high molecular weight polyic  (Takeda)

Journal: bioRxiv

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

doi: 10.1101/2025.10.01.679886

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 triconjugateZ EGFR 1907’ (PPEA) under pseudo native and denatured conditions. Pseudo native and denatured conditions are indicated by -/+ β-mercaptoethanol.

Article Snippet: In contrast, the EGFR-binding affibody molecule (Z EGFR1907’ ) exhibits nanomolar binding affinity to EGFR, does not activate the kinase moiety of the receptor( , ) but its polyIC-polyplex (PPEA) kills EGFR positive tumor cells effectively.

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

Journal: bioRxiv

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

doi: 10.1101/2025.10.01.679886

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 transfected 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 transfected 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 hours after transfection. These experiments were repeated three times with a representative experiment shown.

Article Snippet: In contrast, the EGFR-binding affibody molecule (Z EGFR1907’ ) exhibits nanomolar binding affinity to EGFR, does not activate the kinase moiety of the receptor( , ) but its polyIC-polyplex (PPEA) kills EGFR positive tumor cells effectively.

Techniques: Transfection, In Vitro, Activity Assay, Negative Control

Journal: bioRxiv

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

doi: 10.1101/2025.10.01.679886

Figure Lengend Snippet: Cells were transfected with polyIC at the indicated concentrations using PPEA. Cell viability was measured as described in Figure4. MDA-MB-231 cells express 2.5-3X10 5 EGFRs/cell, SK-BR-3 express 3X10 5 , BT-474 express 10 5 EGFRs/cell and MCF7 cells express 5x10 3 EGFRs/cell.

Article Snippet: In contrast, the EGFR-binding affibody molecule (Z EGFR1907’ ) exhibits nanomolar binding affinity to EGFR, does not activate the kinase moiety of the receptor( , ) but its polyIC-polyplex (PPEA) kills EGFR positive tumor cells effectively.

Techniques: Transfection

Journal: bioRxiv

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

doi: 10.1101/2025.10.01.679886

Figure Lengend Snippet:

Article Snippet: In contrast, the EGFR-binding affibody molecule (Z EGFR1907’ ) exhibits nanomolar binding affinity to EGFR, does not activate the kinase moiety of the receptor( , ) but its polyIC-polyplex (PPEA) kills EGFR positive tumor cells effectively.

Techniques:

Journal: bioRxiv

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

doi: 10.1101/2025.10.01.679886

Figure Lengend Snippet: The PPEA-polyIC-polyplex selectively kills cell lines overexpressing EGFR. Cells were seeded in duplicates into 96-well plate at a density of 5000 cells in 0.1 ml medium per well and grown overnight. Cells were then transfected with polyIC at the indicated concentrations using the PPEA complexes. PEI-PEG ratio=1:1; w/w ratio PEI: Poly(IC) =0.78. U138MG cells do not express EGFR; U87MGwtEGFR cells express 1x10 6 , A431 express 2-3X106 and MDA-MB-468 express 2x10 6 EGFRs/cell.

Article Snippet: In contrast, the EGFR-binding affibody molecule (Z EGFR1907’ ) exhibits nanomolar binding affinity to EGFR, does not activate the kinase moiety of the receptor( , ) but its polyIC-polyplex (PPEA) kills EGFR positive tumor cells effectively.

Techniques: Transfection

Journal: bioRxiv

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

doi: 10.1101/2025.10.01.679886

Figure Lengend Snippet: 50,000 of MDA-MB-231 and SK-BR-3 cells were seeded into 24-well plates in duplicates and grown overnight with 1 ml medium per well. Cells were then transfected with the PPEA-polyplex at the indicated concentrations. To test the “direct” bystander effect, 48 hours following transfection 0.1 ml of medium from challenged cells was exchanged for 0.1 ml medium from non-transfected cells (“indicator cells) seeded on 96 well plates (4000 cell/well) 24 hours earlier. To analyze the PBMC-mediated bystander effect, 5x105 of PBMCs were added to transfected cells 24 hrs following transfection. After 24 hrs of co-culture, 0.15 ml of medium from the “conditioned medium” was added to untreated cells seeded into 96 well plates (4000 cell/well) 24 hours earlier. Survival of untransfected cells was determined by methylene blue assay, 72 hrs after challenge with the conditioned medium. (A) Shows experiment design. (B) Shows the “direct” bystander effect of medium from polyIC-targeted MDA-MB-231 and SK-BR-3 cells on unchallenged MDA-MB-231 and SK-BR-3, respectively. (C) Shows PBMC-mediated bystander effect of PBMCs co-incubated with polyIC-transfected MDA-MB-231 and SK-BR-3 cells on unchallenged MDA-MB-231 and SK-BR-3, respectively.

Article Snippet: In contrast, the EGFR-binding affibody molecule (Z EGFR1907’ ) exhibits nanomolar binding affinity to EGFR, does not activate the kinase moiety of the receptor( , ) but its polyIC-polyplex (PPEA) kills EGFR positive tumor cells effectively.

Techniques: Transfection, Co-Culture Assay, Incubation