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Engineering Protein-Drug Conjugates for Cancer Immunotherapy

Primary Investigators:
John T. Wilson (PI)
Blaise Kimmel (postdoc and primary research mentor)
Neil Chada (graduate student)
Brief Description of Project:
The goal of this project is to engineer a bispecific nanobody for tumor targeted delivery of small molecule immunotherapeutics. The undergraduate student, Jonah Finkelstein, has been working in our lab over the past academic year and has developed skills in cloning and protein engineering and production. During the summer, he will further leverage these skills to conduct an independent project in which he will engineer a bispecific nanobody with one domain that targets serum albumin and another domain that targets B7-H3 and has a tag for site-selective ligation of a small molecule STING agonist. This will provide experience in areas of molecular biology, chemistry, immunology, and pharmaceutical engineering.

Desired Qualifications:
Prior experience in cell and molecular biology, including cloning and protein expression.
Nature of Supervision:
The student will supervised by a postdoc in the lab, Dr. Blaise Kimmel, and a graduate student in the lab, Neil Chada. They will supervise all aspects of the project and will work closely with the student over the course of the summer, interacting almost daily. The PI will meet with the group at least every two weeks to discuss the project and the PI and student will meet individually at the beginning and end of the project period to discuss project and career development goals. The student will have an opportunity to present their work throughout the semester in weekly lab and small group meetings.
A Brief Research Plan (period is for 10 weeks):
The proposed research plan for the 10 week period is:
1. Design and build a plasmid for expression of a fusion protein comprising an anti-albumin and an anti-B7H3 nanobody that also has a site for sortase-mediated ligation of azide groups. 
2. Clone the construct into E. coli. The student has experience with cloning and expression of the anti-albumin nanobody and will use this workflow to clone a fusion protein that contains the anti-B7H3 domain. 
3. Express and purify the fusion protein. The student has experience with this process and so will begin to work independently to express and purify the protein and characterize it by SDS-PAGE and mass spectrometry. 
4. Perform a sortase-mediated ligation of an amino-PEG-azide linker and characterize conjugation efficiency via mass spectrometry. 
5. Conjugate a DBCO-modified dye to the azide group of the protein and characterize via UV-vis spectroscopy. 
6. Evaluate the binding affinity of the fusion protein to albumin via isothermal calorimetry and binding to B7-H3-expressing cells via flow cytometry.

Number of Open Slots: 1
Contact Information:
Name: John T. Wilson
Department: Chemical and Biomolecular Engineering