Browsing by Author "Schardt, John"
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Item ENGINEERED MULTIVALENCY FOR ENHANCED AFFIBODY-BASED HER3 CANCER THERAPY(2018) Schardt, John; Jay, Steven M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The receptor tyrosine kinase HER3 is well established as a compelling therapeutic target in numerous cancers, including ovarian cancer. HER3 potently activates the PI3K/Akt pro-survival pathway, mediates drug resistance, and is implicated in cancer progression and poor clinical outcomes. Yet, conventional small molecule- and monoclonal antibody-based approaches have so far failed to yield a widely used therapeutic that directly targets HER3. Here, we investigated a novel approach involving specific, multivalent engagement of HER3 with affibody molecules as an alternative to existing therapeutics. We established that multivalent HER3-targeted affibodies more effectively inhibit neuregulin 1β-mediated HER3 activation compared to monovalent affibodies; these multivalent ligands induced rapid and prolonged HER3 downregulation, indicating a potentially valuable mechanism of action to limit HER3-mediated pro-mitogenic signaling and acquired resistance. HER3-targeted affibodies also proved highly amenable to molecular engineering approaches, as modulation of linker length, valency, and albumin binding domain (ABD) fusion placement allowed for robust retention of ligand bioactivity. We further report significant mechanistic evidence supporting HER3 downregulation as a highly specific phenomenon prompted by HER3 sequestration by multivalent ligands. Most importantly, we show that both monovalent and bivalent HER3-targeted affibody-ABD fusion proteins significantly reduce tumor burden in an adriamycin-resistant ovarian cancer model in mice. Overall, these data serve as compelling evidence for HER3 multivalent ligands as promising experimental therapeutics for the treatment of ovarian cancer as single agents as well as in combination with other drugs. Further, HER3 affibodies represent a promising template for development of targeted therapies or drug conjugates for more powerful ovarian cancer therapy in the future.Item Linker Domain Size Does Not Impact Bivalent HER3 Targeting Affibody Efficacy(2020) Oubaid, Jinan; Schardt, John; Jay, Steven; Jay, Steven; Schardt, JohnThe Epidermal Growth Factor (EGF) family of receptors, also called ErbB or HER family, is a group of tyrosine kinase transmembrane proteins that have many regulatory purposes including regulating cell proliferation and survival. Members of the HER family rely on forming dimers upon ligand binding to promote downstream signaling. Gene mutations can result in the deregulation of the HER receptors, further resulting in cancer. HER3, a receptor that is deregulated in many cancers including ovarian, breast, and lung cancer, has been found to be responsible for drug resistance to therapeutics that currently exist to target other members of the HER family. This can occur through increased phosphorylation and overexpression of the HER3 receptor. There are many HER3 targeted therapeutics, including monoclonal antibodies (mAbs), that are currently in phase 1 and 2 of clinical studies; however, no HER3 targeted therapeutics have been approved by the FDA. In addition to this, previous studies have demonstrated that not every patient will respond to a specific treatment plan or therapeutic; therefore, the development of various treatment options is essential. An engineered protein known as the affibody, which in previous studies has shown to be highly soluble, thermally stable, and small in size allowing for effective tissue penetration, has emerged as a potential therapeutic agent for cancer. In this study, it was found that multivalent affibodies, which are affibodies with more than one binding domain, are more effective at inhibiting HER3 activation, also known as phosphorylation, and inducing HER3 downregulation than monovalent affibodies in multiple cell lines. Inhibiting receptor activation can be effective at reducing cell proliferation and survivability. In addition, other modifications were made to optimize the affibodies, such as altering the length of the linker that tethers the binding domains in a multivalent affibody together, and to test for their efficacy. Finally, an albumin binding domain was incorporated into the affibody design to help increase affibody half-life, which would be essential for in vivo testing.