARPin G3 HAVD) as the DARPins evolved by SNAP display. Mutations that contribute to binding are coloured brown while neutral mutations are coloured yellow (see Table ). Figures were prepared with Pymol based on PDB 4HRN ( Jost et al. , 2013 ).

Article Snippet: The displayed protein library was incubated with biotinylated HER2 (purchased from R&D Systems, cat. no. 1129-ER-050) and captured on streptavidin-coated beads (Dynal).

Techniques: Mutagenesis, Sequencing, Binding Assay

Journal: Protein Engineering, Design and Selection

Article Title: Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process

doi: 10.1093/protein/gzv029

Figure Lengend Snippet: Affinities and binding kinetics of the selected HER2-binding DARPins a

Article Snippet: The displayed protein library was incubated with biotinylated HER2 (purchased from R&D Systems, cat. no. 1129-ER-050) and captured on streptavidin-coated beads (Dynal).

Techniques: Binding Assay

Journal: Protein Engineering, Design and Selection

Article Title: Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process

doi: 10.1093/protein/gzv029

Figure Lengend Snippet: ( A ) ELISA analysis of selected DARPins. The top 10 DARPins (10 μg/ml of purified DARPin) were analysed for binding to immobilised HER2. All selected DARPins from SNAP display outputs gave a greater binding signal than DARPin G3-HAVD. All evolved DARPins were also analysed for their specificity in binding. No binding was observed to Streptavidin (which was used to capture biotinylated HER2 during selections) or lysozyme indicating the selected DARPins do not non-specifically bind to other proteins. Competition for binding of selected DARPins to HER2 with DARPin H10-2-G3 (10-fold excess DARPin H10-2-G3 over selected DARPins) showed that each selected DARPin competed for the same epitope as DARPin H10-2-G3. ( B ) Correlation between the observed fluorescence values of cell populations and the reciprocal of their binding affinity shows a correlation between the two properties. Most notably, Trastuzumab appears to have a significantly lower affinity on cells, probably as a result of the different binding mode and epitope the antibody binds to on HER2.

Article Snippet: The displayed protein library was incubated with biotinylated HER2 (purchased from R&D Systems, cat. no. 1129-ER-050) and captured on streptavidin-coated beads (Dynal).

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Binding Assay, Fluorescence

Journal: Nuclear medicine and biology

Article Title: A Rapid Bead-Based Radioligand Binding Assay for the Determination of Target-Binding Fraction and Quality Control of Radiopharmaceuticals

doi: 10.1016/j.nucmedbio.2019.04.005

Figure Lengend Snippet: A) Inconsistent results obtained from cell-based assays to determine the target-binding fraction (TBF) of [89Zr]Zr-DFO-SC16.56 (molar activity = 14 MBq/nmol) at end of synthesis (EOS) and 24 h after storage of the sample at −80 °C in 4 consecutive experimental trials done over a period of 4 weeks; B) Demonstration of high TBF for [89Zr]Zr-DFO-SC16.56 using Ni-NTA beads coated with His-tagged DLL3 and minimum non-specific binding (NSB) to naked Ni-NTA beads at (EOS) and 24 h later; C) High TBF demonstrated by [177Lu]Lu-CHX-A”-DTPA-SC16.56 (molar activity = 22.4 MBq/nmol) – a radioimmunotherapeutic variant of the DLL3-targeting antibody. Specificity of binding to antigen-coated beads for both the DLL3-targeting radioligands was validated in the presence of a 3500-fold excess of unlabeled SC16.56 immunoconjugates; D) Evaluation of the TBF of [89Zr]Zr-DFO-Trastuzumab (molar activity = 14 MBq/nmol) using Ni-NTA beads coated with His-tagged Her2 protein showed ~25% NSB of the radioligand to naked Ni-NTA beads; E) Using streptavidin beads coated with biotinylated Her2 to evaluate the TBF of [89Zr]Zr-DFO-Trastuzumab eliminated the NSB and demonstrated high specific binding, which could be blocked by excess unlabeled DFO-Trastuzumab, but not DFO-Pertuzumab; F) Using 131I-labeled biotinylated Her2 to determine the degree of loading of the streptavidin beads demonstrated linearity with increasing concentration of the antigen over at least 4 orders of magnitude. The error bars on each data point represent 95% CI. The plot validates that the bead-based assay is being operated in the pre-saturation concentration range when 1 μg (10 pmoles) of biotinylated Her2 was used with 20 μL (200 μg) of streptavidin beads.

Article Snippet: His-tagged DLL3 protein and the SC16.56 antibody were kindly provided by AbbVie Stemcentrx; polyvalent biotinylated sLea-PAA (01-044) was purchased from Glycotech, and the 5B1 antibody was kindly provided by MabVax Therapeutics Holdings Inc.; biotinylated human Her2 protein (HE2-H822R-200μg) was purchased from Acro Biosystems, and both Trastuzumab and Pertuzumab were purchased from the pharmacy at MSKCC; recombinant human His-tagged B7-H3 protein (1949-B3-50) was purchased from R&D Systems, and Omburtamab was kindly provided by Y-mabs Therapeutics; recombinant human Her2 Fc-chimera His-tagged protein (1129-ER-050) and recombinant human His-tagged PSMA/FOLH1 protein (4234-ZN-010) were purchased from R&D Systems; PSMA-617 (9933) was obtained from ABX.

Techniques: Binding Assay, Activity Assay, Variant Assay, Labeling, Concentration Assay, Bead-based Assay

Journal: Nuclear medicine and biology

Article Title: A Rapid Bead-Based Radioligand Binding Assay for the Determination of Target-Binding Fraction and Quality Control of Radiopharmaceuticals

doi: 10.1016/j.nucmedbio.2019.04.005

Figure Lengend Snippet: A) High TBF of [89Zr]Zr-DFO-5B1 (molar activity =13 MBq/nmol) with polyvalent biotinylated CA19.9 antigen; C) Demonstration of the utility of the bead-based assay with small molecule radioligand – [177Lu]Lu-PSMA-617 (molar activity = 2.6 MBq/nmol) showing high (~80%) TBF and minimum NSB at 24 h post radiosynthesis.

Article Snippet: His-tagged DLL3 protein and the SC16.56 antibody were kindly provided by AbbVie Stemcentrx; polyvalent biotinylated sLea-PAA (01-044) was purchased from Glycotech, and the 5B1 antibody was kindly provided by MabVax Therapeutics Holdings Inc.; biotinylated human Her2 protein (HE2-H822R-200μg) was purchased from Acro Biosystems, and both Trastuzumab and Pertuzumab were purchased from the pharmacy at MSKCC; recombinant human His-tagged B7-H3 protein (1949-B3-50) was purchased from R&D Systems, and Omburtamab was kindly provided by Y-mabs Therapeutics; recombinant human Her2 Fc-chimera His-tagged protein (1129-ER-050) and recombinant human His-tagged PSMA/FOLH1 protein (4234-ZN-010) were purchased from R&D Systems; PSMA-617 (9933) was obtained from ABX.

Techniques: Activity Assay, Bead-based Assay

Journal: iScience

Article Title: Affimer reagents enable targeted delivery of therapeutic agents and RNA via virus-like particles

doi: 10.1016/j.isci.2024.110461

Figure Lengend Snippet: Identification of Affimers that bind cellularly expressed HER2 (A) Phage ELISA of 32 Affimer clones identified as binding the ECD of HER2. Identical clones are shown with the same shading. (B) Phage ELISA of 32 Affimer clones identified as binding the ECD of HER2 in the presence of HER2. Identical clones are shown with the same shading. (C) Affinity precipitation of endogenous HER2 from HER2-overexpressing cell lines (AU-565 and BT-474) and the HER2-negative cell line (MDA-MB-231) using HER2-binding Affimers identified by phage screening against the ECD of HER2. ( n = 3 independent experiments with representative blots shown; white lines delineate individual membranes; all blots were run concurrently). (D) Phage ELISA of 48 Affimer-clones identified as preferential binding fixed HER2-expressing cells (MDA-MB-453) compared to HER2-negative cells (MCF7). An arbitrary cutoff of 3 was used to determine which clones would be taken forward for DNA sequence analysis identifying the two unique binding Affimers D11 and H7. Identical clones are shown with the same shading. ECD, extracellular domain (amino acids 1-652); WCL, whole-cell lysates; Cont., control Affimer (Variable regions AAAA; AAE).

Article Snippet: Target biotinylation, selection of Affimers by phage display and phage ELISA against the ECD of HER2 was as previously described., Briefly, biotinylated HER2 ECD His-tagged (10004-H02H, Sino Biologicals, Eschborn, Germany; EZ-Link NHS-Biotin, Thermo Scientific, Waltham, MA; 5-fold molar excess) was immobilised on blocked (2x blocking buffer, Sigma) streptavidin wells.

Techniques: Enzyme-linked Immunosorbent Assay, Clone Assay, Binding Assay, Affinity Precipitation, Expressing, Sequencing, Control

Journal: iScience

Article Title: Affimer reagents enable targeted delivery of therapeutic agents and RNA via virus-like particles

doi: 10.1016/j.isci.2024.110461

Figure Lengend Snippet: Affimers D11 and H7 colocalize with HER2 and show internalization into HER2-positive cells AlexaFluor 488-conjugated Affimers (green) D11 (A) and H7 (B) were incubated with HER2-positive (AU-565 and BT-474) and -negative (MDA-MB-231) cell lines for 1 h before fixation. Cells were stained for HER2 (pink) and with Hoechst (blue). The internalization of the Affimers D11 and H7 was assessed as mean AlexaFluor 488 intensity per cell (C). The mechanism of internalization was assessed with costaining for EEA1 [pink, (D) and (E)] and LAMP2 [pink, (F) and (G)]. Colocalization of Affimers with HER2/EEA1/LAMP2 is shown by arrows. Images were taken on a Zeiss confocal microscope with a 40x magnification under the same exposure for AlexaFluor 488-conjugated Affimers. Images were analyzed using ZenLite and ImageJ software. Data are mean ± SEM, n = 3 independent experiments, with representative images shown, unpaired t test. ∗ p < 0.05 (C).

Article Snippet: Target biotinylation, selection of Affimers by phage display and phage ELISA against the ECD of HER2 was as previously described., Briefly, biotinylated HER2 ECD His-tagged (10004-H02H, Sino Biologicals, Eschborn, Germany; EZ-Link NHS-Biotin, Thermo Scientific, Waltham, MA; 5-fold molar excess) was immobilised on blocked (2x blocking buffer, Sigma) streptavidin wells.

Techniques: Incubation, Staining, Microscopy, Software

Journal: iScience

Article Title: Affimer reagents enable targeted delivery of therapeutic agents and RNA via virus-like particles

doi: 10.1016/j.isci.2024.110461

Figure Lengend Snippet: Impact of Affimers on the metabolic activity of breast cancer cell lines A range of breast cancer cell lines AU-565, SKBR3, BT-474, MDA-MB-453, MCF-7, MDA-MB-231, and ZR-75-1 were seeded one day prior to the addition of HER2-binding Affimers or HER2-binding Affimer-MMAE conjugates. Cells were incubated for a further 72 h, and metabolic activity was analyzed by AlamarBlue measurement. HER-2-binding Affimers did not affect metabolic activity in either HER2-positive cell lines AU-565 and BT474 or the HER-2-negative cell line MDA-MB-231 [(A) and (B)]. HER2-binding Affimers were conjugated with the cytotoxin MMAE via a cathepsin cleavable group and a PABC spacer (C). Seven breast cancer cell lines with varied HER2 expression level as measured by immunoblotting (D); representative blot shown; dotted line indicates removal of a lane containing lysates from a non-breast cancer cell line were treated with the Affimer-MMAE conjugates and showed dose-dependent inhibition of metabolic activity [(E) and (F)], the efficacy of which varied with HER2 expression level. All values were normalized to cells incubated with media alone. Data are mean ± SEM; dose-response curves were fitted using GraphPad Prism v 9.0, [Inhibitor] vs. response -- Variable slope (four parameters); n = 3 independent experiments for all panels. MMAE, monomethyl auristatin E.

Article Snippet: Target biotinylation, selection of Affimers by phage display and phage ELISA against the ECD of HER2 was as previously described., Briefly, biotinylated HER2 ECD His-tagged (10004-H02H, Sino Biologicals, Eschborn, Germany; EZ-Link NHS-Biotin, Thermo Scientific, Waltham, MA; 5-fold molar excess) was immobilised on blocked (2x blocking buffer, Sigma) streptavidin wells.

Techniques: Activity Assay, Binding Assay, Incubation, Expressing, Western Blot, Inhibition

Journal: iScience

Article Title: Affimer reagents enable targeted delivery of therapeutic agents and RNA via virus-like particles

doi: 10.1016/j.isci.2024.110461

Figure Lengend Snippet: Cytotoxicity of HER2-binding Affimer-MMAE conjugates

Article Snippet: Target biotinylation, selection of Affimers by phage display and phage ELISA against the ECD of HER2 was as previously described., Briefly, biotinylated HER2 ECD His-tagged (10004-H02H, Sino Biologicals, Eschborn, Germany; EZ-Link NHS-Biotin, Thermo Scientific, Waltham, MA; 5-fold molar excess) was immobilised on blocked (2x blocking buffer, Sigma) streptavidin wells.

Techniques:

Journal: iScience

Article Title: Affimer reagents enable targeted delivery of therapeutic agents and RNA via virus-like particles

doi: 10.1016/j.isci.2024.110461

Figure Lengend Snippet: HER2-binding Affimers mediate targeted delivery of VLPs Affimer dimer containing HER2-binding Affimer D11 and CPMV-VLP-binding Affimer were used to target CPMV VLPs containing eGFP RNA to HER2-positive cells (A). Affimer dimer increased cellular uptake of eGFP RNA-containing CPMV VLPs to HER2-positive cell lines (AU-565 and SKBR3) in a dose-dependent manner (Kruskal-Wallis test; p = 0.0109 and p = 0.0099, respectively) while uptake was minimal in HER2-negative cell line MDA-MB-231 [Kruskal-Wallis test; p = 0.3133; (B) and (C)]. Affimer dimer:VLP conjugates were non-toxic at doses used (0.07 μM VLP and increasing ratios of Affimer dimer). Two-way ANOVA; p = 0.5687 (D). Data are mean ± SEM; n = 3 independent experiments. (A) Created with BioRender.com (2023).

Article Snippet: Target biotinylation, selection of Affimers by phage display and phage ELISA against the ECD of HER2 was as previously described., Briefly, biotinylated HER2 ECD His-tagged (10004-H02H, Sino Biologicals, Eschborn, Germany; EZ-Link NHS-Biotin, Thermo Scientific, Waltham, MA; 5-fold molar excess) was immobilised on blocked (2x blocking buffer, Sigma) streptavidin wells.

Techniques: Binding Assay

Journal: iScience

Article Title: Affimer reagents enable targeted delivery of therapeutic agents and RNA via virus-like particles

doi: 10.1016/j.isci.2024.110461

Figure Lengend Snippet: HER2-binding Affimer D11 bind different epitopes to other HER2-binding SBPs The Alphafold2-predicted model shows HER2-binding Affimer D11 level binds subdomains II and III of the HER2 ECD. The prediction is color coded based on pLDDT values that indicate the confidence level of the prediction (A) while Affibody Z HER2:342 (yellow; PDB code: 3MZW ) binds subdomain III and DARPinG3 (gray; PDB code: 4HRN ) binds subdomain IV. Affimer D11 is shown in magenta (B). Replacement of HER2-binding Affimer D11 with either Affibody Z HER2:342 or DARPinG3 in Affimer dimer still results in an increased percentage of eGFP-positive cells over VLP alone (C) but not to the same magnitude as the Affimer dimer (D). Data are mean ± SEM; n = 3 independent experiments, with representative images shown in (C); scale bar is 50 μm. One-way ANOVA with Dunnett’s post hoc test ∗∗∗∗ p < 0.0001 (D). Image created with Pymol v2.5.4 (Schrodinger LLC) (B).

Article Snippet: Target biotinylation, selection of Affimers by phage display and phage ELISA against the ECD of HER2 was as previously described., Briefly, biotinylated HER2 ECD His-tagged (10004-H02H, Sino Biologicals, Eschborn, Germany; EZ-Link NHS-Biotin, Thermo Scientific, Waltham, MA; 5-fold molar excess) was immobilised on blocked (2x blocking buffer, Sigma) streptavidin wells.

Techniques: Binding Assay

Journal: iScience

Article Title: Affimer reagents enable targeted delivery of therapeutic agents and RNA via virus-like particles

doi: 10.1016/j.isci.2024.110461

Figure Lengend Snippet:

Article Snippet: Target biotinylation, selection of Affimers by phage display and phage ELISA against the ECD of HER2 was as previously described., Briefly, biotinylated HER2 ECD His-tagged (10004-H02H, Sino Biologicals, Eschborn, Germany; EZ-Link NHS-Biotin, Thermo Scientific, Waltham, MA; 5-fold molar excess) was immobilised on blocked (2x blocking buffer, Sigma) streptavidin wells.

Techniques: Virus, Recombinant, Software

Journal: Drug delivery

Article Title: Valency-affinity mapping of multivalent liposomes for tunable target cell discrimination.

doi: 10.1080/10717544.2025.2512950

Figure Lengend Snippet: Figure 3. Determination of kinetic rate constants via surface plasmon resonance. Sensorgrams were generated using immobilized HER2 panned with each of the DARPin variants. The sensorgrams were double referenced and fit to a 1:1 binding model from which the kinetic constants were derived. (a) G3 was panned at increasing concentrations (0, 1, 5, 10, 20, 30 nM) at a flow rate of 60 μL/min. (b) G3-V was panned at increasing concentrations (0, 1, 3, 5, 10, 20, 30 nM) at a flow rate of 60 μL/min. (c) G3-D was panned at increasing concentrations (0, 1, 5, 10, 20, 30, 40 nM) at a flow rate of 30 μL/min. (d) G3-HAVD was panned at increasing concentrations (0, 25, 50, 100 500, 1000, 2500 nM) at a flow rate of 30 μL/min.

Article Snippet: Biotin caPture Reagent was immobilized on the chip with a flow rate of 2 μl/min and contact time of 5 min. the ligand, avi-tag biotinylated heR2 (R&D systems, aVi11356), was reconstituted to 250 μg/ml in 1X PBs (cytiva, BR100672) per manufacturer instructions. immediately prior to use it was diluted in 1X hBs-P+ (cytiva, BR100827) to a working concentration of 75 nM. the prepared heR2 was then immobilized on the chip at a flow rate of 10 μl/min and contact time of 3 min. the DaRPin analytes (G3, G3-V, G3-D, G3-haVD) were diluted to their working concentrations, ranging from 0 to 40 nM, in 1X hBs-P+. the DaRPins were analyzed under two different run conditions: 1) with an association time of 10 min followed by a dissociation time of 1 h at a flow rate of 30 μl/min and 2) with an association time of 5 min followed by a dissociation time of 1 h at a flow rate of 60 μl/min. after each cycle, the chip was fully regenerated with three parts 8 M guanidine-hcl to one part 1 M NaOh for 2 minutes at a flow rate of 30 μl/min. hBs-P + was used as the running buffer and all runs were performed at 25 °c. the data were analyzed in BiacoretM insight evaluation software. all cycles were double-referenced against a blank run containing no analyte and a reference channel containing no immobilized ligand, and the data were fit to a 1:1 langmuir binding model.

Techniques: SPR Assay, Generated, Binding Assay, Derivative Assay

Journal: Drug delivery

Article Title: Valency-affinity mapping of multivalent liposomes for tunable target cell discrimination.

doi: 10.1080/10717544.2025.2512950

Figure Lengend Snippet: Figure 5. HEK293T HER2-eGFP Binding metrics. (A) (top panel) Flow cytometry scatter data for HEK293T cells transfected with HER2-EGFP and incubated with Cy5-PEG liposomes (conjugated with G3-D at 3 µM in this representative example) were fitted to a Hill function (red curve) and plotted with HER2αMAX (red dashed line) to note the peak binding selectivity. The Cy5 threshold for positive binding (horizonal blue dashed line) was based on no-protein control particle binding signal, and HER2OPT (vertical blue dashed line) denotes the HER2 threshold which maximizes BPI (iv/(i + iv))×(ii/(ii + iii)). X-axes for all three plots indicate log-transformed HER2-EGFP signal scaled from 0-1 and y-axis for top panel indicates log-transformed Cy5 signal scaled from 0-1. The binding selectivity (middle panel) was calculated by determining the slope of the fitted Hill curve across all HER2 values and was overlayed with HER2αMAX (red dashed line) and HER2OPT (vertical blue dashed line). BPI (bottom panel) was determined across the range of normalized HER2 expression values from 0.1 to 0.9 to find BPIMAX (vertical blue dashed line). (B) HER2αMAX and (C) HER2OPT for all DARPin types tethered at concentration between 0.5 and 5.0 μM of protein (light to dark bars, respectively) demonstrate similar behavior due to changes in DARPin valency and affinity, as increases in both tend to shift optimal binding conditions to lower receptor values. The magnitudes of (E) αMAX and (F) BPIMAX also follow similar trajectories across various DARPin affinities and valences. When examining the contributions of (D) on-target binding and (G) off-target binding to BPIMAX, DARPins achieved similarly high on-target binding across various tethering densities and all except G3-V demonstrated similar magnitude of off-target binding which increased for each DARPin as a function of tethering concentration. G3-V exhibited very strong off-target binding when tethered at high DARPin concentrations despite having a lower affinity than G3, suggesting some degree of nonspecific binding to cells. Two-way ANOVA without replication reveals that both DARPin type and tethering concentration significantly impact HER2OPT, HER2αMAX, and off-target binding but not on-target binding and BPI (DARPin type: p = 1.24E-05, 2.03E-05, 1.69E-03, 2.24E-01, and 1.74E-01, respectively; tethering concentration: p = 5.63E-04, 2.59E-02, 1.14E-02, 8.65E-01, and 6.17E-01, respectively).

Article Snippet: Biotin caPture Reagent was immobilized on the chip with a flow rate of 2 μl/min and contact time of 5 min. the ligand, avi-tag biotinylated heR2 (R&D systems, aVi11356), was reconstituted to 250 μg/ml in 1X PBs (cytiva, BR100672) per manufacturer instructions. immediately prior to use it was diluted in 1X hBs-P+ (cytiva, BR100827) to a working concentration of 75 nM. the prepared heR2 was then immobilized on the chip at a flow rate of 10 μl/min and contact time of 3 min. the DaRPin analytes (G3, G3-V, G3-D, G3-haVD) were diluted to their working concentrations, ranging from 0 to 40 nM, in 1X hBs-P+. the DaRPins were analyzed under two different run conditions: 1) with an association time of 10 min followed by a dissociation time of 1 h at a flow rate of 30 μl/min and 2) with an association time of 5 min followed by a dissociation time of 1 h at a flow rate of 60 μl/min. after each cycle, the chip was fully regenerated with three parts 8 M guanidine-hcl to one part 1 M NaOh for 2 minutes at a flow rate of 30 μl/min. hBs-P + was used as the running buffer and all runs were performed at 25 °c. the data were analyzed in BiacoretM insight evaluation software. all cycles were double-referenced against a blank run containing no analyte and a reference channel containing no immobilized ligand, and the data were fit to a 1:1 langmuir binding model.

Techniques: Binding Assay, Flow Cytometry, Transfection, Incubation, Liposomes, Control, Transformation Assay, Expressing, Concentration Assay

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: Top: Sequence of ADAPT ERBB2-1 and ten representative candidates found after phage display affinity maturation compared to ABD before randomization. One sequence (ADAPT ERBB2-mat10 ) contains a substitution of a scaffold residue that was not intentionally diversified in the library. The frequency of each clone in the sequence data (among 438 sequences) and tracks (A–H) where the corresponding sequence was found are indicated to the right. Track D was underrepresented in the sequence data set. Bottom: Sequences of 21 clones identified after rounds three and four of FACS sorting. The total frequency in the sequence data (278 sequences) is shown and the two numbers in brackets indicate the number of times each sequence was observed in selections without or with HSA present, respectively. Nine of the variants contained scaffold substitutions and modifications in the region spanning helix two and three were common in sequences observed after selections in the presence of HSA. 13 of these candidates were also observed after two rounds of FACS (not shown) and two of them had been observed already among the clones obtained after four rounds of phage display (identical sequences are indicated by asterisks).

Article Snippet: 100 nM HSA-Alexa Fluor 488-conjugate (Invitrogen, labeled according to the supplier's recommendations) or 50 nM biotinylated human ERBB2-His 6 (Sino Biological) was used together with polyclonal human IgG-Alexa Fluor 647 conjugate (Invitrogen).

Techniques: Sequencing, Clone Assay

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: Affinities and melting temperatures (T m ) for the non-randomized albumin-binding domain (ABD), ADAPT ERBB2-1 and variants of ADAPT ERBB2-1 .

Article Snippet: 100 nM HSA-Alexa Fluor 488-conjugate (Invitrogen, labeled according to the supplier's recommendations) or 50 nM biotinylated human ERBB2-His 6 (Sino Biological) was used together with polyclonal human IgG-Alexa Fluor 647 conjugate (Invitrogen).

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: ( A ). ERBB2-binding with and without an excess of HSA. Cells expressing variants from tracks selected without (A, C, E and G) or with (B, D, F and H) 1 µM HSA present were incubated with 50 nM ERBB2 +/− 1 µM HSA and analyzed by flow-cytometry. ERBB2-binding was detected through incubation with streptavidin-R-phycoerythrin (y-axis) and surface expression levels by IgG-Alexa Fluor 647 conjugate (x-axis), data are shown using logarithmic scales. Each diagram presents an overlay of contour plots of the expressing populations of cells from two separate samples from the same batch (A-H) incubated with only ERBB2 (blue) or ERBB2 and HSA (red). The contours represent cell density in the respective regions corresponding to 20, 40, 60 or 80% of the maximum cell density observed. Each sample is represented by 25–30000 events. Corresponding data for ADAPT ERBB2-1 and scaffold ABD are shown in the inlay of panel A to facilitate a comparison. The library each track is derived from, the form of ERBB2 and concentrations used during phage display selection are also indicated. (B ). HSA-binding. Cells from tracks selected without (A, C, E and G) or with (B, D, F and H) 1 µM HSA present were incubated with 50 nM HSA-Alexa Fluor 488 conjugate and analyzed by flow-cytometry. HSA-binding is shown on the y-axis and surface expression level, detected by IgG-Alexa Fluor 647 conjugate, on the x-axis. As controls, cells expressing ADAPT ERBB2-1 or scaffold ABD are included.

Article Snippet: 100 nM HSA-Alexa Fluor 488-conjugate (Invitrogen, labeled according to the supplier's recommendations) or 50 nM biotinylated human ERBB2-His 6 (Sino Biological) was used together with polyclonal human IgG-Alexa Fluor 647 conjugate (Invitrogen).

Techniques: Binding Assay, Expressing, Incubation, Flow Cytometry, Derivative Assay, Selection

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: 21 clones identified after rounds three and four of FACS sorting were screened for ERBB2-binding ( A ), HSA-binding ( B ) and ERBB2-binding in the presence of an excess of HSA ( C ). 5 nM ERBB2 was used in screen A and the binding signal divided by the surface expression level for each clone was normalized against the signal from ADAPT ERBB2-1 . 20 nM HSA was used in screen B and the signals were normalized to scaffold ABD. 5 nM ERBB2 together with 1 µM unlabeled HSA was used in screen C and ADAPT ERBB2-1 was used for normalization. All measurements were done in duplicate on different days and the bars indicate the standard deviation.

Article Snippet: 100 nM HSA-Alexa Fluor 488-conjugate (Invitrogen, labeled according to the supplier's recommendations) or 50 nM biotinylated human ERBB2-His 6 (Sino Biological) was used together with polyclonal human IgG-Alexa Fluor 647 conjugate (Invitrogen).

Techniques: Clone Assay, Binding Assay, Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: Affinities of selected affinity matured ADAPTs after FACS.

Article Snippet: 100 nM HSA-Alexa Fluor 488-conjugate (Invitrogen, labeled according to the supplier's recommendations) or 50 nM biotinylated human ERBB2-His 6 (Sino Biological) was used together with polyclonal human IgG-Alexa Fluor 647 conjugate (Invitrogen).

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

doi: 10.1371/journal.pone.0103094

Figure Lengend Snippet: ( A ). ERBB2-binding at high concentrations of HSA. Four variants of ADAPT ERBB2-FACS-12 (A8) were displayed at the bacterial surface and incubated with labeled ERBB2 (10 nM) in the presence of varying concentrations of HSA. All mutated variants show a retained ability to bind ERBB2 at very high concentrations of albumin, which is in contrast to the parental ADAPT ERBB2-FACS-12 (A8) . Increasing streptavidin-R-phycoerythrin signals were observed at high concentrations of HSA. This effect was only seen for molecules with retained ERBB2-binding and was also observed for an ERBB2-binding Affibody molecule used as control . Hence, this experimental artifact was not ADAPT-specific. All measurements were done in duplicate on different days and the bars indicate the standard deviation. The albumin concentration is shown on a logarithmic scale, the two highest concentrations were 300 and 600 µM, respectively. ( B ). Competition experiment using ERBB2-binding proteins expressed on staphylococcal cells and flow cytometry. ERBB2-binding (5 nM) to cell-displayed binders was always blocked by a 10-fold excess of the same protein in a soluble form. Only ADAPTs and trastuzumab competed with each other for ERBB2-binding, no competition with the ERBB2-binding Affibody molecule Z ERBB2 was observed. All measurements were done in duplicate on different days and the bars indicate the standard deviation. ( C ). Binding to native ERBB2 on SKOV-3 cells. Cells were incubated with 100 nM biotinylated ADAPT ERBB2-1 or ADAPT ERBB2-FACS-12 (A8) and detected with streptavidin-R-phycoerythrin conjugate. Pre-incubating the cells with an excess of either non-labeled ADAPT ERBB2-FACS-12 (A8) or trastuzumab scFv blocked binding of ADAPT ERBB2-FACS-12 (A8) . A two-sample two-tailed t-test demonstrated that the binding for ADAPT ERBB2-1 was significantly stronger than the signal from the scaffold control (p = 0.0067). All measurements were done in duplicate on different days and the bars indicate the standard deviation. Analyses of non-treated cells were included in all experiments (background).

Article Snippet: 100 nM HSA-Alexa Fluor 488-conjugate (Invitrogen, labeled according to the supplier's recommendations) or 50 nM biotinylated human ERBB2-His 6 (Sino Biological) was used together with polyclonal human IgG-Alexa Fluor 647 conjugate (Invitrogen).

Techniques: Binding Assay, Incubation, Labeling, Standard Deviation, Concentration Assay, Flow Cytometry, Two Tailed Test