Shu-ta Wu

A novel association of neuropilin-1 and MUC1 in pancreatic ductal adenocarcinoma: role in induction of VEGF signaling and angiogenesis

We report that Mucin1 (MUC1), a transmembrane glycoprotein that is overexpressed in >80% of pancreatic ductal adenocarcinoma (PDA), induced a pro-angiogenic tumor microenvironment by increasing the levels of neuropilin-1 (NRP1, a co-receptor of vascular endothelial growth factor (VEGF)) and its ligand VEGF. Expression of tumor-associated MUC1 (tMUC1) positively correlated with NRP1 levels in human and mouse PDA. Further, tMUC1hi PDA cells secreted high levels of VEGF and expressed high levels of VEGF receptor 2 (VEGFR2) and its phosphorylated forms as compared with tMUC1low/null PDA. This enabled the tMUC1hi/NRP1hi PDA cells to (a) induce endothelial cell tube formation, (b) generate long ectopic blood vessels and (c) enhance distant metastasis in a zebrafish xenograft model. Concurrently, the proteins associated with epithelial-to-mesenchymal transition, N-cadherin and Vimentin, were highly induced in these tMUC1/NRP1hi PDA cells. Hence, blocking signaling via the NRP1–VEGF axis significantly reduced tube formation, new vessel generation and metastasis induced by tMUC1hi PDA cells. Finally, we show that blocking the interaction between VEGF165 and NRP1 with a NRP1 antagonist significantly reduced VEGFR signaling and PDA tumor growth in vivo. Taken together, our data suggest a novel molecular mechanism by which tMUC1 may modulate NRP1-dependent VEGFR signaling in PDA cells.

Antibody-Guided In Vivo Imaging for Early Detection of Mammary Gland Tumors.

BACKGROUND: Earlier detection of transformed cells using target-specific imaging techniques holds great promise. We have developed TAB 004, a monoclonal antibody highly specific to a protein sequence accessible in the tumor form of MUC1 (tMUC1). We present data assessing both the specificity and sensitivity of TAB 004 in vitro and in genetically engineered mice in vivo. METHODS: Polyoma Middle T Antigen mice were crossed to the human MUC1.Tg mice to generate MMT mice. In MMT mice, mammary gland hyperplasia is observed between 6 and 10 weeks of age that progresses to ductal carcinoma in situ by 12 to 14 weeks and adenocarcinoma by 18 to 24 weeks. Approximately 40% of these mice develop metastasis to the lung and other organs with a tumor evolution that closely mimics human breast cancer progression. Tumor progression was monitored in MMT mice (from ages 8 to 22 weeks) by in vivo imaging following retro-orbital injections of the TAB 004 conjugated to indocyanine green (TAB-ICG). At euthanasia, mammary gland tumors and normal epithelial tissues were collected for further analyses. RESULTS: In vivo imaging following TAB-ICG injection permitted significantly earlier detection of tumors compared with physical examination. Furthermore, TAB-ICG administration in MMT mice enabled the detection of lung metastases while sparing recognition of normal epithelia. CONCLUSIONS: The data highlight the specificity and the sensitivity of the TAB 004 antibody in differentiating normal versus tumor form of MUC1 and its utility as a targeted imaging agent for early detection, tumor monitoring response, as well as potential clinical use for targeted drug delivery.

Early detection of pancreatic cancer in mouse models using a novel antibody, TAB004

Pancreatic ductal adenocarcinoma (PDA) is the fourth-leading cause of cancer death in the United States with a 5-year overall survival rate of 8% for all stages combined. But this decreases to 3% for the majority of patients that present with stage IV PDA at time of diagnosis. The lack of distinct early symptoms for PDA is one of the primary reasons for the late diagnosis. Common symptoms like weight loss, abdominal and back pains, and jaundice are often mistaken for symptoms of other issues and do not appear until the cancer has progressed to a late stage. Thus the development of novel imaging platforms for PDA is crucial for the early detection of the disease. MUC1 is a tumor-associated antigen (tMUC1) expressed on 80% of PDA. The goal of this study was to determine the targeting and detection capabilities of a tMUC1 specific antibody, TAB004. TAB004 antibody conjugated to a near infrared fluorescent probe was injected intraperitoneally into immune competent orthotopic and spontaneous models of PDA. Results show that fluorophore conjugated TAB004 specifically targets a) 1 week old small tumor in the pancreas in an orthotopic PDA model and b) very early pre-neoplastic lesions (PanIN lesions) that develop in the spontaneous PDA model before progression to adenocarcinoma. Thus, TAB004 is a promising antibody to deliver imaging agents directly to the pancreatic tumor microenvironment, significantly affecting early detection of PDA.

Treatment of pancreatic ductal adenocarcinoma with tumor antigen specific-targeted delivery of paclitaxel loaded PLGA nanoparticles

BackgroundPancreatic ductal adenocarcinoma (PDA) remains the most aggressive cancers with a 5-year survival below 10%. Systemic delivery of chemotherapy drugs has severe side effects in patients with PDA and does not significantly improve overall survival rate. It is highly desirable to advance the therapeutic efficacy of chemotherapeutic drugs by targeting their delivery and increasing accumulation at the tumor site. MUC1 is a membrane-tethered glycoprotein that is aberrantly overexpressed in > 80% of PDA thus making it an attractive antigenic target.MethodsPoly lactic-co-glycolic acid nanoparticles (PLGA NPs) conjugated to a tumor specific MUC1 antibody, TAB004, was used as a nanocarrier for targeted delivery into human PDA cell lines in vitro and in PDA tumors in vivo. The PLGA NPs were loaded with fluorescent imaging agents, fluorescein diacetate (FDA) and Nile Red (NR) or isocyanine green (ICG) for in vitro and in vivo imaging respectively or with a chemotherapeutic drug, paclitaxel (PTX) for in vitro cytotoxicity assays. Confocal microscopy was used to visualize internalization of the nanocarrier in vitro in PDA cells with high and low MUC1 expression. The in vivo imaging system (IVIS) was used to visualize in vivo tumor targeting of the nanocarrier. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was used to determine in vitro cell survival of cells treated with PTX-loaded nanocarrier. One-sided t-test comparing treatment groups at each concentration and two-way ANOVAs comparing internalization of antibody and PLGA nanoparticles.ResultsIn vitro, TAB004-conjugated ICG-nanocarriers were significantly better at internalizing in PDA cells than its non-conjugated counterpart. Similarly, TAB004-conjugated PTX-nanocarriers were significantly more cytotoxic in vitro against PDA cells than its non-conjugated counterpart. In vivo, TAB004-conjugated ICG-nanocarriers showed increased accumulation in the PDA tumor compared to the non-conjugated nanocarrier while sparing normal organs.ConclusionsThe study provides promising data for future development of a novel MUC1-targeted nanocarrier for direct delivery of imaging agents or drugs into the tumor microenvironment.

Abstract 4708: A novel anti-MUC1 CAR T cell drives immunity to pancreatic cancer

Background: Pancreatic cancer is the 4th leading cause of cancer deaths in the US with very poor prognosis. Treatment options are limited to surgery and chemo/radiation that often times do not increase survival and are associated with high toxicity. Targeted immune-based therapies have shown some promise but needs further exploration. Mucin 1 (MUC1), a glycoprotein expressed on the apical surface of epithelial cells of most epithelial organs, undergoes hypoglycozylation in tumors. This tumor-form of MUC1 (tMUC1) is over-expressed in 80% of pancreatic ductal adenocarcinomas (PDAC). tMUC1 therefore remains a promising target for therapeutic intervention. We have developed a patented antibody (TAB004) which specifically detects tMUC1 and spares normal MUC1. Using a novel technology, functional fragments of TAB004 antibody (scFv) were incorporated into the chimeric antigen receptor (CAR) construct and used to genetically modify primary human T cells. ScFv domain which recognizes tMUC1 is linked to the co-stimulatory molecules of T cells (CD28 and CD3ζ). When the engineered T cells come in contact with tMUC1 expressing tumor cells, multiple T cell signaling pathways are initiated leading to fully activated cytotoxic T cells that lyse the tumor cells. Methods: Retroviral based technique was used to deliver the CAR gene into human PBMC derived primary T cells. A fluorescent tag (mKate) was fused to the C-terminus of CAR molecules, in order to visualize CAR expression on T cell membrane by fluorescent microscopy and potentially for in vivo tracking. Cytotoxicity was evaluated using co-culture method with varying T cell to target cell ratios followed by MTT assay. Intracellular IFNγ was measured by flow cytometry. Results: tMUC1-CAR-T cells show increased activation and proliferation compared to normal T cells. These cells bind strongly to tMUC1 expressing human pancreatic cancer cells forming immunologic synapse. Minimal binding of the tMUC1-CAR T cells was observed to normal or low MUC1 expressing tumors cells suggesting high specificity of these CAR T cells to tMUC1. CAR expression was distributed evenly on the cell surface of the T cells. Engineered tMUC1-CAR T cells exhibit robust cytotoxicity against a panel of PDA cell lines, associated with high IFNγ release. Fortunately, the same CAR T cells display minimum toxicity against normal epithelial cells. CAR T cell function will be evaluated in the preclinical mouse model of PDA, as single treatment and also in combination with checkpoint inhibitors and chemotherapy drugs. Conclusion: Despite the remarkable successes reported using CAR T cells in clinic, particularly CD19 CAR T for leukemia; some adverse effects have been attributed to this treatment. This highlights the urgent need for developing tumor-specific CAR T cells. This study demonstrates the specificity and effectiveness of tMUC1-CAR T cells against pancreatic cancer cells. Thus, tMUC1 CAR T cells have the potential to be further developed for future clinical use. Citation Format: Mahboubeh Yazdanifar, Ru Zhou, Shu-ta Wu, Priyanka Grover, Pinku Mukherjee. A novel anti-MUC1 CAR T cell drives immunity to pancreatic cancer [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 4708. doi:10.1158/1538-7445.AM2017-4708

Abstract 1323: Targeting tMUC1 for the diagnostics and treatment of pancreatic cancer

Background: Pancreatic Cancer is (PC) is the 4th leading cause of cancer-related deaths in the United States with 94% of PC patients dying within 5 years of diagnosis. Treatment options for PC patients are limited as PC is diagnosed at a late stage from the lack of early symptoms and inaccessibility of the pancreatic region. Typical early detection methods involve ultrasound tests or cholangiopancreatography which can be intrusive or use radioactive materials. Typical treatments involve ablation, chemotherapy, or a combination of chemotherapy with radiation. But these methods can have adverse side and off-target effects due to systemic delivery of drugs There is a need to diagnose pancreatic cancer efficiently and to target the delivery of anti-cancer treatments at optimal dosages to avoid adverse side-effects in patients. Pancreatic Ductal Adenocarcinoma (PDA) represents 90% of PCs and over 80% of PDAs over-express tumor-associated Mucin-1 (tMUC1), a membrane-tethered glycoprotein protein. Tumor-associated MUC1 exhibits changes to its glycosylation pattern that expose its protein core, thus making it identifiable to the TAB004, a specific antibody we developed and have shown to only recognizes this form of MUC1. Thus, we hypothesize that TAB004 antibody can be utilized as a targeting agent to accurately diagnose PDA and to enable the use of highly cytotoxic anti-cancer treatments at localized concentrations, which would make the treatment more effective at lower doses overall. Methods: We determined the specificity and internalization of TAB004 (OncoTab Inc) in several MUC1-expressing PDA cell lines in vitro using confocal and high resolution fluorescence microscopy. NuLink kit (NuChemie) was used in TAB004-PLGA nanoparticle (NPs) conjugation. Confirmation of NPs to TAB004 conjugation (NP-TAB) was determined using flow cytometry. NP-TAB and NPs internalization was compared using confocal microscopy in vitro in several tMUC1-expressing PDA cell lines. Viability of cell lines was determined using the Dojindo CCK -8 Cell Proliferation Assay and Cytotoxicity Assay in response to NPs and NP-TAB treatments. Imaging of mice injected with TAB004 conjugated with ICG (Dojindo ICG labeling Kit-NH2), NPs with ICG, and NP-TAB with ICG was performed using and IVIS Spectrum. Results: TAB004 was specific for and internalized significantly more in tMUC1 expressing cell lines. NP-TAB persist longer in tMUC1 expressing cells in vitro. TAB004 was able to target tMUC1 expressing orthotopic PDA tumors. NP-TAB had significantly increased accumulation at the tumor site than NPs. Further experiments are currently being performed in vivo in an appropriate mouse model of PDA to determine treatment efficacy. Conclusion: TAB004 is a promising targeting agent for diagnosing and treating tMUC1 expressing PDA. Given that tMUC1 is associated with aggressive form of PDA and patients with MUC1-positive PDA have poor prognosis, these results are significant and clinically relevant. Citation Format: Shuta Wu, Anthony J. Fowler, Craig Ogle, Pinku Mukherjee. Targeting tMUC1 for the diagnostics and treatment of pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1323.

Early detection of mammary tumors in vivo using a highly specific tumor antibody.

14 Background: Earlier detection of abnormal cells using target-specific techniques holds great promise. One of those targets Mucin-1 (MUC1) is expressed as a hypo-glycosylated form (i.e., tMUC1) in 95% of breast tumors and plays a crucial role in cancer progression. We have developed Tab004, a monoclonal antibody highly specific to a protein sequence accessible in tMUC1. Here we present data assessing the specificity and sensitivity of Tab004 in vitro and in genetically engineered mice in vivo overtime. METHODS We have developed human MUC1 Tg mice that were bred to mice that carry the oncogene, polyoma middle T antigen driven by the MMTV promoter. These mice designated MMT develop mammary gland tumors spontaneously and expresses the human form of tMUC1. PyVMT and C57Bl/6 served as controls. Mice develop mammary gland hyperplasia between 8-10 weeks of age that progresses to ductal carcinoma in situ by 12-14 weeks and adenocarcinoma by 18-24 weeks. Approximately 40% of the mice develop metastasis to the lung and other organs. The tumor progression appropriately mimics the human disease. MMT mice (n = 20) were injected twice monthly retro-orbitally with 12.5ug (100uL) of Tab004-conjugated to indocyanine green (ICG) and imaged thereafter from 8 to 22 weeks of age. Fluorescence was assessed 4 and 24 hrs post injection using the IVIS system. At euthanasia, tissue was collected for further analyses. Further, human breast tumor and normal mammary epithelial tissues were evaluated by immunohistochemical staining. RESULTS Tab004 specifically recognizes tMUC1 and not normal MUC1. In MMT mice, ICG-conjugated Tab004 allowed early detection of tumors in vivo sparing recognition of normal mammary epithelia in the C57BL/6 mice or in the PyV MT tumors. Detection with ICG-conjugated Tab004 allowed monitoring of tumor progression overtime. Importantly, ICG-conjugated Tab004 permitted significantly earlier detection than physical examination. CONCLUSIONS The data highlight the specificity and the sensitivity of Tab004 in detecting tMUC1 in vitro, in situ and in relevant murine models in vivo. Thus, Tab004 will have significant clinical relevance for development as a targeted imaging agent and in the future for targeted drug delivery.

Abstract 4484: Anti-cancer treatments delivered by antibody guided PLGA nanoparticles

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background Pancreatic Cancer is (PC) is the 4th leading cause of cancer-related deaths in the United States with 94% of PC patients dying within 5 years of diagnosis. There are limited treatment options for PC patients since PC is often diagnosed at a late stage, leaving only chemotherapy or a combination of chemotherapy with radiation as a viable treatment. Standard dosages are usually based on a calculated body surface area (BSA) measurement that correlates with patient blood volume. This type of dosing can lead to suboptimal dosages which can cause overdosing or ineffective treatments. Due to systemic delivery of drugs, current chemotherapies have off-target effects leading to major complications. There is a need to target the delivery of anti-cancer treatments at optimal dosages to avoid adverse side-effects in patients. Pancreatic Ductal Adenocarcinoma (PDA) represents 90% of PCs and over 80% of PDAs over-express tumor-associated Mucin-1 (MUC1), a membrane-tethered glycoprotein protein. Tumor-associated MUC1 exhibits changes to its glycosylation pattern that expose its protein core, thus making it identifiable to the TAB004, a specific antibody we synthesize and have shown to only recognizes this form of MUC1. Poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) are an attractive drug delivery tool. 1) NPs are biodegradable and biocompatible in vivo and are FDA approved, 2) NPs are adaptable to multiple types of drugs/treatments, 3) NPs have the ability to protect drugs from degradation in vivo and allow sustained release, and have modifiable surface properties, which enable site specific targeting. Thus, we hypothesize that PLGA NPs with TAB004 covalently attached as a targeting mechanism will enable the use of highly cytotoxic anti-cancer treatments at localized concentrations that would make the treatment more effective at lower doses, increasing its therapeutic index. Methods We determined the viability of several MUC1-expressing PDA cell lines in vitro in response to treatment with paclitaxel or γ-secretase inhibitor-loaded NPs, drugs alone, and NPs alone, using the MTT assay. Internalization of fluorescein diacetate-NPs (fNPs) was determined using confocal microscopy. Results Efficient internalization of fNPs was observed within 30min of treatment and was saturated at 1hr post treatment. Drug loaded NPs were highly effective in killing MUC1-expressing human and mouse PDA cells lines in vitro and displayed comparable results to drug alone while blank NPs and DMSO controls showed negligible cytotoxicity. The results will be confirmed in vivo in an appropriate mouse model of PDA that expresses human MUC1 transgene. Conclusion TAB004 guided NPs are a promising drug-delivery method for treatment of MUC1-positive PDA. Given that MUC1 is associated with aggressive form of PDA and patients with MUC1-positive PDA have poor prognosis, these results are significant and clinically relevant. Citation Format: Shuta Wu, Anthony Fowler, Craig Ogle, Pinku Mukherjee. Anti-cancer treatments delivered by antibody guided PLGA nanoparticles. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4484. doi:10.1158/1538-7445.AM2014-4484