hfc fusion protein  (BPS Bioscience)

Journal: bioRxiv

Article Title: An Engineered IGF2 Mutant for Lysosomal Targeting Chimeras Development and Membrane Proteins Degradation

doi: 10.1101/2024.02.20.581320

Figure Lengend Snippet: Wild-type IGF2 promotes proliferation of BT474 and SK-Hep1 cells. (A) Percentage of BT474 cell proliferation over 3 days. BT-474 cells were treated with 10 to 1000 nM of hFc-IGF2 on day 0. The proliferation percentage was measured and recorded. (B) Statistical data on cell proliferation on the first and third days. (C) Live BT474 imaging by IncuCyte throughout the 3-day period following treatment with various concentrations of hFc-IGF2. (D) to (F) Proliferation of SK-Hep1 cells was monitored by IncuCyte throughout the 5-day period following treatment with gradient concentrations of hFc-IGF2.

Article Snippet: We incubated SK-BR-3 cells with varying concentrations of hFc-IGF2 fusion protein and continuously monitored cell growth by IncuCyte (Sartorius, Incucyte S3).

Techniques: Imaging

Journal: bioRxiv

Article Title: An Engineered IGF2 Mutant for Lysosomal Targeting Chimeras Development and Membrane Proteins Degradation

doi: 10.1101/2024.02.20.581320

Figure Lengend Snippet: Initial screening of IGF2 mutants by ELISA and antiproliferation assay. (A) Binding affinity of different hFc-IGF2 mutants were analyzed by ELISA. The IGF2 mutants were tested for their ability to bind to domain 11 of IGF2R. (B) The proliferation of different IGF2 mutants, when fused with pertuzumab, was monitored using the IncuCyte machine. The growth and proliferation of SK-BR-3 cells in response to the different mutants were observed and analyzed. (C) Live SK-BR-3 imaging by IncuCyte at day 4 following treatment with various concentrations of hFc-IGF2. (D) The relative proliferation rate of SK-BR-3 cells was determined by comparing their growth at day1 and day4. The proliferation rates were normalized to the control group treated with PBS at day 4.

Article Snippet: We incubated SK-BR-3 cells with varying concentrations of hFc-IGF2 fusion protein and continuously monitored cell growth by IncuCyte (Sartorius, Incucyte S3).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Imaging

Journal: bioRxiv

Article Title: An Engineered IGF2 Mutant for Lysosomal Targeting Chimeras Development and Membrane Proteins Degradation

doi: 10.1101/2024.02.20.581320

Figure Lengend Snippet: IGF2-M5.6 selectively binds to IGF2R and triggers efficient internalization of extracellular IgG-647. A) IGF2R binding properties of IGF2 analogues. Immunocaptured IGF2R was incubated with increasing concentrations of the following IGF2 analogues: wild-type IGF2 (WT) and IGF2 mutants (M5.1 to M5.10). B) IGF1R binding properties of IGF2 analogues. Immunocaptured IGF1R was incubated with increasing concentrations of the different IGF2 analogues listed above. C) and D) Proliferation of BT-474 and SK-Hep1 cells incubated with different concentrations of hFc-IGF2-M5.6 for 5 days. Cell proliferation was detected by CellTiter-Glo assay (Promega). E) Visualization of Ptz-eiLYTAC-mediated IgG-647 internalization in SK-BR-3 cells by confocal microscopy. The cells were incubated at 37 °C for 1 h with 10 nM pertuzumab (Ptz) or Ptz-eiLYTAC and 20 nM of Alexa Fluor 647-labeled goat anti-human secondary antibody in complete growth media. Cells were further labeled with LysoTracker Green for 30 min and Hoechst for 5 min before imaging.

Article Snippet: We incubated SK-BR-3 cells with varying concentrations of hFc-IGF2 fusion protein and continuously monitored cell growth by IncuCyte (Sartorius, Incucyte S3).

Techniques: Binding Assay, Analogues, Incubation, Glo Assay, Confocal Microscopy, Labeling, Imaging

Journal: bioRxiv

Article Title: An Engineered IGF2 Mutant for Lysosomal Targeting Chimeras Development and Membrane Proteins Degradation

doi: 10.1101/2024.02.20.581320

Figure Lengend Snippet: IGF2-M5.6 has no effect on proliferation of SK-BR-3 cells. (A) Percentage of SK-BR-3 cell proliferation was monitored by IncuCyte throughout the 5-day period following treatment with gradient concentrations of hFc-IGF2-M5.6. The proliferation percentage was measured and recorded. (B) Statistical data on cell proliferation on the first and fifth days.

Article Snippet: We incubated SK-BR-3 cells with varying concentrations of hFc-IGF2 fusion protein and continuously monitored cell growth by IncuCyte (Sartorius, Incucyte S3).

Techniques:

Journal: Discover. Oncology

Article Title: Association between response to anti-PD-1 treatment and blood soluble PD-L1 and IL-8 changes in patients with NSCLC

doi: 10.1007/s12672-023-00641-2

Figure Lengend Snippet: Identification of PD-L1 mAbs and sandwich ELISA. A mAb purity identification after affinity chromatography and SDS‒PAGE. BSA was used as the standard protein for concentration comparison. Bio2F1 and 11E3 are prepared anti-PD-L1 mAbs. And PD-L1-His is the standard for sandwich ELISA. B mAb binding and release curves for Kd values analyzed by ForteBio. 11E3 (upper) and 2F1 (bottom). Curves from top to bottom represent concentrations of PD-L1-His of 100, 50, 25, 12.5, 6.25, 3.13, and 1.78 nM. C mAb blocking ability analyzed by ELISA. 11E3 (left panel), 2F1 (right panel). D mAb blocking ability analyzed by flow cytometry. Left, control; middle 2F1, right, 11E3. E Standard curve of sandwich ELISA detecting functional sPD-L1. F ROC curve of sandwich ELISA. Functional sPD-L1 was obtained from 40 healthy controls and 40 lung cancer patients

Article Snippet: Mouse anti-human PD-L1 monoclonal antibodies (mAbs) were established by immunizing BALB/c (female, 6w) mice with 50 μg/mouse of human PD-L1-hFc fusion protein (Sino Biological Inc.) mixed with Freund’s Complete Adjuvant (Sigma, St Louis, MO, USA), i.p. and s.c.), followed by 3 subsequent i.p. and s.c. of 50 μg/mouse of hPD-L1-hFc given every second week together with Freund’s Incomplete Adjuvant (Sigma).

Techniques: Sandwich ELISA, Affinity Chromatography, Concentration Assay, Binding Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Functional Assay

Journal: Discover. Oncology

Article Title: Association between response to anti-PD-1 treatment and blood soluble PD-L1 and IL-8 changes in patients with NSCLC

doi: 10.1007/s12672-023-00641-2

Figure Lengend Snippet: Correlation between baseline functional sPD-L1 and clinical characteristics. A The difference in PFS between patients with low and high functional sPD-L1 levels was compared (P = 0.1766), and dichotomization was based on the median plasma functional sPD-L1 level. B Baseline functional sPD-L1 was compared between patients with low and high tumor tissue PD-L1 expression (P = 0.0162). C Correlation between tissue PD-L1 and baseline functional sPD-L1 (P = 0.0376, r = 0.3581). D , E , F , Patient baseline functional sPD-L1 levels were compared among different T, N, and M stages, and there was a significant difference between the four subsets in N staging (P = 0.0199). G Comparison of baseline functional sPD-L1 between patients with or without lymph node metastasis (N0 vs. N1 + N2 + N3, P = 0.0037). H Comparison of baseline functional sPD-L1 between patients at different TNM stages (II + III vs. stage IV, P = 0.9071). Comparison of baseline functional sPD-L1 in patients with different tissue types (squamous cell carcinoma vs. adenocarcinoma, P = 0.1730)

Article Snippet: Mouse anti-human PD-L1 monoclonal antibodies (mAbs) were established by immunizing BALB/c (female, 6w) mice with 50 μg/mouse of human PD-L1-hFc fusion protein (Sino Biological Inc.) mixed with Freund’s Complete Adjuvant (Sigma, St Louis, MO, USA), i.p. and s.c.), followed by 3 subsequent i.p. and s.c. of 50 μg/mouse of hPD-L1-hFc given every second week together with Freund’s Incomplete Adjuvant (Sigma).

Techniques: Functional Assay, Expressing

Journal: Discover. Oncology

Article Title: Association between response to anti-PD-1 treatment and blood soluble PD-L1 and IL-8 changes in patients with NSCLC

doi: 10.1007/s12672-023-00641-2

Figure Lengend Snippet: Correlation between baseline functional sPD-L1 and clinical characteristics

Article Snippet: Mouse anti-human PD-L1 monoclonal antibodies (mAbs) were established by immunizing BALB/c (female, 6w) mice with 50 μg/mouse of human PD-L1-hFc fusion protein (Sino Biological Inc.) mixed with Freund’s Complete Adjuvant (Sigma, St Louis, MO, USA), i.p. and s.c.), followed by 3 subsequent i.p. and s.c. of 50 μg/mouse of hPD-L1-hFc given every second week together with Freund’s Incomplete Adjuvant (Sigma).

Techniques: Functional Assay, Expressing