Cancer is one of the leading causes of death in the United States. Monoclonal antibody drugs became one of the commercially and clinically successful drugs for many diseases. Among the monoclonal antibodies, T cell-dependent bispecific antibodies directly target tumor cells for cancer treatment, they kill tumor cells by activating T cells through binding to the CD3 (cluster of differentiation 3) receptor on T cells. Many anti-CD3 antibodies bind to the surface exposed CD3 γ, δ, ε subunits for T cell activation. SP34, an anti-CD3ε antibody, specifically binds to the first 27 amino acids of CD3ε. Synthesis of CD3ε peptides proofed to be difficult due to its hydrophobic nature and presence of an N-terminal glutamine that caused many side reactions resulting in very poor peptide quality and purity. For some commercial full-length CD3ε proteins it is unclear whether N-terminal glutamine is present or absent. In cases where N-terminal glutamine is present it is modified to pyroglutamic acid. To study the SP34-CD3ε interaction a reliable and defined source of CD3ε peptide and peptide variants is required. By utilizing the SUMO (Small Ubiquitin-related Modifier) system from yeast, CD3ε1-27 amino acid and a truncated version 2-27 amino acid peptide are expressed in E. coli cells with an SMT3 (Mitotic Fidelity Gene 3) tag. Subsequently, SMT3 tag is removed with SUMO protease and the resulting peptide is further purified. This novel in vitro approach results in high yields of non-modified peptides with great purity (>95%).
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/43859 |
| Date | 10 February 2022 |
| Creators | Kim, Albert |
| Contributors | Abraham, Carmela |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/ |
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