ABSTRACT
Gap junctions, transmembrane protein channels that directly connect the cytoplasm of neighboring cells and enable the exchange of molecules between cells, are a promising new frontier for therapeutic delivery. Specifically, cell-derived lipid vesicles that contain functional gap junction channels, termed Connectosomes, have recently been demonstrated to substantially increase the effectiveness of small molecule chemotherapeutics. However, because gap junctions are present in nearly all tissues, Connectosomes have no intrinsic ability to target specific cell types, which potentially limits their therapeutic effectiveness. To address this challenge, here we display targeting ligands consisting of single-domain antibodies on the surfaces of Connectosomes. We demonstrate that these targeted Connectosomes selectively interact with cells that express a model receptor, promoting the selective delivery of the chemotherapeutic doxorubicin to this target cell population. More generally, our approach has the potential to boost cytoplasmic delivery of diverse therapeutic molecules to specific cell populations while protecting off-target cells, a critical step toward realizing the therapeutic potential of gap junctions.
Subject(s)
Antibodies, Immobilized/metabolism , Cell-Derived Microparticles/metabolism , Gap Junctions/metabolism , Models, Biological , Single-Domain Antibodies/metabolism , Antibiotics, Antineoplastic/administration & dosage , Antibiotics, Antineoplastic/pharmacology , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/genetics , Cell Survival/drug effects , Cell-Derived Microparticles/drug effects , Doxorubicin/administration & dosage , Doxorubicin/adverse effects , Doxorubicin/pharmacology , Drug Compounding , Drug Delivery Systems/adverse effects , Gap Junctions/drug effects , HEK293 Cells , HeLa Cells , Humans , Ligands , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Membrane Fusion , Microscopy, Fluorescence , Protein Transport , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/genetics , Surface PropertiesABSTRACT
T cells recognize and kill a myriad of pathogen-infected or cancer cells using a diverse set of T cell receptors (TCRs). The affinity of TCR to cognate antigen is of high interest in adoptive T cell transfer immunotherapy and antigen-specific T cell repertoire immune profiling because it is widely known to correlate with downstream T cell responses. We introduce the in situ TCR affinity and sequence test (iTAST) for simultaneous measurement of TCR affinity and sequence from single primary CD8(+) T cells in human blood. We demonstrate that the repertoire of primary antigen-specific T cells from pathogen-inexperienced individuals has a surprisingly broad affinity range of 1000-fold composed of diverse TCR sequences. Within this range, samples from older individuals contained a reduced frequency of high-affinity T cells compared to young individuals, demonstrating an age-related effect of T cell attrition that could cause holes in the repertoire. iTAST should enable the rapid selection of high-affinity TCRs ex vivo for adoptive immunotherapy and measurement of T cell response for immune monitoring applications.