Abstract 4699: Single domain antibody (sdAb) localizes in cancer cells to inhibit signal transducer and activator of transcription 3 (STAT3) resulting in therapeutic inhibition of multiple cancers

Abstract

Abstract STAT3 is involved in the pathogenesis of many malignancies, so we developed an anti-STAT3 VHH (variable region of the heavy chain), SBT-100, that internalizes in cancer cells and binds unphosphorylated STAT3 (U-STAT3) and phosphorylated STAT3 (P-STAT3) and results in significant inhibition of multiple cancers. ATCC cell lines for triple negative breast cancers (MDA-MB-231, MDA-MB-468, MDA-MB-453), ER+/PR+ breast cancer (MCF-7), HER2+ breast cancer (BT474), pancreatic cancers (PANC-1, BX-PC3), murine mammary cancer (4T1), STAT3 null cells (PC-3), and castrate-resistant prostate cancer (DU145) were tested. Athymic nude mice were obtained from Envigo. The IL-6 Reporter Cell Assay was obtained from Promega. VEGF inhibition ELISA was done using retinal epithelial cells (ATCC). In vitro growth inhibition was done using a MTT assay. Immunoprecipitation (IP) and western blot studies in MDA-MB-231, PANC-1, HeLa, DU145, 4T1 and PC-3 showed that SBT-100 binds U-STAT3 and P-STAT3. No STAT3 binding was seen in PC-3. Binding to P-STAT3 was seen in lysates from the 4T1 cells, which have constitutively activated STAT3. Since rodent and human STAT3 have a 99% homology, rodents are excellent models for extrapolating to human disease for over production of P-STAT3. Within 24 hrs IL-6 assay showed that SBT-100 blocked the production of IL-6 (p<0.0001) compared to control. The degree of IL-6 suppression was comparable to the negative control, BB1608 (STAT3 inhibitor). VEGF ELISA showed significant (p<0.0001) inhibition of VEGF production within 12 hrs and was maintained for up to 48 hrs. After 3 days with SBT-100 the MTT assay showed growth inhibition (p<0.001) in BT474 (93%), MCF-7 (93%), MDA-MB-231 (77%), MDA-MB-468 (85%), MDA-MB-453 (64%), PANC-1 (79%), BX-PC3 (90%), and DU145 (92%). MDA-MB-231 tumors grown in xenograft athymic mice showed suppression (p<0.001), IHC staining in the cytoplasm, and nucleus after 7 days of SBT-100 treatment. U-STAT3 and P-STAT3 activate genes that promote growth, proliferation, angiogenesis, immune suppression, cancer stem cells, metastasis, and apoptosis inhibition. SBT-100 enters the cancers cells, binds STAT3 and P-STAT3 causing growth inhibition (p<0.001) in MCF-7, BT474, MDA-MB-231, MDA-MB-468, MDA-MB-453, PANC-1, BX-PC3, and DU145. These results suggest that SBT-100 can be developed as a therapeutic for cancers expressing either STAT3 or P-STAT3. Citation Format: Sunanda Singh, Genoveva Murillo, Amanda Rom, Avani Singh, Samara Singh, Meenakshi Parihar, Dong Chen, Rajendra Mehta, Robert Baker, Anjali Singh, Ashutosh S. Parihar. Single domain antibody (sdAb) localizes in cancer cells to inhibit signal transducer and activator of transcription 3 (STAT3) resulting in therapeutic inhibition of multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4699. doi:10.1158/1538-7445.AM2017-4699