The goal of this proposal is to develop a new therapeutic system to improve the treatment of non-Hodgkin's lymphoma (NHL). Cases of NHL have doubled since 1980 and there are an estimated 69,000 new diagnoses in 2013. B-cell lymphomas constitute 85% of NHL cases;therefore, the new anti-cancer treatment relies on the cross-linking of CD20 receptors on the B-cell surface to induce apoptosis. The therapeutic system has already been shown to induce apoptosis of B-cells in vitro and in vivo. To achieve the goal of the project, we will optimize the therapeutic system for the treatment of NHL. The therapeutic system utilizes a pair of oppositely charged peptides that form an antiparallel coiled coil. The coiled coil peptides (CC1/CC2) form physical crosslinks through biorecognition. Biorecognition leads to crosslinking of CD20 receptors on B-cells. A CD20-specific Fab'fragment is attached to one of the coiled coil peptides (Fab'- CC1), and multiple copies of the complementary peptide are attached to a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (P-CC2;P is the polymer backbone). We hypothesize that increasing the crosslinking of CD20 through conjugate structure modifications of our previous design will result in increased apoptosis. To test our hypothesis and accomplish our goals, we propose to design and synthesize conjugates of varying architecture and coiled coil peptides containing L- and/or D-amino acid residues. These conjugates will be highly tunable, and can self-assemble into highly ordered structures due to specific interactions between peptides. The conjugate biorecognition will be evaluated and design parameters will be identified that enhance biorecognition. Structural features to optimize include: Mw of HPMA backbone, spacing of peptide grafts, and spacer length between peptides and polymer/Fab'. These studies will provide valuable insight into specific design features that improve self-assembly of coiled coil systems in general. Self-assembly of the conjugates at the cell surface and the crosslinking of CD20 will be evaluated using super-resolution optical imaging techniques. Super-resolution imaging can be used to quantify the degree of CD20 clustering. Super-resolution imaging of biomaterial systems will more clearly link biological mechanisms to the overall effect induced by the biomaterial. The relationship between the crosslinking of CD20 and induction of apoptosis will be evaluated. Furthermore, the mechanism of action of a biomaterial can be determined by identifying other proteins or cellular structures involved. Optimized conjugates will then be tested in vivo and the results will be correlated with CD20 clustering results. This new therapeutic system will improve response rates in patients with NHL, and also may be applied to other B-cell related diseases such as multiple sclerosis and rheumatoid arthritis. The biorecognition of coiled-coil peptides at the cell surface to induce cellular signaling is an exciting strategy in treating diseases. Furthermore, using super-resolution imaging to evaluate self-assembly and protein crosslinking is a novel approach in evaluating therapeutics.
This proposal describes a novel therapeutic system, and a novel method to optimize the therapeutic system using super-resolution optical imaging. Optical imaging and image analysis will be used to optimize the therapeutic to induce cell death of B-cells. The proposed project intends to develop a novel therapeutic to improve the treatment of non-Hodgkin Lymphoma.
|Hartley, Jonathan M; Zhang, Rui; Gudheti, Manasa et al. (2016) Tracking and quantifying polymer therapeutic distribution on a cellular level using 3D dSTORM. J Control Release 231:50-9|
|Hartley, Jonathan M; Chu, Te-Wei; Peterson, Eric M et al. (2015) Super-Resolution Imaging and Quantitative Analysis of Membrane Protein/Lipid Raft Clustering Mediated by Cell-Surface Self-Assembly of Hybrid Nanoconjugates. Chembiochem 16:1725-9|