Ozay E Ilker, Gonzalez-Perez Gabriela, Torres Joe A, Vijayaraghavan Jyothi, Lawlor Rebecca, Sherman Heather L, Garrigan Daniel T, Burnside Amy S, Osborne Barbara A, Tew Gregory N, Minter Lisa M
Program in Molecular and Cellular Biology, University of Massachusetts Amherst, Amherst, Massachusetts, USA.
Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Columbia University, New York, New York, USA.
Mol Ther. 2016 Dec;24(12):2118-2130. doi: 10.1038/mt.2016.177. Epub 2016 Sep 16.
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.
利用抗体靶向细胞蛋白，以便在疾病调节背景下更好地理解细胞信号通路，是一个快速发展的研究领域。人源化抗体因其治疗潜力越来越受到关注，但细胞靶点的收集仅限于那些从细胞分泌或在细胞表面表达的蛋白。这种方法使得大量细胞内蛋白作为抗体结合的潜在靶点未被探索。蛋白激酶Cθ（PKCθ）对T细胞的激活、增殖和分化至关重要，其特定残基的磷酸化是其活性所必需的。在此，我们报告了一种蛋白转导结构域模拟物的设计、合成和表征，该模拟物能够有效地将抗磷酸化PKCθ（Thr538）抗体递送至人外周血单个核细胞，并改变T细胞激活和分化的下游指标的表达。我们使用移植物抗宿主病的人源化淋巴细胞转移模型，来评估蛋白转导结构域模拟物：抗pPKCθ调节在体外递送至人外周血单个核细胞时的持久性。我们证明，蛋白转导结构域模拟物：抗体复合物能够很容易地以高效方式引入难以转染的人外周血单个核细胞，引发足以改变疾病进展的生物学反应。因此，蛋白转导结构域模拟物：抗体递送可能代表一种通过靶向细胞内蛋白来操纵细胞反应的高效体外方法。
