PMC

Schematic diagram of the cell signaling pathways that are affected by activated STAT3. It is proposed that many of these pathways interacting with activated STAT3 can be suppressed by anti-STAT3 B VHH.

Effects of anti-STAT3 B VHH in MDA-MB231 xenografts in vivo. (A) Effects of SBT-100 on the tumor size after transplantation of MDA-MB231 in athymic mice treated with either vehicle or anti-STAT3 B VHH. (B) Growth curves of the MDA-MB231 tumors in athymic mice treated with either 1 or 2 mg/kg anti-STAT3 B VHH.

Effects of anti-STAT3 B VHH on the proliferation of breast cancer cell lines. (A) Steroid receptor–positive and negative breast cancer cells were incubated with increasing concentrations of anti-STAT3 B VHH. The cell proliferation was determined by MTT assays as described in the “Methods” section. (B) IC₅₀ determination of ST effects for all the cell lines. The IC₅₀ was determined using the IC₅₀ Tool Kit (http://www.ic50.tk/). MTT indicates 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Binding of STAT3 with VHH13 generated in bacteria and VHH14 derived from mammalian source. (A) Schematic comparison of conventional antibodies and small-molecular-weight camelid antibodies devoid of light chain comprising heavy chain with variable region (VHH). (B) The kinetics profiles of bacterial VHH13-human STAT3. Binding of human STAT3 with anti-STAT3 B VHH was determined using Biacore 3000. For scouting, the sample was allowed to flow over the chip and the binding of sample to the ligand was monitored in real time. The affinity constant (K_D) was determined as a ratio of dissociation and association rates. (C) Western blot showing the immunoprecipitation of various STAT3 VHHs to MDA-MB-231 cells using Dynabeads coupled with STAT3 VHHs, commercially available STAT3 was used as a positive control, and STAT1 as a negative control. The band densities were determined using UN-SCAN-IT software (Silk Scientific Inc., Orem, UT, USA). (D) Comparative expression of STAT3 in breast (MDA-MB-231, 4T1), pancreatic (Panc-1), and prostate (DU145) cancer cells and HeLa cells. Western blot showing the immunoprecipitation of bacterial STAT3 VHH13 to cell lysates from breast, pancreatic and prostate cancer cells as well as HeLa + IFN-γ. For this, cell lysate was incubated with Dynabeads coupled with STAT3 VHH13.