Journal: Science Advances

Article Title: Contrasting temporal dynamics of fluorescence and photoacoustic signals from Cetuximab-IRDye800 conjugate in EGFR-overexpressing tumors

doi: 10.1126/sciadv.adv4639

Figure Lengend Snippet: ( A ) Schematic representation of the synthesis of Cetuximab-IRDye800 conjugate. The N -hydroxysuccinimide (NHS) ester of the dye was reacted with the anti-EGFR antibody, Cetuximab, in predefined ratios to develop the conjugate, followed by purification through gel filtration chromatography. “ x ” represents the respective number of dye molecules per antibody. ( B ) Ultraviolet-visible (UV-Vis) absorption spectra of IRDye800 (magenta) and Cetuximab-IRDye800 (cyan) in phosphate-buffered saline (PBS). The spectrum for Cetuximab-IRDye800 shows characteristic absorption peaks for both IRDye800 and Cetuximab along with a peak at 703 nm characteristic of H-aggregate formation. ( C ) SDS-PAGE image of Cetuximab-IRDye800 showing binding of the dye to both the heavy and light chains of the Cetuximab antibody. Free dye, pseudocolored in blue (IRDye800) band, is shown as a reference. HC, heavy chain; LC, light chain. ( D ) Table showing the conjugation ratio of IRDye800 per mole Cetuximab in a reaction to generate Cetuximab-IRDye800 with varying IRDye800 ratios, as mentioned in the sample name. The conjugation ratios for IRDye800 are listed along with the conjugation efficiency. Data are presented as mean ± SD. n ≥ 2 per sample.

Article Snippet: The human anti-EGFR antibody (Cetuximab, ERBITUX from Eli Lilly) was obtained from Premium Health Services (Columbia, MD, USA).

Techniques: Purification, Filtration, Chromatography, Saline, SDS Page, Binding Assay, Conjugation Assay

Journal: Science Advances

Article Title: Contrasting temporal dynamics of fluorescence and photoacoustic signals from Cetuximab-IRDye800 conjugate in EGFR-overexpressing tumors

doi: 10.1126/sciadv.adv4639

Figure Lengend Snippet: ( A ) Hematoxylin and eosin (H&E) and corresponding immunofluorescence image of tumor cross section obtained from a mouse tumor 6 hours after Cetuximab-IRDye-AF injection. ( B ) H&E and corresponding immunofluorescence image of tumor cross section obtained from a mouse tumor 24 hours after Cetuximab-IRDye-AF injection [scale bars, 2.5 mm and 100 μm (inset)]. ( C to G ) Quantification of Cetuximab-IRDye-AF distribution heterogeneity parameters including mean Cetuximab-IRDye-AF intensity (C), %Cetuximab-IRDye-AF coverage area (D), Cetuximab-IRDye-AF–to–CD31 ratio to quantify the amount of Cetuximab-IRDye-AF per blood vessel (E), distance of Cetuximab-IRDye-AF from nearest CD31 + blood vessel (F), and Cetuximab-IRDye-AF–to–EGFR ratio to quantify number of EGFR bound by Cetuximab-IRDye-AF (G). Data are presented as mean ± SD ( n ≥ 10 tumor sections per group from more than five mice each for the 6 and 24 hours and two mice from the untreated group), analyzed using Kruskal-Wallis test, followed by post hoc pairwise comparisons using Dunn’s test, except for Cetuximab-IRDye-AF distance to CD31 (F), which is analyzed using Welch’s t test. P values are provided for each graph.

Article Snippet: The human anti-EGFR antibody (Cetuximab, ERBITUX from Eli Lilly) was obtained from Premium Health Services (Columbia, MD, USA).

Techniques: Immunofluorescence, Injection

Journal: PLOS One

Article Title: EGFR-targeted affibody–polyIC polyplex kills EGFR-overexpressing cancer cells without activating the EGFR

doi: 10.1371/journal.pone.0334584

Figure Lengend Snippet: (A i) SDS-PAGE analysis showing the purity of the Ni-NTA purified Z EGFR 1907’ under pseudo native and denatured conditions. Pseudo native gel bands (boxed) were extracted and analysed by mass spectrometry. Mass spectrometry data confirmed that both bands correspond to one species, (A ii) DNA sequencing of the plasmid revealed the above sequence and this was also confirmed by mass spectrometry analysis (B i) Elution profile of the size exclusion gel filtration chromatography of Z EGFR 1907’ affibody purified via His-Tag Ni-NTA purification. Superdex 75 1660 column equilibrated with 20 mM Hepes pH 7.4, 500 mM NaCl, 10% Glycerol and 2 mM β mercaptoethanol and 1 ml fractions were collected. Insert shows the complete elution profile. (B ii) 20 µl of selected sample fractions from the purification were denatured, run on an SDS-PAGE gel and stained with Coomassie blue. (C) Silver stained SDS-PAGE gel of the concentrated 1:1 triconjugate Z EGFR 1907’ (PPEA) under pseudo native and denatured conditions. Pseudo native and denatured conditions are indicated by -/+ β-mercaptoethanol.

Article Snippet: We engineered an affibody-PEG-polyIC-polyplex to target the EGFR and deliver polyIC to the cytoplasm of cancer cells.

Techniques: SDS Page, Purification, Mass Spectrometry, DNA Sequencing, Plasmid Preparation, Sequencing, Filtration, Chromatography, Staining

Journal: PLOS One

Article Title: EGFR-targeted affibody–polyIC polyplex kills EGFR-overexpressing cancer cells without activating the EGFR

doi: 10.1371/journal.pone.0334584

Figure Lengend Snippet: (A) PPEA-polyplexes selectively kill cell lines overexpressing EGFR. Cells were seeded in duplicates into 96-well plates at a density of 5000 cells in 0.1 ml medium per well and grown overnight. Cells were then treated with polyIC at the indicated concentrations using the PPEA complex. PEI-PEG ratio = 1:1; w/w ratio PEI: polyIC = 0.78. U138MG cells do not express EGFR; U87MGwtEGFR cells express 1x10 6 , A431 express 2-3x10 6 and MDA-MB-468 express 2x10 6 EGFRs/cell. (B,C) In vitro anti-tumor activity of PPEA-polyIC-polyplex is polyIC-specific. A431 and U87MGwtEGFR cell lines were treated with same doses of PPEA-polyIC-polyplex (B) or PPEA polyI polyplex (C) , which served as negative control. Viability was measured by the PrestoBlue Cell Viability Reagent (Invitrogen), according to the manufacturer’s instructions, at 72 hrs after treatment. These experiments were repeated three times with a representative experiment shown.

Article Snippet: We engineered an affibody-PEG-polyIC-polyplex to target the EGFR and deliver polyIC to the cytoplasm of cancer cells.

Techniques: In Vitro, Activity Assay, Negative Control

Journal: PLOS One

Article Title: EGFR-targeted affibody–polyIC polyplex kills EGFR-overexpressing cancer cells without activating the EGFR

doi: 10.1371/journal.pone.0334584

Figure Lengend Snippet: Anti-tumor cell activity of PPEA-polyplex in cell lines with medium levels of EGFR.

Article Snippet: We engineered an affibody-PEG-polyIC-polyplex to target the EGFR and deliver polyIC to the cytoplasm of cancer cells.

Techniques: Activity Assay

Journal: PLOS One

Article Title: EGFR-targeted affibody–polyIC polyplex kills EGFR-overexpressing cancer cells without activating the EGFR

doi: 10.1371/journal.pone.0334584

Figure Lengend Snippet: (A) Cell lines were incubated with anti-EGFR-PE and the PE fluorescence was measured by flow cytometry. The median fluorescence intensity for the respective quadrant was used to calculate relative levels of EGFR expression on cell surface compared to the A431 cell line. Proteomic data was calculated from mass spectrometer peptide counts. (B) Relative levels of EGFR expression on cell surface of several CRC cell lines and breast cancer cell lines, BT-20 and MDA-MB-468, compared to the A431 cell line.

Article Snippet: An analogue of the anti-EGFR affibody [ ], Z EGFR 1907’ -Cys was expressed in E.coli BL21 (DE3) and purified on a Ni-NTA column from lysates as described in the methods section.

Techniques: Incubation, Fluorescence, Flow Cytometry, Expressing, Mass Spectrometry

Journal: PLOS One

Article Title: EGFR-targeted affibody–polyIC polyplex kills EGFR-overexpressing cancer cells without activating the EGFR

doi: 10.1371/journal.pone.0334584

Figure Lengend Snippet: (A i) SDS-PAGE analysis showing the purity of the Ni-NTA purified Z EGFR 1907’ under pseudo native and denatured conditions. Pseudo native gel bands (boxed) were extracted and analysed by mass spectrometry. Mass spectrometry data confirmed that both bands correspond to one species, (A ii) DNA sequencing of the plasmid revealed the above sequence and this was also confirmed by mass spectrometry analysis (B i) Elution profile of the size exclusion gel filtration chromatography of Z EGFR 1907’ affibody purified via His-Tag Ni-NTA purification. Superdex 75 1660 column equilibrated with 20 mM Hepes pH 7.4, 500 mM NaCl, 10% Glycerol and 2 mM β mercaptoethanol and 1 ml fractions were collected. Insert shows the complete elution profile. (B ii) 20 µl of selected sample fractions from the purification were denatured, run on an SDS-PAGE gel and stained with Coomassie blue. (C) Silver stained SDS-PAGE gel of the concentrated 1:1 triconjugate Z EGFR 1907’ (PPEA) under pseudo native and denatured conditions. Pseudo native and denatured conditions are indicated by -/+ β-mercaptoethanol.

Article Snippet: These bands were analysed by mass spectrometry and the resulting sequence for the Z EGFR 1907’ -Cys affibody is shown in Aii.

Techniques: SDS Page, Purification, Mass Spectrometry, DNA Sequencing, Plasmid Preparation, Sequencing, Filtration, Chromatography, Staining

Journal: PLOS One

Article Title: EGFR-targeted affibody–polyIC polyplex kills EGFR-overexpressing cancer cells without activating the EGFR

doi: 10.1371/journal.pone.0334584

Figure Lengend Snippet: SDS-PAGE analysis showing the purity of the proteins used for the Biacore analyses. Expected molecular weights: sEGFR 1–501 56kDA, sEGFR 1–621 69kDa, Z EGFR 1907’ 11kDa and Cetuximab 152kDa. (B) Binding kinetics for the Z EGFR 1907’ and Cetuximab interacting with sEGFR 1–501 and sEGFR 1–621 , used to determine K d values using Biacore S200. (C) Competitive binding kinetics of Z EGFR 1907’ and Cetuximab detected under various conditions shows Cetuximab and Z EGFR 1907’ appear to bind to the same EGFR binding site. Solid and dotted line graphs represent raw and fitted data respectively.

Article Snippet: These bands were analysed by mass spectrometry and the resulting sequence for the Z EGFR 1907’ -Cys affibody is shown in Aii.

Techniques: SDS Page, Binding Assay