Creative Biolabs has built up a novel intrabody generation platform, Super™ Intrabody Platform, based on an innovative combination of Yeast Two-hybrid System with antibody library technology.
Intrabodies are antibodies that work within cells to bind to intracellular antigens. They are directed against intracellular target molecules and expressed within a specific subcellular compartment as directed by the intracellular localization signals genetically fused to N- or C-terminus. For this reason, intrabodies are defined as antibodies that have been modified for intracellular localization. As antibodies ordinarily are designed to be secreted from cells, intrabodies require special alterations, including modification of sequences for hyperstability, selection of antibodies resistant to the more reducing intracellular environment, or expression as a fusion protein with maltose binding protein or other stable intracellular proteins. Intrabodies have wide applications in dissecting target protein function, in target validation and functional genomics, as well as acting as potential therapeutic reagents. Intrabodies have promising applications against hepatitis B, avian influenza, prion diseases,inflammation, Parkinson's disease, and Huntington's disease.
Super™ Intrabody Platform develops intrabodies in the form of single domain antibody, although human antibodies in scFv format have been developed as first generation of intrabodies. Single domain antibodies (also called as sdAbs, VHH antibodies or domain antibodies) are antibody fragments consisting of a single monomeric variable antibody domain. With a molecular weight of only 12–15 kDa, single domain antibodies are much smaller than common antibodies (150–160 kDa), and even smaller than Fab fragments (~50 kDa) and single-chain variable fragments (~25 kDa).
We have created a large single domain llama antibody library using Matchmaker™ Gold Yeast Two-hybrid System (Clontech), a GAL4-based two-hybrid assay, in which an antigen protein is expressed as a fusion to the Gal4 DNA-binding domain (DNA-BD), while single domain antibodies engineered from heavy-chain antibodies found in camelids are expressed as fusions to the Gal4 activation domain (AD). As a result, when antigen (bait) and antibody library (prey) fusion proteins interact, the DNA-BD and AD are brought into proximity to activate transcription of reporter genes, making the discovery of high affinity single domain antibodies specific for the antigen.
Since the antibodies discovered on this platform are expressed and selected inside of yeast cells, there are the best intrabodies in the first place that do not require further sequence modification. Also, this platform does not require antigen in a purified protein form to raise antibodies. Instead, the gene of the antigen is expressed into an intracellular protein to capture its antibodies in the yeast cells. As a result, intrabodies discovered from this platform usually recognize the native conformation of the antigen and are of immediate therapeutic potential.