Abstract 5436: Novel single-domain antibody (sdAb), SBT-100, localizes in the cytoplasm to inhibit IL-6 mediated P-STAT3 nuclear translocation in cancer cells

Abstract BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine that is involved in immune defense and plays an important role in biologic activities of cells including tumor cells. These effects are mediated by several signaling pathways, including the signal transducer and transcription activator 3 (STAT3), which plays a critical role in the development, growth, and metastasis of cancer. Inside cancer cells within the tumor microenvironment IL-6 activates (phosphorylates) STAT3. The P-STAT3 dimers translocate into the nucleus where it turns on many genes (e.g., BCL-XL, MCL-1, Myc, Cyclin D1/D2, VEGF, MMP-2, etc.) that are necessary for tumorigenesis. Since there are no commercially available IL-6 or STAT3 inhibitors, blocking IL-6/STAT3 signaling is a potential therapeutic strategy for cancer. SBT-100 is a novel sdAb which binds P-STAT3 to inhibit its translocation into the nuclei of cancer cells, thereby suppressing the cancers growth. METHODS: Human cancer cell lines obtained from ATCC. The STAT3 reporter assay (Promega, WI). IHC staining with IL-6 stimulation, the primary antibody (Ab) was STAT3 (124H6) mouse mAb (Cell Signaling), secondary Ab was anti-mouse IgG (H&L), Alexa Fluor 488 (Cell Signaling), blocking Ab was SBT-100, and recombinant human IL-6 was from Peprotech. In vitro cancer cell suppression via MTT assay (3 day). Xenograft study, athymic nude mice were obtained from Envigo. RESULTS: The STAT3 reporter assay demonstrates that IL-6 activation of STAT3 is completely inhibited by 100ug/ml of SBT-100 (p<0.0012). SBT-100 shows significant (p<0.001) suppression of ten different human cancers in vitro which have constitutive expression of P-STAT3. Immunohistochemical (IHC) staining demonstrates that SBT-100 crosses the cell membrane and localizes within the cytoplasm of MDA-MB-231 cells. IHC staining of STAT3 shows IL-6 stimulation drives P-STAT3 into the nuclei of HEp-2 and PANC-1 cells and this translocation is inhibited by SBT-100. In vivo xenograft studies demonstrate that SBT-100 gives significant (p<0.001) tumor growth suppression of MDA-MB-231. CONCLUSION: SBT-100 inhibits IL-6 mediated function of P-STAT3 as demonstrated by two different assays. IHC staining demonstrates SBT-100 crosses the cell membrane to localize inside cancer cells. Based on its mechanism of action, SBT-100 can be a potential antibody targeted IL-6/STAT3 therapy for human cancer. Citation Format: Sunanda Singh, Genoveva Murillo, Avani Singh, Samara Singh, Meenakshi Parihar, Rajendra Mehta, Anjali Singh, Ashutosh Parihar. Novel single-domain antibody (sdAb), SBT-100, localizes in the cytoplasm to inhibit IL-6 mediated P-STAT3 nuclear translocation in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5436.