Engineering therapeutic proteins for cell entry: the natural approach.

Owing to the challenges of cell entry, protein-based therapies have so far been restricted to extracellular targets, whereas intracellular targets have been almost exclusively addressed by small molecules. The specificity and potency of proteins would enable them to be effective intracellular drugs, provided that the proteins are delivered efficiently to appropriate intracellular compartments within specific cell types. By mimicking the natural mechanisms of toxins and other natural proteins, new intracellular delivery systems are being developed, the first of which are showing clinical efficacy. This review highlights a range of ingenious approaches designed to adapt natural entry mechanisms to facilitate delivery of proteins and open up a range of validated intracellular targets to modulation by potent and specific therapeutic drugs.

Structural and pharmacological characterization of novel potent and selective monoclonal antibody antagonists of glucose-dependent insulinotropic polypeptide receptor.

Glucose-dependent insulinotropic polypeptide (GIP) is an endogenous hormonal factor (incretin) that, upon binding to its receptor (GIPr; a class B G-protein-coupled receptor), stimulates insulin secretion by beta cells in the pancreas. There has been a lack of potent inhibitors of the GIPr with prolonged in vivo exposure to support studies on GIP biology. Here we describe the generation of an antagonizing antibody to the GIPr, using phage and ribosome display libraries. Gipg013 is a specific competitive antagonist with equally high potencies to mouse, rat, dog, and human GIP receptors with a Ki of 7 nm for the human GIPr. Gipg013 antagonizes the GIP receptor and inhibits GIP-induced insulin secretion in vitro and in vivo. A crystal structure of Gipg013 Fab in complex with the human GIPr extracellular domain (ECD) shows that the antibody binds through a series of hydrogen bonds from the complementarity-determining regions of Gipg013 Fab to the N-terminal α-helix of GIPr ECD as well as to residues around its highly conserved glucagon receptor subfamily recognition fold. The antibody epitope overlaps with the GIP binding site on the GIPr ECD, ensuring competitive antagonism of the receptor. This well characterized antagonizing antibody to the GIPr will be useful as a tool to further understand the biological roles of GIP.

Isolation of a small-molecule inhibitor of the antiapoptotic protein Bcl-xL from a DNA-encoded chemical library.

Bcl-xL is an antiapoptotic member of the Bcl-2 protein family and an attractive target for the development of anticancer agents. Here we describe the isolation of binders to Bcl-xL from a DNA-encoded chemical library using affinity-capture selections and massively parallel high-throughput sequencing of >30,000 sequence tags of library members. The most potent binder identified, compound 19/93 [(R)-3-(amido indomethacin)-4-(naphthalen-1-yl)butanoic acid], bound to Bcl-xL with a dissociation constant (K(d)) of 930 nM and was able to compete with a Bak-derived BH3 peptide, an antagonist of Bcl-xL function.

Affinity maturation of phage display antibody populations using ribosome display.

A comparison has been performed, using phage display or ribosome display, of stringent selections on antibody populations derived from three rounds of phage display selection. Stringent selections were performed by reducing concentrations of the antigen, bovine insulin, down to 1 nM. Higher affinity antibodies were isolated using ribosome display in a process that introduces random mutations across the clone population. Whereas the highest affinity antibody produced by phage display, D3, has a K(d) of 5.8 nM as a scFv fragment, ribosome display generated higher affinity variants of this antibody with K(d) values of 189 pM and 152 pM, without or with the use of error prone mutagenesis, respectively. The affinities were further increased for each antibody on conversion of the scFv fragments to whole IgG format, to a K(d) of less than 21 pM for the highest affinity variant of D3. Mutation of VH D101 of antibody D3 to glycine or valine, removing the salt bridge between K94 and D101 at the base of VHCDR3, was responsible for the enhanced affinity observed. In addition to the variants of D3, other unrelated antibodies of comparable or higher affinity for insulin, were isolated by ribosome display, but not phage display, indicating that ribosome display can enrich for different populations of antibodies. Affinity maturation of phage antibody populations using ribosome display is a valuable method of rapidly generating diverse, high affinity antibodies to antigen and should be readily applicable to the isolation of antibodies for the detection and assay of biomarkers.