Browsing by Subject "Perlecan"

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    Antibodies In Cancer Therapy: New Targets, Applications And Combination Strategies
    (2019-07) Khanna, Vidhi Devendra
    Over the last decade, antibodies have become an important component in the arsenal of cancer therapeutics. Their high-specificity, low-off target effects, desirable pharmacokinetics and high success rate are a few of the many attributes that make them amenable for development as drugs. The work presented here explores the targeting, mechanisms and use of antibody-based cancer therapy. In the first chapter, we used a phage display-based cell panning procedure to develop two fully humanized antibodies, Tw1S4_6 and Tw1S4_AM6, that bind specifically to HSPG2/perlecan, a protein found to be overexpressed on tumor cells. Immunohistochemistry studies revealed high HSPG2 expression across various tumor sub-types including melanoma, bladder cancer, glioblastoma and ovarian cancer. There was significant correlation between high HSPG2 expression and poor survival in triple negative breast cancer, bladder and ovarian cancers. The data presented here points towards the relevance of HSPG2 as a novel target for not only triple negative breast cancer but other malignancies as well. Based on its over-expression in different solid tumors, we evaluated HSPG2 as a therapeutic target in the second chapter. We observed significant tumor growth inhibition with Tw1S4_AM6 in the triple negative MDA-MB-231-LM2 breast cancer xenograft model. This efficacy was reduced in NSG mice, suggesting NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) as a potential mechanism of action. In vitro studies using human PBMCs confirmed induction of ADCC with anti-HSPG2 antibodies. In addition, conjugation of Tw1S4_AM6 on the surface of polymeric nanoparticles enabled increased tumor cell uptake of nanoparticles, suggesting Tw1S4_AM6 could be valuable as a targeting ligand for drug delivery systems. There is a significant interest in designing therapeutic agents that can enhance ADCC and thereby improve clinical responses with approved antibodies. We have developed a suite of highly substituted imidazoquinolines, which activate TLR 7 and/or 8 and induce significantly higher levels of cytokines compared to the FDA-approved TLR7 agonist, imiquimod. In the third chapter, we evaluated our series of TLR7/8 agonists for their ability to improve ADCC. Our studies show that the second generation TLR 7/8 agonists induce robust pro-inflammatory cytokine secretion and activate NK cells. These agonists also enhanced ADCC in vitro. Finally, we found that these agonists significantly improved the anticancer efficacy of two monoclonal antibodies in vivo. Thus, the work presented here encompasses the three critical aspects of antibody therapeutics: identifying the target, understanding their mechanisms, and leveraging these mechanisms to improve their efficacy.
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    Humanized antibody development using phage display: applications to solid tumor metastasis
    (2016-07) Kalscheuer, Stephen
    The outlook for cancer patients who present with evidence of metastasis is best characterized as a precipitous decline in prognosis and overall survival. This dramatic reduction in survival suggests a need to focus on the development of therapeutic and diagnostic reagents that are tailored to cancer in its disseminated form. In pursuit of this goal, a phage display based phenotype screening platform was developed to generate humanized antibodies for use in both circulating tumor cell detection, and therapeutic intervention, using in vivo models of breast cancer metastasis. A broader perspective on this work is that it highlights methods that focus on biologic drug development based on disease phenotype, as opposed to conventional target-based methods. In the context of metastasis, the present work focused on the relevant cancer cell phenotype, termed epithelial to mesenchymal transition, which is believed to be the driver phenotype of cancer dissemination. Phenotype screening approaches do not require prior knowledge of potential targets, and are thus amenable to cancer biomarker discovery, which in turn can lead to innovative, first-in-class approaches to cancer management. A broadly applicable method for deriving humanized antibodies from cell based phenotype screening was developed. Target deconvolution approaches to identify the binding partner of candidate antibodies were also explored. Finally, the fine tuning of antibody binding affinity via affinity maturation methods was also explored through physiologically based pharmacokinetic modelling, in an attempt to establish optimal targeting affinities for both solid tumors and metastases. The work concludes with in vitro and in vivo characterization of two candidate antibodies as therapeutic agents.