Anti-Human Integrin alpha 4 beta 7 Recombinant Antibody (Etrolizumab) (CAT#: TAB-135)

Figure 1 β7 integrin translocates from the cell surface to the inside of the cell upon in vitro treatment of lymphocytes with etrolizumab-s.

(A) Upper panels: Flow cytometry of peripheral blood mononuclear cells (PBMCs) from patients with Crohn's disease (CD), ulcerative colitis (UC), and control donors (CON) treated with etrolizumab-s for 24 h at 4 and 37°C. Left: Representative flow cytometry. The frequency of 9D8+ staining on CD4+ T cells is indicated. Right: Quantitative flow cytometry showing surface expression of 9D8 at 37°C relative to expression at 4°C (n = 5 per group). Lower panels: Representative (left) and quantitative flow cytometry (right) of cells cultured at 4 and 37°C without etrolizumab-s treatment. (B) Flow cytometry of PBMCs from CD, UC, and CON donors treated with AF-647-labeled etrolizumab-s. Left: Representative flow cytometry. The frequency of etrolizumab-s+9D8- cells among CD4+ T cells is indicated. Right: Quantitative flow cytometry showing the proportion of these cells at 4°C in relation to 37°C (n = 5 per group). (C) Quantitative flow cytometry of PBMCs treated with AF647-labeled etrolizumab-s at 4 or 37°C and subsequently subjected to acid wash (left) or not (right). n = 5 per group; data are normalized to expression at 37°C. ∗∗∗p < 0.001; ns – not significant.

Lichnog, C., Klabunde, S., Becker, E., Fuh, F., Tripal, P., Atreya, R.,... & Chung, S. (2019). Cellular mechanisms of etrolizumab treatment in inflammatory bowel disease. Frontiers in pharmacology, 10, 39.

Figure 2 β7 integrin is internalized by treatment of lymphocytes with etrolizumab(-s).

(A) Left: Representative images showing localization of the fluorescence signal of AF647-labeled etrolizumab-s after treatment of cells from CD, UC, and CON donors at 4 or 37°C for 24 h. Scale bar: 3 μm. Right: Quantification of mean fluorescence intensity (MFI) of AF647 signal in the cytosol relative to the membrane at 4 and 37°C; n = 5 per group. ∗∗∗p < 0.001. (B) Assessment of etrolizumab internalization with ImageStream®. Representative flow cytometry and microscopy results after cell incubation with AF488-labeled etrolizumab at 4°C (upper row) or 37°C (lower row) and without (left panels) or with (right panels) quench procedure. BF, bright field. Data are representative for two independent experiments with a total of eight samples. (C) Representative images showing localization of AF647 fluorescence signal of etrolizumab-s and AF488 fluorescence signal of EEA (left) and LAMP-1 (right) after cell treatment with etrolizumab-s at 4 or 37°C for 24 h. Scale bar = 3 μm. Images are representative for three independent experiments.

Lichnog, C., Klabunde, S., Becker, E., Fuh, F., Tripal, P., Atreya, R.,... & Chung, S. (2019). Cellular mechanisms of etrolizumab treatment in inflammatory bowel disease. Frontiers in pharmacology, 10, 39.

Figure 3 Assessment of etrolizumab-s internalization by STED microscopy.

(A) Representative STED microscopy images showing localization and distribution of β7 integrin on the surface of cells from CD, UC, and CON donors. Additionally, a negative control (NEG) without primary antibody staining is shown. Images are representative for seven independent experiments. (B) Representative images showing etrolizumab-s localization on/in cells incubated with etrolizumab-s at 4°C or cells incubated with etrolizumab-s at 37°C and additionally treated with or without Triton-X. Additionally, a negative control without primary antibody staining is shown. Images are representative for five independent experiments.

Lichnog, C., Klabunde, S., Becker, E., Fuh, F., Tripal, P., Atreya, R.,... & Chung, S. (2019). Cellular mechanisms of etrolizumab treatment in inflammatory bowel disease. Frontiers in pharmacology, 10, 39.

Figure 4 β7 internalization minimizes α4β7 expression on the cell surface.

(A) Representative flow cytometry plot showing expression of α4β7 integrin after treatment with etrolizumab at 4 and 37°C. Percentages indicate α4+β7+ and total α4+ cells. (B) Upper panels: Representative flow cytometry plots showing expression of αE integrin after treatment with etrolizumab at 4 and 37°C. Lower panel: Quantification of β7 mean fluorescence intensity (MFI) of β7+ cells (n = 5). ∗∗p < 0.01.

Lichnog, C., Klabunde, S., Becker, E., Fuh, F., Tripal, P., Atreya, R.,... & Chung, S. (2019). Cellular mechanisms of etrolizumab treatment in inflammatory bowel disease. Frontiers in pharmacology, 10, 39.

Figure 1 Anti-alpha 4 beta 7 Antibody (TAB-135) in ELISA

ELISA analysis of TAB-135 was performed by coating with Human alpha 4 beta 7 Protein.

Figure 2 Anti-alpha 4 beta 7 Antibody (TAB-135) in Dot Blot

Dot Blot analysis of TAB-135 was performed by coating with Human alpha 4 beta 7 Protein.