Structural basis of IL-23 antagonism by an Alphabody protein scaffold

Protein scaffolds can provide a promising alternative to antibodies for various biomedical and biotechnological applications, including therapeutics. Here we describe the design and development of the Alphabody, a protein scaffold featuring a single-chain antiparallel triple-helix coiled-coil fold. We report affinity-matured Alphabodies with favourable physicochemical properties that can specifically neutralize human interleukin (IL)-23, a pivotal therapeutic target in autoimmune inflammatory diseases such as psoriasis and multiple sclerosis. The crystal structure of human IL-23 in complex with an affinity-matured Alphabody reveals how the variable interhelical groove of the scaffold uniquely targets a large epitope on the p19 subunit of IL-23 to harness fully the hydrophobic and hydrogen-bonding potential of tryptophan and tyrosine residues contributed by p19 and the Alphabody, respectively. Thus, Alphabodies are suitable for targeting protein-protein interfaces of therapeutic importance and can be tailored to interrogate desired design and binding-mode principles via efficient selection and affinity-maturation strategies

Cell-penetrating Alphabody protein scaffolds for intracellular drug targeting

The therapeutic scope of antibody and nonantibody protein scaffolds is still prohibitively limited against intracellular drug targets. Here, we demonstrate that the Alphabody scaffold can be engineered into a cell-penetrating protein antagonist against induced myeloid leukemia cell differentiation protein MCL-1, an intracellular target in cancer, by grafting the critical B-cell lymphoma 2 homology 3 helix of MCL-1 onto the Alphabody and tagging the scaffold’s termini with designed cell-penetration polypeptides. Introduction of an albumin-binding moiety extended the serum half-life of the engineered Alphabody to therapeutically relevant levels, and administration thereof in mouse tumor xenografts based on myeloma cell lines reduced tumor burden. Crystal structures of such a designed Alphabody in complex with MCL-1 and serum albumin provided the structural blueprint of the applied design principles. Collectively, we provide proof of concept for the use of Alphabodies against intracellular disease mediators, which, to date, have remained in the realm of small-molecule therapeutics

Frameshifting in the P6 cDNA Phage Display System

Phage display is a powerful technique that enables easy identification of targets for any type of ligand. Targets are displayed at the phage surface as a fusion protein to one of the phage coat proteins. By means of a repeated process of affinity selection on a ligand, specific enrichment of displayed targets will occur. In our studies using C-terminal display of cDNA fragments to phage coat protein p6, we noticed the occasional enrichment of targets that do not contain an open reading frame. This event has previously been described in other phage display studies using N-terminal display of targets to phage coat proteins and was due to uncommon translational events like frameshifting. The aim of this study was to examine if C-terminal display of targets to p6 is also subjected to frameshifting. To this end, an enriched target not containing an open reading frame was selected and an E-tag was coupled at the C-terminus in order to measure target display at the surface of the phage. The tagged construct was subsequently expressed in 3 different reading frames and display of both target and E-tag measured to detect the occurrence of frameshifting. As a result, we were able to demonstrate display of the target both in the 0 and in the +1 reading frame indicating that frameshifting can also take place when C-terminal fusion to minor coat protein p6 is applied

Autoantibodies to two novel peptides in seronegative and early rheumatoid arthritis

Pathophysiology and treatment of rheumatic disease