Ru Zhou

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.

SMAD4-independent activation of TGF-β signaling by MUC1 in a human pancreatic cancer cell line

Pancreatic Ductal Adenocarcinoma (PDA) has a mortality rate that nearly matches its incidence rate. Transforming Growth Factor Beta (TGF-β) is a cytokine with a dual role in tumor development switching from a tumor suppressor to a tumor promoter. There is limited knowledge of how TGF-β function switches during tumorigenesis. Mucin 1 (MUC1) is an aberrantly glycosylated, membrane-bound, glycoprotein that is overexpressed in >80% of PDA cases and is associated with poor prognosis. In PDA, MUC1 promotes tumor progression and metastasis via signaling through its cytoplasmic tail (MUC1-CT) and interacting with other oncogenic signaling molecules. We hypothesize that high levels of MUC1 in PDA may be partly responsible for the TGF-β functional switch during oncogenesis. We report that overexpression of MUC1 in BxPC3 human PDA cells (BxPC3.MUC1) enhances the induction of epithelial to mesenchymal transition leading to increased invasiveness in response to exogenous TGF-β1. Simultaneously, these cells resist TGF-β induced apoptosis by downregulating levels of cleaved caspases. We show that mutating the tyrosines in MUC1-CT to phenylalanine reverses the TGF-β induced invasiveness. This suggests that the tyrosine residues in MUC1-CT are required for TGF-β induced invasion. Some of these tyrosines are phosphorylated by the tyrosine kinase c-Src. Thus, treatment of BxPC3.MUC1 cells with a c-Src inhibitor (PP2) significantly reduces TGF-β induced invasiveness. Similar observations were confirmed in the Chinese hamster ovarian (CHO) cell line. Data strongly suggests that MUC1 may regulate TGF-β function in PDA cells and thus have potential clinical relevance in the use of TGF-β inhibitors in clinical trials.

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 A37: Development and future of CAR T cell therapy for pancreatic ductal adenocarcinoma and triple negative breast cancer

Rationale and Background: Immunotherapy of cancer has gained much attention in the past decade with the development of immune checkpoint inhibitors and chimeric antigen receptor (CAR) technology that can activate and redirect patient T cells to kill tumors that over-express a specific antigen. CARs are fusion receptors that are comprised of an antibody-derived single-chain variable fragment (scFv) coupled via hinge and transmembrane elements to a T cell signaling and co-stimulatory domain. This technology is in its early stages of development and has not been fully exploited for the treatment of metastatic epithelial cancers. We focus our studies on Pancreatic Ductal Adenocarcinomas (PDAC) and Triple Negative Breast Cancer (TNBC). However, if successful, the studies will be applicable to other epithelial tumors. For CAR-T cells to work, and to avoid off target toxicities, both the target antigen and the antibody recognizing the target have to be highly specific. The challenge is that there are few such antigen-antibody combinations for solid tumors. We have recently developed a novel patented antibody (designated TAB 004) that specifically recognizes ONLY the tumor-associated form of MUC1 (tMUC1) but not the normal form of MUC1 (nMUC1) in several subtypes of breast cancers including TNBC and in PDAC. We show compelling data that TAB004 recognizes tMUC1 in >90% of human TNBCs and 85% of PDAC but spares all normal epithelial tissues. The antigenic isoform that TAB004 recognizes is completely hidden in normal epithelia making it extremely safe for development of CAR-T cells. Hypothesis: TNBC and PDAC can be specifically targeted with the tMUC1-CAR-engineered T cells, whilst sparing normal organs. Methodology and Results: We have engineered several TAB-specific CAR constructs using the scFv fragment of TAB 004. Six constructs are developed, 3 for human T cells and 3 for mouse T cells: 1. TAB- CD28-CD3zeta (2nd generation CAR), 2. TAB-CD28-41BB-CD3zeta (3rd generation CAR), and 3. TAB-CD28-OX40-CD3zeta (3rd generation CAR). Data shows that we can successfully engineer human T cells to express the TAB-CAR on their surface and that these engineered T cells can bind specifically to tumor cells expressing the unique tMUC1 epitope, become activated, and effectively kill the tumor cells. We show that these engineered T cells only minimally bind and kill normal epithelial cell lines. However, some of the cell lines are more resistant than others. We are therefore conducting combination therapy with various drugs that are known to enhance immune based therapies including checkpoint inhibitors, COX-2 inhibitors, cyclophosphamide and others. We may also have data to show that the engineered TAB-CAR T cells kill tMUC1-expressing TNBC and PDAC cells in vivo. We will show data that TAB-CAR-T cell kill human PDAC and TNBC cells in vitro and propose to conduct the same in vivo xenograft model of human PDAC and metastatic TNBC. In the following months, we will test if TAB-CAR-T cell retards tumor growth in an orthotopic syngeneic mouse model of PDAC and metastatic BC in human MUC1.Tg immune competent mice. We further propose to demonstrate that the TAB-CAR-T cell can mediate apoptosis in the immune competent KCM mice (KC X human MUC1.Tg mice) that develop spontaneous PDAC and the MMT mice (PyVMT X human MUC1.Tg mice) that develop spontaneous mammary gland tumors. Both models mimic the human disease progression and express human MUC1 in a tissue specific manner. This is important since all normal epithelia in these mice express the nMUC1 except the tumors that expresses the target, tMUC1. Impact: If successful, this project will have a major impact and accelerate progress toward a clinical trial for PDAC and metastatic TNBC. This will be the first attempt to test the efficacy of a CAR-T cell in an immune competent, human MUC1.Tg mouse model that develops spontaneous tumors within the appropriate stromal and hormonal microenvironment. Citation Format: Pinku Mukherjee, Ru Zhou, Mahboubeh Yazdanifar, Das Roy Lopamudra. Development and future of CAR T cell therapy for pancreatic ductal adenocarcinoma and triple negative breast cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A37.

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 P4-09-16: A monoclonal antibody with exceptional specificity across major breast cancer subtypes

Background: Breast cancer (BC) remains the second leading cause of cancer-related deaths for women in the United States and is recognized to be a heterogeneous disease. Advances in technologies such as whole genome sequencing are leading the way to precision medicine and the leading researchers are envisioning personalized therapies in the not too distant future. However, given the diversity of cancer cell populations, that remains a challenging task at best. The tumor form of MUC1 (designated tMUC1), a transmembrane glycoprotein, is aberrantly glycosylated and overexpressed in ∼95% of BC. We have developed an antibody (TAB004) that specifically recognizes tMUC1 across all major subtypes of BC and importantly does not recognize normal breast epithelia. This is a significant development in light of the challenges faced in treating triple negative BC. Methods: A panel of thirty BC cell lines was obtained from ATCC. The following techniques were used to assess the specificity of TAB 004 to the major subtypes based on ER, PR and Her2 expression: 1) Flow cytometry to quantify membrane bound expression of tMUC1 using Cy7-conjugated TAB004; 2) Western blotting to detect molecular weight patterns of tMUC1 in whole cell lysate; 3) A TAB004 based GMP-grade ELISA kit to measure shed tMUC1 in the supernatant and 4) In vivo imaging of tumors in mice using TAB 004 conjugated to Indocyanine Green (ICG). Specificity and sensitivity was further confirmed using primary human serum and tissue samples from all major BC subtypes obtained from bio-repositories at Duke University Cancer Center, Fox Chase Cancer Center and Carolinas Health Care System. Shed tMUC1 in serum samples were tested using the TAB 004 ELISA kit and tissue sections were analyzed using immunohistochemical (IHC) staining with TAB 004 conjugated to HRP. Results: 1) Flow cytometry data shows that TAB 004 recognized tMUC1 on all major BC subtypes: 25 out of 30 BC cell lines tested had higher expression than a normal epithelial breast cell line; 2) Western blotting also detected tMUC1 on all BC subtypes with distinct molecular weight patterns; 3) ELISA showed high levels of shed tMUC1 by most BC cells and correlated with bound/cytoplasmic levels. 4) In vivo imaging shows clear localization of TAB004-ICG to the tumors expressing tMUC1. Primary human breast cancer patient data shows that shed tMUC1 was detected in the serum obtained from all major BC subtypes and showed statistically significant differentiation from normal/benign. IHC results show strong tMUC1 expression in malignant tissue with excellent differentiation from adjacent normal tissue. Conclusion: TAB004 antibody9s extraordinary specificity across major BC subtypes has been confirmed with flow cytometry, western blotting, ELISA and Immunohistochemistry. A number of clinical applications are under development: (a) An ELISA test as a supplement to mammography for the early detection of BC in women with dense breasts; (b) serum monitoring during treatment and to detect disease recurrence; and, (c) targeted antibody-drug/antibody-imaging agent based therapies and imaging modalities particularly for triple negative BC. Citation Format: Das Roy L, Zhou R, Dillon L, Moore LJ, Puri R, Marks JR, Lyerly HK, Mukherjee P. A monoclonal antibody with exceptional specificity across major breast cancer subtypes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-16.

Abstract 26: MUC1 enhances neuropilin-1 signaling in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) has the worst prognosis of all cancers and is the 4th leading cause of cancer-related deaths in the United States. Mucin1 (MUC1) is a transmembrane glycoprotein over-expressed in more than 60% of PDA and its expression correlates with high metastases and poor prognosis. In PDA, there is a correlation between blood vessel density, tumor levels of VEGF, and disease progression. We have recently discovered a novel association between MUC1 and neuropilin-1 (NRP-1) expression. Neuropilin-1 (NRP-1) is a co-receptor for VEGF165 and blockade of NRP-1-VEGF165 interaction has been shown to inhibit angiogenesis and tumor growth in several other cancer types. Thus, we hypothesize that MUC1 induces a pro-angiogenic tumor microenvironment by increasing levels of VEGF and NRP-1, thus enhancing disease progression and metastases. We have generated Muc1-expressing KC and Muc1-null KCKO mouse PDA cell lines for this study. Compared to KCKO, KC cells secrete more VEGF and express higher levels of NRP-1 as well as VEGF receptors (1/2/3). Although the cell culture supernatants from KC do not alter the NRP-1 level on the endothelial line 2H11 cells, they significantly promote the 2H11 cell tube formation. Consistent with the in vitro results, tumor lysates from KC-bearing mice have higher levels of NRP-1 and VEGFR2 and its phosphorylations at Tyr residues 951, 996, and 1175. In human PDA cell lines, inhibition of MUC1 expression by specific siRNA reduced NRP-1 level while overexpression of full-length MUC1 increases NRP-1 expression. This suggests a direct regulation of NRP-1 by MUC1. Furthermore, the over-expression of MUC1 in BxPC3 human PDA cell line induces strong angiogenesis and promotes tumor metastasis in a zebrafish tumor xenograft model. Finally, we report that inhibition of NRP-1 by its specific blocking peptide suppresses BxPC3.MUC1 tumor growth in vivo. Taking together, MUC1 in PDA cells can enhance their own NRP-1 levels and promote the VEGF-VEGFR-NRP-1 signaling, which may favor the creation of a pro-angiogenic microenvironment for tumor growth and metastases. Our findings highlight the potential for MUC1 targeted blockade of VEGF-NRP-1 interaction in PDA by conjugating the NRP-1 inhibitory agents to a MUC1 targeting antibody. Citation Format: Ru Zhou, Jennifer Curry, Priyanka Grover, Lopamudra Das Roy, TinChung Leung, Pinku Mukherjee. MUC1 enhances neuropilin-1 signaling in pancreatic ductal adenocarcinoma. [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 26. doi:10.1158/1538-7445.AM2014-26