Fig 1.
Expression and purification of the Muc1-Bi-1 and Muc1-Bi-2 from E. coli.
a). The sequences of Muc1-Bi-1 and humanized Muc1-Bi-2. The CDRs of Muc1-VHH and CD16-VHH was grafted to DP-47 reference framework and only a few amino acids were changed (shown as *). b). The diagram of Muc1-Bi-1 and Muc1-Bi-2 constructs. To facilitate protein detection and purification, a His6-tag was added to the c-terminal end. Two amino acids GS were added between anti-Muc1 and anti-CD16 VHHs. c). Coomassie blue–staining of purified proteins (Muc1-Bi-1 or Muc1-Bi-2) after Ni-NTA affinity chromatography. d). Size exclusion chromatography of protein marker (top panel), Muc1-Bi-1 (middle panel), and Muc1-Bi-2 (bottom panel).
Fig 2.
Muc1-Bi-1 and Muc1-Bi-2 can recognize Muc1 positive tumor cells.
a). Flow cytometry analysis of Muc1-Bi-1 and Muc1-Bi-2 on Muc1-negative cell lines HePG2 and CHO, Muc1-positive cell lines, HT29, LS174T, and SKOV3. Red line indicates cells with no staining; green line indicates cells with only anti-His-FITC staining; blue line indicates cells with positive control anti-Muc1 antibody; purple line indicates cells with Muc1-Bi-1 protein; orange line indicates cells with Muc1-Bi-2 protein. b). Quantitative analysis of Muc1-Bi-1 and Muc1-Bi-2 binding of LS174T cells by flow cytometry. c). Western blot analysis of Muc1 expression in different cell lines using Muc1-Bi-1 (top) and Muc1-Bi-2 (bottom). d). ELISA analysis of Muc1-Bi-1 and Muc1-Bi-2 binding on recombinant huMuc1.
Fig 3.
Muc1-Bi-1 and Muc1-Bi-2 mediate specific cytotoxic activities (E:T ratio = 1:1).
Cytotoxicity assays were performed as described in the Materials and Method. Muc1-Bi-1 and Muc1-Bi-2 were incubated with different cell lines in the presence of NK cells. The effector cells (NK cells) (2500 cells/well) and target cells, CHO, HT29, LS174T, and SKOV3 (2500 cells/well) at ratio of 1:1 (E:T ratio = 1:1). Data are means ± SD, * P < 0.05; ** P < 0.01 compared with control.
Fig 4.
Muc1-Bi-1 and Muc1-Bi-2 mediate specific cytotoxic activities (E:T ratio = 10:1).
Cytotoxicity assays were performed as described in the Materials and Method. Muc1-Bi-1 and Muc1-Bi-2 were incubated with different cell lines in the presence of NK cells. The effector cells (NK cells) (25000 cells/well) and target cells, CHO, HT29, LS174T, and SKOV3 (2500 cells/well) at ratio of 10:1 (E:T ratio = 10:1). Data are means ± SD, * P < 0.05; ** P < 0.01 compared with control.
Fig 5.
Muc1-Bi-1 and Muc1-Bi-2 mediate specific cytotoxic activities (E:T ratio = 10:1).
Cytotoxicity assays were performed as described in the Materials and Method. Muc1-Bi-1 and Muc1-Bi-2 were incubated with different cell lines in the presence of PBMCs. The effector cells (PBMCs) (25000 cells/well) and target cells, CHO, HT29, LS174T, and SKOV3 (2500 cells/well) at ratio of 10:1 (E:T ratio = 10:1). Data are means ± SD, * P < 0.05; ** P < 0.01 compared with control.
Fig 6.
Muc1-Bi-1 and Muc1-Bi-2 mediate specific cytotoxic activities in a dose dependent manner.
Cytotoxicity assays were performed as described in the Materials and Method. Muc1-Bi-1 and Muc1-Bi-2 were incubated with different cell lines with or without the presence of NK cells (E:T ratio = 10:1). a). CHO, b). HT29, c). LS174T, d). SKOV3). The concentrations of Muc1-Bi-1 or Muc1-Bi-2 were ranging from 0.1 ng/ml to 10 μg/ml.
Fig 7.
Muc1-Bi-1 and Muc1-Bi-2 inhibit tumor growth in vivo.
NOD/SCID mice (n = 7/group, female) were engrafted subcutaneously with LS174T cells (1×106 cells/mice) (circle, solid line) with or without freshly isolated human PBMCs (5×106 cells/mice). The mice were then treated with Muc1-Bi-1 (20μg/mouse), or Muc1-Bi-2 (5 and 20μg/mouse) as described in the Materials and Methods. For the mice transplanted with LS174T and PBMCs simultaneously, a). LS174T only (circle, solid line), LS174T with PBMCs (square, dash line), LS174T and PBMCs treated with Muc1-Bi-1 (20μg/mouse) (circle, dash line). b). LS174T only (circle, solid line), LS174T with PBMCs (square, dash line), LS174T and PBMCs treated with Muc1-Bi-2 (5μg/mouse) (triangle, dash line), Muc1-Bi-2 (20μg/mouse) (circle, dash line). c). PBMCs were transplanted after tumor size reaches 50–100 mm3, PBS (circle, solid line), GPC3 (square, dash line), Muc1-Bi-1 (20μg/mouse) (circle, dash line). The data represent the average tumor volumes of 6 mice. Error bars represent the standard deviation (**P< 0.01, t test, Muc1-Bi-1 and Muc1-Bi-2 (20μg) vs the other groups).