Student Presentation -- Lucas Bennink
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Ph.D. Dissertation Defense, Friday October 19, 2018 -- Engineering Next Generation Collagen Hybridizing Peptides for Targeted Delivery

SMBB 3250, 8:00 am

Speaker: Lucas Bennink. Advisor: Dr. Michael Yu


Abstract:

Collagen is found ubiquitously in the extracellular matrix (ECM) of animals. Although collagen remodeling is tightly controlled in healthy tissues, excessive collagen remodeling is associated with various pathological conditions. Therefore, degraded collagens in the ECMs of pathologic tissues could be a useful biomarker for diseases, but there is a lack of acceptable probes that can target regions of collagen turnover. This is due in part to the degraded unstructured collagen being a poor epitope for conventional protein recognition (e.g., mAbs). Previously, our research group developed collagen hybridizing peptides (short peptide sequences with a high propensity to fold into a triple helix) that can bind to the unstructured collagens by folding into the triple helical secondary protein structure. This unique targeting strategy allowed CHPs to bind to almost any type of denatured collagen regardless of its degradation pathway. This dissertation focuses on developing the next generation CHPs for in vivo collagen targeting, which is made possible by understanding the advantages and drawbacks of conventional CHPs for in vivo use, followed by designing new CHP derivatives and evaluating their performance in vitro and in vivo.

To better understand the in vivo stability of CHPs, we evaluated their proteolytic stability in biological fluids. We demonstrated that CHPs have high stability due to their neutral, hydrophilic, and repetitive sequence containing a high content of proline derivatives. We also discovered that fluorophores that are conjugated to CHPs by common thioether linker could undergo a retro-Michael reaction resulting in off-target accumulation.

The major weakness of CHPs is their inherent ability to self-trimerize which eliminates their propensity to hybridize with denatured collagens. This poses a significant challenge for accurate determination of CHP's pharmacokinetic and biodistribution profiles. We developed a new, non-trimerizing CHP that maintains binding affinity to denatured collagen by incorporating an artificial amino acid, fluoroproline (flp). CD spectroscopy and in vitro collagen binding assays demonstrated that the new (GfO)9 CHP tagged with a near-infrared fluorophore, enables in vivo imaging and semi-quantitative assessment of osteolytic bone lesions in multiple myeloma mouse models.

Lastly, we produced multivalent CHPs based on multi-arm PEG platform for increased collagen binding affinity. Since conventional CHPs quickly refold when templated by a multi-arm backbone, non-trimerizing (GfO)9 was conjugated to 4- and 8-arm PEGs to create multivalent CHP platforms. The CHP-PEG conjugates exhibited increased collagen binding affinity due to the multi-ligand effect and also demonstrated increased mean residence time in target tissues after IV injection. We envision CHPs and CHP-conjugates becoming a powerful new tool for a wide range of biomedical sciences, from the study of ECM biology to the development of clinical imaging probes.