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

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 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

Humanized anti-Sialyl-Tn antibodies for the treatment of ovarian carcinoma.

The expression of Sialyl-Tn (STn) in tumors is associated with metastatic disease, poor prognosis, and reduced overall survival. STn is expressed on ovarian cancer biomarkers including CA-125 (MUC16) and MUC1, and elevated serum levels of STn in ovarian cancer patients correlate with lower five-year survival rates. In the current study, we humanized novel anti-STn antibodies and demonstrated the retention of nanomolar (nM) target affinity while maintaining STn antigen selectivity. STn antibodies conjugated to Monomethyl Auristatin E (MMAE-ADCs) demonstrated in vitro cytotoxicity specific to STn-expressing ovarian cancer cell lines and tumor growth inhibition in vivo with both ovarian cancer cell line- and patient-derived xenograft models. We further validated the clinical potential of these STn-ADCs through tissue cross-reactivity and cynomolgus monkey toxicity studies. No membrane staining for STn was present in any organs of human or cynomolgus monkey origin, and the toxicity profile was favorable and only revealed MMAE-class associated events with none being attributed to the targeting of STn. The up-regulation of STn in ovarian carcinoma in combination with high affinity and STn-specific selectivity of the mAbs presented herein warrant further investigation for anti-STn antibody-drug conjugates in the clinical setting

Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity

ABSTRACT Targeted therapeutics that can differentiate between normal and malignant tumor cells represent the ideal standard for the development of a successful anti-cancer strategy. The Sialyl-Thomsen-nouveau antigen (STn or Sialyl-Tn, also known as CD175s) is rarely seen in normal adult tissues, but it is abundantly expressed in many types of human epithelial cancers. We have identified novel antibodies that specifically target with high affinity the STn glycan independent of its carrier protein, affording the potential to recognize a wider array of cancer-specific sialylated proteins. A panel of murine monoclonal anti-STn therapeutic antibodies were generated and their binding specificity and efficacy were characterized in vitro and in in vivo murine cancer models. A subset of these antibodies were conjugated to monomethyl auristatin E (MMAE) to generate antibody-drug conjugates (ADCs). These ADCs demonstrated in vitro efficacy in STn-expressing cell lines and significant tumor growth inhibition in STn-expressing tumor xenograft cancer models with no evidence of overt toxicity

Binding affinity, cytotoxicity and selectivity of humanized mAbs.

(A) 2G12-2B2 L0H3 (left) and 5G2-1B3 L1H2 (right) flow cytometric results show binding to STn expressing MDA-MB-231 STn+ cells. (B) Humanized mAbs 2G12-2B2 L0H3 (top) and 5G2-1B3 L1H2 (bottom) binding selectivity for STn and Tn by glycan array.</p