Growth signalobody selects functional intrabodies in the mammalian cytoplasm

Abstract

A versatile strategy to inhibit protein functions in the cytoplasmic environment is eagerly anticipated for drug discovery. In this study, we demonstrate a novel system to directly select functional intrabodies from a library in the mammalian cytoplasm. In this system, a target homo-oligomeric antigen is expressed together with a single-chain Fv (scFv) library that is linked to the cytoplasmic domain of a receptor tyrosine kinase (RTK) in the cytoplasm of murine interleukin-3 (IL-3)-dependent cells. As the tyrosine kinase is activated by dimerization, only scFv-RTK clones that can bind to the target antigen would be oligomerized and transduce a growth signal under the IL-3-deprived condition, which leads to selection of functional intrabodies. To demonstrate this system, we used rabies virus phosphoprotein (RV-P) that forms dimers in the cytoplasm as a target antigen. As a result, functional intrabodies were selected using our system from a naïve scFv library as well as from a pre-selected anti-RV-P library generated by phage display. This system may be applied for screening intrabodies that can prevent progression of various severe diseases.

Targeted Intracellular Delivery of Antibodies: The State of the Art

“…To overcome this limitation, many different strategies for the generation and/or selection of suitable antibodies for cytosolic expression have been developed. These approaches include: use of single-domain antibody variants [camelid VHH, shark-derived variable new antigen receptors, or variable domain of the heavy chain (VH) or light chain (VL) selected from human antibodies] ( Boldicke, 2017 ); fusion with different protein domains [maltose binding protein ( Bach et al, 2001 ), Fc-domain ( Strube and Chen, 2004 ), proteasome-targeting PEST motif ( Joshi et al, 2012 ), and others ( Jurado et al, 2006 ); grafting of complementary determining regions ( Donini et al, 2003 ); construction and selection of intrabodies lacking S–S bonds ( Colby et al, 2004 ; Cetin et al, 2017 ); numerous eukaryotic in vitro selection-based strategies aimed at isolating a functional and soluble antibody fragment ( Guglielmi et al, 2011 ; Matz et al, 2014 ; Lee et al, 2016 ) and even electrostatic manipulation via the introduction of negative charges ( Kvam et al, 2010 ; Liu et al, 2015 ). In addition to direct interaction with the antigen located in the cytosol, simple fusion with an appropriate signal peptide ( Biocca et al, 1991 ) can redirect intrabodies expressed in the cytosol to bind the antigens within the cell nucleus ( Verheesen et al, 2006 ) or mitochondrion ( Biocca et al, 1995 ).…”

Biomolecular engineering for nanobio/bionanotechnology

“…Inspired by natural fusion proteins, synthetic fusion proteins have been designed to achieve synergistically improved bioactivities or to generate novel functional combinations derived from each of their component moieties, which are integrated into one molecule by peptide linkers. The fusion proteins have been widely applied in various areas, including recombinant protein production by the tag-mediated enhancement of protein expression, solubility and high-throughput purification [291, 292], fluorescent protein-mediated molecular imaging [293], advanced biocatalysis [101, 108, 111, 115, 164, 290, 294–297], biosensing and bioelectronic materials [290, 298–300], pharmaceuticals, diagnostics and therapeutics [208, 290, 301, 302], reporter protein-mediated immunoassays [303–310], the chimeric receptor-mediated control of cell fate, e.g., growth, death, migration or differentiation [311–319], the library selection of antibodies [203, 320, 321] and antibody-mediated drug delivery [218, 322, 323]. …”

Antibodies inside of a cell can change its outside: Can intrabodies provide a new therapeutic paradigm?

“…antibodies that are stable even in the absence of disulfide bonds, various approaches relying on two hybrid-like and other in-cell interaction analysis techniques or quality control screenings have been developed (for review see [32] ). Some of these screening technologies allow selection of intrabodies that are stable in the cytosol in an antigen-specific way [28] , [48] , [50] , [51] , [54] , while other screening technologies allow to more generally screen for antibodies that are able to fold correctly in the cytosol independently of their antigen-specificity [3] , [19] .…”