http://hdl.handle.net/1721.1/7788 2013-05-21T14:37:14Z http://hdl.handle.net/1721.1/76957 Engineering persistent interleukin-2 for cancer immunotherapy Gai, Shuning Mobilizing the immune system to recognize and destroy tumor cells is a promising strategy for treating cancer. In contrast to standard therapeutic approaches such as surgery, radiation, and chemotherapy, immunotherapy offers the possibility of systemic yet tumor-specific cell killing as well as long-lasting cancer protection. A significant mode of tumor rejection is direct tumor cell killing by immune cells, such as cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. These cell types are stimulated to proliferate by the cytokine interleukin-2 (IL-2). Consequently, IL-2 has been actively pursued as an agent for immunotherapy, either alone or in combination with other therapeutic strategies. IL-2 is characterized by rapid systemic clearance, with a fast-phase serum half-life of 13 minutes and a slow-phase half-life of 85 minutes. We hypothesized that prolonging the persistence of IL-2 at the cell surface or extending its circulation lifetime would increase its immunostimulatory potency. Therefore, we evolved murine IL-2 to bind the alpha subunit of its receptor, known as IL-2Ra or CD25, with 500-fold higher affinity; tethered IL-2 to the surface of T cells via streptavidin sandwiches; and fused IL-2 to the antibody Fc fragment, designated Fc/ IL-2, which extended the slow-phase serum half-life by 15 hours. Compared to free IL-2, Fc/IL-2 fusions induced superior control of solid tumors in mice. Interestingly, combining Fc/IL-2 with an anti-tumor antibody led to potent suppression of tumor growth during treatment. Furthermore, combination therapy protected two of three mice from subsequent tumor re-challenge. Depletion of CTLs or NK cells completely or partially, respectively, abrogated treatment efficacy, suggesting these immune cell types contribute to the anti-tumor response. In the context of Fc fusion, increasing the affinity of IL-2 for CD25 did not further improve efficacy. Ablation of CD25 binding, however, significantly reduced efficacy and also increased treatment toxicity. Since we employed a mutant Fc with disrupted FcyR binding, and hence reduced effector function, and fused IL-2 to mutant Fc monovalently, the significant therapeutic benefit of Fc/IL-2 over free IL-2 likely results from the extension of IL-2 circulation lifetime. We hypothesize that long-circulating IL-2 would potently synergize with other anti-tumor antibodies for effective cancer immunotherapy. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2012.; Cataloged from PDF version of thesis.; Includes bibliographical references (p. 102-109). 2012-01-01T05:00:00Z http://hdl.handle.net/1721.1/76904 Biomaterials for protection and repair of the central nervous system Pritchard, Christopher D., (Christopher David) An injectable hydrogel for controlled release of methylprednisolone was designed based on the inflammatory response during acute spinal cord injury. The gel is injectable through a small gauge needle, cross-links under physiological conditions, and releases methylprednisolone over a time period on the order of weeks. Swelling properties were characterized to address potential safety concerns for potential clinical use. Two studies are presented towards the development of a model Brown-Sequard syndrome and accompanying behavioral and pathological outcome measures for evaluation of biomaterials in vivo. A modified poly(glycerol-co-sebacic acid) membrane was developed using electrospun poly(s-caprolactone) nanofibers. Retinal adhesion and histology was evaluated in vitro. Membranes were evaluated in vivo for their ability to selectively remove photoreceptors in situ and promote survival and integration of retinal transplants. Viscoelastic poly(ethylene glycol) sols were evaluated as potential vitreous substitutes. Finally, a business plan outlines the strategy towards clinical trials for a hydrogel vitreous substitute. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2012.; "June 2012." Cataloged from PDF version of thesis.; Includes bibliographical references. 2012-01-01T05:00:00Z http://hdl.handle.net/1721.1/76903 Controlled release of gentamicin from polyelectrolyte multilayers to treat implant-related infection Moskowitz, Joshua Seth Polyelectrolyte multilayered (PEM) coatings were fabricated to incorporate and release the small, hydrophilic antibiotic gentamicin from implant surfaces for infection control. The use of a cationic hydrolytically cleavable poly(p-amino ester) rendered these films biodegradable, yielding both diffusion-based and surface-erosion based release of this therapeutic. The Layer-by- Layer (LbL) assembly platform was used to create conformal, micron scale reservoirs with highly tunable drug release. Film release profiles were engineered through film architecture design and post-processing crosslinking techniques. Delivery of gentamicin was sustained for weeks, which is a significant improvement from previous gentamicin-releasing LbL systems. To gain better insight on the mechanisms of release and aid in rational film design, a theoretical treatment of the physical system was performed. These results include an analytical mathematical model describing the release of drug per surface area of film as a function of time as well as a computational model that simulates the time-dependent concentration profiles in these LbL systems. These erodible, antibiotic coatings were demonstrated to be bactericidal against Staphylococcus aureus, an infectious microorganism that is highly relevant to implant-related infections. Film degradation products were generally nontoxic toward MC3T3-E1 osteoprogenitor cells. A reproducible in vivo rabbit bone infection model was developed to test the PEM coatings against sterile, uncoated placebos; subsequent in vivo experimentation demonstrated the proof-of-principle that an antibiotic-eluting LbL film can efficaciously treat a pre-existing implant-related infection. One further application was studied which combined the release-based mechanism of these erodible films with a permanent, contact-killing LbL film. This combination has the treatment benefit of an initial burst release of antibiotic, prevents biofilm formation, and reduces the probability of developing antibiotic resistance due to the prolonged presence of sublethal concentrations of gentamicin. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2012.; Cataloged from PDF version of thesis.; Includes bibliographical references (p. 186-203). 2012-01-01T05:00:00Z http://hdl.handle.net/1721.1/76565 Encoded hydrogel microparticles for high-throughput molecular diagnostics and personalized medicine Chapin, Stephen Clifford The ability to accurately detect and quantify biological molecules in complex mixtures is crucial in basic research as well as in clinical settings. Advancements in genetic analysis, molecular diagnostics, and patient-tailored medicine require robust detection technologies that can obtain high-density information from a range of physiological samples in a rapid and cost-effective manner. Compared to conventional microarrays and methods based on polymerase chain reaction (PCR), suspension (particle-based) arrays offer several advantages in the multiplexed detection of biomolecules, including higher rates of sample processing, reduced consumption of sample and reagent, and rapid probe-set modification for customizable assays. This thesis expands the utility of a novel hydrogel-based microparticle array through (1) the creation of a microfluidic, flow-through fluorescence scanner for high-throughput particle analysis, (2) the development of a suite of techniques for the highly sensitive and specific detection of microRNA (miRNA) biomarkers, and (3) the investigation of new methods for directly measuring biomolecules at the single-cell level. Graphically-encoded hydrogel microparticles synthesized from non-fouling, bioinert poly(ethylene glycol) (PEG) and functionalized with biomolecule probes offer great promise in the development of high-performance, multiplexed bioassays. To extend this platform to applications in high-throughput analysis, particle design was optimized to ensure mechanical stability in high-velocity flow systems, and a single-color microfluidic scanner was constructed for the rapid fluorescence interrogation of each particle's spatially-segregated "code" and "probe" regions. The detection advantages of three-dimensional, probe-laden hydrogel scaffolds and the operational efficiencies of suspension array technology were then leveraged for the rapid multiplexed expression profiling of miRNA. The graphical encoding method and ligationbased labeling scheme implemented here allowed for scalable multiplexing with a simple workflow and an unprecedented combination of sensitivity, flexibility, and throughput. Through the rolling circle amplification of a labeling oligonucleotide, it was possible to further enhance the system's sensitivity and resolve single-molecule miRNA binding events on particle surfaces, enabling the first direct detection of low-abundance miRNA in human serum without the need for RNA extraction or target amplification. Finally, by arraying cells and gel particles in polydimethylsiloxane (PDMS) microwells, it was possible to dramatically improve the particles' target capture efficiency and thereby move closer to a regime in which miRNAs and other biological molecules may be directly detected without target amplification from single cells. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2012.; Cataloged from PDF version of thesis.; Includes bibliographical references (p. 141-161). 2012-01-01T05:00:00Z