Advisor: Dr. John Blazeck (School of Chemical and Biomolecular Engineering, Georgia Institute of Technology)

Committee Members:

Dr. Julie Champion (School of Chemical and Biomolecular Engineering, Georgia Institute of Technology)

Dr. Gabe Kwong (Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology & Emory University)

Dr. Raquel Lieberman (School of Chemistry and Biochemistry, Georgia Institute of Technology)

Dr. Amit Reddi (School of Chemistry and Biochemistry, Georgia Institute of Technology)

Creating and enhancing protease-responsive masked chimeric antigen receptors using engineering platforms in yeast

Immunotherapies such as chimeric antigen receptor (CAR)-T cell therapy have emerged as promising cancer treatments that can target tumors by binding to specific antigen targets. While these antigens are highly overexpressed in solid tumors, they are also expressed at low levels in healthy tissue, leading to the ‘on-target, off-tumor’ effect, in which CAR-T cells kill both healthy and cancerous cells. One strategy to combat this issue is through the conditional activation of therapeutics by the fusion of peptide masks to a protein drug. Masks block the binding of a protein to its intended target, and can be removed by proteases, which are overexpressed by cancers. 

We have developed a yeast display-based platform for the discovery of protease-removable peptide masks and have successfully isolated masks for a variety of cancer-relevant targets. However, some of these peptides have weak masking ability, only partially blocking target binding, while others do not fully unmask or allow for adequate restoration of binding upon protease treatment. Thus, we will first create a diversifying, dual adenine/cytosine base editor in yeast that can be used as a tool for in vivo mutagenesis for the improvement of mask affinity and ability to block antigen binding. Next, we will study the activity and specificity of proteases to allow for cancer-relevant proteolytic removal of masks. Finally, we will improve the efficiency of mask removal with the incorporation of protease exosite-binders into our masked CAR design. Overall, this project aims to create strong masks for cancer-targeting CARs which can be efficiently removed by tumor-associated proteases.