# Designing a Cleavable Cell Surface Protein for Cytotherapy and Drug Delivery Applications
###### Abstract  
Many cytotherapy applications focus on delivering a therapeutic molecule or nanoparticle to a disease site. One challenging step in this delivery is releasing the therapeutic molecule from the delivery cell upon arrival at the delivery sight. Here a protein is designed and expressed that can bind a biotin-labeled cargo and release that cargo in response to the presence of urokinase plasminogen activator. A gene was designed that coded for a protein that contained a streptavidin domain for binding biotin-labeled cargo, a urokinase cleavage domain for release by urokinase plasminogen activator, and a PLAP domain for cell-surface expression. The utility of the resultant protein was tested with biotin (5-fluorescein) and a biotinylated PLGA nanoparticle to test the performance of the delivery systems with models for small molecule drugs and nanoformulations. When expressed in neural progenitor cells (C17.2), the designed protein was able to bind both the biotin (5-fluorescein) and the biotinylated PLGA nanoparticles and was able to release the biotin (5-fluorescein) in response to urokinase plasminogen activator. This designed, multi-domain protein may prove useful as a method for specifically releasing a cargo from delivery cells at a target site. synthetic biology; cytotherapy; PLGA nanoparticles; streptavidin-biotin +
Footnote †: journal:  
the plasmin system and cleaves the inactive plasminogen into active plasmin which has cascade effects modifying the extracellular matrix, vascular development, and growth factor availability. uPA also cleaves its cell-surface receptor, the urokinase plasminogen activator receptor (uPAR). A consensus sequence for uPA has been identified [17; 18], SGRSA, that has been used for a variety of medicinal applications [18; 19]. If a payload was connected to a delivery cell with a linker that was cleavable by uPA the payload would remain attached to the delivery cell until it reached an area with high uPA activity at which point it would be released. Streptavidin [20] is a well-known bacterial protein that binds biotin with high affinity. This binding affinity makes streptavidin an attractive possibility for engineering a protein that would attach a biotin-labeled payload to the surface of a cell. Unfortunately, while streptavidin binds biotin in a 1:1 ratio, the actual binding event occurs between four molecules of streptavidin cooperatively binding four molecules of biotin. This 4:4 correspondence creates challenges for successfully engineering a hybrid protein containing a streptavidin domain. Recently, a modified streptavidin protein has been engineered that acts as a monomer, binding biotin on a 1:1 basis (referred to as mono-streptavidin, mSA) [21; 22]. This expands its potential for engineering into a designed protein as a biotin binding moiety. Placent alkaline phosphatase (PLAP) [23] is a membrane anchored protein that is used as a model to understand phosphatidylcholine-glycan (GPI) anchored proteins. A sequence (PLAP domain) has been identified at the C-terminal end of the PLAP protein which signals the attachment of the protein to the GPI anchor and expression on the surface of the cell. This domain has been engineered into other proteins to cause their cell surface expression through GPI anchors [23]. Here, a cleavable mono-streptavidin (cmSA) delivery protein is designed and tested that incorporates an mSA domain for binding biotin-labeled payloads, a uPA cleavage domain for efficient release, and a PLAP domain for cell surface anchoring (Figure 1). This protein could be one method for attaching and delivering a payload using cytotherapy.