To access this work you must either be on the Smith College campus OR have valid Smith login credentials.

On Campus users: To access this work if you are on campus please Select the Download button.

Off Campus users: To access this work from off campus, please select the Off-Campus button and enter your Smith username and password when prompted.

Non-Smith users: You may request this item through Interlibrary Loan at your own library.

Publication Date

2023-05-15

First Advisor

Sarah J. Moore

Document Type

Honors Project

Degree Name

Bachelor of Arts

Department

Biochemistry

Keywords

protein engineering, cancer precision medicine

Abstract

Although cancer has been researched for decades with the hopes of finding a cure, there are currently only a few targeted cancer therapeutics that are approved for routine clinical use. Cancer is a devastating disease that can rapidly metastasize without early detection and treatment. There is a critical unmet need for precision medicine which allows patients to receive treatment based on the molecular characteristics of their disease. The benefits of molecular targeting include higher efficacy rates and fewer side effects for patients. While there are antibody therapeutics used as targeting agents, their complex structure and high production costs limit their applications for various diseases. Antibodies have long circulation times in the human body, are poor molecular imaging agents, and have diffusion limitations where they often do not reach the center of a dense tumor. Non-antibody protein scaffolds have a high potential for solving these issues due to their smaller size, range of possible modifications, and lower production costs. Fibronectin type III (Fn3) proteins, specifically, are desirable imaging and targeting agents because of their ability to clear from non-targeted tissues rapidly while accumulating in the tumor. The motivation for this research is to develop a protein-drug conjugate targeted for cancer cells using an Fn3 scaffold. Past students in the Moore Lab have engineered Fn3 proteins that have a high binding affinity to mesothelin (MSLN) and induce apoptosis of MSLN-positive tumor cells. MSLN is a cell surface protein that is highly expressed in cancerous tissues compared to normal tissues and correlates with poor prognosis. When MSLN interacts with tumor cell surface biomarker MUC16, there is an increase in metastasis. The goals of this thesis were to utilize thiol chemistries to conjugate a maleimide fluorophore to engineered Fn3 proteins as a model for protein-drug conjugates and establish a spheroid model system for in vitro characterization of such conjugates. A cysteine-containing tag (cys-tag) was added to the C-terminus of the Fn3 protein to ensure MSLN binding, mediated by loops proximal to the N-terminus, was unaffected. The cys-tag sequence was added to various generations of Fn3 proteins using recombinant DNA technology. Transformations into two E. coli strains were performed to generate high-quality plasmid DNA and for high-yield protein expression. After large-scale protein purification and lyophilization, concentrated proteins were reacted with Alexa Fluor 488 (AF488) to confirm thiol reactivity. Flow cytometry assays and immunohistochemistry staining of triple-negative breast cancer cell line, HCC1806, confirmed MSLN expression and successful generation of spheroids. Future work will include trafficking assays to visualize the internalization of potential anti-MSLN Fn3 molecules.

Rights

©2023 Vivien Qiao. Access limited to the Smith College community and other researchers while on campus. Smith College community members also may access from off-campus using a Smith College log-in. Other off-campus researchers may request a copy through Interlibrary Loan for personal use.

Language

English

Download

Smith Only:
Off Campus Download

Share

COinS