Intracellular delivery of anti-pPKCθ (T538) via protein transduction domain mimics for T cell immunomodulation

• Published in: The Journal of immunology (1950) 2016-05, Vol.196 (1_Supplement), p.214-214.5
• Main Author: Ozay, Emrah Ilker
• Format: Article
• Language: English
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.196.Supp.214.5

Humanized antibodies are increasingly gaining attention for their therapeutic potential in immunotherapy but, to date, the collection of cellular targets has been limited to those that are secreted from cells or are expressed on the cell surface. This approach leaves a wealth of intracellular proteins unexplored as potential targets. We evaluated the potential for delivering a phospho-specific antibody into human peripheral blood mononuclear cells (hPBMCs) as a means to interrogate and modulate their response to activating stimuli. Protein kinase C-theta (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 (PTDM) capable of efficiently delivering the antibody against phosphorylated PKCθ (T538) into hPBMCs. We designed an in vitro delivery method for the PTDM:anti-pPKCθ complex then assessed T cell activation and the expression of various downstream indicators of T cell activation and differentiation. In proof-of-principle experiments using a humanized, lymphocyte transfer model of graft-vs-host disease, we evaluated the durability of PTDM:anti-pPKCθ modulation, when delivered into hPBMCs ex vivo. Altogether, these results demonstrate that PTDM:antibody complexes can be readily introduced with high efficacy into traditionally hard-to-transfect human PBMCs to modulate immune function, eliciting a biological response sufficient to alter disease progression. Thus, PTDM:antibody delivery may represent an efficient ex vivo approach to manipulating cellular responses by targeting intracellular proteins.