Intracellular Delivery of Anti-pPKCθ (Thr538) via Protein Transduction Domain Mimics for Immunomodulation

Abstract

Targeting cellular proteins with antibodies, to better understand cellular signaling pathways in the context of disease modulation, is a fast-growing area of investigation. Humanized antibodies are increasingly gaining attention for their therapeutic potential, but the collection of cellular targets is limited to those secreted from cells or expressed on the cell surface. This approach leaves a wealth of intracellular proteins unexplored as putative targets for antibody binding. Protein kinase Cθ (PKCθ) is essential to T cell activation, proliferation, and differentiation, and its phosphorylation at specific residues is required for its activity. Here we report on the design, synthesis, and characterization of a protein transduction domain mimic capable of efficiently delivering an antibody against phosphorylated PKCθ (Thr538) into human peripheral mononuclear blood cells and altering expression of downstream indicators of T cell activation and differentiation. We used a humanized, lymphocyte transfer model of graft-versus-host disease, to evaluate the durability of protein transduction domain mimic:Anti-pPKCθ modulation, when delivered into human peripheral mononuclear blood cells ex vivo. We demonstrate that protein transduction domain mimic:Antibody complexes can be readily introduced with high efficacy into hard-to-transfect human peripheral mononuclear blood cells, eliciting a biological response sufficient to alter disease progression. Thus, protein transduction domain mimic:Antibody delivery may represent an efficient ex vivo approach to manipulating cellular responses by targeting intracellular proteins.

Protein Mimic Hydrophobicity Affects Intracellular Delivery but not Cargo Binding

“…Herein, a series of variably hydrophobic PTDMs accessed through controlled polymerization were used to probe how hydrophobicity modulates cargo binding and how both parameters affect EGFP delivery in Jurkat T cells . Being able to access intracellular targets in vital human immune cells, such as T cells, will aid researchers studying new biochemical pathways and immunotherapies …”

Associative and Dissociative Processes in Non‐Covalent Polymer‐Mediated Intracellular Protein Delivery

“…Our group has developed and studied PTDMs capable of non‐covalently binding and delivering protein cargo into difficult‐to‐transfect cell lines . Most of this work has centered on chemically optimizing PTDMs through structure–activity relationships, and special attention has been given to optimizing the ratio of hydrophilic to hydrophobic monomers and the overall polymer hydrophobicity (Figure ) .…”

“…While the cellular membrane is passively permeable to small, usually hydrophobic molecules, it generally restricts the passage of large, charged macromolecules such as peptides and proteins . For researchers seeking to deliver proteins intracellularly for fundamental studies or therapeutic development, the membrane imposes a barrier that must be surmounted . The rising interest in and success using proteins as therapeutics also creates an urgent need to optimize and develop new delivery reagents .…”

“…The rising interest in and success using proteins as therapeutics also creates an urgent need to optimize and develop new delivery reagents . Among the various classes of proteins, antibodies are gaining major recognition due to their use as therapeutics . Though antibodies are successful as therapeutics, they are restricted to the extracellular environment, thereby requiring the use of delivery reagents if their intended target resides inside the cell .…”

“…[2] For researchers seeking to deliver proteins intracellularly for fundamentals tudies or therapeutic development, the membrane imposesabarrier that must be surmounted. [3,4] The rising interest in and success using proteins as therapeutics also createsa nu rgent need to optimize and develop new delivery reagents. [3,[5][6][7][8] Among the various classes of proteins, antibodies are gaining major recognition due to their use as therapeutics.…”