title: Conditional Cell Penetration of a Masked CPP and Optimization of Multimeric CPP-Delivery Modules for Intracellular Cargo Delivery
Creators: name: Hofmann, Sarah
Publisher: name: UNSPECIFIED
Year: 2024

description: The development of biotherapeutics has evolved from less specific small molecule drugs to targeted delivery systems using receptor-specific ligands, lowering therapeutic doses and reducing off-target effects. Most protein therapeutics are developed to target specific proteins on the cell surface, modulating cellular pathways downstream of target binding. Due to their large size and hydrophilic nature, these therapeutics are often limited to extracellular targets, as they cannot permeate the cell membrane. However, since many oncogenic disorders result from dysregulated intracellular protein-protein interactions, targeted intracellular delivery of biomolecules remains a highly desired strategy for therapeutic efficiency. Cell-penetrating peptides (CPPs) are promising molecules for intracellular targeting, as they can translocate themselves and various cargos across cell membranes. However, CPPs would benefit from increased selectivity for tumor cells. This also applies for the intracellular delivery of larger cargos which may require multimerization of CPPs on a scaffold to enhance their ability to interact with the cell membrane for efficient permeation of the cell membrane.
This doctoral thesis presents a series of studies aimed at developing conditionally activated CPPs for cell-targeted intracellular delivery and optimizing multimeric CPP delivery modules.
In the first part, the cationic and amphiphilic L17E peptide was used as model CPP to generate an activatable CPP and demonstrate the conditional and selective intracellular delivery an attached cargo. The under physiological conditions positively charged lysine residues of the L17E peptide were masked with protecting groups to investigate their role in cellular uptake. An enzymatic cleavage system was then developed to activate the peptide conditionally. Protecting all five lysine residues of the peptide showed impaired cellular uptake, while enzymatic cleavage of the protecting groups resulted in the reactivation of intracellular delivery. A following uptake assay by an ADEPT (Antibody Dependent Enzyme Prodrug Therapy)-like delivery procedure using a HER2-targeting antibody-enzyme conjugate demonstrated the selective delivery of a toxin with increased cell killing observed on the HER2-positive cell line compared to the HER2-negative cell line. 
Secondly, encouraged by the results on the conditional delivery by the activatable L17E peptide, the concept was extended to a dextran scaffold carrying multiple L17E peptides. However, the delivery of a fluorophore showed ambiguous results and demasking of the protecting groups of the L17E peptide on dextran could not be verified.
The third part investigated streptavidin as a versatile multifaceted delivery platform. With its four binding sites, it serves as a scaffold for binding up to four biotinylated components simultaneously. For example, any cargo or dextran-conjugate carrying CPPs, therapeutic drugs, receptor-targeting ligands or any other effector molecule would only need to be biotinylated to be assembled on streptavidin. Since previous investigations using the L17E-dextran conjugates on streptavidin showed significant cytotoxicity, optimization was required. The number of L17E peptides per dextran molecule was optimized and further CPPs were investigated. These included a cell lytic attenuated variant of the L17E peptide, the L17E/Q21E peptide, a stapled peptide ATSP-7041 and an α-helical peptide apCC Di B. Of these new dextran-CPP conjugates, at least two L17E- and L17E/Q21E-dextran conjugates per streptavidin molecule with an eGFP as cargo was required for intracellular delivery of the architecture. Cellular proliferation assays of these streptavidin architectures indicated for successful cellular internalization some cytotoxicity needs to be accepted.