Journal: Cell Death Discovery

Article Title: Antibody and antibody fragments site-specific conjugation using new Q-tag substrate of bacterial transglutaminase

doi: 10.1038/s41420-024-01845-3

Figure Lengend Snippet: A Schematic representation of antibody with engineered glutamine tag (Q-Tag) and microbial transglutaminase mediated conjugation. B SDS-PAGE analysis of AlexaFluor488-cadaverine or MMAE conjugated engineered Trastuzumab-Qtag antibodies: 1-LLQG, 2-Tag2, 3-Tag3, 4-Tag4, 5-Tag5. ID 6: Trastuzumab-Qtag2-MMAE; ID 7: TrastuzumabK453del-Qtag2-MMAE. The reaction products were separated by 10% SDS-PAGE, then visualized by fluorescent detection and silver-nitrate staining. LC light chain, HC heavy chain, HC dimer heavy chain dimer.

Article Snippet: Staining with 0.5 μg of AlexaFluor488 labeled anti-DM1 mouse monoclonal antibody (mab0160, Covalab, France) diluted in PBS was carried out at RT for 1 h in a humid chamber and the slides were subsequently washed with PBS.

Techniques: Conjugation Assay, SDS Page, Staining

Journal: Cell Death Discovery

Article Title: Antibody and antibody fragments site-specific conjugation using new Q-tag substrate of bacterial transglutaminase

doi: 10.1038/s41420-024-01845-3

Figure Lengend Snippet: DAR evaluation by mass analysis of various trastuzumab constructs and payloads.

Article Snippet: Staining with 0.5 μg of AlexaFluor488 labeled anti-DM1 mouse monoclonal antibody (mab0160, Covalab, France) diluted in PBS was carried out at RT for 1 h in a humid chamber and the slides were subsequently washed with PBS.

Techniques: Construct

Journal: Cell Death Discovery

Article Title: Antibody and antibody fragments site-specific conjugation using new Q-tag substrate of bacterial transglutaminase

doi: 10.1038/s41420-024-01845-3

Figure Lengend Snippet: Trastuzumab-Qtag 2 , Fab-Qtag 2 , scFv-Qtag 2 and VHH-Qtag 2 were conjugated with AlexaFluor488-cadaverine and the antigen binding profile was compared to the commercially available Neu-antibody(24D2)-PE. Green, AlexaFluor488 or PE signal; blue, DAPI (nucleus).

Article Snippet: Staining with 0.5 μg of AlexaFluor488 labeled anti-DM1 mouse monoclonal antibody (mab0160, Covalab, France) diluted in PBS was carried out at RT for 1 h in a humid chamber and the slides were subsequently washed with PBS.

Techniques: Binding Assay

Journal: Cell Death Discovery

Article Title: Antibody and antibody fragments site-specific conjugation using new Q-tag substrate of bacterial transglutaminase

doi: 10.1038/s41420-024-01845-3

Figure Lengend Snippet: In vitro killing of ADCs.

Article Snippet: Staining with 0.5 μg of AlexaFluor488 labeled anti-DM1 mouse monoclonal antibody (mab0160, Covalab, France) diluted in PBS was carried out at RT for 1 h in a humid chamber and the slides were subsequently washed with PBS.

Techniques: In Vitro

Journal: Cell Death Discovery

Article Title: Antibody and antibody fragments site-specific conjugation using new Q-tag substrate of bacterial transglutaminase

doi: 10.1038/s41420-024-01845-3

Figure Lengend Snippet: A 2D in vivo fluorescent images of NMRI Nudes mice and B ex vivo fluorescent signal of organs after intravenous injection of AlexaFluor 680 labeled Trastuzumab-Qtag2-MMAE (Mean ± SEM ( n = 4)). Immunofluorescent analysis of BT-474 tumor sections after intraperitoneal injection of C vehicle control or Trastuzumab-Qtag2-DM1 D on day 1 and E day 3. The green signal represents the homing of ADC using AlexaFluor488 labeled anti-DM1 mouse monoclonal antibody. Blue, DAPI (nucleus). F Quantitation of the ADCs concentrations in mouse serum (15 mg/kg of ADC, BT-474-Her2 positive tumor) was performed by competition ELISA assay. 96-well plates were coated with anti-DM1 mAb to capture ADC-DM1 and HRP-labeled DM1. The detected absorbance is inversely proportional to the amount of ADC-DM1 conjugate in the test sample (Mean ± SEM ( n = 3). Statistical significance was evaluated using GraphPad Prism 7.0b (GraphPad Software Inc., San Diego, CA, USA) software using unpaired one tailed t -test, N.S. non significative.

Article Snippet: Staining with 0.5 μg of AlexaFluor488 labeled anti-DM1 mouse monoclonal antibody (mab0160, Covalab, France) diluted in PBS was carried out at RT for 1 h in a humid chamber and the slides were subsequently washed with PBS.

Techniques: In Vivo, Ex Vivo, Injection, Labeling, Control, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Software, One-tailed Test

Journal: Cell Death Discovery

Article Title: Antibody and antibody fragments site-specific conjugation using new Q-tag substrate of bacterial transglutaminase

doi: 10.1038/s41420-024-01845-3

Figure Lengend Snippet: The in vivo efficacy was investigated in SCID mice implanted subcutaneous with BT-474 HER2-positive mammary tumors. A Tumor response was evaluated using single administration of 3 mg/kg dose (trastuzumab ADC or T-DM1) and tumor volume was measured at different time intervals. B Tumor response was evaluated using several administrations of various dose of antibody fragments (trastuzumab -Qtag2 -DM1 or T-DM1) and tumor volume was measured at different time intervals Mean ± SEM ( n = 6). Tumor size was followed over 3 weeks. Statistical significance was calculated by unpaired one tailed t -test using GraphPad Prism 7.0b (GraphPad Software Inc., San Diego, CA, USA) software;. n.s : non significative, * P < 0.05 *** P < 0.001.

Article Snippet: Staining with 0.5 μg of AlexaFluor488 labeled anti-DM1 mouse monoclonal antibody (mab0160, Covalab, France) diluted in PBS was carried out at RT for 1 h in a humid chamber and the slides were subsequently washed with PBS.

Techniques: In Vivo, One-tailed Test, Software