egfr binding Affibody Search Results


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  • 91
    Affibody egfr binding affibody
    (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of the <t>affibody</t> as indicated. All samples were treated with PNGase to degloycosylate and visualize <t>EGFR</t> extracellular domain (∼70 kDa) as a distinct band. Each sample contained both an affibody and EGFR fragment unless otherwise indicated. Note that only the N23BP mutant gave a photoproduct, which corresponded to a mass increase of 10 kDa. Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) N23BP and EGFR mixed as in (A) or with the equimolar concentration (260 nM) bovine serum albumin (BSA) as indicated and irradiated for the time listed. A band corresponding to the EGFR-affibody conjugate appears at 5, 15, and 30 min (red arrows). Ladder proteins are (top to bottom) 100, 75, and 50 kDa.
    Egfr Binding Affibody, supplied by Affibody, used in various techniques. Bioz Stars score: 91/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Affibody dimeric egfr binding affibody
    (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of the <t>affibody</t> as indicated. All samples were treated with PNGase to degloycosylate and visualize <t>EGFR</t> extracellular domain (∼70 kDa) as a distinct band. Each sample contained both an affibody and EGFR fragment unless otherwise indicated. Note that only the N23BP mutant gave a photoproduct, which corresponded to a mass increase of 10 kDa. Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) N23BP and EGFR mixed as in (A) or with the equimolar concentration (260 nM) bovine serum albumin (BSA) as indicated and irradiated for the time listed. A band corresponding to the EGFR-affibody conjugate appears at 5, 15, and 30 min (red arrows). Ladder proteins are (top to bottom) 100, 75, and 50 kDa.
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    Affibody egfr binding affibody molecules
    (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of the <t>affibody</t> as indicated. All samples were treated with PNGase to degloycosylate and visualize <t>EGFR</t> extracellular domain (∼70 kDa) as a distinct band. Each sample contained both an affibody and EGFR fragment unless otherwise indicated. Note that only the N23BP mutant gave a photoproduct, which corresponded to a mass increase of 10 kDa. Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) N23BP and EGFR mixed as in (A) or with the equimolar concentration (260 nM) bovine serum albumin (BSA) as indicated and irradiated for the time listed. A band corresponding to the EGFR-affibody conjugate appears at 5, 15, and 30 min (red arrows). Ladder proteins are (top to bottom) 100, 75, and 50 kDa.
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    Affibody egfr binding affibody molecule zegfr
    PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z <t>EGFR:2377</t> -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting <t>Affibody</t> increasing as the tumors grow from time from inoculation
    Egfr Binding Affibody Molecule Zegfr, supplied by Affibody, used in various techniques. Bioz Stars score: 84/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Affibody compact egfr binding affibody zegfr
    Modular capacity of probes for labeling <t>EGFR</t> on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with <t>FAP–affibody</t> fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.
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    86
    Affibody bp group inhibited affibody egfr binding
    (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of the <t>affibody</t> as indicated. All samples were treated with PNGase to degloycosylate and visualize <t>EGFR</t> extracellular domain (∼70 kDa) as a distinct band. Each sample contained both an affibody and EGFR fragment unless otherwise indicated. Note that only the N23BP mutant gave a photoproduct, which corresponded to a mass increase of 10 kDa. Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) N23BP and EGFR mixed as in (A) or with the equimolar concentration (260 nM) bovine serum albumin (BSA) as indicated and irradiated for the time listed. A band corresponding to the EGFR-affibody conjugate appears at 5, 15, and 30 min (red arrows). Ladder proteins are (top to bottom) 100, 75, and 50 kDa.
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    Affibody egfr specific affibody
    Figure 3. Competition of Aff800, Pan800, and EGF800 binding by unlabeled molecules. Aff800 ( A and B ), Pan800 ( C and D ), and EGF800 ( E and F ) were incubated with <t>F98-EGFR</t> and F98-vIII in the presence of different concentrations (ranging from 0.49 to 500 nM) of unlabeled nanobody 7D12, <t>affibody,</t> EGF, or panitumumab. The fluorescence signals were determined after removal of unbound probes ( n = 3 for each data point).
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    Affibody bispecific affibody against egfr
    Figure 3. Competition of Aff800, Pan800, and EGF800 binding by unlabeled molecules. Aff800 ( A and B ), Pan800 ( C and D ), and EGF800 ( E and F ) were incubated with <t>F98-EGFR</t> and F98-vIII in the presence of different concentrations (ranging from 0.49 to 500 nM) of unlabeled nanobody 7D12, <t>affibody,</t> EGF, or panitumumab. The fluorescence signals were determined after removal of unbound probes ( n = 3 for each data point).
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    Affibody egfr targeted affibody ea68
    Binding characterization. (A) <t>EGFR</t> high A431 cells were mixed with the indicated concentration of <t>affibody</t> (EA26S (blue circles) or EA62S (green squares)). Binding was detected with fluorophore-conjugated anti-His6 antibody via flow cytometry. Equilibrium dissociation constants were calculated assuming a 1:1 binding model. n = 3 measurements per variant. (B) EGFR high A431 cells (white) or EGFR low MCF7 cells (black) were incubated with 500 nM affibody. Binding was detected as in (A). n = 3 measurements per variant.
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    94
    Affibody anti egfr affibody molecules
    Peptide-based chelators used for labeling with 99m Tc. a General structure of the N 3 S chelators formed by a C-terminal cysteine and three adjacent amino acids. X 1 , X 2 and X 3 denote the side chains of the amino acids. b Sequences of <t>EGFR-binding</t> <t>affibody</t> molecules evaluated in this study. The variable amino acids are marked in bold
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    Affibody anti egfr affibody alexa 488
    Mean instantaneous D fit for different <t>anti-EGFR</t> <t>Affibody</t> conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.
    Anti Egfr Affibody Alexa 488, supplied by Affibody, used in various techniques. Bioz Stars score: 78/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Affibody anti egfr affibody irdye 800cw conjugate
    Mean instantaneous D fit for different <t>anti-EGFR</t> <t>Affibody</t> conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.
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    Affibody alexa 680 anti egfr affibody conjugates
    Mean instantaneous D fit for different <t>anti-EGFR</t> <t>Affibody</t> conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.
    Alexa 680 Anti Egfr Affibody Conjugates, supplied by Affibody, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Affibody anti human epidermal growth factor receptor egfr affibody
    Side-by-side comparison of mean squared displacement (MSD) curves and diffusion coefficient (D) histograms from <t>CHO-EGFR-eGFP</t> cells grown on uncoated glass vs. linear-PEG+0.4 mM GRGDS-coated glass. Data were plotted from at least 15 areas acquired from 3 independent samples. Each MSD value comes from at least 6500 (ranging up to 300,000) individual separations, resulting in very small standard error in the MSD. Error bars are plotted but too small to be visible. Panels A (uncoated) and B (linear PEG + GRGDS): diffusion coefficient histogram of tracked spots. EGFR-eGFP (red), anti-EGFR <t>Affibody</t> Alexa 546 (magenta), anti-EGFR Affibody Atto 647N (green). Dotted lines show the mean D coefficient extrapolation. Panels C (uncoated) and D (linear PEG + GRGDS): Mean Square displacement plot. EGFR-eGFP (red), anti-EGFR Affibody Alexa 546 (magenta), anti-EGFR Affibody Atto 647N (green). Dotted lines show the mean D coefficient extrapolation.
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    Affibody dye conjugated anti egfr affibody species
    Mean instantaneous D fit for different <t>anti-EGFR</t> <t>Affibody</t> conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.
    Dye Conjugated Anti Egfr Affibody Species, supplied by Affibody, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Affibody egfr expression
    Mean instantaneous D fit for different <t>anti-EGFR</t> <t>Affibody</t> conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.
    Egfr Expression, supplied by Affibody, used in various techniques. Bioz Stars score: 99/100, based on 194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Affibody anti egfr
    Specific binding and uptake of IRDye800CW-labeled <t>Affibody</t> molecules. (A) The protein expression levels of <t>EGFR</t> and HER2 in MDA-MB-231 (MDA231), A431, SKOV3, and SKBR3 cells. Actin served as an internal control. The relative expression levels were calculated
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    Affibody egfr labeling
    Modular capacity of probes for labeling <t>EGFR</t> on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with <t>FAP–affibody</t> fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.
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    Affibody molecules against egfr
    Binding dynamics of monomeric and heptameric targeting ligands by BIAcore analysis. The extracellular domain of (A) <t>EGFR</t> and (B) <t>HER2</t> receptors were immobilized on the CM5 chip. Different concentrations of monomer or heptamer proteins were injected into the channels. Analyses were performed at room temperature at a flow rate of 20 µl/min.
    Molecules Against Egfr, supplied by Affibody, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Affibody nh2 nde i h6 p1 capsid subunit
    Binding dynamics of monomeric and heptameric targeting ligands by BIAcore analysis. The extracellular domain of (A) <t>EGFR</t> and (B) <t>HER2</t> receptors were immobilized on the CM5 chip. Different concentrations of monomer or heptamer proteins were injected into the channels. Analyses were performed at room temperature at a flow rate of 20 µl/min.
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    Affibody conjugate targeting egfr
    Expression of <t>EGFR</t> in <t>GBM</t> cell lines and specificity of the Z EGFR:03115 –IR700DX binding to EGFR. ( a ) Varying EGFR expression in the selected cancer cell lines was confirmed by Western blot. The numbers in the brackets represent the relative EGFR expression as determined by densitometric analysis. ( b ) Z EGFR:03115 –IR700DX (30 nM) binding as assessed by flow cytometry in the selected cancer cells with varying EGFR expression and after blocking with 100‐fold excess of unlabeled Z EGFR:03115 . Data are presented as mean ± SEM ( n = 3). ( c – f ) Confocal microscopy images demonstrating target‐specific binding (4°C) and internalization (37°C) of either the Z EGFR:03115 –IR700DX (1 µM), anti‐EGFR‐FITC antibody (25 nM for visualization purposes) or IR700DX alone (1 µM): ( c ) U251 cell lines (1 h incubation time), ( d + e ) U87‐MGvIII or MCF7 cell lines (1–6 h incubation time), ( f ) U87‐MGvIII spheroids (6 h incubation time). Hoechst®33342 (blue) and LysoTracker™Green DND‐26 (green) were used for counterstaining. ( g ) Quantification of fluorescence intensity (median fluorescence intensity) of ∼8 µm slices through U87‐MGvIII spheroids following a 6 h incubation with Z EGFR:03115 –IR700DX (500 nM) or an anti‐EGFR‐FITC antibody (500 nM). Data are presented as mean ± SEM ( n = 3). ( h ) H E, EGFR and Ki67 immunostaining of U87‐MGvIII spheroid (400–500 µm) sections 72 h after seeding.
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    Affibody spherical nanoparticle shape present cancer cell receptor
    Genetic encapsulation and assembly to produce fluorescently engineered HBV capsids with cancer targeting capability. a) Encapsulation of FPs surface‐display of cancer targeting peptides. b) Linker‐length dependent localization of fluorophores. c) Construction of double‐layered fluorescent protein <t>nanoparticle</t> with cancer targeting capability.
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    Affibody human egfr
    Genetic encapsulation and assembly to produce fluorescently engineered HBV capsids with cancer targeting capability. a) Encapsulation of FPs surface‐display of cancer targeting peptides. b) Linker‐length dependent localization of fluorophores. c) Construction of double‐layered fluorescent protein <t>nanoparticle</t> with cancer targeting capability.
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    Affibody egfr gfp
    Genetic encapsulation and assembly to produce fluorescently engineered HBV capsids with cancer targeting capability. a) Encapsulation of FPs surface‐display of cancer targeting peptides. b) Linker‐length dependent localization of fluorophores. c) Construction of double‐layered fluorescent protein <t>nanoparticle</t> with cancer targeting capability.
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    Affibody egfr dependent specificity
    CPP screening and selection process. ( a ) Selection begins with a T7 phage library displaying a fusion of a cargo (in this example, an <t>EGFR</t> receptor binding domain, EBD), an Avitag and <t>Phylomer</t> peptides; ( b ) Avitagged-Phylomer sequences with potential for CPP activity are internalized into cells; this uptake can also be facilitated by binding to specific cell types via a cell surface receptor (CSR) and uptake into endosomes by receptor-mediated endocytosis; ( c ) peptides with capacity for cytosolic delivery allow the phage to enter the cytoplasm; ( d ) selection is performed in mammalian cells expressing the bacterial biotin ligase BirA in the cytoplasm, which ligates free biotin to the lysine residue of the phage-displayed Avitag sequence; this step produces selectively labeled T7 phage that have internalized into the cytoplasm by virtue of the CPP; ( e ) sodium pyrophosphate (PPi), a specific inhibitor of BirA, is added to the cells to terminate the biotinylation reaction; ( f ) cells are lysed and streptavidin-coated magnetic beads (SAV) are added to the lysate to selectively capture and concentrate biotinylated T7 phage. Enriched phage are then amplified in E . coli and subjected to further rounds of selection. Identification of specific CPPs is achieved by deep sequencing of early selection rounds or by Sanger-sequencing of individual phage clones after 3–4 rounds of selection.
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    Affibody aby 029
    Experimental image collection for SCC-15 tumor demonstrating the experimental flow. Mice with subcutanteous SCC-15 tumors were injected with <t>ABY-029</t> and IRDye 680RD-Affibody Control Imaging Agent. Fluorescent images were collected on the Odyssey CLx over an hour. The final images collected at 60 minutes are shown here for both ABY-029 and IRDye 680RD-Aff ctrl . The corresponding and inherently aligned binding potential (BP) map is shown next to the aligned EGFR IHC performed in Matlab. The EGFR IHC image was color separated to isolate the brown stain only, and this brown stain was compared to the BP map, ABY-029 fluorescence and IRDye 680-Aff ctrl fluorescence.
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    Image Search Results


    (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of the affibody as indicated. All samples were treated with PNGase to degloycosylate and visualize EGFR extracellular domain (∼70 kDa) as a distinct band. Each sample contained both an affibody and EGFR fragment unless otherwise indicated. Note that only the N23BP mutant gave a photoproduct, which corresponded to a mass increase of 10 kDa. Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) N23BP and EGFR mixed as in (A) or with the equimolar concentration (260 nM) bovine serum albumin (BSA) as indicated and irradiated for the time listed. A band corresponding to the EGFR-affibody conjugate appears at 5, 15, and 30 min (red arrows). Ladder proteins are (top to bottom) 100, 75, and 50 kDa.

    Journal: Journal of the American Chemical Society

    Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

    doi: 10.1021/jacs.8b07601

    Figure Lengend Snippet: (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of the affibody as indicated. All samples were treated with PNGase to degloycosylate and visualize EGFR extracellular domain (∼70 kDa) as a distinct band. Each sample contained both an affibody and EGFR fragment unless otherwise indicated. Note that only the N23BP mutant gave a photoproduct, which corresponded to a mass increase of 10 kDa. Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) N23BP and EGFR mixed as in (A) or with the equimolar concentration (260 nM) bovine serum albumin (BSA) as indicated and irradiated for the time listed. A band corresponding to the EGFR-affibody conjugate appears at 5, 15, and 30 min (red arrows). Ladder proteins are (top to bottom) 100, 75, and 50 kDa.

    Article Snippet: The coding sequence for the EGFR binding affibody designated as ZEGFR:1907 or the nonbinding parent Z domain was synthesized by Integrated DNA Technologies and ligated into the pET21b+ vector, attaching an N-terminal T7 epitope tag and C-terminal 6xHis tag.

    Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Mutagenesis, Concentration Assay, Irradiation

    (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.

    Journal: Journal of the American Chemical Society

    Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

    doi: 10.1021/jacs.8b07601

    Figure Lengend Snippet: (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.

    Article Snippet: The coding sequence for the EGFR binding affibody designated as ZEGFR:1907 or the nonbinding parent Z domain was synthesized by Integrated DNA Technologies and ligated into the pET21b+ vector, attaching an N-terminal T7 epitope tag and C-terminal 6xHis tag.

    Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Irradiation, Produced, Labeling, Transfection, Fluorescence, Incubation

    Left: Spheroids were treated with 1 μ M N23BP (top) or WT (bottom) affibody for a total of 4 h (left column) and 20 h (middle and right columns) and additionally irradiated with 365 nm light for 30 min after 3.5 h incubation (right column). These spheroids were sectioned at approximately the same depth and imaged for Rhodamine and GFP distribution. Micrographs show Rhodamine signal within each section normalized by the average GFP intensity. Scale bars are 200 μ m. Right: Five spheroids were grown and irradiated for 1 h with the indicated N23BP affibody concentration in growth media. Affibody containing media was removed, and spheroids were lysed and loaded onto a PAGE gel and probed for affibody conjugates using an anti-T7 antibody. High molecular weight bands around the expected molecular weight of EGFR were observed only when the spheroid-affibody mixtures were irradiated, indicating photo-cross-linking.

    Journal: Journal of the American Chemical Society

    Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

    doi: 10.1021/jacs.8b07601

    Figure Lengend Snippet: Left: Spheroids were treated with 1 μ M N23BP (top) or WT (bottom) affibody for a total of 4 h (left column) and 20 h (middle and right columns) and additionally irradiated with 365 nm light for 30 min after 3.5 h incubation (right column). These spheroids were sectioned at approximately the same depth and imaged for Rhodamine and GFP distribution. Micrographs show Rhodamine signal within each section normalized by the average GFP intensity. Scale bars are 200 μ m. Right: Five spheroids were grown and irradiated for 1 h with the indicated N23BP affibody concentration in growth media. Affibody containing media was removed, and spheroids were lysed and loaded onto a PAGE gel and probed for affibody conjugates using an anti-T7 antibody. High molecular weight bands around the expected molecular weight of EGFR were observed only when the spheroid-affibody mixtures were irradiated, indicating photo-cross-linking.

    Article Snippet: The coding sequence for the EGFR binding affibody designated as ZEGFR:1907 or the nonbinding parent Z domain was synthesized by Integrated DNA Technologies and ligated into the pET21b+ vector, attaching an N-terminal T7 epitope tag and C-terminal 6xHis tag.

    Techniques: Irradiation, Incubation, Concentration Assay, Polyacrylamide Gel Electrophoresis, Molecular Weight

    Specific binding and uptake of IRDye800CW-labeled Affibody molecules. (A) The protein expression levels of EGFR and HER2 in MDA-MB-231 (MDA231), A431, SKOV3, and SKBR3 cells. Actin served as an internal control. The relative expression levels were calculated

    Journal: Neoplasia (New York, N.Y.)

    Article Title: In Vivo Imaging of Xenograft Tumors Using an Epidermal Growth Factor Receptor-Specific Affibody Molecule Labeled with a Near-infrared Fluorophore 1

    doi:

    Figure Lengend Snippet: Specific binding and uptake of IRDye800CW-labeled Affibody molecules. (A) The protein expression levels of EGFR and HER2 in MDA-MB-231 (MDA231), A431, SKOV3, and SKBR3 cells. Actin served as an internal control. The relative expression levels were calculated

    Article Snippet: Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding Affibody molecule.

    Techniques: Binding Assay, Labeling, Expressing, Multiple Displacement Amplification

    The effect of EGF and EGFR-specific Affibody (Eaff) on EGFR-mediated phosphorylation of EGFR and ERK1/2 (P44/42 MAPK) proteins. A431 cells were treated with either Eaff or EGF. Two concentrations (5 and 20 nM) for both Eaff (Eaff5 and Eaff20) and EGF

    Journal: Neoplasia (New York, N.Y.)

    Article Title: In Vivo Imaging of Xenograft Tumors Using an Epidermal Growth Factor Receptor-Specific Affibody Molecule Labeled with a Near-infrared Fluorophore 1

    doi:

    Figure Lengend Snippet: The effect of EGF and EGFR-specific Affibody (Eaff) on EGFR-mediated phosphorylation of EGFR and ERK1/2 (P44/42 MAPK) proteins. A431 cells were treated with either Eaff or EGF. Two concentrations (5 and 20 nM) for both Eaff (Eaff5 and Eaff20) and EGF

    Article Snippet: Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding Affibody molecule.

    Techniques:

    Affibody-fused FAP for targeted in vivo tumor imaging. Affibody is a protein that specifically binds EGFR. Adapted with permission from Ref. 60 . Copyright (2017) Royal Society of Chemistry.

    Journal: Acta Pharmaceutica Sinica. B

    Article Title: Fluorogen-activating proteins: beyond classical fluorescent proteins

    doi: 10.1016/j.apsb.2018.02.001

    Figure Lengend Snippet: Affibody-fused FAP for targeted in vivo tumor imaging. Affibody is a protein that specifically binds EGFR. Adapted with permission from Ref. 60 . Copyright (2017) Royal Society of Chemistry.

    Article Snippet: Remarkably, Bruchez and co-workers developed a new tumor-targeting probe, affiFAP, containing a protein that specifically binds EGFR (affibody) and dL5** FAP.

    Techniques: In Vivo, Imaging

    PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

    Journal: EJNMMI Research

    Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

    doi: 10.1186/s13550-016-0213-8

    Figure Lengend Snippet: PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

    Article Snippet: The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377 -ST and ZTaq:3638 -ST).

    Techniques: Positron Emission Tomography

    PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

    Journal: EJNMMI Research

    Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

    doi: 10.1186/s13550-016-0213-8

    Figure Lengend Snippet: PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

    Article Snippet: The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377 -ST and ZTaq:3638 -ST).

    Techniques: Positron Emission Tomography

    Modular capacity of probes for labeling EGFR on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with FAP–affibody fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.

    Journal: Bioconjugate Chemistry

    Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors

    doi: 10.1021/bc500525b

    Figure Lengend Snippet: Modular capacity of probes for labeling EGFR on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with FAP–affibody fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.

    Article Snippet: A protein domain (FAP dL5** ) that binds to malachite-green (MG) derivatives for fluorescence activation was expressed as a recombinant fusion to one or two copies of the compact EGFR binding affibody ZEGFR:1907 .

    Techniques: Labeling, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Incubation, Imaging

    Characterization of probes binding on A431 cell surface. (A) Dissociation constant analysis of probes on cell surface. 5 × 10 5 /mL quantities of cells were incubated with probes for 1 h at 37 °C followed by 2 μM MG-B-tau for 5 min. Then cells were kept on ice for flow cytometry. The mean fluorescence intensity was corrected with background of cells incubating with F/MG and then normalized to mean fluorescence at 250 nM of probes. (B) Competition assay of nonfluorescent affibody A binding to the cell surface. Cells were labeled with 250 nM AFA or F and a serial dilution of A followed by 100 nM of MG-B-tau added prior to measurement. (C) Detection of receptor activation by Western blots. Starved cells were labeled with 250 nM probes followed by 100 nM of MG-B-tau and then cells were treated with 100 ng/mL EGF. Then cells were lysed for Western blot in order to detect phosphorylated EGFR and total EGFR. (D) Live-cell fluorescence microscopy of A431 cells labeled by various probes. Cells were labeled with 250 nM of probe and 100 nM of MG-B-tau prior to imaging or 100 nM of Cy5 conjugated affibody dimer. Scale bar 20 μm.

    Journal: Bioconjugate Chemistry

    Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors

    doi: 10.1021/bc500525b

    Figure Lengend Snippet: Characterization of probes binding on A431 cell surface. (A) Dissociation constant analysis of probes on cell surface. 5 × 10 5 /mL quantities of cells were incubated with probes for 1 h at 37 °C followed by 2 μM MG-B-tau for 5 min. Then cells were kept on ice for flow cytometry. The mean fluorescence intensity was corrected with background of cells incubating with F/MG and then normalized to mean fluorescence at 250 nM of probes. (B) Competition assay of nonfluorescent affibody A binding to the cell surface. Cells were labeled with 250 nM AFA or F and a serial dilution of A followed by 100 nM of MG-B-tau added prior to measurement. (C) Detection of receptor activation by Western blots. Starved cells were labeled with 250 nM probes followed by 100 nM of MG-B-tau and then cells were treated with 100 ng/mL EGF. Then cells were lysed for Western blot in order to detect phosphorylated EGFR and total EGFR. (D) Live-cell fluorescence microscopy of A431 cells labeled by various probes. Cells were labeled with 250 nM of probe and 100 nM of MG-B-tau prior to imaging or 100 nM of Cy5 conjugated affibody dimer. Scale bar 20 μm.

    Article Snippet: A protein domain (FAP dL5** ) that binds to malachite-green (MG) derivatives for fluorescence activation was expressed as a recombinant fusion to one or two copies of the compact EGFR binding affibody ZEGFR:1907 .

    Techniques: Binding Assay, Incubation, Flow Cytometry, Cytometry, Fluorescence, Competitive Binding Assay, Labeling, Serial Dilution, Activation Assay, Western Blot, Microscopy, Imaging

    (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of the affibody as indicated. All samples were treated with PNGase to degloycosylate and visualize EGFR extracellular domain (∼70 kDa) as a distinct band. Each sample contained both an affibody and EGFR fragment unless otherwise indicated. Note that only the N23BP mutant gave a photoproduct, which corresponded to a mass increase of 10 kDa. Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) N23BP and EGFR mixed as in (A) or with the equimolar concentration (260 nM) bovine serum albumin (BSA) as indicated and irradiated for the time listed. A band corresponding to the EGFR-affibody conjugate appears at 5, 15, and 30 min (red arrows). Ladder proteins are (top to bottom) 100, 75, and 50 kDa.

    Journal: Journal of the American Chemical Society

    Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

    doi: 10.1021/jacs.8b07601

    Figure Lengend Snippet: (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of the affibody as indicated. All samples were treated with PNGase to degloycosylate and visualize EGFR extracellular domain (∼70 kDa) as a distinct band. Each sample contained both an affibody and EGFR fragment unless otherwise indicated. Note that only the N23BP mutant gave a photoproduct, which corresponded to a mass increase of 10 kDa. Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) N23BP and EGFR mixed as in (A) or with the equimolar concentration (260 nM) bovine serum albumin (BSA) as indicated and irradiated for the time listed. A band corresponding to the EGFR-affibody conjugate appears at 5, 15, and 30 min (red arrows). Ladder proteins are (top to bottom) 100, 75, and 50 kDa.

    Article Snippet: In testing varying irradiation times, cross-linking between EGFR and N23BP affibody was observed to occur within 15 min. Affibodies containing BP at different amino acid sites may not have shown photo-cross-linking to EGFR because the BP group inhibited affibody-EGFR binding or the BP group was not properly oriented relative to EGFR once bound.

    Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Mutagenesis, Concentration Assay, Irradiation

    (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.

    Journal: Journal of the American Chemical Society

    Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

    doi: 10.1021/jacs.8b07601

    Figure Lengend Snippet: (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.

    Article Snippet: In testing varying irradiation times, cross-linking between EGFR and N23BP affibody was observed to occur within 15 min. Affibodies containing BP at different amino acid sites may not have shown photo-cross-linking to EGFR because the BP group inhibited affibody-EGFR binding or the BP group was not properly oriented relative to EGFR once bound.

    Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Irradiation, Produced, Labeling, Transfection, Fluorescence, Incubation

    Left: Spheroids were treated with 1 μ M N23BP (top) or WT (bottom) affibody for a total of 4 h (left column) and 20 h (middle and right columns) and additionally irradiated with 365 nm light for 30 min after 3.5 h incubation (right column). These spheroids were sectioned at approximately the same depth and imaged for Rhodamine and GFP distribution. Micrographs show Rhodamine signal within each section normalized by the average GFP intensity. Scale bars are 200 μ m. Right: Five spheroids were grown and irradiated for 1 h with the indicated N23BP affibody concentration in growth media. Affibody containing media was removed, and spheroids were lysed and loaded onto a PAGE gel and probed for affibody conjugates using an anti-T7 antibody. High molecular weight bands around the expected molecular weight of EGFR were observed only when the spheroid-affibody mixtures were irradiated, indicating photo-cross-linking.

    Journal: Journal of the American Chemical Society

    Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

    doi: 10.1021/jacs.8b07601

    Figure Lengend Snippet: Left: Spheroids were treated with 1 μ M N23BP (top) or WT (bottom) affibody for a total of 4 h (left column) and 20 h (middle and right columns) and additionally irradiated with 365 nm light for 30 min after 3.5 h incubation (right column). These spheroids were sectioned at approximately the same depth and imaged for Rhodamine and GFP distribution. Micrographs show Rhodamine signal within each section normalized by the average GFP intensity. Scale bars are 200 μ m. Right: Five spheroids were grown and irradiated for 1 h with the indicated N23BP affibody concentration in growth media. Affibody containing media was removed, and spheroids were lysed and loaded onto a PAGE gel and probed for affibody conjugates using an anti-T7 antibody. High molecular weight bands around the expected molecular weight of EGFR were observed only when the spheroid-affibody mixtures were irradiated, indicating photo-cross-linking.

    Article Snippet: In testing varying irradiation times, cross-linking between EGFR and N23BP affibody was observed to occur within 15 min. Affibodies containing BP at different amino acid sites may not have shown photo-cross-linking to EGFR because the BP group inhibited affibody-EGFR binding or the BP group was not properly oriented relative to EGFR once bound.

    Techniques: Irradiation, Incubation, Concentration Assay, Polyacrylamide Gel Electrophoresis, Molecular Weight

    Figure 3. Competition of Aff800, Pan800, and EGF800 binding by unlabeled molecules. Aff800 ( A and B ), Pan800 ( C and D ), and EGF800 ( E and F ) were incubated with F98-EGFR and F98-vIII in the presence of different concentrations (ranging from 0.49 to 500 nM) of unlabeled nanobody 7D12, affibody, EGF, or panitumumab. The fluorescence signals were determined after removal of unbound probes ( n = 3 for each data point).

    Journal: Cancer Biology & Therapy

    Article Title: A comparative study of affibody, panitumumab, and EGF for near-infrared fluorescence imaging of EGFR- and EGFRvIII-expressing tumors

    doi: 10.4161/cbt.26719

    Figure Lengend Snippet: Figure 3. Competition of Aff800, Pan800, and EGF800 binding by unlabeled molecules. Aff800 ( A and B ), Pan800 ( C and D ), and EGF800 ( E and F ) were incubated with F98-EGFR and F98-vIII in the presence of different concentrations (ranging from 0.49 to 500 nM) of unlabeled nanobody 7D12, affibody, EGF, or panitumumab. The fluorescence signals were determined after removal of unbound probes ( n = 3 for each data point).

    Article Snippet: A dimeric form of EGFR-specific affibody (13.7 kDa) was labeled with the near-infrared (NIR) fluorescent dye IRDye® 800CW and used for imaging A431 xenograft tumors.

    Techniques: Binding Assay, Incubation, Fluorescence

    Testing Z EGFR:03115 –IR700DX specificity in vivo and studying the effect of functional groups on dye pharmacokinetics. ( a ) The U87‐MGvIII tumor could easily be differentiated as early as 1 h post Z EGFR:03115 –IR700DX (6 µg/mouse) being intravenously injected, whereas minimal tumor uptake was observed when administering the same amount of the non‐specific Z Taq ‐IR700DX. ( b ) Fluorescence imaging of Z EGFR:03115 –IR700DX uptake in excised tissues (1 h post‐injection) and respective tumor‐to‐organ ratios. ( c ) Mean radiant efficiency in U87‐MGvIII tumors 1 h after administering either 6 µg Z EGFR:03115 –IR700DX, 18 µg Z EGFR:03115 –IR700DX or 6 µg of the non‐specific Z Taq ‐IR700DX. ( d ) Tumor‐to‐background ratio comparison when altering the injected dose of Z EGFR:03115 –IR700DX. ( e , f ) Fluorescence intensity and tumor‐to‐background ratio in the U87‐MGvIII tumors over time after 18 µg Z EGFR:03115 –IR700DX. ( g , h ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors. Images were acquired 30 min, 1 h or 3 h post IR700DX–maleimide, IR800CW–maleimide, IR700DX–NHS ester and IR700DX–carboxylate injection and the mean radiant efficiency was determined for each of the dyes. ( i ) An SDS‐PAGE gel of mouse blood serum imaged using the IVIS/Spectrum imaging system to visualize the fluorescent dyes’ association with blood proteins. All data are presented as mean ± SD ( n ≥ 3).

    Journal: International Journal of Cancer

    Article Title: Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment

    doi: 10.1002/ijc.31246

    Figure Lengend Snippet: Testing Z EGFR:03115 –IR700DX specificity in vivo and studying the effect of functional groups on dye pharmacokinetics. ( a ) The U87‐MGvIII tumor could easily be differentiated as early as 1 h post Z EGFR:03115 –IR700DX (6 µg/mouse) being intravenously injected, whereas minimal tumor uptake was observed when administering the same amount of the non‐specific Z Taq ‐IR700DX. ( b ) Fluorescence imaging of Z EGFR:03115 –IR700DX uptake in excised tissues (1 h post‐injection) and respective tumor‐to‐organ ratios. ( c ) Mean radiant efficiency in U87‐MGvIII tumors 1 h after administering either 6 µg Z EGFR:03115 –IR700DX, 18 µg Z EGFR:03115 –IR700DX or 6 µg of the non‐specific Z Taq ‐IR700DX. ( d ) Tumor‐to‐background ratio comparison when altering the injected dose of Z EGFR:03115 –IR700DX. ( e , f ) Fluorescence intensity and tumor‐to‐background ratio in the U87‐MGvIII tumors over time after 18 µg Z EGFR:03115 –IR700DX. ( g , h ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors. Images were acquired 30 min, 1 h or 3 h post IR700DX–maleimide, IR800CW–maleimide, IR700DX–NHS ester and IR700DX–carboxylate injection and the mean radiant efficiency was determined for each of the dyes. ( i ) An SDS‐PAGE gel of mouse blood serum imaged using the IVIS/Spectrum imaging system to visualize the fluorescent dyes’ association with blood proteins. All data are presented as mean ± SD ( n ≥ 3).

    Article Snippet: The EGFR‐specific (ZEGFR:03115 ) and non‐specific (ZTaq ) affibody molecules were successfully conjugated via the maleimide group to IR700DX and the fluorescent‐SDS‐PAGE as well as silver staining confirmed labeling of the conjugates (Supporting Information Figs. 1a and 1b ).

    Techniques: In Vivo, Functional Assay, Injection, Fluorescence, Imaging, Mouse Assay, SDS Page

    Expression of EGFR in GBM cell lines and specificity of the Z EGFR:03115 –IR700DX binding to EGFR. ( a ) Varying EGFR expression in the selected cancer cell lines was confirmed by Western blot. The numbers in the brackets represent the relative EGFR expression as determined by densitometric analysis. ( b ) Z EGFR:03115 –IR700DX (30 nM) binding as assessed by flow cytometry in the selected cancer cells with varying EGFR expression and after blocking with 100‐fold excess of unlabeled Z EGFR:03115 . Data are presented as mean ± SEM ( n = 3). ( c – f ) Confocal microscopy images demonstrating target‐specific binding (4°C) and internalization (37°C) of either the Z EGFR:03115 –IR700DX (1 µM), anti‐EGFR‐FITC antibody (25 nM for visualization purposes) or IR700DX alone (1 µM): ( c ) U251 cell lines (1 h incubation time), ( d + e ) U87‐MGvIII or MCF7 cell lines (1–6 h incubation time), ( f ) U87‐MGvIII spheroids (6 h incubation time). Hoechst®33342 (blue) and LysoTracker™Green DND‐26 (green) were used for counterstaining. ( g ) Quantification of fluorescence intensity (median fluorescence intensity) of ∼8 µm slices through U87‐MGvIII spheroids following a 6 h incubation with Z EGFR:03115 –IR700DX (500 nM) or an anti‐EGFR‐FITC antibody (500 nM). Data are presented as mean ± SEM ( n = 3). ( h ) H E, EGFR and Ki67 immunostaining of U87‐MGvIII spheroid (400–500 µm) sections 72 h after seeding.

    Journal: International Journal of Cancer

    Article Title: Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment

    doi: 10.1002/ijc.31246

    Figure Lengend Snippet: Expression of EGFR in GBM cell lines and specificity of the Z EGFR:03115 –IR700DX binding to EGFR. ( a ) Varying EGFR expression in the selected cancer cell lines was confirmed by Western blot. The numbers in the brackets represent the relative EGFR expression as determined by densitometric analysis. ( b ) Z EGFR:03115 –IR700DX (30 nM) binding as assessed by flow cytometry in the selected cancer cells with varying EGFR expression and after blocking with 100‐fold excess of unlabeled Z EGFR:03115 . Data are presented as mean ± SEM ( n = 3). ( c – f ) Confocal microscopy images demonstrating target‐specific binding (4°C) and internalization (37°C) of either the Z EGFR:03115 –IR700DX (1 µM), anti‐EGFR‐FITC antibody (25 nM for visualization purposes) or IR700DX alone (1 µM): ( c ) U251 cell lines (1 h incubation time), ( d + e ) U87‐MGvIII or MCF7 cell lines (1–6 h incubation time), ( f ) U87‐MGvIII spheroids (6 h incubation time). Hoechst®33342 (blue) and LysoTracker™Green DND‐26 (green) were used for counterstaining. ( g ) Quantification of fluorescence intensity (median fluorescence intensity) of ∼8 µm slices through U87‐MGvIII spheroids following a 6 h incubation with Z EGFR:03115 –IR700DX (500 nM) or an anti‐EGFR‐FITC antibody (500 nM). Data are presented as mean ± SEM ( n = 3). ( h ) H E, EGFR and Ki67 immunostaining of U87‐MGvIII spheroid (400–500 µm) sections 72 h after seeding.

    Article Snippet: The EGFR‐specific (ZEGFR:03115 ) and non‐specific (ZTaq ) affibody molecules were successfully conjugated via the maleimide group to IR700DX and the fluorescent‐SDS‐PAGE as well as silver staining confirmed labeling of the conjugates (Supporting Information Figs. 1a and 1b ).

    Techniques: Expressing, Binding Assay, Western Blot, Flow Cytometry, Cytometry, Blocking Assay, Confocal Microscopy, Incubation, Fluorescence, Immunostaining

    Z EGFR:03115 –IR700DX‐mediated PIT causes cellular death selectively in EGFR+ve cells. Decrease in cell viability as assessed by the CellTiter‐Glo® luminescent cell viability assay 24 or 96 h post‐PIT in 2D cells and 3D spheroids, following 6 h incubation with the Z EGFR:03115 –IR700DX and irradiation with a light dose of 8 or 16 J/cm 2 , was confirmed to be dose dependent and receptor mediated. ( a ) U87‐MGvIII cells 24 h post‐PIT. ( b ) MCF7 cells 24 h post‐PIT. ( c , d ) U87‐MGvIII spheroids 24 and 96 h post‐PIT. ( e ) WSz4 spheroids 96 h post‐PIT. Data are presented as mean ± SEM ( n = 3). Statistical significance in comparison to the control group was determined using an unpaired two‐tailed Student's t‐ test with Welch's correction. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001. [Color figure can be viewed at http://wileyonlinelibrary.com ]

    Journal: International Journal of Cancer

    Article Title: Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment

    doi: 10.1002/ijc.31246

    Figure Lengend Snippet: Z EGFR:03115 –IR700DX‐mediated PIT causes cellular death selectively in EGFR+ve cells. Decrease in cell viability as assessed by the CellTiter‐Glo® luminescent cell viability assay 24 or 96 h post‐PIT in 2D cells and 3D spheroids, following 6 h incubation with the Z EGFR:03115 –IR700DX and irradiation with a light dose of 8 or 16 J/cm 2 , was confirmed to be dose dependent and receptor mediated. ( a ) U87‐MGvIII cells 24 h post‐PIT. ( b ) MCF7 cells 24 h post‐PIT. ( c , d ) U87‐MGvIII spheroids 24 and 96 h post‐PIT. ( e ) WSz4 spheroids 96 h post‐PIT. Data are presented as mean ± SEM ( n = 3). Statistical significance in comparison to the control group was determined using an unpaired two‐tailed Student's t‐ test with Welch's correction. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001. [Color figure can be viewed at http://wileyonlinelibrary.com ]

    Article Snippet: The EGFR‐specific (ZEGFR:03115 ) and non‐specific (ZTaq ) affibody molecules were successfully conjugated via the maleimide group to IR700DX and the fluorescent‐SDS‐PAGE as well as silver staining confirmed labeling of the conjugates (Supporting Information Figs. 1a and 1b ).

    Techniques: Cell Viability Assay, Incubation, Irradiation, Two Tailed Test

    In vivo Z EGFR:03115 –IR700DX‐mediated PIT studies. ( a ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors 1 h after injecting 18 μg of Z EGFR:03115 –IR700DX or IR700DX–maleimide (top row). Subsequently, mice were irradiated with an optical dose of 100 J/cm 2 by a red LED and, immediately after, imaged again (bottom row). ( b ) Tumor growth inhibition of the Z EGFR:03115 –IR700DX‐targeted PIT in U87‐MGvIII tumors after administering three doses of 18 µg of the conjugate and irradiating with 100 J/cm 2 at days 1, 3 and 5 in comparison to control groups. Data are presented as mean ± SD ( n = 6 for each group, ** p ≤ 0.01 as assessed by the Kruskal–Wallis test). ( c ) Visual observation of normal tissue damage in the PDT treated mice, while no skin damage was present in the Z EGFR:03115 –IR700DX PIT mice. These were the appearances seen in all mice. ( d ) H E staining of treated and untreated U87‐MGvIIII tumors (arrows indicate regions of tissue necrosis).

    Journal: International Journal of Cancer

    Article Title: Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment

    doi: 10.1002/ijc.31246

    Figure Lengend Snippet: In vivo Z EGFR:03115 –IR700DX‐mediated PIT studies. ( a ) Fluorescence imaging of mice bearing subcutaneous U87‐MGvIII tumors 1 h after injecting 18 μg of Z EGFR:03115 –IR700DX or IR700DX–maleimide (top row). Subsequently, mice were irradiated with an optical dose of 100 J/cm 2 by a red LED and, immediately after, imaged again (bottom row). ( b ) Tumor growth inhibition of the Z EGFR:03115 –IR700DX‐targeted PIT in U87‐MGvIII tumors after administering three doses of 18 µg of the conjugate and irradiating with 100 J/cm 2 at days 1, 3 and 5 in comparison to control groups. Data are presented as mean ± SD ( n = 6 for each group, ** p ≤ 0.01 as assessed by the Kruskal–Wallis test). ( c ) Visual observation of normal tissue damage in the PDT treated mice, while no skin damage was present in the Z EGFR:03115 –IR700DX PIT mice. These were the appearances seen in all mice. ( d ) H E staining of treated and untreated U87‐MGvIIII tumors (arrows indicate regions of tissue necrosis).

    Article Snippet: The EGFR‐specific (ZEGFR:03115 ) and non‐specific (ZTaq ) affibody molecules were successfully conjugated via the maleimide group to IR700DX and the fluorescent‐SDS‐PAGE as well as silver staining confirmed labeling of the conjugates (Supporting Information Figs. 1a and 1b ).

    Techniques: In Vivo, Fluorescence, Imaging, Mouse Assay, Irradiation, Inhibition, Staining

    Z EGFR:03115 –IR700DX accumulates in U87‐MGvIII orthotopic glioma tumors. ( a ) T 2 ‐weighted MRI images of an intracranial brain tumor model 11 days post‐cell implantation. ( b ) Photographic image of the brain and the corresponding Z EGFR:03115 –IR700DX fluorescent image demonstrates predominant accumulation of the conjugate within the brain tumor mass. ( c ) Transaxial brain histological sections (10μm) containing tumor tissue were obtained for ex vivo analysis immediately after 1 h in vivo image acquisition. Z EGFR:03115 –IR700DX clearly delineated tumor mass from the surrounding normal tissues which correlated well with H E and EGFR staining of the consecutive sections.

    Journal: International Journal of Cancer

    Article Title: Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment

    doi: 10.1002/ijc.31246

    Figure Lengend Snippet: Z EGFR:03115 –IR700DX accumulates in U87‐MGvIII orthotopic glioma tumors. ( a ) T 2 ‐weighted MRI images of an intracranial brain tumor model 11 days post‐cell implantation. ( b ) Photographic image of the brain and the corresponding Z EGFR:03115 –IR700DX fluorescent image demonstrates predominant accumulation of the conjugate within the brain tumor mass. ( c ) Transaxial brain histological sections (10μm) containing tumor tissue were obtained for ex vivo analysis immediately after 1 h in vivo image acquisition. Z EGFR:03115 –IR700DX clearly delineated tumor mass from the surrounding normal tissues which correlated well with H E and EGFR staining of the consecutive sections.

    Article Snippet: The EGFR‐specific (ZEGFR:03115 ) and non‐specific (ZTaq ) affibody molecules were successfully conjugated via the maleimide group to IR700DX and the fluorescent‐SDS‐PAGE as well as silver staining confirmed labeling of the conjugates (Supporting Information Figs. 1a and 1b ).

    Techniques: Magnetic Resonance Imaging, Ex Vivo, In Vivo, Staining

    In vitro morphological changes following affibody‐based PIT. ( a ) Incubation of U87‐MGvIII spheroids with the Z EGFR:03115 –IR700DX for 6 h and irradiation with a red LED (16 J/cm 2 ) induced phototoxic cell death and disintegration of the architectural structure of the spheroid population. ( b ) U87‐MGvIII cells grown as a monolayer culture showed rapid cell swelling and bleb formation (see arrows) as visualized by a phase‐contrast image 1 h post Z EGFR:03115 –IR700DX (red) irradiation with the 639 nm laser on a confocal microscope. ( c ) Following methanol fixation of U87‐MGvIII cells, either treated by PIT or just irradiated, and staining with an anti‐calreticulin‐AlexaFluor488 antibody overnight (4°C), images were acquired by confocal microscopy. ( d ) Cell membrane disruption was monitored by propidium iodide (1 µg/mL) staining. U87‐MGvIII cells irradiated only or treated with Z EGFR:03115 –IR700DX‐based PIT were analyzed by flow cytometry 1 and 24 h post‐treatment. ( e ) Reactive oxygen species production was assessed using the DCFDA cellular ROS detection assay kit using U87‐MGvIII cells treated with affibody‐based PIT (15 min after light exposure). The results were normalized to the control cells. Data are presented as mean ± SEM ( n = 3).

    Journal: International Journal of Cancer

    Article Title: Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment

    doi: 10.1002/ijc.31246

    Figure Lengend Snippet: In vitro morphological changes following affibody‐based PIT. ( a ) Incubation of U87‐MGvIII spheroids with the Z EGFR:03115 –IR700DX for 6 h and irradiation with a red LED (16 J/cm 2 ) induced phototoxic cell death and disintegration of the architectural structure of the spheroid population. ( b ) U87‐MGvIII cells grown as a monolayer culture showed rapid cell swelling and bleb formation (see arrows) as visualized by a phase‐contrast image 1 h post Z EGFR:03115 –IR700DX (red) irradiation with the 639 nm laser on a confocal microscope. ( c ) Following methanol fixation of U87‐MGvIII cells, either treated by PIT or just irradiated, and staining with an anti‐calreticulin‐AlexaFluor488 antibody overnight (4°C), images were acquired by confocal microscopy. ( d ) Cell membrane disruption was monitored by propidium iodide (1 µg/mL) staining. U87‐MGvIII cells irradiated only or treated with Z EGFR:03115 –IR700DX‐based PIT were analyzed by flow cytometry 1 and 24 h post‐treatment. ( e ) Reactive oxygen species production was assessed using the DCFDA cellular ROS detection assay kit using U87‐MGvIII cells treated with affibody‐based PIT (15 min after light exposure). The results were normalized to the control cells. Data are presented as mean ± SEM ( n = 3).

    Article Snippet: The EGFR‐specific (ZEGFR:03115 ) and non‐specific (ZTaq ) affibody molecules were successfully conjugated via the maleimide group to IR700DX and the fluorescent‐SDS‐PAGE as well as silver staining confirmed labeling of the conjugates (Supporting Information Figs. 1a and 1b ).

    Techniques: In Vitro, Incubation, Irradiation, Microscopy, Staining, Confocal Microscopy, Flow Cytometry, Cytometry, Detection Assay

    Binding characterization. (A) EGFR high A431 cells were mixed with the indicated concentration of affibody (EA26S (blue circles) or EA62S (green squares)). Binding was detected with fluorophore-conjugated anti-His6 antibody via flow cytometry. Equilibrium dissociation constants were calculated assuming a 1:1 binding model. n = 3 measurements per variant. (B) EGFR high A431 cells (white) or EGFR low MCF7 cells (black) were incubated with 500 nM affibody. Binding was detected as in (A). n = 3 measurements per variant.

    Journal: Molecular systems design & engineering

    Article Title: Evaluation of affibody charge modification identified by synthetic consensus design in molecular PET imaging of epidermal growth factor receptor

    doi: 10.1039/C7ME00095B

    Figure Lengend Snippet: Binding characterization. (A) EGFR high A431 cells were mixed with the indicated concentration of affibody (EA26S (blue circles) or EA62S (green squares)). Binding was detected with fluorophore-conjugated anti-His6 antibody via flow cytometry. Equilibrium dissociation constants were calculated assuming a 1:1 binding model. n = 3 measurements per variant. (B) EGFR high A431 cells (white) or EGFR low MCF7 cells (black) were incubated with 500 nM affibody. Binding was detected as in (A). n = 3 measurements per variant.

    Article Snippet: In a previous study , 3 basic and 3 acidic residues from the EGFR-targeted affibody EA68 were neutralized via mutation yielding 19 mutants of which clones ‘EA35S’ and ‘EA35C’ retained the greatest combination of target affinity, thermal stability, and recombinant yield.

    Techniques: Binding Assay, Concentration Assay, Flow Cytometry, Cytometry, Variant Assay, Incubation

    Peptide-based chelators used for labeling with 99m Tc. a General structure of the N 3 S chelators formed by a C-terminal cysteine and three adjacent amino acids. X 1 , X 2 and X 3 denote the side chains of the amino acids. b Sequences of EGFR-binding affibody molecules evaluated in this study. The variable amino acids are marked in bold

    Journal: Amino Acids

    Article Title: Influence of composition of cysteine-containing peptide-based chelators on biodistribution of 99mTc-labeled anti-EGFR affibody molecules

    doi: 10.1007/s00726-018-2571-1

    Figure Lengend Snippet: Peptide-based chelators used for labeling with 99m Tc. a General structure of the N 3 S chelators formed by a C-terminal cysteine and three adjacent amino acids. X 1 , X 2 and X 3 denote the side chains of the amino acids. b Sequences of EGFR-binding affibody molecules evaluated in this study. The variable amino acids are marked in bold

    Article Snippet: Hence, liver acts as depots for anti-EGFR affibody molecules.

    Techniques: Labeling, Binding Assay

    Mean instantaneous D fit for different anti-EGFR Affibody conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.

    Journal: PLoS ONE

    Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

    doi: 10.1371/journal.pone.0074200

    Figure Lengend Snippet: Mean instantaneous D fit for different anti-EGFR Affibody conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.

    Article Snippet: Anti-EGFR Affibody-Alexa 488 has been demonstrated to be a specific probe for EGFR and displays a very low non-specific binding on PEG-BSA nanogel surfaces ( ).

    Techniques:

    Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.

    Journal: PLoS ONE

    Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

    doi: 10.1371/journal.pone.0074200

    Figure Lengend Snippet: Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.

    Article Snippet: Anti-EGFR Affibody-Alexa 488 has been demonstrated to be a specific probe for EGFR and displays a very low non-specific binding on PEG-BSA nanogel surfaces ( ).

    Techniques: Fluorescence, Confocal Microscopy, Labeling, Derivative Assay

    Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

    Journal: PLoS ONE

    Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

    doi: 10.1371/journal.pone.0074200

    Figure Lengend Snippet: Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

    Article Snippet: Anti-EGFR Affibody-Alexa 488 has been demonstrated to be a specific probe for EGFR and displays a very low non-specific binding on PEG-BSA nanogel surfaces ( ).

    Techniques:

    Analysis of definitely mobile vs immobile or very slow moving spots. A) Mean instantaneous D fit for different anti-EGFR Affibody conjugates, after removing data for spots with D values below 0.1 µm 2 /s. Each datapoint corresponds to mean ± SD of of the tracks contained in at least 10 different areas containing a minimum of 50 different cells. Blue bars indicate dyes excited at 491 nm, green at 561 nm, and red at 638 nm. B) Percentages of spots for each dye with D values below 0.1 µm 2 /s. C) Plot of mean instantaneous D fit for different anti-EGFR Affibody conjugates (calculated from all spots) vs percentage of spots with D values

    Journal: PLoS ONE

    Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

    doi: 10.1371/journal.pone.0074200

    Figure Lengend Snippet: Analysis of definitely mobile vs immobile or very slow moving spots. A) Mean instantaneous D fit for different anti-EGFR Affibody conjugates, after removing data for spots with D values below 0.1 µm 2 /s. Each datapoint corresponds to mean ± SD of of the tracks contained in at least 10 different areas containing a minimum of 50 different cells. Blue bars indicate dyes excited at 491 nm, green at 561 nm, and red at 638 nm. B) Percentages of spots for each dye with D values below 0.1 µm 2 /s. C) Plot of mean instantaneous D fit for different anti-EGFR Affibody conjugates (calculated from all spots) vs percentage of spots with D values

    Article Snippet: Anti-EGFR Affibody-Alexa 488 has been demonstrated to be a specific probe for EGFR and displays a very low non-specific binding on PEG-BSA nanogel surfaces ( ).

    Techniques:

    Mean instantaneous D fit for different anti-EGFR Affibody conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.

    Journal: PLoS ONE

    Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

    doi: 10.1371/journal.pone.0074200

    Figure Lengend Snippet: Mean instantaneous D fit for different anti-EGFR Affibody conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.

    Article Snippet: Conversely, Qi et al determine that Cy5.5 and Alexa 680 anti-EGFR Affibody conjugates display equally specific binding in vivo, while SR680 and IRDye 800 CW perform remarkably poorer, however Ogawa et al label a humanised full-length antibody (148 kDa) at multiple sites, while Qi et al label the affibody (Mw ca.

    Techniques:

    Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.

    Journal: PLoS ONE

    Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

    doi: 10.1371/journal.pone.0074200

    Figure Lengend Snippet: Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.

    Article Snippet: Conversely, Qi et al determine that Cy5.5 and Alexa 680 anti-EGFR Affibody conjugates display equally specific binding in vivo, while SR680 and IRDye 800 CW perform remarkably poorer, however Ogawa et al label a humanised full-length antibody (148 kDa) at multiple sites, while Qi et al label the affibody (Mw ca.

    Techniques: Fluorescence, Confocal Microscopy, Labeling, Derivative Assay

    Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

    Journal: PLoS ONE

    Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

    doi: 10.1371/journal.pone.0074200

    Figure Lengend Snippet: Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

    Article Snippet: Conversely, Qi et al determine that Cy5.5 and Alexa 680 anti-EGFR Affibody conjugates display equally specific binding in vivo, while SR680 and IRDye 800 CW perform remarkably poorer, however Ogawa et al label a humanised full-length antibody (148 kDa) at multiple sites, while Qi et al label the affibody (Mw ca.

    Techniques:

    Analysis of definitely mobile vs immobile or very slow moving spots. A) Mean instantaneous D fit for different anti-EGFR Affibody conjugates, after removing data for spots with D values below 0.1 µm 2 /s. Each datapoint corresponds to mean ± SD of of the tracks contained in at least 10 different areas containing a minimum of 50 different cells. Blue bars indicate dyes excited at 491 nm, green at 561 nm, and red at 638 nm. B) Percentages of spots for each dye with D values below 0.1 µm 2 /s. C) Plot of mean instantaneous D fit for different anti-EGFR Affibody conjugates (calculated from all spots) vs percentage of spots with D values

    Journal: PLoS ONE

    Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

    doi: 10.1371/journal.pone.0074200

    Figure Lengend Snippet: Analysis of definitely mobile vs immobile or very slow moving spots. A) Mean instantaneous D fit for different anti-EGFR Affibody conjugates, after removing data for spots with D values below 0.1 µm 2 /s. Each datapoint corresponds to mean ± SD of of the tracks contained in at least 10 different areas containing a minimum of 50 different cells. Blue bars indicate dyes excited at 491 nm, green at 561 nm, and red at 638 nm. B) Percentages of spots for each dye with D values below 0.1 µm 2 /s. C) Plot of mean instantaneous D fit for different anti-EGFR Affibody conjugates (calculated from all spots) vs percentage of spots with D values

    Article Snippet: Conversely, Qi et al determine that Cy5.5 and Alexa 680 anti-EGFR Affibody conjugates display equally specific binding in vivo, while SR680 and IRDye 800 CW perform remarkably poorer, however Ogawa et al label a humanised full-length antibody (148 kDa) at multiple sites, while Qi et al label the affibody (Mw ca.

    Techniques:

    Side-by-side comparison of mean squared displacement (MSD) curves and diffusion coefficient (D) histograms from CHO-EGFR-eGFP cells grown on uncoated glass vs. linear-PEG+0.4 mM GRGDS-coated glass. Data were plotted from at least 15 areas acquired from 3 independent samples. Each MSD value comes from at least 6500 (ranging up to 300,000) individual separations, resulting in very small standard error in the MSD. Error bars are plotted but too small to be visible. Panels A (uncoated) and B (linear PEG + GRGDS): diffusion coefficient histogram of tracked spots. EGFR-eGFP (red), anti-EGFR Affibody Alexa 546 (magenta), anti-EGFR Affibody Atto 647N (green). Dotted lines show the mean D coefficient extrapolation. Panels C (uncoated) and D (linear PEG + GRGDS): Mean Square displacement plot. EGFR-eGFP (red), anti-EGFR Affibody Alexa 546 (magenta), anti-EGFR Affibody Atto 647N (green). Dotted lines show the mean D coefficient extrapolation.

    Journal: PLoS ONE

    Article Title: A Systematic Investigation of Differential Effects of Cell Culture Substrates on the Extent of Artifacts in Single-Molecule Tracking

    doi: 10.1371/journal.pone.0045655

    Figure Lengend Snippet: Side-by-side comparison of mean squared displacement (MSD) curves and diffusion coefficient (D) histograms from CHO-EGFR-eGFP cells grown on uncoated glass vs. linear-PEG+0.4 mM GRGDS-coated glass. Data were plotted from at least 15 areas acquired from 3 independent samples. Each MSD value comes from at least 6500 (ranging up to 300,000) individual separations, resulting in very small standard error in the MSD. Error bars are plotted but too small to be visible. Panels A (uncoated) and B (linear PEG + GRGDS): diffusion coefficient histogram of tracked spots. EGFR-eGFP (red), anti-EGFR Affibody Alexa 546 (magenta), anti-EGFR Affibody Atto 647N (green). Dotted lines show the mean D coefficient extrapolation. Panels C (uncoated) and D (linear PEG + GRGDS): Mean Square displacement plot. EGFR-eGFP (red), anti-EGFR Affibody Alexa 546 (magenta), anti-EGFR Affibody Atto 647N (green). Dotted lines show the mean D coefficient extrapolation.

    Article Snippet: For cell culture experiments, we studied the effect of surface treatments on non-specific binding of anti-human epidermal growth factor receptor (EGFR) Affibody labelled with two different fluorescent probes.

    Techniques: Diffusion-based Assay

    Histogram showing percentage of tracks with diffusion coefficient falling in the D = 0 bin of the D distribution histogram in the three acquisition channels on CHO-EGFR-eGFP cells grown on differently coated glass surfaces and labelled with anti-EGFR Affibody Alexa 546 and Atto 647N for 15 minutes at 37°C. Each datapoint corresponds to mean ± SEM of 15 areas acquired from 3 independent samples.

    Journal: PLoS ONE

    Article Title: A Systematic Investigation of Differential Effects of Cell Culture Substrates on the Extent of Artifacts in Single-Molecule Tracking

    doi: 10.1371/journal.pone.0045655

    Figure Lengend Snippet: Histogram showing percentage of tracks with diffusion coefficient falling in the D = 0 bin of the D distribution histogram in the three acquisition channels on CHO-EGFR-eGFP cells grown on differently coated glass surfaces and labelled with anti-EGFR Affibody Alexa 546 and Atto 647N for 15 minutes at 37°C. Each datapoint corresponds to mean ± SEM of 15 areas acquired from 3 independent samples.

    Article Snippet: For cell culture experiments, we studied the effect of surface treatments on non-specific binding of anti-human epidermal growth factor receptor (EGFR) Affibody labelled with two different fluorescent probes.

    Techniques: Diffusion-based Assay

    Representative images of cells exposed to labelled proteins. Panel A–D: representative images of CHO-EGFR-eGFP cells grown on uncoated glass. Whitelight (A), anti-EGFR Affibody Atto 647N (B), anti-EGFR Affibody Alexa 546 (C), and EGFR-eGFP (D). Panels E–H: representative images of CHO-EGFR-eGFP cells grown on linear-PEG +0.4 mM GRGDS peptide-coated glass. Whitelight (E), Anti-EGFR Affibody Atto 647N (F), anti-EGFR Affibody Alexa 546 (G), and EGFR-eGFP (H) (bar 8 µm).

    Journal: PLoS ONE

    Article Title: A Systematic Investigation of Differential Effects of Cell Culture Substrates on the Extent of Artifacts in Single-Molecule Tracking

    doi: 10.1371/journal.pone.0045655

    Figure Lengend Snippet: Representative images of cells exposed to labelled proteins. Panel A–D: representative images of CHO-EGFR-eGFP cells grown on uncoated glass. Whitelight (A), anti-EGFR Affibody Atto 647N (B), anti-EGFR Affibody Alexa 546 (C), and EGFR-eGFP (D). Panels E–H: representative images of CHO-EGFR-eGFP cells grown on linear-PEG +0.4 mM GRGDS peptide-coated glass. Whitelight (E), Anti-EGFR Affibody Atto 647N (F), anti-EGFR Affibody Alexa 546 (G), and EGFR-eGFP (H) (bar 8 µm).

    Article Snippet: For cell culture experiments, we studied the effect of surface treatments on non-specific binding of anti-human epidermal growth factor receptor (EGFR) Affibody labelled with two different fluorescent probes.

    Techniques:

    Mean instantaneous D fit for different anti-EGFR Affibody conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.

    Journal: PLoS ONE

    Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

    doi: 10.1371/journal.pone.0074200

    Figure Lengend Snippet: Mean instantaneous D fit for different anti-EGFR Affibody conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.

    Article Snippet: Direct Measurement by Spot Density of Dye-substrate Binding 0.5 nM of dye-conjugated anti-EGFR affibody species in Serum-Free Medium +25 mM HEPES pH 7.2 were reacted with PEG-BSA nanogel-coated dishes for 10′ at 37°C, under the same experimental conditions used for cell tracking experiments, then rinsed twice with SFM+HEPES and imaged at 37°C as described above.

    Techniques:

    Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.

    Journal: PLoS ONE

    Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

    doi: 10.1371/journal.pone.0074200

    Figure Lengend Snippet: Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.

    Article Snippet: Direct Measurement by Spot Density of Dye-substrate Binding 0.5 nM of dye-conjugated anti-EGFR affibody species in Serum-Free Medium +25 mM HEPES pH 7.2 were reacted with PEG-BSA nanogel-coated dishes for 10′ at 37°C, under the same experimental conditions used for cell tracking experiments, then rinsed twice with SFM+HEPES and imaged at 37°C as described above.

    Techniques: Fluorescence, Confocal Microscopy, Labeling, Derivative Assay

    Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

    Journal: PLoS ONE

    Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

    doi: 10.1371/journal.pone.0074200

    Figure Lengend Snippet: Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

    Article Snippet: Direct Measurement by Spot Density of Dye-substrate Binding 0.5 nM of dye-conjugated anti-EGFR affibody species in Serum-Free Medium +25 mM HEPES pH 7.2 were reacted with PEG-BSA nanogel-coated dishes for 10′ at 37°C, under the same experimental conditions used for cell tracking experiments, then rinsed twice with SFM+HEPES and imaged at 37°C as described above.

    Techniques:

    Analysis of definitely mobile vs immobile or very slow moving spots. A) Mean instantaneous D fit for different anti-EGFR Affibody conjugates, after removing data for spots with D values below 0.1 µm 2 /s. Each datapoint corresponds to mean ± SD of of the tracks contained in at least 10 different areas containing a minimum of 50 different cells. Blue bars indicate dyes excited at 491 nm, green at 561 nm, and red at 638 nm. B) Percentages of spots for each dye with D values below 0.1 µm 2 /s. C) Plot of mean instantaneous D fit for different anti-EGFR Affibody conjugates (calculated from all spots) vs percentage of spots with D values

    Journal: PLoS ONE

    Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

    doi: 10.1371/journal.pone.0074200

    Figure Lengend Snippet: Analysis of definitely mobile vs immobile or very slow moving spots. A) Mean instantaneous D fit for different anti-EGFR Affibody conjugates, after removing data for spots with D values below 0.1 µm 2 /s. Each datapoint corresponds to mean ± SD of of the tracks contained in at least 10 different areas containing a minimum of 50 different cells. Blue bars indicate dyes excited at 491 nm, green at 561 nm, and red at 638 nm. B) Percentages of spots for each dye with D values below 0.1 µm 2 /s. C) Plot of mean instantaneous D fit for different anti-EGFR Affibody conjugates (calculated from all spots) vs percentage of spots with D values

    Article Snippet: Direct Measurement by Spot Density of Dye-substrate Binding 0.5 nM of dye-conjugated anti-EGFR affibody species in Serum-Free Medium +25 mM HEPES pH 7.2 were reacted with PEG-BSA nanogel-coated dishes for 10′ at 37°C, under the same experimental conditions used for cell tracking experiments, then rinsed twice with SFM+HEPES and imaged at 37°C as described above.

    Techniques:

    Specific binding and uptake of IRDye800CW-labeled Affibody molecules. (A) The protein expression levels of EGFR and HER2 in MDA-MB-231 (MDA231), A431, SKOV3, and SKBR3 cells. Actin served as an internal control. The relative expression levels were calculated

    Journal: Neoplasia (New York, N.Y.)

    Article Title: In Vivo Imaging of Xenograft Tumors Using an Epidermal Growth Factor Receptor-Specific Affibody Molecule Labeled with a Near-infrared Fluorophore 1

    doi:

    Figure Lengend Snippet: Specific binding and uptake of IRDye800CW-labeled Affibody molecules. (A) The protein expression levels of EGFR and HER2 in MDA-MB-231 (MDA231), A431, SKOV3, and SKBR3 cells. Actin served as an internal control. The relative expression levels were calculated

    Article Snippet: Imaging of EGFR expression in murine xenografts using site-specifically labelled anti-EGFR (111)In-DOTA-Z (EGFR:2377) Affibody molecule: aspect of the injected tracer amount.

    Techniques: Binding Assay, Labeling, Expressing, Multiple Displacement Amplification

    The effect of EGF and EGFR-specific Affibody (Eaff) on EGFR-mediated phosphorylation of EGFR and ERK1/2 (P44/42 MAPK) proteins. A431 cells were treated with either Eaff or EGF. Two concentrations (5 and 20 nM) for both Eaff (Eaff5 and Eaff20) and EGF

    Journal: Neoplasia (New York, N.Y.)

    Article Title: In Vivo Imaging of Xenograft Tumors Using an Epidermal Growth Factor Receptor-Specific Affibody Molecule Labeled with a Near-infrared Fluorophore 1

    doi:

    Figure Lengend Snippet: The effect of EGF and EGFR-specific Affibody (Eaff) on EGFR-mediated phosphorylation of EGFR and ERK1/2 (P44/42 MAPK) proteins. A431 cells were treated with either Eaff or EGF. Two concentrations (5 and 20 nM) for both Eaff (Eaff5 and Eaff20) and EGF

    Article Snippet: Imaging of EGFR expression in murine xenografts using site-specifically labelled anti-EGFR (111)In-DOTA-Z (EGFR:2377) Affibody molecule: aspect of the injected tracer amount.

    Techniques:

    Modular capacity of probes for labeling EGFR on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with FAP–affibody fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.

    Journal: Bioconjugate Chemistry

    Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors

    doi: 10.1021/bc500525b

    Figure Lengend Snippet: Modular capacity of probes for labeling EGFR on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with FAP–affibody fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.

    Article Snippet: This affinity probe preserves the capacity of the FAP to enhance fluorescence upon fluorogen binding, along with the specificity and sensitivity of EGFR labeling by affibody.

    Techniques: Labeling, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Incubation, Imaging

    Characterization of probes binding on A431 cell surface. (A) Dissociation constant analysis of probes on cell surface. 5 × 10 5 /mL quantities of cells were incubated with probes for 1 h at 37 °C followed by 2 μM MG-B-tau for 5 min. Then cells were kept on ice for flow cytometry. The mean fluorescence intensity was corrected with background of cells incubating with F/MG and then normalized to mean fluorescence at 250 nM of probes. (B) Competition assay of nonfluorescent affibody A binding to the cell surface. Cells were labeled with 250 nM AFA or F and a serial dilution of A followed by 100 nM of MG-B-tau added prior to measurement. (C) Detection of receptor activation by Western blots. Starved cells were labeled with 250 nM probes followed by 100 nM of MG-B-tau and then cells were treated with 100 ng/mL EGF. Then cells were lysed for Western blot in order to detect phosphorylated EGFR and total EGFR. (D) Live-cell fluorescence microscopy of A431 cells labeled by various probes. Cells were labeled with 250 nM of probe and 100 nM of MG-B-tau prior to imaging or 100 nM of Cy5 conjugated affibody dimer. Scale bar 20 μm.

    Journal: Bioconjugate Chemistry

    Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors

    doi: 10.1021/bc500525b

    Figure Lengend Snippet: Characterization of probes binding on A431 cell surface. (A) Dissociation constant analysis of probes on cell surface. 5 × 10 5 /mL quantities of cells were incubated with probes for 1 h at 37 °C followed by 2 μM MG-B-tau for 5 min. Then cells were kept on ice for flow cytometry. The mean fluorescence intensity was corrected with background of cells incubating with F/MG and then normalized to mean fluorescence at 250 nM of probes. (B) Competition assay of nonfluorescent affibody A binding to the cell surface. Cells were labeled with 250 nM AFA or F and a serial dilution of A followed by 100 nM of MG-B-tau added prior to measurement. (C) Detection of receptor activation by Western blots. Starved cells were labeled with 250 nM probes followed by 100 nM of MG-B-tau and then cells were treated with 100 ng/mL EGF. Then cells were lysed for Western blot in order to detect phosphorylated EGFR and total EGFR. (D) Live-cell fluorescence microscopy of A431 cells labeled by various probes. Cells were labeled with 250 nM of probe and 100 nM of MG-B-tau prior to imaging or 100 nM of Cy5 conjugated affibody dimer. Scale bar 20 μm.

    Article Snippet: This affinity probe preserves the capacity of the FAP to enhance fluorescence upon fluorogen binding, along with the specificity and sensitivity of EGFR labeling by affibody.

    Techniques: Binding Assay, Incubation, Flow Cytometry, Cytometry, Fluorescence, Competitive Binding Assay, Labeling, Serial Dilution, Activation Assay, Western Blot, Microscopy, Imaging

    Binding dynamics of monomeric and heptameric targeting ligands by BIAcore analysis. The extracellular domain of (A) EGFR and (B) HER2 receptors were immobilized on the CM5 chip. Different concentrations of monomer or heptamer proteins were injected into the channels. Analyses were performed at room temperature at a flow rate of 20 µl/min.

    Journal: PLoS ONE

    Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

    doi: 10.1371/journal.pone.0043077

    Figure Lengend Snippet: Binding dynamics of monomeric and heptameric targeting ligands by BIAcore analysis. The extracellular domain of (A) EGFR and (B) HER2 receptors were immobilized on the CM5 chip. Different concentrations of monomer or heptamer proteins were injected into the channels. Analyses were performed at room temperature at a flow rate of 20 µl/min.

    Article Snippet: The previously reported affibody molecules against EGFR and HER2, ZEGFR and ZHER2 , were used as target binding moieties.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Injection, Flow Cytometry

    Analysis of the protease resistance of the monomer and the heptamer by thermolysin. (A) About 5 µg of monomeric and heptameric Z EGFR and (B) monomeric and heptameric Z HER2 targeting ligands were incubated with 100 ng of thermolysin at different temperatures for 20 min. After incubation, reaction was stopped by adding SDS sample buffer and each reaction mixture was separated on a 10% SDS-PAGE to examine protein degradation.

    Journal: PLoS ONE

    Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

    doi: 10.1371/journal.pone.0043077

    Figure Lengend Snippet: Analysis of the protease resistance of the monomer and the heptamer by thermolysin. (A) About 5 µg of monomeric and heptameric Z EGFR and (B) monomeric and heptameric Z HER2 targeting ligands were incubated with 100 ng of thermolysin at different temperatures for 20 min. After incubation, reaction was stopped by adding SDS sample buffer and each reaction mixture was separated on a 10% SDS-PAGE to examine protein degradation.

    Article Snippet: The previously reported affibody molecules against EGFR and HER2, ZEGFR and ZHER2 , were used as target binding moieties.

    Techniques: Incubation, SDS Page

    Co-localization of EEA1 and heptameric targeting ligands. (A) Two different concentrations of the FITC-labeled heptameric Z EGFR targeting ligands were incubated with A431 cells for 2 h at 37°C. (B) FITC labeled heptameric Z HER2 targeting ligands at two concentrations were incubated with SK-OV3 cells for 2 h at 37°C. EEA1 proteins were detected by Alexa 555-conjugated secondary antibody. Top left panels: cell nuclei stained with DAPI (blue); Top right panels: FITC labeled heptamer (green); bottom left panels: EEA1 antibody (red); bottom right panels: merged image of the three stainings.

    Journal: PLoS ONE

    Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

    doi: 10.1371/journal.pone.0043077

    Figure Lengend Snippet: Co-localization of EEA1 and heptameric targeting ligands. (A) Two different concentrations of the FITC-labeled heptameric Z EGFR targeting ligands were incubated with A431 cells for 2 h at 37°C. (B) FITC labeled heptameric Z HER2 targeting ligands at two concentrations were incubated with SK-OV3 cells for 2 h at 37°C. EEA1 proteins were detected by Alexa 555-conjugated secondary antibody. Top left panels: cell nuclei stained with DAPI (blue); Top right panels: FITC labeled heptamer (green); bottom left panels: EEA1 antibody (red); bottom right panels: merged image of the three stainings.

    Article Snippet: The previously reported affibody molecules against EGFR and HER2, ZEGFR and ZHER2 , were used as target binding moieties.

    Techniques: Labeling, Incubation, Staining

    Heat stability assessment of the monomer and the heptamer by circular dichroism analysis. (A) Monomeric and heptameric Z EGFR , (B) monomeric and heptameric Z HER2 targeting ligands, and (C) heptameric core itself were prepared in a 10 mM phosphate buffer, pH 7.4. Temperature was increased from 25°C to 94°C. Spectra were recorded at various temperatures. The ellipticity at 220 nm was used for the analysis.

    Journal: PLoS ONE

    Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

    doi: 10.1371/journal.pone.0043077

    Figure Lengend Snippet: Heat stability assessment of the monomer and the heptamer by circular dichroism analysis. (A) Monomeric and heptameric Z EGFR , (B) monomeric and heptameric Z HER2 targeting ligands, and (C) heptameric core itself were prepared in a 10 mM phosphate buffer, pH 7.4. Temperature was increased from 25°C to 94°C. Spectra were recorded at various temperatures. The ellipticity at 220 nm was used for the analysis.

    Article Snippet: The previously reported affibody molecules against EGFR and HER2, ZEGFR and ZHER2 , were used as target binding moieties.

    Techniques:

    Cell-based surface receptor binding properties of the monomer and heptamer. (A) EGFR-positive A431 cells were grown on coverslips. Different concentration of FITC-labeled monomeric and heptameric Z EGFR ligands was incubated with A431 cells for 30 min at 25°C. (B) HER2-positive SK-OV3 cells were grown on coverslips. FITC-labeled monomeric and heptameric Z HER2 ligands were incubated with SK-OV3 cells for 30 min at 25°C. (C) EGFR-negative Jurkat cells and HER2-low expressing MCF7 cells were grown on coverslips. 100 nM of FITC-labeled monomeric and heptameric ligands were incubated with Jurkat and MCF cells for 30 min at 25°C.

    Journal: PLoS ONE

    Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

    doi: 10.1371/journal.pone.0043077

    Figure Lengend Snippet: Cell-based surface receptor binding properties of the monomer and heptamer. (A) EGFR-positive A431 cells were grown on coverslips. Different concentration of FITC-labeled monomeric and heptameric Z EGFR ligands was incubated with A431 cells for 30 min at 25°C. (B) HER2-positive SK-OV3 cells were grown on coverslips. FITC-labeled monomeric and heptameric Z HER2 ligands were incubated with SK-OV3 cells for 30 min at 25°C. (C) EGFR-negative Jurkat cells and HER2-low expressing MCF7 cells were grown on coverslips. 100 nM of FITC-labeled monomeric and heptameric ligands were incubated with Jurkat and MCF cells for 30 min at 25°C.

    Article Snippet: The previously reported affibody molecules against EGFR and HER2, ZEGFR and ZHER2 , were used as target binding moieties.

    Techniques: Cell Surface Receptor Assay, Concentration Assay, Labeling, Incubation, Expressing

    Native gel separation of monomeric and heptameric targeting ligands. The purified monomeric Z EGFR , heptameric Z EGFR , monomeric Z HER2 and heptameric Z HER2 ligands were separated on an 8% native gel. About 5 µg of the purified monomer or 20 µg heptamer was loaded to the appropriate lane.

    Journal: PLoS ONE

    Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

    doi: 10.1371/journal.pone.0043077

    Figure Lengend Snippet: Native gel separation of monomeric and heptameric targeting ligands. The purified monomeric Z EGFR , heptameric Z EGFR , monomeric Z HER2 and heptameric Z HER2 ligands were separated on an 8% native gel. About 5 µg of the purified monomer or 20 µg heptamer was loaded to the appropriate lane.

    Article Snippet: The previously reported affibody molecules against EGFR and HER2, ZEGFR and ZHER2 , were used as target binding moieties.

    Techniques: Purification

    Cell binding analysis by flow cytometry. (A) 100 nM FITC-monomeric and heptameric Z EGFR ligands were used for labeling of EGFR positive A431 and negative Jurkat cells, and analyzed by flow cytometry. Cells incubated with PBS were served as negative control. (B) 100 nM FITC-monomeric and heptameric Z HER2 ligands were used for labeling of HER2 positive SK-OV3 and HER2 low expressing MCF7 cells, and analyzed by flow cytometry. Cells incubated with PBS were served as negative control.

    Journal: PLoS ONE

    Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

    doi: 10.1371/journal.pone.0043077

    Figure Lengend Snippet: Cell binding analysis by flow cytometry. (A) 100 nM FITC-monomeric and heptameric Z EGFR ligands were used for labeling of EGFR positive A431 and negative Jurkat cells, and analyzed by flow cytometry. Cells incubated with PBS were served as negative control. (B) 100 nM FITC-monomeric and heptameric Z HER2 ligands were used for labeling of HER2 positive SK-OV3 and HER2 low expressing MCF7 cells, and analyzed by flow cytometry. Cells incubated with PBS were served as negative control.

    Article Snippet: The previously reported affibody molecules against EGFR and HER2, ZEGFR and ZHER2 , were used as target binding moieties.

    Techniques: Binding Assay, Flow Cytometry, Cytometry, Labeling, Incubation, Negative Control, Expressing

    SDS-PAGE analysis of the purified monomeric and heptameric targeting ligands. The purified heptameric Z EGFR , monomeric Z EGFR , heptameric Z HER2 , and monomeric Z HER2 ligands were separated on a 10% SDS-PAGE gel. About 5 µg of each protein was applied to each lane.

    Journal: PLoS ONE

    Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

    doi: 10.1371/journal.pone.0043077

    Figure Lengend Snippet: SDS-PAGE analysis of the purified monomeric and heptameric targeting ligands. The purified heptameric Z EGFR , monomeric Z EGFR , heptameric Z HER2 , and monomeric Z HER2 ligands were separated on a 10% SDS-PAGE gel. About 5 µg of each protein was applied to each lane.

    Article Snippet: The previously reported affibody molecules against EGFR and HER2, ZEGFR and ZHER2 , were used as target binding moieties.

    Techniques: SDS Page, Purification

    Determination of the molecular weights of the heptameric targeting ligands by analytical ultracentrifugation analysis. Purified heptameric Z EGFR and heptameric Z HER2 ligands were centrifuged at 10,000 g for 20 h. Absorbances at 280 nm were recorded every two hours.

    Journal: PLoS ONE

    Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

    doi: 10.1371/journal.pone.0043077

    Figure Lengend Snippet: Determination of the molecular weights of the heptameric targeting ligands by analytical ultracentrifugation analysis. Purified heptameric Z EGFR and heptameric Z HER2 ligands were centrifuged at 10,000 g for 20 h. Absorbances at 280 nm were recorded every two hours.

    Article Snippet: The previously reported affibody molecules against EGFR and HER2, ZEGFR and ZHER2 , were used as target binding moieties.

    Techniques: Purification

    Expression of EGFR in GBM cell lines and specificity of the Z EGFR:03115 –IR700DX binding to EGFR. ( a ) Varying EGFR expression in the selected cancer cell lines was confirmed by Western blot. The numbers in the brackets represent the relative EGFR expression as determined by densitometric analysis. ( b ) Z EGFR:03115 –IR700DX (30 nM) binding as assessed by flow cytometry in the selected cancer cells with varying EGFR expression and after blocking with 100‐fold excess of unlabeled Z EGFR:03115 . Data are presented as mean ± SEM ( n = 3). ( c – f ) Confocal microscopy images demonstrating target‐specific binding (4°C) and internalization (37°C) of either the Z EGFR:03115 –IR700DX (1 µM), anti‐EGFR‐FITC antibody (25 nM for visualization purposes) or IR700DX alone (1 µM): ( c ) U251 cell lines (1 h incubation time), ( d + e ) U87‐MGvIII or MCF7 cell lines (1–6 h incubation time), ( f ) U87‐MGvIII spheroids (6 h incubation time). Hoechst®33342 (blue) and LysoTracker™Green DND‐26 (green) were used for counterstaining. ( g ) Quantification of fluorescence intensity (median fluorescence intensity) of ∼8 µm slices through U87‐MGvIII spheroids following a 6 h incubation with Z EGFR:03115 –IR700DX (500 nM) or an anti‐EGFR‐FITC antibody (500 nM). Data are presented as mean ± SEM ( n = 3). ( h ) H E, EGFR and Ki67 immunostaining of U87‐MGvIII spheroid (400–500 µm) sections 72 h after seeding.

    Journal: International Journal of Cancer

    Article Title: Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment

    doi: 10.1002/ijc.31246

    Figure Lengend Snippet: Expression of EGFR in GBM cell lines and specificity of the Z EGFR:03115 –IR700DX binding to EGFR. ( a ) Varying EGFR expression in the selected cancer cell lines was confirmed by Western blot. The numbers in the brackets represent the relative EGFR expression as determined by densitometric analysis. ( b ) Z EGFR:03115 –IR700DX (30 nM) binding as assessed by flow cytometry in the selected cancer cells with varying EGFR expression and after blocking with 100‐fold excess of unlabeled Z EGFR:03115 . Data are presented as mean ± SEM ( n = 3). ( c – f ) Confocal microscopy images demonstrating target‐specific binding (4°C) and internalization (37°C) of either the Z EGFR:03115 –IR700DX (1 µM), anti‐EGFR‐FITC antibody (25 nM for visualization purposes) or IR700DX alone (1 µM): ( c ) U251 cell lines (1 h incubation time), ( d + e ) U87‐MGvIII or MCF7 cell lines (1–6 h incubation time), ( f ) U87‐MGvIII spheroids (6 h incubation time). Hoechst®33342 (blue) and LysoTracker™Green DND‐26 (green) were used for counterstaining. ( g ) Quantification of fluorescence intensity (median fluorescence intensity) of ∼8 µm slices through U87‐MGvIII spheroids following a 6 h incubation with Z EGFR:03115 –IR700DX (500 nM) or an anti‐EGFR‐FITC antibody (500 nM). Data are presented as mean ± SEM ( n = 3). ( h ) H E, EGFR and Ki67 immunostaining of U87‐MGvIII spheroid (400–500 µm) sections 72 h after seeding.

    Article Snippet: Herein, we demonstrate that administration of an affibody‐infrared light‐activated conjugate targeting EGFR selectively induces cell death in EGFR+ve GBM cells, while limiting toxicity in normal tissues.

    Techniques: Expressing, Binding Assay, Western Blot, Flow Cytometry, Cytometry, Blocking Assay, Confocal Microscopy, Incubation, Fluorescence, Immunostaining

    In vitro morphological changes following affibody‐based PIT. ( a ) Incubation of U87‐MGvIII spheroids with the Z EGFR:03115 –IR700DX for 6 h and irradiation with a red LED (16 J/cm 2 ) induced phototoxic cell death and disintegration of the architectural structure of the spheroid population. ( b ) U87‐MGvIII cells grown as a monolayer culture showed rapid cell swelling and bleb formation (see arrows) as visualized by a phase‐contrast image 1 h post Z EGFR:03115 –IR700DX (red) irradiation with the 639 nm laser on a confocal microscope. ( c ) Following methanol fixation of U87‐MGvIII cells, either treated by PIT or just irradiated, and staining with an anti‐calreticulin‐AlexaFluor488 antibody overnight (4°C), images were acquired by confocal microscopy. ( d ) Cell membrane disruption was monitored by propidium iodide (1 µg/mL) staining. U87‐MGvIII cells irradiated only or treated with Z EGFR:03115 –IR700DX‐based PIT were analyzed by flow cytometry 1 and 24 h post‐treatment. ( e ) Reactive oxygen species production was assessed using the DCFDA cellular ROS detection assay kit using U87‐MGvIII cells treated with affibody‐based PIT (15 min after light exposure). The results were normalized to the control cells. Data are presented as mean ± SEM ( n = 3).

    Journal: International Journal of Cancer

    Article Title: Near‐infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment

    doi: 10.1002/ijc.31246

    Figure Lengend Snippet: In vitro morphological changes following affibody‐based PIT. ( a ) Incubation of U87‐MGvIII spheroids with the Z EGFR:03115 –IR700DX for 6 h and irradiation with a red LED (16 J/cm 2 ) induced phototoxic cell death and disintegration of the architectural structure of the spheroid population. ( b ) U87‐MGvIII cells grown as a monolayer culture showed rapid cell swelling and bleb formation (see arrows) as visualized by a phase‐contrast image 1 h post Z EGFR:03115 –IR700DX (red) irradiation with the 639 nm laser on a confocal microscope. ( c ) Following methanol fixation of U87‐MGvIII cells, either treated by PIT or just irradiated, and staining with an anti‐calreticulin‐AlexaFluor488 antibody overnight (4°C), images were acquired by confocal microscopy. ( d ) Cell membrane disruption was monitored by propidium iodide (1 µg/mL) staining. U87‐MGvIII cells irradiated only or treated with Z EGFR:03115 –IR700DX‐based PIT were analyzed by flow cytometry 1 and 24 h post‐treatment. ( e ) Reactive oxygen species production was assessed using the DCFDA cellular ROS detection assay kit using U87‐MGvIII cells treated with affibody‐based PIT (15 min after light exposure). The results were normalized to the control cells. Data are presented as mean ± SEM ( n = 3).

    Article Snippet: Herein, we demonstrate that administration of an affibody‐infrared light‐activated conjugate targeting EGFR selectively induces cell death in EGFR+ve GBM cells, while limiting toxicity in normal tissues.

    Techniques: In Vitro, Incubation, Irradiation, Microscopy, Staining, Confocal Microscopy, Flow Cytometry, Cytometry, Detection Assay

    Genetic encapsulation and assembly to produce fluorescently engineered HBV capsids with cancer targeting capability. a) Encapsulation of FPs surface‐display of cancer targeting peptides. b) Linker‐length dependent localization of fluorophores. c) Construction of double‐layered fluorescent protein nanoparticle with cancer targeting capability.

    Journal: Advanced Science

    Article Title: Genetic Assembly of Double‐Layered Fluorescent Protein Nanoparticles for Cancer Targeting and Imaging

    doi: 10.1002/advs.201600471

    Figure Lengend Snippet: Genetic encapsulation and assembly to produce fluorescently engineered HBV capsids with cancer targeting capability. a) Encapsulation of FPs surface‐display of cancer targeting peptides. b) Linker‐length dependent localization of fluorophores. c) Construction of double‐layered fluorescent protein nanoparticle with cancer targeting capability.

    Article Snippet: As described in Scheme a,c, mC‐DL‐HBVC, mCout ‐HBVC, and mCin ‐HBVC that have all spherical nanoparticle shape present cancer cell receptor (EGFR)‐binding affibody peptides on their outer surface (Figure d–f; Table ).

    Techniques:

    CPP screening and selection process. ( a ) Selection begins with a T7 phage library displaying a fusion of a cargo (in this example, an EGFR receptor binding domain, EBD), an Avitag and Phylomer peptides; ( b ) Avitagged-Phylomer sequences with potential for CPP activity are internalized into cells; this uptake can also be facilitated by binding to specific cell types via a cell surface receptor (CSR) and uptake into endosomes by receptor-mediated endocytosis; ( c ) peptides with capacity for cytosolic delivery allow the phage to enter the cytoplasm; ( d ) selection is performed in mammalian cells expressing the bacterial biotin ligase BirA in the cytoplasm, which ligates free biotin to the lysine residue of the phage-displayed Avitag sequence; this step produces selectively labeled T7 phage that have internalized into the cytoplasm by virtue of the CPP; ( e ) sodium pyrophosphate (PPi), a specific inhibitor of BirA, is added to the cells to terminate the biotinylation reaction; ( f ) cells are lysed and streptavidin-coated magnetic beads (SAV) are added to the lysate to selectively capture and concentrate biotinylated T7 phage. Enriched phage are then amplified in E . coli and subjected to further rounds of selection. Identification of specific CPPs is achieved by deep sequencing of early selection rounds or by Sanger-sequencing of individual phage clones after 3–4 rounds of selection.

    Journal: Scientific Reports

    Article Title: A platform for discovery of functional cell-penetrating peptides for efficient multi-cargo intracellular delivery

    doi: 10.1038/s41598-018-30790-2

    Figure Lengend Snippet: CPP screening and selection process. ( a ) Selection begins with a T7 phage library displaying a fusion of a cargo (in this example, an EGFR receptor binding domain, EBD), an Avitag and Phylomer peptides; ( b ) Avitagged-Phylomer sequences with potential for CPP activity are internalized into cells; this uptake can also be facilitated by binding to specific cell types via a cell surface receptor (CSR) and uptake into endosomes by receptor-mediated endocytosis; ( c ) peptides with capacity for cytosolic delivery allow the phage to enter the cytoplasm; ( d ) selection is performed in mammalian cells expressing the bacterial biotin ligase BirA in the cytoplasm, which ligates free biotin to the lysine residue of the phage-displayed Avitag sequence; this step produces selectively labeled T7 phage that have internalized into the cytoplasm by virtue of the CPP; ( e ) sodium pyrophosphate (PPi), a specific inhibitor of BirA, is added to the cells to terminate the biotinylation reaction; ( f ) cells are lysed and streptavidin-coated magnetic beads (SAV) are added to the lysate to selectively capture and concentrate biotinylated T7 phage. Enriched phage are then amplified in E . coli and subjected to further rounds of selection. Identification of specific CPPs is achieved by deep sequencing of early selection rounds or by Sanger-sequencing of individual phage clones after 3–4 rounds of selection.

    Article Snippet: Specifically, the Phylomer CPP retained EGFR-dependent specificity when combined with a targeting Affibody, and also largely maintained potency after PASylation .

    Techniques: Conditioned Place Preference, Selection, Binding Assay, Activity Assay, Cell Surface Receptor Assay, Expressing, Sequencing, Labeling, Magnetic Beads, Amplification, Clone Assay

    Phylomer CPP delivery is compatible with cell specific targeting and half-life extension approaches. ( a ) CHO-K1 cells stably expressing EGFR receptor or ( b ) CHO-K1 cells were treated with 1746del_SpyT conjugated to EGFRAffibody_Bouganin_SpyC (EGFRAffbd_Boug_SpyC) toxin. After 48 h incubation, the cell viability was assessed according to the resazurin reduction potential. Comparison of 100 nM immunotoxin treatment in CHO-K1_EGFR ( a , right) vs CHO-K1 ( b , right) cells shows that conjugation to 1746del improved delivery compared to the EGFRAffibody alone, and that the 1746del-conjugate retains EGFRAffibody-encoded specificity. Results are representative of 3 independent experiments. Error bars represent standard deviation from the mean of duplicate samples. Significance was assessed by one-way ANOVA with Dunnett’s multiple comparison test (**p

    Journal: Scientific Reports

    Article Title: A platform for discovery of functional cell-penetrating peptides for efficient multi-cargo intracellular delivery

    doi: 10.1038/s41598-018-30790-2

    Figure Lengend Snippet: Phylomer CPP delivery is compatible with cell specific targeting and half-life extension approaches. ( a ) CHO-K1 cells stably expressing EGFR receptor or ( b ) CHO-K1 cells were treated with 1746del_SpyT conjugated to EGFRAffibody_Bouganin_SpyC (EGFRAffbd_Boug_SpyC) toxin. After 48 h incubation, the cell viability was assessed according to the resazurin reduction potential. Comparison of 100 nM immunotoxin treatment in CHO-K1_EGFR ( a , right) vs CHO-K1 ( b , right) cells shows that conjugation to 1746del improved delivery compared to the EGFRAffibody alone, and that the 1746del-conjugate retains EGFRAffibody-encoded specificity. Results are representative of 3 independent experiments. Error bars represent standard deviation from the mean of duplicate samples. Significance was assessed by one-way ANOVA with Dunnett’s multiple comparison test (**p

    Article Snippet: Specifically, the Phylomer CPP retained EGFR-dependent specificity when combined with a targeting Affibody, and also largely maintained potency after PASylation .

    Techniques: Conditioned Place Preference, Stable Transfection, Expressing, Incubation, Conjugation Assay, Standard Deviation

    Experimental image collection for SCC-15 tumor demonstrating the experimental flow. Mice with subcutanteous SCC-15 tumors were injected with ABY-029 and IRDye 680RD-Affibody Control Imaging Agent. Fluorescent images were collected on the Odyssey CLx over an hour. The final images collected at 60 minutes are shown here for both ABY-029 and IRDye 680RD-Aff ctrl . The corresponding and inherently aligned binding potential (BP) map is shown next to the aligned EGFR IHC performed in Matlab. The EGFR IHC image was color separated to isolate the brown stain only, and this brown stain was compared to the BP map, ABY-029 fluorescence and IRDye 680-Aff ctrl fluorescence.

    Journal: Proceedings of SPIE--the International Society for Optical Engineering

    Article Title: Paired-agent imaging for detection of head and neck cancers

    doi: 10.1117/12.2510897

    Figure Lengend Snippet: Experimental image collection for SCC-15 tumor demonstrating the experimental flow. Mice with subcutanteous SCC-15 tumors were injected with ABY-029 and IRDye 680RD-Affibody Control Imaging Agent. Fluorescent images were collected on the Odyssey CLx over an hour. The final images collected at 60 minutes are shown here for both ABY-029 and IRDye 680RD-Aff ctrl . The corresponding and inherently aligned binding potential (BP) map is shown next to the aligned EGFR IHC performed in Matlab. The EGFR IHC image was color separated to isolate the brown stain only, and this brown stain was compared to the BP map, ABY-029 fluorescence and IRDye 680-Aff ctrl fluorescence.

    Article Snippet: We have validated PAI using two different paired-agent sets: 1) IRDye 800CW-epidermal growth factor and IRDye 700DX [ ]; and 2) ABY-029 (anti-EGFR Affibody molecule labeled with IRDye 800CW, Food and Drug Administration (FDA) exploratory Investigational New Drug (eIND; 122681) and Affibody Control Imaging Agent molecule labeled with IRDye 680RD (IR680-Affctrl ) [ ].

    Techniques: Flow Cytometry, Mouse Assay, Injection, Imaging, Binding Assay, Immunohistochemistry, Staining, Fluorescence

    Selection of clinically relevant agents for PAI. A ) ABY-029 and IRDye 700DX are both in clinical trials and display very similar plasma excretion curves. B ], and anti-EGFR Affibody molecule labeled with IRDye 800CW and IR680-Aff ctrl ]. The binding potential calculated in the same 4 tumor lines are linearly correlated with a strong, positive Pearson’s correlation ( r = 0.98). C ) The normalized fluorescence uptake in normal tissues (muscle, skin, fat) are similar. D ) The binding potential map of normal tissue in a naïve mouse indicates that ABY-029 and IRDye 700DX be success paired-agents.

    Journal: Proceedings of SPIE--the International Society for Optical Engineering

    Article Title: Paired-agent imaging for detection of head and neck cancers

    doi: 10.1117/12.2510897

    Figure Lengend Snippet: Selection of clinically relevant agents for PAI. A ) ABY-029 and IRDye 700DX are both in clinical trials and display very similar plasma excretion curves. B ], and anti-EGFR Affibody molecule labeled with IRDye 800CW and IR680-Aff ctrl ]. The binding potential calculated in the same 4 tumor lines are linearly correlated with a strong, positive Pearson’s correlation ( r = 0.98). C ) The normalized fluorescence uptake in normal tissues (muscle, skin, fat) are similar. D ) The binding potential map of normal tissue in a naïve mouse indicates that ABY-029 and IRDye 700DX be success paired-agents.

    Article Snippet: We have validated PAI using two different paired-agent sets: 1) IRDye 800CW-epidermal growth factor and IRDye 700DX [ ]; and 2) ABY-029 (anti-EGFR Affibody molecule labeled with IRDye 800CW, Food and Drug Administration (FDA) exploratory Investigational New Drug (eIND; 122681) and Affibody Control Imaging Agent molecule labeled with IRDye 680RD (IR680-Affctrl ) [ ].

    Techniques: Selection, Labeling, Binding Assay, Fluorescence

    Correlation of EGFR stain intensity from immunohistochemistry with binding potential ( left ) and targeted ABY-029 (green, right ) and non-targeted IR680-Aff ctrl (red, right ). BP displays a strong positive linear correlation with EGFR expression, while ABY-029 and IR680-Aff ctrl display moderate and weak, negative linear correlations, respectively, with EGFR.

    Journal: Proceedings of SPIE--the International Society for Optical Engineering

    Article Title: Paired-agent imaging for detection of head and neck cancers

    doi: 10.1117/12.2510897

    Figure Lengend Snippet: Correlation of EGFR stain intensity from immunohistochemistry with binding potential ( left ) and targeted ABY-029 (green, right ) and non-targeted IR680-Aff ctrl (red, right ). BP displays a strong positive linear correlation with EGFR expression, while ABY-029 and IR680-Aff ctrl display moderate and weak, negative linear correlations, respectively, with EGFR.

    Article Snippet: We have validated PAI using two different paired-agent sets: 1) IRDye 800CW-epidermal growth factor and IRDye 700DX [ ]; and 2) ABY-029 (anti-EGFR Affibody molecule labeled with IRDye 800CW, Food and Drug Administration (FDA) exploratory Investigational New Drug (eIND; 122681) and Affibody Control Imaging Agent molecule labeled with IRDye 680RD (IR680-Affctrl ) [ ].

    Techniques: Staining, Immunohistochemistry, Binding Assay, Expressing