Development of a novel intracellular targeting nanobody that blocks Cx43-mediated pathological proliferation

|| Project Summary This R21 is in response to FOA PA-20-195/ NOT-HL-21-024: development of new ideas and first-generation prototypes for therapeutics and nanotechnologies as applied to the cardiovascular mission areas of the NHLBI. Our proposed research aims to design and test novel small antibodies (12-15 kDa) called nanobodies.

Nanobodies are unique monoclonal antibodies, found in camelid species, that have the specific advantage of being able to enter the cell. The developed nanobodies will target a specific protein interaction inside smooth muscle cells (SMC) with the aim of disrupting their pathological proliferation. We have identified connexin 43

(Cx43)-cyclin E interactions as a significant contributor to pathological SMC proliferation and cardiovascular disease progression. This interaction occurs inside the cell, so therapeutic approaches to disrupting interactions are very limited. The focus of the research will be to develop Cx43-cyclin E targeting nanobodies as potential

therapeutics in the treatment of cardiovascular disease. Acquired injuries are associated with pathological proliferation. For example, stent placement surgery damages the blood vessel wall, leading to rapid SMC proliferation, a hallmark of stent failure. SMC proliferation thickens the blood vessel walls, called neointima formation, eventually blocking the artery. Current therapies target

general proliferation pathways using adapted chemotherapeutic agents. However, these non-specific agents kill healthy cells as well, reducing vascular healing and leading to long-term failure rates of 25%. By targeting the mediators of only pathological proliferation in SMC, it may be possible to stop neointima formation and promote

the healthy recovery of endothelial cells on the blood vessel wall. In large vessels, Cx43 is expressed only in SMC, and the target site is a post-translational modification that we have shown does not alter Cx43 channel conduction. Preliminary data in this proposal suggest the Cx43-cyclin E interactions occur in models of human SMC proliferation. We have generated novel disruptor peptides

replicating the Cx43-cyclin E binding site and provide data that they can limit neointima in mice and humans. As peptides are of limited therapeutic use, due to in vivo stability issues, we have adapted these peptides for injection into llama, generating nanobodies. The project objectives are to define the best binding nanobodies

from screening, alter these to enable cell entry, validate functions in vitro, and demonstrate the translatable nature in mouse and human models of neointima. This research is specifically responsive to NHLBI-NOT-HL-21-024. Our study aims to produce prototype nanotechnologies (nanobodies) that significantly improve cellular therapeutics (intracellular targets), and

address gaps in the repair and regeneration of vascular disease states. Doing this will achieve more effective therapies for future translation and our data will feed into future NHLBI Catalyze Program applications.