Sunanda Singh1, Genoveva Murillo2, Avani Singh1, Samara Singh1, Meenakshi S. Parihar1, Anjali H. Singh1, Rajendra Mehta2, and Ashutosh S. Parihar1
1Singh Biotechnology, LLC, 14153 Yosemite Drive, Suite 101, Bayonet Point Hospital Medical Complex, Hudson, FL 34667; 2IIT Research Institute, 10 W. 35th Street, Chicago IL 60616.
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.
Sunanda Singh1, Genoveva Murillo2, Avani Singh1, Samara Singh1, Meenakshi S. Parihar1, Anjali H. Singh1, Rajendra Mehta2, and Ashutosh S. Parihar1
1Singh Biotechnology, LLC, 14153 Yosemite Drive, Suite 101, Bayonet Point Hospital Medical Complex, Hudson, FL 34667; 2IIT Research Institute, 10 W. 35th Street, Chicago IL 60616.
Background: Over 90% of pancreatic cancers have KRAS mutations and hyper-expression of P-STAT3 oncoproteins, which if specifically targeted may help treatment of pancreatic cancers. Singh Biotechnology’s proprietary technology engineered SBT-100, a single domain antibody that is bispecific for KRAS & STAT3, which can cross the cell membranes and bind to these intracellular oncoproteins. Combining this targeted therapy with an established chemotherapy, such as gemcitabine, may improve patient’s response to treatment.
Methods: Human pancreatic cancer cells (PANC-1 and BX-PC3) were used. Biacore assay demonstrates SBT-100 binding to KRAS, KRAS (G12D), and STAT3. Immunoprecipitation (IP) and western blot analysis confirmed binding to STAT3 by SBT-100. Pancreatic cancer cells were treated at varying doses of SBT-100 ranging from 0µg/ml to 200µg/ml ± gemcitabine, and after 72 hours growth inhibition was determined by a MTT assay. PANC-1 tumors were grown in athymic nude mice, divided into four groups and staged to a range of 100-150mm3 before treatment. Groups were: vehicle only, SBT-100, gemcitabine, and SBT-100 & gemcitabine. Animals received treatments for 14 days, then monitored for 7 days.
Results: Biacore study shows SBT-100 binds KRAS with an affinity of 10-9M, KRAS (G12D) with 10-8M, and STAT3 with 10-8M. IP and western blot analysis demonstrates that SBT-100 binds P-STAT3. MTT assay demonstrates SBT-100 inhibits the growth of PANC-1 and BX-PC3 (p < 0.001). In PANC1 cells a combination of SBT-100 & gemcitabine demonstrates synergism in inhibiting growth of PANC-1, even at 1/8th the gemcitabine IC50 concentration. PANC-1 xenograft study demonstrates that combination therapy of SBT-100 & gemcitabine is superior to either SBT-100 or gemcitabine alone. Compared to the vehicle group, SBT-100 & gemcitabine is far superior (p < 0.001) and gives statistically significant suppression of pancreatic cancer growth in vivo.
Conclusion: Targeted therapy for KRAS and P-STAT3 expressing tumors with SBT-100 & gemcitabine is synergistic for the treatment of pancreatic cancer. This study suggests that synergism maybe achieved with lower doses of gemcitabine, thereby reducing toxicity in patients.
Sunanda Singh1, Genoveva Murillo2, Avani Singh1, Samara Singh1, Meenakshi S. Parihar1, Anjali H. Singh1, Rajendra Mehta2, and Ashutosh S. Parihar1
1Singh Biotechnology, LLC, 14153 Yosemite Drive, Suite 101, Bayonet Point Hospital Medical Complex, Hudson, FL 34667; 2IIT Research Institute, 10 W. 35th Street, Chicago IL 60616.
Background: Glioblastoma is difficult to treat malignancy that has a high incidence of KRAS mutations (>90%) and hyper-expression of P-STAT3 (>90%). Most chemotherapeutic agents and large biologics (four chain heterotetrameric IgG) cannot cross the BBB. Therefore Singh Biotechnology’s (SBT) propriety technology has developed a novel targeted therapy and engineered SBT-100 a 15 kD single domain antibody (sdAb) that is bispecific for KRAS and STAT3, which can cross the BBB to target gliablastomas.
Methods: Human glioblastoma (U87MG) cell line was used and obtained from ATCC. Biacore affinity assay was used to demonstrate SBT-100 binding to KRAS, KRAS (G12D), and STAT3. The glioblastoma cells were incubated with 0µg/ml to 200µg/ml of SBT-100. MTT assay was performed after 3 days of treatment with SBT-100 and the growth inhibition was calculated. Xenograft (athymic nude mouse) with a well established tumor growing for 6 weeks was injected with SBT-100 intraperitoneally (IP). After 15 minutes the brain of this animal was harvested for immunohistochemical staining.
Results: Biacore studies showed SBT-100 binds KRAS with affinity constant of 10-9M, KRAS (G12D) at 10-8M, and STAT3 at 10-8M. MTT assay reveals 62% (p < 0.01) growth inhibition of U87MG within 3 days. A xenograft mouse with a well established tumor (>150mm3), when injected IP with 5mg/kg of SBT-100 showed localization in the brain within 15 minutes. Therefore SBT-100 (sdAb) crosses the BBB, and shows intracellular localization in the animal’s neurons and glial cells.
Conclusion: SBT-100 significantly inhibits the growth of glioblastoma. KRAS mutations and/or over expression of P-STAT3 in glioblastomas are promising targets for sdAbs like SBT-100, which is bispecific for KRAS and STAT3. SBT-100 crosses the BBB and localizes within the neurons and glial cells of the brain. SBT’s proprietary single domain antibody technology platform used to engineer SBT-100 holds promise for targeting primary brain malignancies, metastatic cancers that go to the brain, and for neurologic disease amenable to targeted therapy. Many chemo-resistant and radiation-resistant cancers use STAT3 as an escape mechanism.
Sunanda Singh1, Genoveva Murillo2, Avani Singh1, Samara Singh1, Meenakshi S. Parihar1, Anjali H. Singh1, Rajendra Mehta2, and Ashutosh S. Parihar1
1Singh Biotechnology, LLC, Tampa Bay Technology Incubator at The University of South Florida, 3802 Spectrum Blvd., Suite 115, Tampa Bay, FL 33612; 2IIT Research Institute, 10 W. 35th Street, Chicago IL 60616; 3Creative Biolabs, 41-1 Ramsey Road, Shirley, NY 11967
Background: 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.
Methods: 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.
Results: 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.
Conclusion: 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.
Sunanda Singh1, Genoveva Murillo2, Avani Singh1, Samara Singh1, Meenakshi S. Parihar1, Anjali H. Singh1, Rajendra Mehta2, and Ashutosh S. Parihar1
1Singh Biotechnology, LLC, Tampa Bay Technology Incubator at The University of South Florida, 3802 Spectrum Blvd., Suite 115, Tampa Bay, FL 33612; 2IIT Research Institute, 10 W. 35th Street, Chicago IL 60616; 3Creative Biolabs, 41-1 Ramsey Road, Shirley, NY 11967
Body: SBT-100 is a single domain antibody (sdAb), developed by Singh Biotechnology, that binds unphosphorylated signal transducer and activator of transcription 3 (STAT3) and phosphorylated STAT3 (P-STAT3). SBT-100 is approximately 13 kD or less than 1/10th the size of a human IgG molecule, and is able to cross the cell membrane to bind intracellular STAT3 and P-STAT3. This in turn inhibits its effects on genes that promote malignant behavior of cancer cells. SBT-100 has a short serum half-life but a long biological half-life. Since certain types of human breast cancers express P-STAT3, we wanted to determine if SBT-100 could inhibit the growth of human breast cancers in vitro and in vivo by studying its effects on MCF-7 (ER+/PR+), BT474 (HER2+), and MDA-MB-231 (TNBC) cells.
Background: Many different types of human cancers (solid tumors, leukemias, and lymphomas) are dependent on constitutive expression of (P-STAT3) for their malignant phenotype. Growth factors, tyrosine kinase receptors, cytokines (IL-6, IL-11, IL-12, IL-23), BCR-ABL, and Src are some ways that STAT3 can be activated. In turn P-STAT3 turns on genes such as Cyclin D1 & D3, MMPs, Bcl-xL, Mcl-1, survivin, VEGF, and HIF-1 alpha. Constitutive expression of P-STAT3 has been shown to promote cancer cell proliferation, survival, angiogenesis, immune suppression, and metastasis. Additionally there is increasing evidence suggesting that unphosphorylated STAT3 contributes to malignant phenotype of cancers. STAT3 is also important for the survival of cancer stem cells as well as for some human breast cancers.
Methods: Immunoprecipitation and Western blot analyses were carried out to test whether SBT-100 binds cytoplasmic STAT3 and P-STAT3 in various malignant cell lines (e.g., MDA-MB-231, PANC-1, DU145, and HeLa). MTT assays were done to determine if SBT-100 could suppress the growth of different types of human breast cancers in vitro. Xenograft cancer models using ER+/PR+ (MCF-7), HER2+ (BT474), and TNBC (MDA-MB-231) cancer cells were used to evaluate treatment with SBT-100 1mg/kg/BID (IV and/or IP route).
Results: Immunoprecipitation and Western blot studies demonstrated that SBT-100 binds to both STAT3 and P-STAT3 in human cancers cells (MDA-MB-231, PANC-1, DU145, and HeLa). In a three day MTT assay, at least 90% growth suppression was achieved for all three subtypes of human breast cancer, which is highly significant. In the xenograft cancer models, SBT-100 (1mg/kg/BID) treatment for 28 days, yield growth suppression as follows: MDA-MB-231 44.8% (p< 0.05) versus its control group and BT474 52% (p< 0.07). While the MCF-7 xenograft cancer model showed no suppression.
Conclusion: Singh Biotechnology’s novel sdAb, SBT-100 suppresses growth of TNBC and HER2+ human breast cancers in vivo and suppresses growth of ER+/PR+, HER2+, and TNBC cells in vitro. The most significant anti-cancer effects of SBT-100 is observed against human TNBC.
Sunanda Singh1, Genoveva Murillo2, Avani Singh1, Samara Singh1, Meenakshi S. Parihar1, Anjali H. Singh1, Rajendra Mehta2, and Ashutosh S. Parihar1
1Singh Biotechnology, LLC, Tampa Bay Technology Incubator at The University of South Florida, 3802 Spectrum Blvd., Suite 115, Tampa Bay, FL 33612; 2IIT Research Institute, 10 W. 35th Street, Chicago IL 60616; 3Creative Biolabs, 41-1 Ramsey Road, Shirley, NY 11967
The transcription factor STAT3 activation, as phosphorylated STAT3 (P-STAT3), is important in the regulation of numerous genes. Some genes once activated produce proteins that promote angiogenesis via proteins such as (VEGF), which are down regulated when STAT3 is blocked. VEGF plays a role in the pathogenesis of the neovascular ("wet") form age-related macular degeneration (AMD), a leading cause of blindness in the elderly. Anti-VEGF treatments offers hope to neovascular AMD patients since it allows a more direct approach to choroidal neovascularization and its selective inhibition. Currently Lucentis® is used as an intraocular injection to treat AMD, a version of Avastin® which is a recombinant, humanized, monoclonal immunoglobulin G1 antibody (149 kD) that inhibits extracellular human VEGF. Singh Biotechnology’s (SBT) sdAb SBT-100 is a biologic that is one-tenth the size (15kD) of a human IgG antibody that binds specifically to intracellular STAT3 which in turn blocks the production of VEGF. In retinal epithelial cells in vitro highly statistical significant (p< 0.0001) inhibition of VEGF is seen within 12 hours and persists for 48 hours with a single treatment. Additionally, SBT-100 has demonstrated significant inhibition of human malignancies in mouse xenografts studies. Therefore, studies are under way to test SBT-100 in vivo for the treatment of AMD in a mouse model.
Sunanda Singh1, Genoveva Murillo2, Avani Singh1, Samara Singh1, Meenakshi S. Parihar1, Anjali H. Singh1, Rajendra Mehta2, and Ashutosh S. Parihar1
1Singh Biotechnology, LLC, Tampa Bay Technology Incubator at The University of South Florida, 3802 Spectrum Blvd., Suite 115, Tampa Bay, FL 33612; 2IIT Research Institute, 10 W. 35th Street, Chicago IL 60616; 3Creative Biolabs, 41-1 Ramsey Road, Shirley, NY 11967
Background: TNBC has limited response to chemotherapy, and poor prognosis when it metastasizes. Activated STAT3 has been shown to play an important role in the pathogenesis of TNBC. We evaluated the hypothesis that inhibition of STAT3 by a sdAb would inhibit growth of TNBC cells in vitro and in mouse xenograft studies.
Methods: Human TNBC cell lines MDA-MB-231, MDA-MB-468, and MDA-MB-453 were grown in culture and treated with varying doses of SBT-100, which specifically binds human STAT3. On day 3 MTT assay were performed to assess growth inhibition. In mouse xenograft studies, human MDA-MB- 231 cells were grown as tumors (size range: 77±7.17 - 104.71±14.64 mm3) in athymic mice and treated with varying doses of SBT-100 or with vehicle intraperitoneally for 14 days and observed for 7 days post treatment. Immunoprecipitation (IP) and western blot studies were performed to assess binding of STAT3 by SBT-100.
Results: After 3 days of SBT-100 treatment in vitro growth suppression was as observed follows: MDA-MB-231, 90% inhibition (p < 0.001); MDA-MB-468, 85% inhibition (p < 0.001), and MDA-MB-453, 64% inhibition(p < 0.001).In the mouse xenograft experiment using MDA-MB-231 tumors,the control group received vehicle only, Group 1 received 1mg/kg/BID, Group 2 received 2mg/kg/BID, and Group 3 received 2mg/kg/day. All treatment groups showed significant tumor size growth suppression and, Groups 1 & 2 showed regression during the 14 day treatment period, which was maintained during the 1 week observation period. No toxicity was observed in the treated mice. IP studies showed that SBT- 100 binds both unphosphorylated and phosphorylated STAT3.
Conclusion: The single domain anti body SBT-100 binds human STAT3,and significantly suppresses the growth of TNBC cells in vitro and in vivo.
Sunanda Singh1, Genoveva Murillo2, Avani Singh1, Samara Singh1, Meenakshi S. Parihar1, Anjali H. Singh1, Rajendra Mehta2, and Ashutosh S. Parihar1
1Singh Biotechnology, LLC, Tampa Bay Technology Incubator at The University of South Florida, 3802 Spectrum Blvd., Suite 115, Tampa Bay, FL 33612; 2IIT Research Institute, 10 W. 35th Street, Chicago IL 60616; 3Creative Biolabs, 41-1 Ramsey Road, Shirley, NY 11967
Background: The primary role of ER+/PR+ and HER2+ in breast cancers is well known. It has been well established that antiestrogens and Herceptin are very efficacious for ER+/PR+ and HER2+ cancers respectively. However, the management of triple negative breast cancer (TNBC) has been challenging. In literature there is strong evidence for the role of activated STAT3 in the pathogenesis of TNBC. What is less understood is whether activated STAT3 plays a significant role in the malignant phenotype of ER+/PR+ and HER2+ breast cancers. Therefore, we evaluated effects of SBT-100, a novel single domain antibody, on ER+/PR+, HER2+, and TNBC subtypes of breast cancers.
Methods: Human breast cancers cells MCF-7 (ER+/PR+), BT474 (HER2+), and MDA-MB-231 (TNBC) were grown in culture. The breast cancers cells were treated at varying doses of SBT-100, that specifically binds to human STAT3, ranging from 0ug/ml to 200ug/ml. MTT assays were performed after 3 days of treatment with SBT-100 and the growth inhibition was calculated. Immunoprecipitation (IP) and western blot analyses were carried out to confirm the binding of STAT3 to SBT-100.
Results: Compared to controls, which received only vehicle, all evaluated cell lines, MCF-7, BT474 and MDA-MB-231(TNBC)cells,demonstrated>90% inhibition of cell proliferation(p< 0.001) at 100ug/ml. The IC50’s of SBT-100 for MCF-7 is 986nM,BT474 is 1.68uM, and MDA-MB-231 is 666nM. Immunoprecipitation and western blot analyses showed that SBT-100 binds to both the unphosphorylated and phosphorylated STAT3.
Conclusion: These results indicated that the novel single domain antibody, SBT-100, binds to human STAT3 resulting in statistically significant growth suppression in ER+/PR+, HER2+, and TNBC breast cancers. SBT-100 is being further evaluated in depth for possible management of breast cancers.
Sunanda Singh1, Genoveva Murillo2, Avani Singh1, Samara Singh1, Meenakshi S. Parihar1, Anjali H. Singh1, Rajendra Mehta2, and Ashutosh S. Parihar1
1Singh Biotechnology, LLC, 14153 Yosemite Drive, Suite 101, Bayonet Point Hospital Medical Complex, Hudson, FL 34667; 2IIT Research Institute, 10 W. 35th Street, Chicago IL 60616.
BACKGROUND: Despite nearly forty years of research, scientists have failed to develop a clinically viable therapy against KRAS, one of the deadliest families of cancer-causing proteins. Mutations in KRAS are prevalent amongst the top three most deadly cancer types in the United States: pancreatic (95%), colorectal (45%), and lung (35%). KRAS has been thought to be undruggable due to: 1) its intracellular location and lack of binding pockets for small molecules; 2) the high (pM) affinity of RAS for GTP precludes direct targeting of the nucleotide binding pocket; 3) high intracellular concentrations of GTP (uM) inhibits competition for the nucleotide-binding pocket by small molecules; and 4) possible toxicity. Mutations of KRAS result in it being perpetually turned on to propagate signal down the MAPK pathway. This results in constant production of P-ERK and plays an important role in malignant development. To overcome these challenges, Singh Biotechnology has developed SBT-100 a first in class & best in class novel sdAb that penetrates the cell membrane to bind KRAS to inhibit its GTPase activity.
METHODS: Human cancer cell lines were purchased from ATCC. BIAcore affinity studies were conducted by Precision Antibody. KRAS GTPase assay was purchased from Promega. Levels of P-ERK were determined using western blots. In vitro cell growth suppression was tested with MTT assay. Athymic nude mice for xenograft studies were purchased from Envigo.
RESULTS: SBT-100 binds KRAS with KD=10-9 and KRAS(G12D) with KD=10-7 as demonstrated by BIAcore affinity assay. Both SBT-100 and SBT-102 significantly inhibit KRAS GTPase activity in vitro and inhibition is comparable to polyclonal antibody to KRAS. Growth of MDA-MB-231 cells with KRAS(G13D) mutation and PANC-1 cells with KRAS(G12D) mutation are significantly decreased in the MTT assay when incubated with SBT-100. Additionally, same cell lines have significantly decreased P-ERK expression when cultured with SBT-100. Xenograft studies demonstrate significant growth suppression of MDA-MB-231 and PANC-1 when treated with SBT-100 in vivo.
CONCLUSION: SBT-100, crosses the cell membrane, binds to KRAS intracellularly and its most common mutant with nanomolar affinity, inhibits KRAS GTPase activity, downregulates P-ERK signaling, and suppresses the growth of cancers cells in vitro and in vivo without showing any toxic effects.
Sunanda Singh1, Genoveva Murillo2, Avani Singh1, Samara Singh1, Meenakshi S. Parihar1, Anjali H. Singh1, Rajendra Mehta2, and Ashutosh S. Parihar1
1Singh Biotechnology, LLC, Tampa Bay Technology Incubator at The University of South Florida, 3802 Spectrum Blvd., Suite 115, Tampa Bay, FL 33612; 2IIT Research Institute, 10 W. 35th Street, Chicago IL 60616; 3Creative Biolabs, 41-1 Ramsey Road, Shirley, NY 11967
Background: TNBC has limited response to chemotherapy, and poor prognosis when it metastasizes. Activated STAT3 has been shown to play an important role in the pathogenesis of TNBC. We evaluated the hypothesis that inhibition of STAT3 by a sdAb would inhibit growth of TNBC cells in vitro and in mouse xenograft studies. Methods: Human TNBC cell lines MDA-MB-231, MDA-MB-468, and MDA-MB-453 were grown in culture and treated with varying doses of SBT-100, which specifically binds human STAT3. On day 3 MTT assay were performed to assess growth inhibition. In mouse xenograft studies, human MDA-MB- 231 cells were grown as tumors (size range: 77±7.17 - 104.71±14.64 mm3) in athymic mice and treated with varying doses of SBT-100 or with vehicle intraperitoneally for 14 days and observed for 7 days post treatment. Immunoprecipitation (IP) and western blot studies were performed to assess binding of STAT3 by SBT-100.Results: After 3 days of SBT-100 treatment in vitro growth suppression was as observed follows: MDA-MB-231, 90% inhibition (p < 0.001); MDA-MB-468, 85% inhibition (p < 0.001), and MDA-MB-453, 64% inhibition(p < 0.001).In the mouse xenograft experiment using MDA-MB-231 tumors,the control group received vehicle only, Group 1 received 1mg/kg/BID, Group 2 received 2mg/kg/BID, and Group 3 received 2mg/kg/day. All treatment groups showed significant tumor size growth suppression and, Groups 1 & 2 showed regression during the 14 day treatment period, which was maintained during the 1 week observation period. No toxicity was observed in the treated mice. IP studies showed that SBT- 100 binds both unphosphorylated and phosphorylated STAT3.
Conclusion: The single domain anti body SBT-100 binds human STAT3,and significantly suppresses the growth of TNBC cells in vitro and in vivo.
Sunanda Singh1, Genoveva Murillo2, Avani Singh1, Samara Singh1, Meenakshi S. Parihar1, Anjali H. Singh1, Rajendra Mehta2, and Ashutosh S. Parihar1
1Singh Biotechnology, LLC, Tampa Bay Technology Incubator at The University of South Florida, 3802 Spectrum Blvd., Suite 115, Tampa Bay, FL 33612; 2IIT Research Institute, 10 W. 35th Street, Chicago IL 60616; 3Creative Biolabs, 41-1 Ramsey Road, Shirley, NY 11967
Background: The primary role of ER+/PR+ and HER2+ in breast cancers is well known. It has been well established that antiestrogens and Herceptin are very efficacious for ER+/PR+ and HER2+ cancers respectively. However, the management of triple negative breast cancer (TNBC) has been challenging. In literature there is strong evidence for the role of activated STAT3 in the pathogenesis of TNBC. What is less understood is whether activated STAT3 plays a significant role in the malignant phenotype of ER+/PR+ and HER2+ breast cancers. Therefore, we evaluated effects of SBT-100, a novel single domain antibody, on ER+/PR+, HER2+, and TNBC subtypes of breast cancers.
Methods: Human breast cancers cells MCF-7 (ER+/PR+), BT474 (HER2+), and MDA-MB-231 (TNBC) were grown in culture. The breast cancers cells were treated at varying doses of SBT-100, that specifically binds to human STAT3, ranging from 0ug/ml to 200ug/ml. MTT assays were performed after 3 days of treatment with SBT-100 and the growth inhibition was calculated. Immunoprecipitation (IP) and western blot analyses were carried out to confirm the binding of STAT3 to SBT-100.
Results: Compared to controls, which received only vehicle, all evaluated cell lines, MCF-7, BT474 and MDA-MB-231(TNBC)cells,demonstrated>90% inhibition of cell proliferation(p< 0.001) at 100ug/ml. The IC50’s of SBT-100 for MCF-7 is 986nM,BT474 is 1.68uM, and MDA-MB-231 is 666nM. Immunoprecipitation and western blot analyses showed that SBT-100 binds to both the unphosphorylated and phosphorylated STAT3.
Conclusion: These results indicated that the novel single domain antibody, SBT-100, binds to human STAT3 resulting in statistically significant growth suppression in ER+/PR+, HER2+, and TNBC breast cancers. SBT-100 is being further evaluated in depth for possible management of breast cancers.
Sunanda Singh1, Genoveva Murillo2, Avani Singh1, Samara Singh1, Meenakshi S. Parihar1, Anjali H. Singh1, Rajendra Mehta2, and Ashutosh S. Parihar1
1Singh Biotechnology, LLC, Tampa Bay Technology Incubator at The University of South Florida, 3802 Spectrum Blvd., Suite 115, Tampa Bay, FL 33612; 2IIT Research Institute, 10 W. 35th Street, Chicago IL 60616; 3Creative Biolabs, 41-1 Ramsey Road, Shirley, NY 11967
Background: The primary role of ER+/PR+ and HER2+ in breast cancers is well known. It has been well established that antiestrogens and Herceptin are very efficacious for ER+/PR+ and HER2+ cancers respectively. However, the management of triple negative breast cancer (TNBC) has been challenging. In literature there is strong evidence for the role of activated STAT3 in the pathogenesis of TNBC. What is less understood is whether activated STAT3 plays a significant role in the malignant phenotype of ER+/PR+ and HER2+ breast cancers. Therefore, we evaluated effects of SBT-100, a novel single domain antibody, on ER+/PR+, HER2+, and TNBC subtypes of breast cancers.
Methods: Human breast cancers cells MCF-7 (ER+/PR+), BT474 (HER2+), and MDA-MB-231 (TNBC) were grown in culture. The breast cancers cells were treated at varying doses of SBT-100, that specifically binds to human STAT3, ranging from 0ug/ml to 200ug/ml. MTT assays were performed after 3 days of treatment with SBT-100 and the growth inhibition was calculated. Immunoprecipitation (IP) and western blot analyses were carried out to confirm the binding of STAT3 to SBT-100.
Results: Compared to controls, which received only vehicle, all evaluated cell lines, MCF-7, BT474 and MDA-MB-231(TNBC)cells,demonstrated>90% inhibition of cell proliferation(p< 0.001) at 100ug/ml. The IC50’s of SBT-100 for MCF-7 is 986nM,BT474 is 1.68uM, and MDA-MB-231 is 666nM. Immunoprecipitation and western blot analyses showed that SBT-100 binds to both the unphosphorylated and phosphorylated STAT3.
Conclusion: These results indicated that the novel single domain antibody, SBT-100, binds to human STAT3 resulting in statistically significant growth suppression in ER+/PR+, HER2+, and TNBC breast cancers. SBT-100 is being further evaluated in depth for possible management of breast cancers.