Conditional Cell Penetration of Masked CPPs by an ADEPT-like Approach

Area of Science:

• Biotechnology
• Molecular Biology
• Drug Delivery Systems

Background:

• Cell-penetrating peptides (CPPs) facilitate intracellular cargo delivery but lack cell-type specificity, causing off-target effects.
• Developing targeted CPPs is crucial for safe and effective drug delivery applications.

Purpose of the Study:

• To engineer a conditional and selective activation of cellular uptake using a model CPP, the L17E peptide.
• To demonstrate enzyme-mediated deprotection for controlled CPP activation and targeted delivery.

Main Methods:

• Lysine residues of the L17E peptide were masked with enzyme-cleavable acetyl protecting groups.
• Cellular uptake of a conjugated fluorophore (TAMRA) was assessed with masked and unmasked peptides.
• In vitro deacetylation was performed using NAD-dependent sirtuin 2 (SirT2) deacetylase.
• Antibody-enzyme conjugates (trastuzumab-SirT2, anti-B7H3-SirT2) were created for targeted delivery of a cytotoxin.

Main Results:

• Masking the L17E peptide significantly reduced fluorophore delivery to HeLa cells.
• SirT2 deacetylase restored cellular uptake of the masked L17E peptide.
• Antibody-enzyme conjugates enabled conditional and selective delivery of a cryptophycin cytotoxin.
• Selective cell killing was observed with antibody-enzyme conjugates, despite some residual cytotoxicity of the masked peptide.

Conclusions:

• Enzyme-cleavable masking of CPPs offers a strategy for conditional and selective intracellular delivery.
• Antibody-targeted enzyme-mediated deprotection can achieve specific drug delivery and cell killing.
• This approach holds promise for enhancing the safety and efficacy of CPP-based therapeutics.