Journal: Scientific Reports

Article Title: Targeting Tn-positive tumors with an afucosylated recombinant anti-Tn IgG

doi: 10.1038/s41598-023-31195-6

Figure Lengend Snippet: Establishment of FX knockout cell line ( FXKO ) and generation of glycoengineered anti-Tn mAb. ( A ) A depiction of the L-fucose metabolic pathway in mammalian cells. ( B ) The FX gene knockout HEK293 free style cell line ( FXKO ) generated using Crispr/Cas9 system. Total cell extracts ( a ), or cultured media ( b ) from WT and FXKO cells analyzed on SDS-PAGE gel and Western and lectin blots with FX, β-actin, AAL, or ConA. SDS-PAGE gel for cultured media stained with Coomassie brilliant blue (CBB) as a loading control. ( C ) Fucosylation levels on cell surface analyzed by flow cytometry with AAL lectin (a 1:3 dilution ratio starting at 10 μg/mL); ConA used as a control. ( D ) Remab6, a chimeric anti-Tn IgG1 mAb, produced in WT and FXKO cell lines (Remab6-AF), and analyzed on SDS-PAGE gel under reducing (βME+) or non-reducing condition (βME−) stained by CBB (a) , and fucosylation levels on their Remab6 analyzed by Western and lectin blots with human IgG or AAL (b) . ( E ) N-glycome analysis of WT- or Remab6-AF produced as in ( D ) by MALDI-TOF–MS. The relative intensity of the most abundant peak in each spectrum set as 100% ( m/z 1836, WT; m/z 1662, Remab6-AF), and selected peak masses annotated as structural features such as fucosylated (red) and non-fucosylated glycans (black). All images except Western and lectin blot (n = 3) are shown as one representative of two independent experiments (n = 2).

Article Snippet: Both total cell extract and cultured media (~ 30 μg) were boiled in Laemmli sample buffer (Cat#1610747, Bio-Rad) containing 2.5% β-mercaptoethanol, and analyzed on SDS-PAGE gel (Cat#M42012, ExpressPlus™ PAGE Gel, 10 × 8, 4–20%, Genscript), and stained with Coomassie, or transferred to a nitrocellulose membrane (Cat#1704158, Bio-Rad) using Trans-Blot Turbo Transfer System (Bio-Rad).

Techniques: Knock-Out, Gene Knockout, Generated, CRISPR, Cell Culture, SDS Page, Western Blot, Staining, Flow Cytometry, Produced

Journal: Scientific Reports

Article Title: Targeting Tn-positive tumors with an afucosylated recombinant anti-Tn IgG

doi: 10.1038/s41598-023-31195-6

Figure Lengend Snippet: Refucosylation of anti-Tn mAb in FXKO cell line via salvage pathway. ( A ) Remab6-expressing FXKO cells fed with different concentration of L-fucose (starting at 0.1 μM) for 4 days in culture, and analyzed refucosylation levels by flow cytometry with AAL; Remab6-expressing WT transfectant used as a control. One representative of two independent experiments (n = 2). (B) A series of Remab6 produced by cells used in ( A ) analyzed by CBB on SDS-PAGE gel ( a ), or Western and lectin blots with human IgG or AAL ( b ). Remab6 from WT was used as a control. One representative of three independent experiments (n = 3). (C) A series of Remab6 produced and characterized in ( A ) analyzed by MALDI-TOF–MS. The relative intensity of the most abundant peak in each spectrum is set as 100% (m/z 1662, 0.1–5 μM; m/z 1836, 10 or 50 μM), and selected peak masses were annotated to note structural features: fucosylated (red) and non-fucosylated glycans (black) (n = 1). ( D ) % of Refucosylation levels on Remab6 analyzed in ( C ). EC 50 calculated with Prism; see Table for further information.

Article Snippet: Both total cell extract and cultured media (~ 30 μg) were boiled in Laemmli sample buffer (Cat#1610747, Bio-Rad) containing 2.5% β-mercaptoethanol, and analyzed on SDS-PAGE gel (Cat#M42012, ExpressPlus™ PAGE Gel, 10 × 8, 4–20%, Genscript), and stained with Coomassie, or transferred to a nitrocellulose membrane (Cat#1704158, Bio-Rad) using Trans-Blot Turbo Transfer System (Bio-Rad).

Techniques: Expressing, Concentration Assay, Flow Cytometry, Transfection, Produced, SDS Page, Western Blot