A novel nanodelivery system for combination tumor therapy

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004.Includes bibliographical references (leaves 36-38).Anti-angiogenic therapy offers many benefits over traditional cytotoxic chemotherapy including fewer toxic side effects and the reduced development of drug resistance. Anti-angiogenics alone have not proven effective in inducing tumor regression in the clinic due to both the cytostatic nature of anti-angiogenic therapy and the potential formation of new regions of hypoxia within the tumor after therapy. The new therapeutic paradigm is for combining both anti-angiogenics and traditional cytotoxic agents for a synergistic effect. The efficacy of cytotoxic agents may be reduced after anti-angiogenic therapy, however, due to limited access to tumor vasculature and hypoxia-induced drug resistance. We propose that loading cytotoxic agents within the tumor prior to blood vessel collapse will enable both greater drug accumulation within the tumor as well as a reduction in the formation of therapy-induced regions of hypoxia. We present here a novel nanodelivery vehicle termed a 'nanocell' for the spatio-temporal recruitment of both anti-angiogenics and cytotoxic agents within the solid tumor to achieve this goal. Nanocells consist of a polymeric nanocore encapsulating the cytostatic agent doxorubicin surrounded by a lipid vesicle containing the anti-angiogenic agent combretastatin A4.(cont.) Nanocell treatment resulted in an 88% reduction in tumor size in vivo, compared to a 66% reduction in tumor size after delivering combretastatin A4 lipid vesicles and doxorubicin nanocores simultaneously but separately. Nanocell treatment also resulted in a significant reduction in systemic toxicity, fewer metastases to the lung and liver, and a greater degree of tumor apoptosis.by David A. Eavarone.S.M

A Voxel-Based Monte Carlo Model of Drug Release from Bulk Eroding Nanoparticles

The use of polymeric nanoparticles as drug delivery devices is becoming increasingly prevalent in a variety of therapeutic applications. Despite their widespread clinical use, the factors influencing the release profiles of nanoparticle-encapsulated drugs are still not quantitatively understood. We present here a new, semi-empirical model of drug release from polymeric nanoparticles using a formulation of dexamethasone encapsulated within poly(lactic-co-glycolic acid) to set model parameters. We introduce a three-dimensional voxel-based framework for Monte Carlo simulations that enables direct investigation of the entire spherical nanoparticle during particle degradation and drug release. Due to implementation of this model at the nanoscale, we utilize assumptions that simplify the model while still allowing multi-phase drug release to be simulated with good correlation to experimental results. In the future, emerging mechanistic understandings of nanoparticle drug release may be integrated into this simulation framework to increase predictive power.National Institutes of Health (U.S.) (GM57073

Glycome and Transcriptome Regulation of Vasculogenesis

Background— Therapeutic vasculogenesis is an emerging concept that can potentially be harnessed for the management of ischemic pathologies. The present study elucidates the potential coregulation of vasculogenesis by the heparan sulfate glycosaminoglycan–rich cell-surface glycome and the transcriptome. Methods and Results— Differentiation of embryonic stem cells into endothelial cells in an in vitro embryoid body is paralleled by an amplification of heparan sulfate glycosaminoglycan sulfation, which correlates with the levels of the enzyme N-deacetylase/N-sulfotransferase 1 (NDST1). Small hairpin RNA–mediated knockdown of NDST1 or modification of heparan sulfate glycosaminoglycans in embryonic stem cells with heparinases or sodium chlorate inhibited differentiation of embryonic stem cells into endothelial cells. This was translated to an in vivo zebrafish embryo model, in which the genetic knockdown of NDST1 resulted in impaired vascularization characterized by a concentration-dependent decrease in intersegmental vessel lumen and a large tail-vessel configuration, which could be rescued by use of exogenous sulfated heparan sulfate glycosaminoglycans. To explore the cross talk between the glycome and the transcriptome during vasculogenesis, we identified by microarray and then validated wild-type and NDST1 knockdown–associated gene-expression patterns in zebrafish embryos. Temporal analysis at 3 developmental stages critical for vasculogenesis revealed a cascade of pathways that may mediate glycocalyx regulation of vasculogenesis. These pathways were intimately connected to cell signaling, cell survival, and cell fate determination. Specifically, we demonstrated that forkhead box O3A/5 proteins and insulin-like growth factor were key downstream signals in this process. Conclusions— The present study for the first time implicates interplay between the glycome and the transcriptome during vasculogenesis, revealing the possibility of harnessing specific cellular glyco-microenvironments for therapeutic vascularization

A novel nanoscale delivery system for spatio-temporal delivery of combination chemotherapy

Thesis (Ph. D. in Biomedical Engineering)--Harvard-MIT Division of Health Sciences and Technology, 2009.Cataloged from PDF version of thesis.Includes bibliographical references.In the continuing search for effective treatments for cancer, the emerging model is the combination of traditional chemotherapy with anti-angiogenesis agents that inhibit blood vessel growth. However, the implementation of this strategy has faced two major obstacles. First, the long-term shutdown of tumor blood vessels by the anti-angiogenesis agent can prevent the tumor from receiving a therapeutic concentration of the chemotherapy agent. Second, inhibiting blood supply drives the formation of intra-tumoral hypoxia, or a lack of oxygen, which has been correlated with increased tumor invasiveness and resistance to chemotherapy. In this thesis we report the disease-driven engineering of a drug delivery system, a 'nanocell', which overcomes these barriers unique to solid tumors. The nanocell comprises a nuclear nanoparticle encapsulated within a lipid membrane and is preferentially taken up by the tumor. The nanocell delivers a temporal release of two drugs within the tumor core: the outer lipid envelope first releases an anti-angiogenesis agent, causing a vascular shutdown; the inner nanoparticle, which is trapped inside the tumor, then releases a chemotherapy agent. This focal release within the tumor targets cells most at risk for hypoxia and results in improved therapeutic index with reduced toxicity. The technology can be extended to additional agents, so as to target multiple signaling pathways or distinct tumor compartments, enabling the model of an 'integrative' approach in cancer therapy.(cont.) In the second part of the thesis we report new tools for the optimization of nanocell formulations. We present a new, three-dimensional, voxel-based computational model for Monte Carlo simulations of nanoparticle delivery systems that enables direct investigation of the entire vehicle during particle degradation and drug release. Use of this model in combination with emerging mechanistic understandings of nanoparticle drug release will facilitate optimization of nanocell combination therapy release profiles. We additionally report the generation and characterization of a set of carbohydrate-based chemotherapeutic agents that have the potential for use in nanocells as reduced toxicity alternatives to traditional chemotherapy agents.by David A. Eavarone.Ph.D.in Biomedical Engineerin

Nanomaterials for Neural Interfaces

This review focuses on the application of nanomaterials for neural interfacing. The junction between nanotechnology and neural tissues can be particularly worthy of scientific attention for several reasons: (i) Neural cells are electroactive, and the electronic properties of nanostructures can be tailored to match the charge transport requirements of electrical cellular interfacing. (ii) The unique mechanical and chemical properties of nanomaterials are critical for integration with neural tissue as long-term implants. (iii) Solutions to many critical problems in neural biology/medicine are limited by the availability of specialized materials. (iv) Neuronal stimulation is needed for a variety of common and severe health problems. This confluence of need, accumulated expertise, and potential impact on the well-being of people suggests the potential of nanomaterials to revolutionize the field of neural interfacing. In this review, we begin with foundational topics, such as the current status of neural electrode (NE) technology, the key challenges facing the practical utilization of NEs, and the potential advantages of nanostructures as components of chronic implants. After that the detailed account of toxicology and biocompatibility of nanomaterials in respect to neural tissues is given. Next, we cover a variety of specific applications of nanoengineered devices, including drug delivery, imaging, topographic patterning, electrode design, nanoscale transistors for high-resolution neural interfacing, and photoactivated interfaces. We also critically evaluate the specific properties of particular nanomaterials—including nanoparticles, nanowires, and carbon nanotubes—that can be taken advantage of in neuroprosthetic devices. The most promising future areas of research and practical device engineering are discussed as a conclusion to the review.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/64336/1/3970_ftp.pd

Abstract 3640: Novel humanized anti-Sialyl-Tn, anti-CD3 bispecific antibodies demonstrate tumor and T-cell specificity for immune activation at the tumor site

Abstract Tumor-associated carbohydrate antigens (TACAs) historically have been challenging targets for antibody therapeutics. Sialyl-Tn (STn) is a cancer specific antigen that is expressed on the surface of carcinomas including ovarian, colon, prostate, and pancreatic tumors but is rarely present in normal tissue. STn expression has been linked to innate immune suppression, a chemoresistant phenotype, metastasis, and poor prognosis. Previous attempts to target this antigen in the clinic with synthetic glycan vaccines proved safe but lacked efficacy. We have developed humanized bispecific antibodies targeting STn and CD3 for T-cell recruitment and activation at the tumor site. These bispecific antibodies were selected for optimal tumor targeting using our glycan microarray that enriches for candidates whose binding is protein-independent and glycan specific. STn-selective binding was demonstrated. Current lead candidates exhibited low nanomolar EC50 binding in flow cytometric assays against both STn expressing tumor cells and T cells. Quantification of T-cell activation and T-cell induced tumor killing in vitro provides a basis for the further clinical development of these bispecific antibody candidates. Citation Format: David A. Eavarone, Jillian Prendergast, Patricia E. Rao, Jenna Stein, Jeff Behrens, Daniel T. Dransfield. Novel humanized anti-Sialyl-Tn, anti-CD3 bispecific antibodies demonstrate tumor and T-cell specificity for immune activation at the tumor site [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 3640. doi:10.1158/1538-7445.AM2017-3640</jats:p

Abstract MIP-071: TARGETING A CHEMORESISTANT OVARIAN CANCER CELL POPULATION VIA THE CARBOHYDRATE ANTIGEN SIALYL TN

Abstract Key words: Sialyl Tn, drug resistance, antibody-drug conjugates, cancer stem cells OBJECTIVES: A successful therapeutic strategy for ovarian cancer will require direct targeting of inherently chemoresistant tumor cells which are comprised in part of cancer stem cells (CSCs) that survive current cytotoxic treatment regimes and drive tumor resurgence. The sialyl-Tn (STn) antigen is a carbohydrate moiety present on tumor cells but rarely seen in normal adult tissue. Importantly, STn has been shown to be present on CSCs in pancreatic, colon, and gastric malignancies. Our objective was to assess the expression of STn and the known CSC marker CD133 in human ovarian cancer (OvCa) cell lines and primary serous carcinomas, and evaluate the ability of STn+ and STn- cells to both grow in an anchorage independent manner and survive standard-of-care cytotoxic therapy. Furthermore, we sought to assess the effect of murine and humanized α -STn antibody-drug conjugates (ADCs) on OvCa cells in vitro and tumor viability in vivo. METHODS: STn and CD133 expression in established OvCa cell lines was analyzed by flow cytometry. STn-CD133-, STn+CD133-, STn-CD133+ and STn+CD133+ cells were purified from OVCAR3 and OVCAR4 by FACS, plated in soft agar, and incubated for 21 days. Colony forming efficiency of each sub-population was calculated. Unsorted cells were treated in vitro with either murine α -STn-monomethyl auristatin E (MMAE) ADC or vehicle control and cell viability was assessed by MTT assay. Subsequently, cells were treated in vitro with α -STn-MMAE, paclitaxel and carboplatin, or appropriate controls, and the profile of cells surviving 72 hours post-treatment was determined by flow cytometric analysis. Finally, OVCAR3-derived mouse xenografts were treated with murine and humanized α-STn-MMAE, unconjugated mAbs alone, and vehicle control. Mice were assessed regularly for tumor growth and cytotoxic effects. RESULTS: In the OvCa cell lines OV90, OVCAR3 and OVCAR4, when grown in traditional 2D culture, STn+ cells comprised 98.4%, 40.0%, and 26.4% of the total cell population, respectively. In each of these cell lines, we readily detected STn+CD133+ sub-populations suggesting that STn is expressed on CD133+ ovarian CSCs. Colony formation assays analyzing FACS-purified STn-CD133-, STn+CD133-, STn-CD133+ and STn+ CD133+ sub-populations suggest that STn expression correlates with anchorage independent growth, a characteristic of cell stemness. Paclitaxel and carboplatin treatment in vitro significantly increased the proportion of STn+ and CD133+ cells, demonstrating the chemoresistant characteristics of these cells. Treatment with the murine α-STn-MMAE ADCs reduced the viability of OvCa cell lines in vitro in a dose-dependent manner. Treatment with murine and humanized α -STn- MMAE antibodies in vivo reduced tumor volumes, whereas vehicle treatment did not impede tumor growth. Interestingly, the unconjugated antibody also had a modest negative impact on tumor volume. CONCLUSION: A novel, highly specific STn antibody identifies the STn antigen in OvCa cell lines and patient samples. STn+ and CD133+ cells demonstrate stem-like characteristics such as anchorage-independent growth and chemoresistance. STn ADCs decreased cell viability in vitro and reduced tumor volumes in vivo, suggesting that specific therapeutic targeting of STn in ovarian tumors may be an effective clinical strategy to eliminate quiescent CSCs. Citation Format: B.R. Rueda, K. Starbuck, D. Eavarone, J. Prendergast, J. Stein, R. Foster, J. Behrens. TARGETING A CHEMORESISTANT OVARIAN CANCER CELL POPULATION VIA THE CARBOHYDRATE ANTIGEN SIALYL TN [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-071.</jats:p

Abstract 36: Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates (ADCs) demonstrate tumor specificity <i>in vitro</i> and <i>in vivo</i> antitumor efficacy

Abstract Tumor-associated carbohydrate antigens (TACAs) historically have been challenging targets for antibody therapeutics. Sialyl-Tn (STn) is a cancer specific antigen that is expressed on the cell surface of carcinomas including ovarian, colon, prostate, and pancreatic tumors but is rarely present in normal tissue. STn expression has been linked to innate immune suppression, a chemoresistant phenotype, metastasis, and poor prognosis. Previous attempts to target this antigen in the clinic with synthetic glycan vaccines proved safe but lacked efficacy. We have developed highly selective humanized monoclonal antibodies and antibody drug conjugates (ADCs) targeting TACAs, such as STn. Remarkable sequence homology across all anti-STn mAbs was observed in both heavy and light chains, and hot spots for hypermutation were identified. These antibodies were selected using our glycan microarray that enriches for candidates whose binding is protein-independent, highly selective and demonstrates exceptional target affinity. Lead humanized candidates demonstrated single digit nanomolar EC50s in ELISA/flow cytometric assays, STn selective cell internalization, and STn specific glycan binding on Siamab’s proprietary glycan array. STn binding sites in common tumor lines (ovarian, gastric and breast) were determined per cell and subsequent cytotoxicity assays in these lines demonstrated in vitro efficacy. Tumor microarray experiments revealed membranous staining in cancerous tissues of various indications. Binding studies of anti-STn antibodies to primary human cancer samples by flow cytometry demonstrated that both tumor and Myeloid-Derived Suppressor Cells (MDSC, both myeloid and granulocytic) express STn. In an OVCAR3 xenograft model, 30 days after the last anti-STn ADC dose was given, groups treated (Q7Dx4) exhibited mean tumor volumes below the Day 1 pre-treatment mean tumor volumes (155mm3). Flow cytometric analysis of tumors from these mice demonstrated that anti-STn ADC treatment reduces STn expression on the primary tumor in a dose-dependent manner (Q7Dx4 vs. single dose) compared to the isotype-ADC control. Our data demonstrates that high-affinity, STn-selective mAbs show promise as therapies for solid tumors and could also target MDSCs to promote antitumor immune responses. Citation Format: Jillian M. Prendergast, David A. Eavarone, Patricia E. Rao, Adam D. Curtis, Lindsay S. Shopland, Todd A. Hoffert, Jenna Stein, Jeff Behrens, Daniel T. Dransfield. Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates (ADCs) demonstrate tumor specificity in vitro and in vivo antitumor efficacy [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 36. doi:10.1158/1538-7445.AM2017-36</jats:p