CPP-mediated Intracellular Delivery of Antibodies
Disciplines
Cell Biology | Molecular Biology | Pharmaceutics and Drug Design
Abstract (300 words maximum)
Possible new therapeutic drugs often fail because they cannot cross cell membranes to reach intracellular locations where they are needed, a particularly vexing problem for large molecules such as therapeutic antibodies. A possible solution to this problem, and the source of this research project, is using cell-penetrating peptides (CPPs) to deliver macromolecules across membranes. CPPs can cross membranes, carrying with them molecules to which they are attached. In this study, a CPP-adaptor protein, TAT-CaM, was used to deliver a truncated form of Staphylococcal Protein A (CBS-SpAtr) to the cytoplasm of living mammalian cells. SpAtr binds the Fc region of IgGs with pM affinity. Our proximal goal is to use TAT-CaM to mediate delivery of CBS-SpAtr-bound IgG, which would potentially allow delivery of a specific antibodies across cell membranes for the purposes of labelling subcellular structures, neutralizing intracellular pathogens, etc. To test this possibility, we designed, expressed, and purified CBS-SpAtr. Binding kinetics of the purified protein showed fast-on, slow-off kinetics, nM in the presence of calcium, and negligible affinity in its absence. Antibody binding showed the anticipated pM binding and was unhindered in the presence of TAT-CaM binding. Cell-penetration experiments were performed with baby hamster kidney (BHK) cells to assay SpAtr deliveries. Experiments are underway to deliver SpAtr into cells with an attached fluorescent antibody to label subcellular structures, e.g. nuclear pore proteins, to demonstrate proof-of-concept. The results show hope for future success in delivering SpAtr with antibodies into cells for possible new therapeutics
Academic department under which the project should be listed
CSM - Molecular and Cellular Biology
Primary Investigator (PI) Name
Jonathan McMurry
Additional Faculty
Daniel P Morris, Molecular and Cellular Biology, dpmorris9@gmail.com
CPP-mediated Intracellular Delivery of Antibodies
Possible new therapeutic drugs often fail because they cannot cross cell membranes to reach intracellular locations where they are needed, a particularly vexing problem for large molecules such as therapeutic antibodies. A possible solution to this problem, and the source of this research project, is using cell-penetrating peptides (CPPs) to deliver macromolecules across membranes. CPPs can cross membranes, carrying with them molecules to which they are attached. In this study, a CPP-adaptor protein, TAT-CaM, was used to deliver a truncated form of Staphylococcal Protein A (CBS-SpAtr) to the cytoplasm of living mammalian cells. SpAtr binds the Fc region of IgGs with pM affinity. Our proximal goal is to use TAT-CaM to mediate delivery of CBS-SpAtr-bound IgG, which would potentially allow delivery of a specific antibodies across cell membranes for the purposes of labelling subcellular structures, neutralizing intracellular pathogens, etc. To test this possibility, we designed, expressed, and purified CBS-SpAtr. Binding kinetics of the purified protein showed fast-on, slow-off kinetics, nM in the presence of calcium, and negligible affinity in its absence. Antibody binding showed the anticipated pM binding and was unhindered in the presence of TAT-CaM binding. Cell-penetration experiments were performed with baby hamster kidney (BHK) cells to assay SpAtr deliveries. Experiments are underway to deliver SpAtr into cells with an attached fluorescent antibody to label subcellular structures, e.g. nuclear pore proteins, to demonstrate proof-of-concept. The results show hope for future success in delivering SpAtr with antibodies into cells for possible new therapeutics