title: 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.
authors.affiliation: Program in Molecular and Cellular Biology, University of Massachusetts Amherst, Amherst, Massachusetts, USA.
authors.forename: E Ilker
authors.initials: EI
authors.lastname: Ozay
authors.affiliation: Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Columbia University, New York, New York, USA.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Gonzalez-Perez
authors.affiliation: Program in Molecular and Cellular Biology, University of Massachusetts Amherst, Amherst, Massachusetts, USA.
authors.forename: Joe A
authors.initials: JA
authors.lastname: Torres
authors.affiliation: Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, Amherst, Massachusetts, USA.
authors.forename: Jyothi
authors.initials: J
authors.lastname: Vijayaraghavan
authors.affiliation: Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, Amherst, Massachusetts, USA.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Lawlor
authors.affiliation: Program in Molecular and Cellular Biology, University of Massachusetts Amherst, Amherst, Massachusetts, USA.
authors.forename: Heather L
authors.initials: HL
authors.lastname: Sherman
authors.affiliation: Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, Amherst, Massachusetts, USA.
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Garrigan
authors.affiliation: Institute for Applied Life Sciences, University of Massachusetts Amherst, Amherst, Massachusetts, USA.
authors.forename: Amy S
authors.initials: AS
authors.lastname: Burnside
authors.affiliation: Program in Molecular and Cellular Biology, University of Massachusetts Amherst, Amherst, Massachusetts, USA.|Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, Amherst, Massachusetts, USA.
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Osborne
authors.affiliation: Program in Molecular and Cellular Biology, University of Massachusetts Amherst, Amherst, Massachusetts, USA.|Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, Amherst, Massachusetts, USA.|Department of Polymer Science and Engineering, University of Massachusetts Amherst, Amherst, Massachusetts, USA.
authors.forename: Gregory N
authors.initials: GN
authors.lastname: Tew
authors.affiliation: Program in Molecular and Cellular Biology, University of Massachusetts Amherst, Amherst, Massachusetts, USA.|Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, Amherst, Massachusetts, USA.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Minter
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000008:administration & dosage* / Q000737:chemistry / Q000494:pharmacology; D002454:Cell Differentiation; D049109:Cell Proliferation; D057846:Cell-Penetrating Peptides / Q000138:chemical synthesis* / Q000737:chemistry; D006086:Graft vs Host Disease / Q000276:immunology*; D006801:Humans; D056747:Immunomodulation; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000276:immunology; D008213:Lymphocyte Activation; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D000074121:Protein Kinase C-theta; D015398:Signal Transduction / Q000187:drug effects; D018417:Th1 Cells / Q000276:immunology
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D057846:Cell-Penetrating Peptides; D007527:Isoenzymes; C076233:PRKCQ protein, human; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta